Validating Enteroid-Derived Monolayers from Murine Gut Organoids for Toxicological Testing of Inorganic Particles: Proof-of-Concept with Food-Grade Titanium Dioxide.

Human exposure to foodborne inorganic nanoparticles (NPs) is a growing concern. However, identifying potential hazards linked to NP ingestion often requires long-term exposure in animals. Owing these constraints, intestinal organoids are a promising alternative to in vivo experiments; as such, an in vitro approach should enable a rapid and reliable assessment of the effects of ingested chemicals on the gut. However, this remains to be validated for inorganic substances. In our study, a transcriptomic analysis and immunofluorescence staining were performed to compare the effects of food-grade TiO2 (fg-TiO2) on enteroid-derived monolayers (EDMs) from murine intestinal organoids to the known impacts of TiO2 on intestinal epithelium. After their ability to respond to a pro-inflammatory cytokine cocktail was validated, EDMs were exposed to 0, 0.1, 1, or 10 µg fg-TiO2/mL for 24 h. A dose-related increase of the muc2, vilin 1, and chromogranin A gene markers of cell differentiation was observed. In addition, fg-TiO2 induced apoptosis and dose-dependent genotoxicity, while a decreased expression of genes encoding for antimicrobial peptides, and of genes related to tight junction function, was observed. These results validated the use of EDMs as a reliable model for the toxicity testing of foodborne NPs likely to affect the intestinal barrier.

Evaluating the Effects of Chronic Oral Exposure to the Food Additive Silicon Dioxide on Oral Tolerance Induction and Food Sensitivities in Mice.

BACKGROUND: The increasing prevalence of food sensitivities has been attributed to changes in gut microenvironment; however, ubiquitous environmental triggers such as inorganic nanoparticles (NPs) used as food additives have not been thoroughly investigated. OBJECTIVES: We explored the impact of the NP-structured food-grade silicon dioxide (fg-SiO2) on intestinal immune response involved in oral tolerance (OT) induction and evaluated the consequences of oral chronic exposure to this food-additive using a mouse model of OT to ovalbumin (OVA) and on gluten immunopathology in mice expressing the celiac disease risk gene, HLA-DQ8. METHODS: Viability, proliferation, and cytokine production of mesenteric lymph node (MLN) cells were evaluated after exposure to fg-SiO2. C57BL/6J mice and a mouse model of OT to OVA were orally exposed to fg-SiO2 or vehicle for 60 d. Fecal lipocalin-2 (Lcn-2), anti-OVA IgG, cytokine production, and immune cell populations were analyzed. Nonobese diabetic (NOD) mice expressing HLA-DQ8 (NOD/DQ8), exposed to fg-SiO2 or vehicle, were immunized with gluten and immunopathology was investigated. RESULTS: MLN cells exposed to fg-SiO2 presented less proliferative T cells and lower secretion of interleukin 10 (IL-10) and transforming growth factor beta (TGF-ß) by T regulatory and CD45+ CD11b+ CD103+ cells compared to control, two factors mediating OT. Mice given fg-SiO2 exhibited intestinal Lcn-2 level and interferon gamma (IFN-γ) secretion, showing inflammation and less production of IL-10 and TGF-ß. These effects were also observed in OVA-tolerized mice exposed to fg-SiO2, in addition to a breakdown of OT and a lower intestinal frequency of T cells. In NOD/DQ8 mice immunized with gluten, the villus-to-crypt ratio was decreased while the CD3+ intraepithelial lymphocyte counts and the Th1 inflammatory response were aggravated after fg-SiO2 treatment. DISCUSSION: Our results suggest that chronic oral exposure to fg-SiO2 blocked oral tolerance induction to OVA, and worsened gluten-induced immunopathology in NOD/DQ8 mice. The results should prompt investigation on the link between SiO2 exposure and food sensitivities in humans. https://doi.org/10.1289/EHP12758.

Long-term exposure from perinatal life to food-grade TiO2 alters intestinal homeostasis and predisposes to food allergy in young mice.

BACKGROUND: Food allergy (FA) is an inappropriate immunological response to food proteins resulting from an impaired induction of oral tolerance. Various early environmental factors can affect the establishment of intestinal homeostasis, predisposing to FA in early life. In this context, we aimed to assess the effect of chronic perinatal exposure to food-grade titanium dioxide (fg-TiO2 ), a common food additive. METHODS: Dams were fed a control versus fg-TiO2 -enriched diet from preconception to weaning, and their progeny received the same diet at weaning. A comprehensive analysis of baseline intestinal and systemic homeostasis was performed in offspring 1 week after weaning by assessing gut barrier maturation and microbiota composition, and local and systemic immune system and metabolome. The effect of fg-TiO2 on the susceptibility of progeny to develop oral tolerance versus FA to cow's milk proteins (CMP) was performed starting at the same baseline time-point, using established models. Sensitization to CMP was investigated by measuring ß-lactoglobulin and casein-specific IgG1 and IgE antibodies, and elicitation of the allergic reaction by measuring mouse mast cell protease (mMCP1) in plasma collected after an oral food challenge. RESULTS: Perinatal exposure to fg-TiO2 at realistic human doses led to an increased propensity to develop FA and an impaired induction of oral tolerance only in young males, which could be related to global baseline alterations in intestinal barrier, gut microbiota composition, local and systemic immunity, and metabolism. CONCLUSIONS: Long-term perinatal exposure to fg-TiO2 alters intestinal homeostasis establishment and predisposes to food allergy, with a clear gender effect.

A 90-day oral exposure to food-grade gold at relevant human doses impacts the gut microbiota and the local immune system in a sex-dependent manner in mice.

BACKGROUND: Edible gold (Au) is commonly used as a food additive (E175 in EU) for confectionery and cake decorations, coatings and in beverages. Food-grade gold is most often composed of thin Au sheets or flakes exhibiting micro- and nanometric dimensions in their thickness. Concerns about the impact of mineral particles used as food additives on human health are increasing with respect to the particular physico-chemical properties of nanosized particles, which enable them to cross biological barriers and interact with various body cell compartments. In this study, male and female mice were exposed daily to E175 or an Au nanomaterial (Ref-Au) incorporated into food at relevant human dose for 90 days in order to determine the potential toxicity of edible gold. RESULTS: E175 or Ref-Au exposure in mice did not induce any histomorphological damage of the liver, spleen or intestine, nor any genotoxic effects in the colon and liver despite an apparent higher intestinal absorption level of Au particles in mice exposed to Ref-Au compared to the E175 food additive. No changes in the intestinal microbiota were reported after treatment with Ref-Au, regardless of sex. In contrast, after E175 exposure, an increase in the Firmicutes/Bacteroidetes ratio and in the abundance of Proteobacteria were observed in females, while a decrease in the production of short-chain fatty acids occurred in both sexes. Moreover, increased production of IL-6, TNFα and IL-1ß was observed in the colon of female mice at the end of the 90-day exposure to E175, whereas, decreased IL-6, IL-1ß, IL-17 and TGFß levels were found in the male colon. CONCLUSIONS: These results revealed that a 90-day exposure to E175 added to the diet alters the gut microbiota and intestinal immune response in a sex-dependent manner in mice. Within the dose range of human exposure to E175, these alterations remained low in both sexes and mostly appeared to be nontoxic. However, at the higher dose, the observed gut dysbiosis and the intestinal low-grade inflammation in female mice could favour the occurrence of metabolic disorders supporting the establishment of toxic reference values for the safe use of gold as food additive.

The food additive titanium dioxide hinders intestinal production of TGF-ß and IL-10 in mice, and long-term exposure in adults or from perinatal life blocks oral tolerance to ovalbumin.

Food hypersensitivities are increasing in industrialized countries, and foodborne nanoparticles (NPs) are suspected as co-factors in their aetiology. Food-grade titanium dioxide (fg-TiO2), a food colouring agent, is composed of NPs with immunomodulatory properties. We investigated whether fg-TiO2 may compromise the establishment of oral tolerance (OT) to food proteins using a model of OT induction to ovalbumin (OVA) in mice, and whether a perinatal exposure could trigger this effect. In pregnant mice fed a TiO2-enriched diet, ICP-MS and TEM-EDX analyses showed passage of TiO2 NPs into the foetus. When their weaned offspring were fed the same diet, a breakdown in OT to OVA was observed at adulthood, characterized by a high anti-OVA IgG production compared to controls. However, adult mice directly exposed to fg-TiO2 did not induce OT to OVA either, ruling out a developmental origin for these effects. When these mice were orally challenged with OVA, intestinal inflammation demonstrated hypersensitivity to OVA. In OVA-naïve mice, fg-TiO2 exposure impaired intestinal TGF-ß and IL-10 production, of key role in OT induction and maintenance. These findings showed that long-term exposure to TiO2 as food additive alters anti-inflammatory cytokine profile, and leads to OT failure regardless of the timing of TiO2 exposure throughout life.

Food-grade titanium dioxide translocates across the buccal mucosa in pigs and induces genotoxicity in an in vitro model of human oral epithelium.

The whitening and opacifying agent titanium dioxide (TiO2) is used worldwide in various foodstuffs, toothpastes and pharmaceutical tablets. Its use as a food additive (E171 in EU) has raised concerns for human health. Although the buccal mucosa is the first area exposed, oral transmucosal passage of TiO2 particles has not been documented. Here we analyzed E171 particle translocation in vivo through the pig buccal mucosa and in vitro on human buccal TR146 cells, and the effects on proliferating and differentiated TR146 cells. In the buccal floor of pigs, isolated TiO2 particles and small aggregates were observed 30 min after sublingual deposition, and were recovered in the submandibular lymph nodes at 4 h. In TR146 cells, kinetic analyses showed high absorption capacities of TiO2 particles. The cytotoxicity, genotoxicity and oxidative stress were investigated in TR146 cells exposed to E171 in comparison with two TiO2 size standards of 115 and 21 nm in diameter. All TiO2 samples were reported cytotoxic in proliferating cells but not following differentiation. Genotoxicity and slight oxidative stress were reported for the E171 and 115 nm TiO2 particles. These data highlight the buccal mucosa as an absorption route for the systemic passage of food-grade TiO2 particles. The greater toxicity on proliferating cells suggest potential impairement of oral epithelium renewal. In conclusion, this study emphasizes that buccal exposure should be considered during toxicokinetic studies and for risk assessment of TiO2 in human when used as food additive, including in toothpastes and pharmaceutical formulations.

Exposure to polyethylene microplastics alters immature gut microbiome in an infant in vitro gut model.

Infants are characterized by an immaturity of the gut ecosystem and a high exposure to microplastics (MPs) through diet, dust and suckling. However, the bidirectional interactions between MPs and the immature infant intestinal microbiota remain unknown. Our study aims to investigate the impact of chronic exposure to polyethylene (PE) MPs on the gut microbiota and intestinal barrier of infants, using the new Toddler mucosal Artificial Colon coupled with a co-culture of epithelial and mucus-secreting cells. Gut microbiota composition was determined by 16S metabarcoding and microbial activities were evaluated by gas, short chain fatty acid and volatolomics analyses. Gut barrier integrity was assessed via evaluation of intestinal permeability, inflammation and mucus synthesis. Exposure to PE MPs induced gut microbial shifts increasing α-diversity and abundance of potentially harmful pathobionts, such as Dethiosulfovibrionaceae and Enterobacteriaceae. Those changes were associated to butyrate production decrease and major changes in volatile organic compounds profiles. In contrast, no significant impact of PE MPs on the gut barrier, as mediated by microbial metabolites, was reported. For the first time, this study indicates that ingestion of PE MPs can induce perturbations in the gut microbiome of infants. Next step would be to further investigate the potential vector effect of MPs.

Microplastics: What happens in the human digestive tract? First evidences in adults using in vitro gut models.

Microplastics (MPs) are ubiquitous in the environment and humans are inevitably exposed to them. However, the effects of MPs in the human digestive environment are largely unknown. The aim of our study was to investigate the impact of repeated exposure to polyethylene (PE) MPs on the human gut microbiota and intestinal barrier using, under adult conditions, the Mucosal Artificial Colon (M-ARCOL) model, coupled with a co-culture of intestinal epithelial and mucus-secreting cells. The composition of the luminal and mucosal gut microbiota was determined by 16S metabarcoding and microbial activities were characterized by gas, short chain fatty acid, volatolomic and AhR activity analyses. Gut barrier integrity was assessed via intestinal permeability, inflammation and mucin synthesis. First, exposure to PE MPs induced donor-dependent effects. Second, an increase in abundances of potentially harmful pathobionts, Desulfovibrionaceae and Enterobacteriaceae, and a decrease in beneficial bacteria such as Christensenellaceae and Akkermansiaceae were observed. These bacterial shifts were associated with changes in volatile organic compounds profiles, notably characterized by increased indole 3-methyl- production. Finally, no significant impact of PE MPs mediated by changes in gut microbial metabolites was reported on the intestinal barrier. Given these adverse effects of repeated ingestion of PE MPs on the human gut microbiota, studying at-risk populations like infants would be a valuable advance.

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.

BACKGROUND: Food-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. RESULTS: The 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. CONCLUSIONS: After 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.

Pharmacological restoration of gut barrier function in stressed neonates partially reverses long-term alterations associated with maternal separation.

RATIONALE: Intestinal permeability plays an important role in gut-brain axis communication. Recent studies indicate that intestinal permeability increases in neonate pups during maternal separation (MS). OBJECTIVES: The present study aims to determine whether pharmacological inhibition of myosin light chain kinase (MLCK), which regulates tight junction contraction and controls intestinal permeability, in stressed neonates, protects against the long-term effects of MS. METHODS: Male Wistar rats were exposed to MS (3 h per day from post-natal day (PND)2 to PND14) or left undisturbed and received daily intraperitoneal injection of a MLCK inhibitor (ML-7, 5 mg/kg) or vehicle during the same period. At adulthood, emotional behaviors, corticosterone response to stress, and gut microbiota composition were analyzed. RESULTS: ML-7 restored gut barrier function in MS rats specifically during the neonatal period. Remarkably, ML-7 prevented MS-induced sexual reward-seeking impairment and reversed the alteration of corticosterone response to stress at adulthood. The effects of ML-7 were accompanied by the normalization of the abundance of members of Lachnospiraceae, Clostridiales, Desulfovibrio, Bacteroidales, Enterorhabdus, and Bifidobacterium in the feces of MS rats at adulthood. CONCLUSIONS: Altogether, our work suggests that improvement of intestinal barrier defects during development may alleviate some of the long-term effects of early-life stress and provides new insight on brain-gut axis communication in a context of stress.

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.

BACKGROUND: Titanium 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. RESULTS: We 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. CONCLUSIONS: Food-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.

Consequences of bisphenol a perinatal exposure on immune responses and gut barrier function in mice.

The potent immunomodulatory effect of the endocrine disruptor bisphenol A during development and consequences during life span are of increasing concern. Particular interests have been raised from animal studies regarding the risk of developing food intolerance and infection. We aimed to identify immune disorders in mice triggered by perinatal exposure to bisphenol A. Gravid mice were orally exposed to bisphenol (50 µg/kg body weight/day) from day 15 of pregnancy until weaning. Gut barrier function, local and systemic immunity were assessed in adult female offspring. Mice perinatally exposed to bisphenol showed a decrease in ileal lysozyme expression and a fall of fecal antimicrobial activity. In offspring mice exposed to bisphenol, an increase in colonic permeability was observed associated with an increase in interferon-γ level and a drop of colonic IgA+ cells and fecal IgA production. Interestingly, altered frequency of innate lymphoid cells type 3 occurred in the small intestine, with an increase in IgG response against commensal bacteria in sera. These effects were related to a defect in dendritic cell maturation in the lamina propria and spleen. Activated and regulatory T cells were decreased in the lamina propria. Furthermore, perinatal exposure to bisphenol promoted a sharp increase in interferon-γ and interleukin-17 production in the intestine and elicited a T helper 17 profile in the spleen. To conclude, perinatal exposure to bisphenol weakens protective and regulatory immune functions in the intestine and at systemic level in adult offspring. The increased susceptibility to inflammatory response is an interesting lead supporting bisphenol-mediated adverse consequences on food reactions and infections.

Gut dysbiosis and impairment of immune system homeostasis in perinatally-exposed mice to Bisphenol A precede obese phenotype development.

Epidemiology evidenced the Bisphenol A (BPA), a chemical found in daily consumer products, as an environmental contributor to obesity and type II diabetes (T2D) in Humans. However, the BPA-mediated effects supporting these metabolic disorders are still unknown. Knowing that obesity and T2D are associated with low-grade inflammation and gut dysbiosis, we performed a longitudinal study in mice to determine the sequential adverse effects of BPA on immune system and intestinal microbiota that could contribute to the development of metabolic disorders. We observed that perinatal exposure to BPA (50 µg/kg body weight/day) induced intestinal and systemic immune imbalances at PND45, through a decrease of Th1/Th17 cell frequencies in the lamina propria concomitant to an increase of splenic Th1/Th17 immune responses. These early effects are associated with an altered glucose sensitivity, a defect of IgA secretion into faeces and a fall of faecal bifidobacteria relative to control mice. Such BPA-mediated events precede infiltration of pro-inflammatory M1 macrophages in gonadal white adipose tissue appearing with ageing, together with a decreased insulin sensitivity and an increased weight gain. Our findings provide a better understanding of the sequential events provoked by perinatal exposure to BPA that could support metabolic disorder development in later life.

Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon.

Food-grade titanium dioxide (TiO2) containing a nanoscale particle fraction (TiO2-NPs) is approved as a white pigment (E171 in Europe) in common foodstuffs, including confectionary. There are growing concerns that daily oral TiO2-NP intake is associated with an increased risk of chronic intestinal inflammation and carcinogenesis. In rats orally exposed for one week to E171 at human relevant levels, titanium was detected in the immune cells of Peyer's patches (PP) as observed with the TiO2-NP model NM-105. Dendritic cell frequency increased in PP regardless of the TiO2 treatment, while regulatory T cells involved in dampening inflammatory responses decreased with E171 only, an effect still observed after 100 days of treatment. In all TiO2-treated rats, stimulation of immune cells isolated from PP showed a decrease in Thelper (Th)-1 IFN-γ secretion, while splenic Th1/Th17 inflammatory responses sharply increased. E171 or NM-105 for one week did not initiate intestinal inflammation, while a 100-day E171 treatment promoted colon microinflammation and initiated preneoplastic lesions while also fostering the growth of aberrant crypt foci in a chemically induced carcinogenesis model. These data should be considered for risk assessments of the susceptibility to Th17-driven autoimmune diseases and to colorectal cancer in humans exposed to TiO2 from dietary sources.

Dietary polyunsaturated fatty acids and heme iron induce oxidative stress biomarkers and a cancer promoting environment in the colon of rats.

The end products of polyunsaturated fatty acid (PUFA) peroxidation, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE), and isoprostanes (8-iso-PGF2α), are widely used as systemic lipid oxidation/oxidative stress biomarkers. However, some of these compounds have also a dietary origin. Thus, replacing dietary saturated fat by PUFAs would improve health but could also increase the formation of such compounds, especially in the case of a pro-oxidant/antioxidant imbalanced diet. Hence, the possible impact of dietary fatty acids and pro-oxidant compounds was studied in rats given diets allowing comparison of the effects of heme iron vs. ferric citrate and of ω-6- vs. ω-3-rich oil on the level of lipid peroxidation/oxidative stress biomarkers. Rats given a heme iron-rich diet without PUFA were used as controls. The results obtained have shown that MDA and the major urinary metabolite of HNE (the mercapturic acid of dihydroxynonane, DHN-MA) were highly dependent on the dietary factors tested, while 8-iso-PGF2α was modestly but significantly affected. Intestinal inflammation and tissue fatty acid composition were checked in parallel and could only explain the differences we observed to a limited extent. Thus, the differences in biomarkers were attributed to the formation of lipid oxidation compounds in food or during digestion, their intestinal absorption, and their excretion into urine. Moreover, fecal extracts from the rats fed the heme iron or fish oil diets were highly toxic for immortalized mouse colon cells. Such toxicity can eventually lead to promotion of colorectal carcinogenesis, supporting the epidemiological findings between red meat intake and colorectal cancer risk. Therefore, the analysis of these biomarkers of lipid peroxidation/oxidative stress in urine should be used with caution when dietary factors are not well controlled, while control of their possible dietary intake is needed also because of their pro-inflammatory, toxic, and even cocarcinogenic effects.

Perinatal exposure to a low dose of bisphenol A impaired systemic cellular immune response and predisposes young rats to intestinal parasitic infection.

Perinatal exposure to the food contaminant bisphenol A (BPA) in rats induces long lasting adverse effects on intestinal immune homeostasis. This study was aimed at examining the immune response to dietary antigens and the clearance of parasites in young rats at the end of perinatal exposure to a low dose of BPA. Female rats were fed with BPA [5 µg/kg of body weight/day] or vehicle from gestational day 15 to pup weaning. Juvenile female offspring (day (D)25) were used to analyze immune cell populations, humoral and cellular responses after oral tolerance or immunization protocol to ovalbumin (OVA), and susceptibility to infection by the intestinal nematode Nippostrongylus brasiliensis (N. brasiliensis). Anti-OVA IgG titers following either oral tolerance or immunization were not affected after BPA perinatal exposure, while a sharp decrease in OVA-induced IFNγ secretion occurred in spleen and mesenteric lymph nodes (MLN) of OVA-immunized rats. These results are consistent with a decreased number of helper T cells, regulatory T cells and dendritic cells in spleen and MLN of BPA-exposed rats. The lack of cellular response to antigens questioned the ability of BPA-exposed rats to clear intestinal infections. A 1.5-fold increase in N. brasiliensis living larvae was observed in the intestine of BPA-exposed rats compared to controls due to an inappropriate Th1/Th2 cytokine production in infected jejunal tissues. These results show that perinatal BPA exposure impairs cellular response to food antigens, and increases susceptibility to intestinal parasitic infection in the juveniles. This emphasized the maturing immune system during perinatal period highly sensitive to low dose exposure to BPA, altering innate and adaptative immune response capacities in early life.

Food intolerance at adulthood after perinatal exposure to the endocrine disruptor bisphenol A.

The food contaminant bisphenol A (BPA) is pointed out as a risk factor in development of food allergy and food intolerance, two adverse food reactions increasing worldwide. We evaluated the consequences of perinatal exposure to low doses of BPA on immune-specific response to the food antigen ovalbumin (OVA) at adulthood. Perinatal exposure to BPA (0.5, 5, or 50 µg/kg/d) from 15th day of gravidity to pups weaning resulted in an increase of anti-OVA IgG titers at all BPA dosages in OVA-tolerized rats, and at 5 µg/kg/d in OVA-immunized rats compared to control rats treated with vehicle. In BPA-treated and OVA-tolerized rats, increased anti-OVA IgG titers were associated with higher IFNγ secretion by the spleen. This result is in accordance with the increase of activated CD4(+)CD44(high)CD62L(low) T lymphocytes observed in spleen of BPA-exposed rats compared to controls. Finally, when BPA-treated OVA-tolerized rats were orally challenged with OVA, colonic inflammation occurred, with neutrophil infiltration, increased IFNγ, and decreased TGFß. We show that perinatal exposure to BPA altered oral tolerance and immunization to dietary antigens (OVA). In summary, the naive immune system of neonate is vulnerable to low doses of BPA that trigger food intolerance later in life.

Genotoxicity of Escherichia coli Nissle 1917 strain cannot be dissociated from its probiotic activity.

Oral administration of the probiotic bacterium Escherichia coli Nissle 1917 improves chronic inflammatory bowel diseases, but the molecular basis for this therapeutic efficacy is unknown. E. coli Nissle 1917 harbors a cluster of genes coding for the biosynthesis of hybrid nonribosomal peptide-polyketide(s). This biosynthetic pathway confers the ability for bacteria to induce DNA double strand breaks in eukaryotic cells. Here we reveal that inactivation of the clbA gene within this genomic island abrogated the ability for the strain to induce DNA damage and chromosomal abnormalities in non-transformed cultured rat intestinal epithelial cells but is required for the probiotic activity of E. coli Nissle 1917. Thus, evaluation of colitis severity induced in rodent fed with E. coli Nissle 1917 or an isogenic non-genotoxic mutant demonstrated the need for a functional biosynthetic pathway both in the amelioration of the disease and in the modulation of cytokine expression. Feeding rodents with a complemented strain for which genotoxicity was restored confirmed that this biosynthetic pathway contributes to the health benefits of the probiotic by modulating its immunomodulatory properties. Our data provide additional evidence for the benefit of this currently used probiotic in colitis but remind us that an efficient probiotic may also have side effects as any other medication.

Proinflammatory role of vasopressin through V1b receptors in hapten-induced experimental colitis in rodents: implication in IBD.

Vasopressin and its receptors modulate several gut functions, but their role in intestinal inflammation is unknown. Our aims were to determine 1) the localization of V1b receptors in human and rodent colon, 2) the role of vasopressin and V1b receptors in experimental colitis using two approaches: V1b⁻(/)⁻ mice and a selective V1b receptor antagonist, SSR149415, and 3) the mechanisms involved. V1b receptors were localized in normal and inflamed colon from humans and rats. Experimental colitis was induced in rats and mice and some groups were treated before or after colitis induction with oral SSR149415 (3-30 mg/kg). Other groups of mice were submitted to dehydration to increase vasopressin plasma levels, prior to colitis induction. Body weight, damage scores, MPO, and TNF-α tissue levels were determined. Finally, colonic segments of wild-type (WT) and V1b⁻(/)⁻ mice were mounted in Ussing chambers and paracellular permeability in response to vasopressin was studied. V1b receptors were expressed in enterocytes and ganglia cells of the enteric nervous system of human and rat intestine. Expression levels were independent from inflammatory status. Colitis was less severe in rodents treated by either preventive or curative SSR149415 and in V1b⁻(/)⁻ mice. 2,4,6-Trinitrobenzene sulfonic acid induced a strong mortality in dehydrated animals that was reversed by preventive SSR149415 or mast cell stabilizer. Vasopressin significantly increased paracellular permeability in WT, but not in V1b⁻(/)⁻ mice. Preincubation of colon tissues with SSR149415 abolished the vasopressin effect. Similarly, vasopressin had no effect in colonic preparations from WT mice pretreated with mast cell stabilizers. Vasopressin, through V1b receptor interaction, has proinflammatory properties linked to mast cell activation and downstream alterations of the colonic epithelial barrier. These findings underline the potential interest of V1b receptor blockers in gut inflammatory diseases.

Impact of oral bisphenol A at reference doses on intestinal barrier function and sex differences after perinatal exposure in rats.

Bisphenol A (BPA), a chemical estrogen widely used in the food-packaging industry and baby bottles, is recovered in human fluids (0.1-10 nM). Recent studies have reported that BPA is hormonally active at low doses, emphasizing the debate of a risk for human health. Estrogen receptors are expressed in the colon, and although the major route of BPA exposure is food, the effects on gut have received no attention. We first examined the endocrine disrupting potency of BPA on colonic paracellular permeability (CPP), experimental colitis, and visceral sensitivity in ovariectomized rats orally exposed to 5 mg/kg/d BPA (i.e., the no observed adverse effect level), 50 microg/kg/d BPA (i.e., tolerable daily intake), or lower doses. BPA dose-dependently decreased basal CPP, with a half-maximal inhibitory dose of 5.2 microg/kg/d, 10-fold below the tolerable daily intake. This correlated with an increase in epithelial tight junction sealing, also observed in Caco-2 cells exposed to 10 nM BPA. When ovariectomized rats were fed with BPA at the no observed adverse effect level, the severity of colitis was reduced, whereas the same dose increased pain sensitivity to colorectal stimuli. We then examined the impact of perinatal exposure to BPA on intestinal permeability and inflammatory response in the offspring. In female rats, but not in male rats, perinatal BPA evoked a decrease of CPP in adulthood, whereas the proinflammatory response of colonic mucosa was strengthened. This study first demonstrates that the xenoestrogen BPA at reference doses influences intestinal barrier function and gut nociception. Moreover, perinatal exposure promotes the development of severe inflammation in adult female offspring only.