Food-grade TiO2 Research Articles

Biotransformation of Food-Grade and Nanometric TiO2 in the Oral-Gastro-Intestinal Tract: Driving Forces and Effect on the Toxicity toward Intestinal Epithelial Cells.

Background: Oral exposure to titanium dioxide (TiO2) is common since it is widely used in food and pharmaceutical products. Concern on the safety of this substance has been recently raised, due to the presence of an ultrafine fraction in food-grade TiO2. Discrepancy exists among data reported in in vitro and in vivo studies on intestinal acute/chronic toxicity of TiO2. This might be due to the different biological identity of TiO2 in traditional in vitro test by respect in vivo conditions. Methods: One food-grade TiO2 and two nanometric TiO2 samples were treated with a simulated human digestive dystem (SHDS) in order to investigate the bio-transformation occurring to the particles once ingested in term of size distribution (Dynamic Light Scattering—DLS-, Flow Particle Imaging, Asymmetric Flow Field Flow Fractionation-AF4-) and surface modification (Electrophoretic Light Scattering—ELS-, Electron Paramagnetic Resonance Spectroscopy—EPR-). The effect of SHDS on the cyto-, genotoxicity and potential to induce oxidative stress towards human colorectal carcinoma HCT116 cells was also assessed. Results: Aggregation as a consequence of the high ionic strength of the gastric and intestinal simulated fluids was observed, together with the formation of a partially irreversible bio-corona containing phosphate ions and proteins. Such bio-corona led to a partial masking of the TiO2 particles surface and reactivity. Pristine and treated TiO2 nanoparticles showed comparable acute toxicity and genotoxicity toward HCT116 cells, whereas a small decrease of the induction of oxidative stress after treatment was observed. Conclusions: Overall the results underline the importance of SHDS as a tool to improve the predictive power of in vitro tests towards intestinal nanomaterial toxicity.

Food-Grade TiO2 Particles Generate Intracellular Superoxide and Alter Epigenetic Modifiers in Human Lung Cells.

Titanium dioxide (TiO2) particles are a common ingredient in food, providing the bright white color for many candies, gums, and frostings. While ingestion of these materials has been examined previously, few studies have examined the effect of these particles on lung cells. Inhalation is an important exposure pathway for workers processing these foods and, more recently, home users who purchase these particles directly. We examine the response of lung cells to food-grade TiO2 particles using a combination of fluorescence microscopy and RT-PCR. These experiments show that TiO2 particles generate intracellular reactive oxygen species, specifically superoxide, and alter expression of two epigenetic modifiers, histone deacetylase 9 (HDAC9) and HDAC10. We use a protein corona formed from superoxide dismutase (SOD), an enzyme that scavenges superoxide, to probe the relationship between TiO2 particles and superoxide generation. These experiments show that low, non-cytotoxic, concentrations of food-grade TiO2 particles lead to cellular responses, including altering two enzymes responsible for epigenetic modifications. This production of superoxide and change in epigenetic modifiers could affect human health following inhalation. We expect this research will motivate future in vivo experiments examining the pulmonary response to food-grade TiO2 particles.

Basal Ti level in the human placenta and meconium and evidence of a materno-foetal transfer of food-grade TiO2 nanoparticles in an ex vivo placental perfusion model

BackgroundTitanium dioxide (TiO2) is broadly used in common consumer goods, including as a food additive (E171 in Europe) for colouring and opacifying properties. The E171 additive contains TiO2 nanoparticles (NPs), part of them being absorbed in the intestine and accumulated in several systemic organs. Exposure to TiO2-NPs in rodents during pregnancy resulted in alteration of placental functions and a materno-foetal transfer of NPs, both with toxic effects on the foetus. However, no human data are available for pregnant women exposed to food-grade TiO2-NPs and their potential transfer to the foetus. In this study, human placentae collected at term from normal pregnancies and meconium (the first stool of newborns) from unpaired mothers/children were analysed using inductively coupled plasma mass spectrometry (ICP-MS) and scanning transmission electron microscopy (STEM) coupled to energy-dispersive X-ray (EDX) spectroscopy for their titanium (Ti) contents and for analysis of TiO2 particle deposition, respectively. Using an ex vivo placenta perfusion model, we also assessed the transplacental passage of food-grade TiO2 particles.ResultsBy ICP-MS analysis, we evidenced the presence of Ti in all placentae (basal level ranging from 0.01 to 0.48 mg/kg of tissue) and in 50% of the meconium samples (0.02–1.50 mg/kg), suggesting a materno-foetal passage of Ti. STEM-EDX observation of the placental tissues confirmed the presence of TiO2-NPs in addition to iron (Fe), tin (Sn), aluminium (Al) and silicon (Si) as mixed or isolated particle deposits. TiO2 particles, as well as Si, Al, Fe and zinc (Zn) particles were also recovered in the meconium. In placenta perfusion experiments, confocal imaging and SEM-EDX analysis of foetal exudate confirmed a low transfer of food-grade TiO2 particles to the foetal side, which was barely quantifiable by ICP-MS. Diameter measurements showed that 70 to 100% of the TiO2 particles recovered in the foetal exudate were nanosized.ConclusionsAltogether, these results show a materno-foetal transfer of TiO2 particles during pregnancy, with food-grade TiO2 as a potential source for foetal exposure to NPs. These data emphasize the need for risk assessment of chronic exposure to TiO2-NPs during pregnancy.

Lipid and protein corona of food-grade TiO2 nanoparticles in simulated gastrointestinal digestion

In the presence of biological matrices, engineered nanomaterials, such as TiO2, develop a biomolecular corona composed of lipids, proteins, etc. In this study, we analyzed the biocorona formed on the food grade TiO2 (E171) going through an in vitro simulated gastrointestinal digestion system in either a fasting food model (FFM), a standardized food model (SFM), or a high fat food model (HFFM). Lipids and proteins were extracted from the biocorona and underwent untargeted lipidomic and label-free shotgun proteomic analyses. Our results showed that the biocorona composition was different before and after food digestion. After digestion, more diverse lipids were adsorbed compared to proteins, most of which were the enzymes added to the simulated digestion system. The corona lipid profile was distinct from the digested food model they presented in, although similarity in the lipid profiles between the corona and the food matrix increased with the fat content in the food model. The corona formed in the two low-fat environments of FFM and SFM shared a higher degree of similarity while very different from their corresponding matrix, with some lipid species adsorbed with high enrichment factors, indicating specific interaction with the TiO2 surface outperforming lipid matrix concentration in determination of corona formation. Formation of the biocorona may have contributed to the reduced oxidative stress as well as toxicological impacts observed in cellular studies. The present work is the first to confirm persistent adsorption of biomolecules could occur on ingested nanomaterials in food digestae. More future studies are needed to study the in vivo impacts of the biocorona, and shed light on how the biocorona affects the biotransformations and fate of the ingested nanomaterials, which may impose impacts on human health.

Toxic effects of the food additives titanium dioxide and silica on the murine intestinal tract: Mechanisms related to intestinal barrier dysfunction involved by gut microbiota

This study aimed to compare the effects of three food-grade particles (micro-TiO2, nano-TiO2, and nano-SiO2) on the murine intestinal tract and to investigate their potential mechanisms of action. A 28-day oral exposure murine model was established. Samples of blood, intestinal tissues and colon contents were collected for detection. The results showed that all three particles could cause inflammatory damage to the intestine, with nano-TiO2 showing the strongest effects. Exposure also led to changes in gut microbiota, especially mucus-associated bacteria. Our results suggest that the toxic effects on the intestine were due to reduced intestinal mucus barrier function and an increase in metabolite lipopolysaccharides which activated the expression of inflammatory factors downstream. In mice exposed to nano-TiO2, the intestinal PKC/TLR4/NF-κB signalling pathway was activated. These findings will raise awareness of toxicities associated with the use of food-grade TiO2 and SiO2.

Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO2 particles: an in vivo and ex vivo study in mice

Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO2 particles: an in vivo and ex vivo study in mice

Jejunal villus absorption and paracellular tight junction permeability are major routes for early intestinal uptake of food-grade TiO2 particles: an in vivo and ex vivo study in mice

BackgroundFood-grade TiO2 (E171 in the EU) is widely used as a coloring agent in foodstuffs, including sweets. Chronic dietary exposure raises concerns for human health due to proinflammatory properties and the ability to induce and promote preneoplastic lesions in the rodent gut. Characterization of intestinal TiO2 uptake is essential for assessing the health risk in humans. We studied in vivo the gut absorption kinetics of TiO2 in fasted mice orally given a single dose (40 mg/kg) to assess the ability of intestinal apical surfaces to absorb particles when available without entrapment in the bolus. The epithelial translocation pathways were also identified ex vivo using intestinal loops in anesthetized mice.ResultsThe absorption of TiO2 particles was analyzed in gut tissues by laser-reflective confocal microscopy and ICP-MS at 4 and 8 h following oral administration. A bimodal pattern was detected in the small intestine: TiO2 absorption peaked at 4 h in jejunal and ileal villi before returning to basal levels at 8 h, while being undetectable at 4 h but significantly present at 8 h in the jejunal Peyer’s patches (PP). Lower absorption occurred in the colon, while TiO2 particles were clearly detectable by confocal microscopy in the blood at 4 and 8 h after treatment. Ex vivo, jejunal loops were exposed to the food additive in the presence and absence of pharmacological inhibitors of paracellular tight junction (TJ) permeability or of transcellular (endocytic) passage. Thirty minutes after E171 addition, TiO2 absorption by the jejunal villi was decreased by 66% (p < 0.001 vs. control) in the presence of the paracellular permeability blocker triaminopyrimidine; the other inhibitors had no significant effect. Substantial absorption through a goblet cell (GC)-associated pathway, insensitive to TJ blockade, was also detected.ConclusionsAfter a single E171 dose in mice, early intestinal uptake of TiO2 particles mainly occurred through the villi of the small intestine, which, in contrast to the PP, represent the main absorption surface in the small intestine. A GC-associated passage and passive diffusion through paracellular TJ spaces between enterocytes appeared to be major absorption routes for transepithelial uptake of dietary TiO2.

Mechanism of Action of TiO2: Recommendations to Reduce Uncertainties Related to Carcinogenic Potential.

The Risk Assessment Committee of the European Chemicals Agency issued an opinion on classifying titanium dioxide (TiO2) as a suspected human carcinogen upon inhalation. Recent animal studies indicate that TiO2 may be carcinogenic through the oral route. There is considerable uncertainty on the carcinogenicity of TiO2, which may be decreased if its mechanism of action becomes clearer. Here we consider adverse outcome pathways and present the available information on each of the key events (KEs). Inhalation exposure to TiO2 can induce lung tumors in rats via a mechanism that is also applicable to other poorly soluble, low-toxicity particles. To reduce uncertainties regarding human relevance, we recommend gathering information on earlier KEs such as oxidative stress in humans. For oral exposure, insufficient information is available to conclude whether TiO2 can induce intestinal tumors. An oral carcinogenicity study with well-characterized (food-grade) TiO2 is needed, including an assessment of toxicokinetics and early KEs.

Evaluation of the cytotoxic and cellular proteome impacts of food-grade TiO2 (E171) using simulated gastrointestinal digestions and a tri-culture small intestinal epithelial model

Engineered nanomaterials (ENMs) are widely used in the food industry; however, regulations for ENMs in food are still in the early stages of development due to insufficient health data. This study investigated the cytotoxicity and changes to the proteomic profile in an in vitro small intestinal epithelium model after exposure to digested food models containing the ubiquitous engineered particulate food additive, TiO2 (E171) with an average size around 110 nm. TiO2 at 0.75% or 1.5% (w/w) concentrations in either a fasting food model (FFM) or a standardized food model (SFM) based on American diet were digested using an in vitro oral-gastric-small intestinal simulator, and the resulting digestas were applied to a small intestinal epithelium tri-culture cellular model. Effects on cell layer integrity, cytotoxicity, and oxidative stress were assessed. In order to explore the impact on cellular processes beyond basic cytotoxicity, mass spectrometry-based quantitative proteomic analyses of control and exposed tri-culture cells was performed. TiO2 in FFM, but not in SFM, produced significant, dose-dependent cytotoxicity (24%, p < 0.001), and at the higher dose caused significant oxidative stress (1.24-fold, p < 0.01), indicative of a food matrix effect. No significant perturbations of the cellular proteome were observed with TiO2 in either FFM or SFM food models. However, proteins involved in energy metabolism and protein synthesis were up-regulated by digestas from SFM compared to those from FFM, indicative of a food matrix effect on the cellular proteome. Interestingly, the differences in profiles between the two food models were more pronounced in the presence of TiO2. Together, these results indicate that TiO2 in a fasting diet may be slightly cytotoxic, and that ingested TiO2 does not significantly alter the epithelial proteome, whereas the food matrix alone can have a dramatic effect on the proteome.

The mechanism-based toxicity screening of particles with use in the food and nutrition sector via the ToxTracker reporter system

The rapid expansion of the incorporation of nano-sized materials in consumer products overlaps with the necessity for high-throughput reliable screening tools for the identification of the potential hazardous properties of the nanomaterials. The ToxTracker assay (mechanism-based reporter assay based on embryonic stem cells that uses GFP-tagged biomarkers for detection of DNA damage, oxidative stress and general cellular stress) is one such tool, which could prove useful in the field of particle toxicology allowing for high throughput screening. Here, ToxTracker was utilised to evaluate the potential hazardous properties of two particulates currently used in the food industry (vegetable carbon (E153) and food-grade TiO2 (E171)). Due to the fact that ToxTracker is based on a stem cell format, it is crucial that the data generated is assessed for its suitability and comparability to more conventionally used relevant source of cells - in this case cells from the gastrointestinal tract and the liver. Therefore, the cell reporter findings were compared to data from traditional assays (cytotoxicity, anti-oxidant depletion and DNA damage) and tissue relevant cell types. The data showed E171 to be the most cytotoxic, decreased intracellular glutathione and the most significant with regards to genotoxic effects. The ToxTracker data showed comparability to conventional toxicity and oxidative stress assays; however, some discrepancies were evident between the findings from ToxTracker and the comet assay.

Impact of the Food Additive Titanium Dioxide (E171) on Gut Microbiota-Host Interaction.

The interaction between gut microbiota and host plays a central role in health. Dysbiosis, detrimental changes in gut microbiota and inflammation have been reported in non-communicable diseases. While diet has a profound impact on gut microbiota composition and function, the role of food additives such as titanium dioxide (TiO2), prevalent in processed food, is less established. In this project, we investigated the impact of food grade TiO2 on gut microbiota of mice when orally administered via drinking water. While TiO2 had minimal impact on the composition of the microbiota in the small intestine and colon, we found that TiO2 treatment could alter the release of bacterial metabolites in vivo and affect the spatial distribution of commensal bacteria in vitro by promoting biofilm formation. We also found reduced expression of the colonic mucin 2 gene, a key component of the intestinal mucus layer, and increased expression of the beta defensin gene, indicating that TiO2 significantly impacts gut homeostasis. These changes were associated with colonic inflammation, as shown by decreased crypt length, infiltration of CD8+ T cells, increased macrophages as well as increased expression of inflammatory cytokines. These findings collectively show that TiO2 is not inert, but rather impairs gut homeostasis which may in turn prime the host for disease development.

High-throughput analysis of photocatalytic reactivity of differing TiO2 formulations using 96-well microplate reactors

The rapid development of photocatalysts for water decontamination benefits from availability of sensitive platforms for screening photocatalytic reactivity. The standard approach typically involves quantifying the degradation of a single dye compound in a slurry system in individual beakers, which requires tedious photocatalyst separation and long operation time. We present a simple and efficient method for assessing the photocatalytic activity of different photocatalyst nanomaterials that eliminates the solid separation process. The 96-well microplate method demonstrated an improved applicability as a high-throughput screening method for photocatalytic reaction mechanisms using a wide range of chemical substrates (i.e., methyl orange, methylene blue, terephthalic acid, and β-nicotinamide adenine dinucleotide coenzyme) and photocatalyst concentrations (1–100 mg/L). By employing photocatalysts at lower concentrations compared to the slurry system, rapid screening was accomplished through direct spectrophotometric or spectrofluorometric measurements. The mass-normalized rate constants of dye degradation were used to determine the photocatalytic reactivity of three commercial TiO2 nanomaterials, which followed an order of SRM TiO2 1898 ≈ Degussa TiO2 P90 > Food-grade TiO2 E171. The extent of hydroxyl radical involvement in methyl orange degradation was estimated to be ∼74% by using radical scavengers in the microplate reactor. Given the utilization of low-concentration photocatalyst, this protocol may be used for evaluating photocatalytic reactivity and oxidative stress caused by photocatalyst exposure in an aquatic environment. We further evaluated photocatalytic reaction kinetics with respect to energetic and photonic efficiency. The method could greatly facilitate comparisons across different laboratories when quantifying photocatalytic reactivity and efficiency, which would aid in standardizing bench-scale photocatalysis testing.

Comparative toxicity of a food additive TiO2, a bulk TiO2, and a nano-sized P25 to a model organism the nematode C. elegans.

To help fill the knowledge gap regarding the potential human health impacts of food pigment TiO2, a comparative toxicity study was performed on a food-grade TiO2 (f-TiO2), a bulk TiO2 (b-TiO2), and a nano-sized TiO2 (Degussa P25), and in the nematode Caenorhabditis elegans. Acute phototoxicity and chronic toxicity effects including reproduction, lifespan, and vulval integrity were evaluated. The f-TiO2, b-TiO2, and P25 had a primary particle size (size range) of 149 (53-308) nm, 129 (64-259) nm, and 26 (11-52) nm, respectively. P25 showed the greatest phototoxicity with a 24-h LC50 of 6.0mg/L (95% CI 5.95, 6.3), followed by the f-TiO2 (LC50 = 6.55mg/L (95% CI 6.35, 6.75)), and b-TiO2 was the least toxic. All three TiO2 (1-10mg/L) induced concentration-dependent effects on the worm's reproduction, with a reduction in brood size by 8.5 to 34%. They all caused a reduction of worm lifespan, accompanied by an increased frequency of age-associated vulval integrity defects (Avid). The impact on lifespan and Avid phenotype was more notable for P25 than the f-TiO2 or b-TiO2. Ingestion and accumulation of TiO2 particles in the worm intestine was observed for all three materials by light microscopy. These findings demonstrate that the food pigment TiO2 induces toxicity effects in the worm and further studies are needed to elucidate the human health implication of such toxicities.

Biomolecular interaction and kinematics differences between P25 and E171 TiO2 nanoparticles

Titanium dioxide (TiO2) nanoparticles (NPs) are used abundantly as food additives (E171). For the purpose of risk assessment, it is imperative to understand the behavior of these nanoparticles in a food relevant environment, and their consequent toxicology impacts. However, most of such studies use model TiO2 NPs (P25) as substitutes for E171. To understand the suitability of this approach, we investigated the functional behavior of E171 and P25 in solutions of bovine serum albumin (BSA) and sucrose as model food ingredients. Our data showed that E171 were better dispersed in BSA than P25. In sucrose, E171 displayed a reduction in agglomerated size while P25 agglomerated extensively. Adsorption studies showed that P25 attracted more pronounced corona formation per unit mass of material compared to E171. In vitro sedimentation, diffusion and dosimetry (ISDD) results demonstrated that the time-weighted dosage of E171 was more than two-folds higher than P25, implying that any test performed using P25 to model E171 would underestimate actual dosage and potential toxicity. Taken collectively, this study demonstrated the specificity of TiO2 nanoparticle interaction with food ingredients, and the importance of using food-grade E171 TiO2 for food-relevant toxicological assessments.

Impact of Titanium Dioxide on the Bioaccessibility of β-Carotene in Emulsions with Different Particle Sizes

Commercial, titanium dioxide (TiO2) ingredients used as color additives (E171) in foods and beverages contain an appreciable fraction of particles in the nanoscale range. At present, little information is available regarding the potential impact of food-grade TiO2 nanoparticles on the gastrointestinal fate of co-ingested bioactives, such as nutraceuticals. In this study, the impact of TiO2 on the bioaccessibility of β-carotene solubilized in model food emulsions was investigated using a simulated gastrointestinal tract model. Raman spectroscopy showed that there was no charge transfer between β-carotene and TiO2 but that some β-carotene absorbed to the surface of TiO2 particles. The initial particle size of the food emulsion did not significantly affect β-carotene bioaccessibility, probably because the same amount of free fatty acids (FFAs) was released by the end of digestion. The addition of TiO2 at levels typically found in foods also had no significant impact on β-carotene bioaccessibility and FFA release, which suggested that this type of inorganic particle does not interfere with the gastrointestinal fate of these lipophilic bioactive agents. This information is important for ensuring the safety of inorganic nanoparticle utilization within the food industry.

Vasomotor dysfunction in human subcutaneous arteries exposed ex vivo to food-grade titanium dioxide

Animal studies have shown that titanium dioxide (TiO2) exposure affects arterial vasomotor function, whereas little is known about the effects in arteries from humans. This study investigated vasomotor responses after direct exposure of human subcutaneous arteries to food-grade TiO2 (E171) (14 or 140 μg/ml) for 30 min and 18 h. Vasomotor responses to bradykinin, 5-hydroxytryptamine (5-HT), sarafotoxin 6c (S6c) and nitroglycerin were recorded in wire-myographs. Vasoconstrictor responses to 5-HT were increased in arteries exposed to E171 for 18 h (P < 0.05). Furthermore, an increase in S6c responses was seen in low concentration E171 exposed arteries (30 min exposure; P < 0.05). The vasorelaxation response to nitroglycerin was increased in low concentration E171 exposed arteries (30 min exposure; P < 0.05). Vasorelaxation responses to bradykinin were unaffected after treatment with E171. There was no difference in gene expression levels of intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1, 5-hydroxytryptamine receptor 1B, 5-hydroxytryptamine receptor 2A, endothelin receptor A and endothelin receptor B in E171 exposed arteries after exposure to TiO2 for 30 min or 18 h. In conclusion, this study shows that the same type of vasomotor dysfunction is found in artery segments of rats and humans following ex vivo exposure to E171.

Food-grade TiO2 is trapped by intestinal mucus in vitro but does not impair mucin O-glycosylation and short-chain fatty acid synthesis in vivo: implications for gut barrier protection

BackgroundTitanium dioxide (TiO2) particles are commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, the risk of gut barrier disruption is an increasing concern because of the presence of a nano-sized fraction. Food-grade E171 may interact with mucus, a gut barrier protagonist still poorly explored in food nanotoxicology. To test this hypothesis, a comprehensive approach was performed to evaluate in vitro and in vivo interactions between TiO2 and intestinal mucus, by comparing food-grade E171 with NM-105 (Aeroxyde P25) OECD reference nanomaterial.ResultsWe tested E171-trapping properties of mucus in vitro using HT29-MTX intestinal epithelial cells. Time-lapse confocal laser scanning microscopy was performed without labeling to avoid modification of the particle surface. Near-UV irradiation of E171 TiO2 particles at 364 nm resulted in fluorescence emission in the visible range, with a maximum at 510 nm. The penetration of E171 TiO2 into the mucoid area of HT29-MTX cells was visualized in situ. One hour after exposure, TiO2 particles accumulated inside “patchy” regions 20 µm above the substratum. The structure of mucus produced by HT29-MTX cells was characterized by MUC5AC immunofluorescence staining. The mucus layer was thin and organized into regular “islands” located approximately 20 µm above the substratum. The region-specific trapping of food-grade TiO2 particles was attributed to this mucus patchy structure. We compared TiO2-mediated effects in vivo in rats after acute or sub-chronic oral daily administration of food-grade E171 and NM-105 at relevant exposure levels for humans. Cecal short-chain fatty acid profiles and gut mucin O-glycosylation patterns remained unchanged, irrespective of treatment.ConclusionsFood-grade TiO2 is trapped by intestinal mucus in vitro but does not affect mucin O-glycosylation and short-chain fatty acid synthesis in vivo, suggesting the absence of a mucus barrier impairment under “healthy gut” conditions.

Toxicity of Food-Grade TiO2 to Commensal Intestinal and Transient Food-Borne Bacteria: New Insights Using Nano-SIMS and Synchrotron UV Fluorescence Imaging.

Titanium dioxide (TiO2) is commonly used as a food additive (E171 in the EU) for its whitening and opacifying properties. However, a risk of intestinal barrier disruption, including dysbiosis of the gut microbiota, is increasingly suspected because of the presence of a nano-sized fraction in this additive. We hypothesized that food-grade E171 and Aeroxyde P25 (identical to the NM-105 OECD reference nanomaterial in the European Union Joint Research Centre) interact with both commensal intestinal bacteria and transient food-borne bacteria under non-UV-irradiated conditions. Based on differences in their physicochemical properties, we expect a difference in their respective effects. To test these hypotheses, we chose a panel of eight Gram-positive/Gram-negative bacterial strains, isolated from different biotopes and belonging to the species Escherichia coli, Lactobacillus rhamnosus, Lactococcus lactis (subsp. lactis and cremoris), Streptococcus thermophilus, and Lactobacillus sakei. Bacterial cells were exposed to food-grade E171 vs. P25 in vitro and the interactions were explored with innovative (nano)imaging methods. The ability of bacteria to trap TiO2 was demonstrated using synchrotron UV fluorescence imaging with single cell resolution. Subsequent alterations in the growth profiles were shown, notably for the transient food-borne L. lactis and the commensal intestinal E. coli in contact with food-grade TiO2. However, for both species, the reduction in cell cultivability remained moderate, and the morphological and ultrastructural damages, observed with electron microscopy, were restricted to a small number of cells. E. coli exposed to food-grade TiO2 showed some internalization of TiO2 (7% of cells), observed with high-resolution nano-secondary ion mass spectrometry (Nano-SIMS) chemical imaging. Taken together, these data show that E171 may be trapped by commensal and transient food-borne bacteria within the gut. In return, it may induce some physiological alterations in the most sensitive species, with a putative impact on gut microbiota composition and functioning, especially after chronic exposure.

Vasomotor function in rat arteries after ex vivo and intragastric exposure to food-grade titanium dioxide and vegetable carbon particles

BackgroundHumans are continuously exposed to particles in the gastrointestinal tract. Exposure may occur directly through ingestion of particles via food or indirectly by removal of inhaled material from the airways by the mucociliary clearance system. We examined the effects of food-grade particle exposure on vasomotor function and systemic oxidative stress in an ex vivo study and intragastrically exposed rats.MethodsIn an ex vivo study, aorta rings from naïve Sprague-Dawley rats were exposed for 30 min to food-grade TiO2 (E171), benchmark TiO2 (Aeroxide P25), food-grade vegetable carbon (E153) or benchmark carbon black (Printex 90). Subsequently, the vasomotor function was assessed in wire myographs. In an in vivo study, lean Zucker rats were exposed intragastrically once a week for 10 weeks to vehicle, E171 or E153. Doses were comparable to human daily intake. Vasomotor function in the coronary arteries and aorta was assessed using wire myographs. Tetrahydrobiopterin, ascorbate, malondialdehyde and asymmetric dimethylarginine were measured in blood as markers of oxidative stress and vascular function.ResultsDirect exposure of E171 to aorta rings ex vivo increased the acetylcholine-induced vasorelaxation and 5-hydroxytryptamine-induced vasocontraction. E153 only increased acetylcholine-induced vasorelaxation, and Printex 90 increased the 5-hydroxytryptamine-induced vasocontraction, whereas Aeroxide P25 did not affect the vasomotor function. In vivo exposure showed similar results as ex vivo exposure; increased acetylcholine-induced vasorelaxation in coronary artery segments of E153 and E171 exposed rats, whereas E171 exposure altered 5-hydroxytryptamine-induced vasocontraction in distal coronary artery segments. Plasma levels of markers of oxidative stress and vascular function showed no differences between groups.ConclusionGastrointestinal tract exposure to E171 and E153 was associated with modest albeit statistically significant alterations in the vasocontraction and vasorelaxation responses. Direct particle exposure to aorta rings elicited a similar type of response. The vasomotor responses were not related to biomarkers of systemic oxidative stress.

Single particle analysis of TiO2 in candy products using triple quadrupole ICP-MS

Titanium dioxide (TiO2) belongs to the materials that have gained great importance in many applications. In its particulate form (micro- or nanoparticles), it has entered a huge number of consumer products and food-grade TiO2, better known as E171 within the European Union, represents an important food additive. Thus, there is an increasing need for analytical methods able to detect and quantify such particles. In this regard, inductively coupled-mass spectrometry (ICP-MS), in particular single particle ICP-MS (spICP-MS), has gained importance due to its simplicity and ease of use. Nevertheless, the number of applications for Ti nanoparticles is rather limited. In this study, we have applied the spICP-MS strategy by comparing different measuring modes available in triple quadrupole ICP-MS. First, single quadrupole mode using the collision/reaction cell system was selected for monitoring the isotope 47Ti. Different cell gases like He, O2 and NH3 were tested under optimised conditions for its applicability in spICP-MS of standard suspensions of TiO2. The determined analytical figures of merit were compared to those obtained by triple quadrupole mode using the 47Ti or 48Ti reaction products using O2 and NH3 as reaction gases. This comparison demonstrated that the triple quadrupole mode (TQ mode) was superior in terms of sensitivity due to the more efficient removal of spectral interferences. Particle size detection limits down to 26nm were obtained using the best instrumental conditions for TiO2 particles at a dwell time of 10ms. Finally, the different measuring modes were applied to the analysis of chewing gum samples after a simple extraction procedure using an ultrasonic bath. The obtained results showed a good agreement for the detected particle size range using the different TQ modes. The size range of TiO2 particles was determined to be between approximately 30 and 200nm, whereas roughly 40% of the particles were smaller than 100nm. For the determination of the particle number concentration in these real samples, we suggest CeO2 particles as internal standard.