Viorica Braniste

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 μg/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 μg/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.

Oestradiol decreases colonic permeability through oestrogen receptor β-mediated up-regulation of occludin and junctional adhesion molecule-A in epithelial cells

Oestradiol modulates paracellular permeability and tight junction (TJ) function in endothelia and reproductive tissues, but whether the ovarian hormones and cycle affect the paracellular pathway in the intestinal epithelium remains unclear. Oestrogen receptors (ERs) are expressed in intestinal epithelial cells, and oestradiol regulates epithelium formation. We examined the effects of oestrous cycle stage, oestradiol benzoate (EB), and progesterone (P) on colonic paracellular permeability (CPP) in the female rat, and whether EB affects expression of the TJ proteins in the rat colon and the human colon cell line Caco‐2. In cyclic rats, CPP was determined through lumen‐to‐blood 51Cr‐labelled EDTA clearance, and in Ussing chambers for dextran permeability. CPP was also examined in ovariectomized (OVX) rats treated with P or EB, with and without the ER antagonist ICI 182,780, or with the selective agonists for ERα (propyl pyrazole triol; PPT) or ERβ (diarylpropionitrile; DPN). In oestrus rats, CPP was reduced (P < 0.01) relative to dioestrus. In OVX rats, EB dose‐dependently decreased CPP, an effect mimicked by DPN and blocked by ICI 182,780, whereas P had no effect. Oestradiol increased occludin mRNA and protein in the colon (P < 0.05), but not zona occludens (ZO)‐1. Further, EB and DPN enhanced occludin and junctional adhesion molecule (JAM)‐A expression in Caco‐2 cells without change in ZO‐1, an effect blocked by ICI 182,780. These data show that oestrogen reinforces intestinal epithelial barrier through ERβ‐mediated up‐regulation of the transmembrane proteins occludin and JAM‐A determining paracellular spaces. These findings highlight the importance of the ERβ pathway in the control of colonic paracellular transport and mucosal homeostasis.

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+CD44high CD62Llow 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 TGFβ. 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.—Menard, S., Guzylack‐Piriou, L., Leveque, M., Braniste, V., Lencina, C., Naturel, M., Moussa, L., Sekkal, S., Harkat, C., Gaultier, E., Theodorou, V., Houdeau, E., Food intolerance at adulthood after perinatal exposure to the endocrine disruptor bisphenol A. FASEB J. 28, 4893–4900 (2014). www.fasebj.org

Changes in intestinal glucocorticoid sensitivity in early life shape the risk of epithelial barrier defect in maternal-deprived rats.

Glucocorticoids (GC) contribute to human intestine ontogeny and accelerate gut barrier development in preparation to birth. Rat gut is immature at birth, and high intestinal GC sensitivity during the first two weeks of life resembles that of premature infants. This makes suckling rats a model to investigate postpartum impact of maternal separation (MS)-associated GC release in preterm babies, and whether GC sensitivity may shape MS effects in immature gut. A 4 hours-MS applied once at postnatal day (PND)10 enhanced plasma corticosterone in male and female pups, increased by two times the total in vivo intestinal permeability (IP) to oral FITC-Dextran 4 kDa (FD4) immediately after the end of MS, and induced bacterial translocation (BT) to liver and spleen. Ussing chamber experiments demonstrated a 2-fold increase of permeability to FD4 in the colon immediately after the end of MS, but not in the ileum. Colonic permeability was not only increased for FD4 but also to intact horseradish peroxidase 44 kDa in MS pups. In vivo, the glucocorticoid receptor (GR) antagonist RU486 or ML7 blockade of myosin light chain kinase controlling epithelial cytoskeleton contraction prevented MS-induced IP increase to oral FD4 and BT. In addition, the GR agonist dexamethasone dose-dependently mimicked MS-increase of IP to oral FD4. In contrast, MS effects on IP to oral FD4 and BT were absent at PND20, a model for full-term infant, characterized by a marked drop of IP to FD4 in response to dexamethasone, and decreased GR expression in the colon only compared to PND10 pups. These results show that high intestinal GC responsiveness in a rat model of prematurity defines a vulnerable window for a post-delivery MS, evoking immediate disruption of epithelial integrity in the large intestine, and increasing susceptibility to macromolecule passage and bacteremia.

A Low Dose of Fermented Soy Germ Alleviates Gut Barrier Injury, Hyperalgesia and Faecal Protease Activity in a Rat Model of Inflammatory Bowel Disease

Pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF), IL-1β and TNF-α predominate in inflammatory bowel diseases (IBD) and TNBS colitis. Increased levels of serine proteases activating protease-activated receptor 2 (PAR-2) are found in the lumen and colonic tissue of IBD patients. PAR-2 activity and pro-inflammatory cytokines impair epithelial barrier, facilitating the uptake of luminal aggressors that perpetuate inflammation and visceral pain. Soy extracts contain phytoestrogens (isoflavones) and serine protease inhibitors namely Bowman-Birk Inhibitors (BBI). Since estrogens exhibit anti-inflammatory and epithelial barrier enhancing properties, and that a BBI concentrate improves ulcerative colitis, we aimed to evaluate if a fermented soy germ extract (FSG) with standardized isoflavone profile and stable BBI content exert cumulative or synergistic protection based on protease inhibition and estrogen receptor (ER)-ligand activity in colitic rats. Female rats received orally for 15 d either vehicle or FSG with or without an ER antagonist ICI 182.780 before TNBS intracolonic instillation. Macroscopic and microscopic damages, myeloperoxidase activity, cytokine levels, intestinal paracellular permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression were assessed 24 h, 3 d and 5 d post-TNBS. FSG treatment improved the severity of colitis, by decreasing the TNBS-induced rise in gut permeability, visceral sensitivity, faecal proteolytic activity and PAR-2 expression at all post-TNBS points. All FSG effects were reversed by the ICI 182.780 except the decrease in faecal proteolytic activity and PAR-2 expression. In conclusion, the anti-inflammatory properties of FSG treatment result from two distinct but synergic pathways i.e an ER-ligand and a PAR-2 mediated pathway, providing rationale for potential use as adjuvant therapy in IBD.

Bisphenol A in the Gut: Another Break in the Wall?

From animal studies, a consensus exists that the synthetic estrogen bisphenol A (BPA), a plastic monomer widely used in the food-packaging industry, is able to disrupt endocrine signalling pathways during development, with persisting effects later in life. Although the fetal and then the adult gut expresses functional estrogen receptors (ERs), the endocrine impact of BPA on the intestinal barrier function remains largely unexplored. The intestinal epithelium and mucosal immune cells provide a first line of defence designed to restrict the passage of harmful substances from the lumen. Intestinal permeability is high at birth, permitting lumen-to-mucosa exchanges involved in the maturation process of the gut immune system. As a barrier to the external environment, gut epithelium is renewed constantly during life. Renewal of gut epithelial cells occurs in less than ~96 h, starting from fetal stage, and is dependent on controlled cell stimulation and proliferation by various signalling pathways. Lessons learned from ER-deficient mice underline the importance of estrogen signalling in growth, organization and maintenance of a normal epithelial barrier. In rats, BPA was recently shown to interact with ERs in the adult gut by mimicking the estradiol-mediated decrease of epithelial permeability through genomic pathways. This effect also occurs in neonates when low doses of BPA (5 μg/kg BW/day: tenfold below the tolerable daily intake for humans) are orally administered to pregnant and then lactating rats. A perinatal exposure to BPA also reduces epithelial cell proliferation in the colon of neonates, while the overall decrease of intestinal permeability remains apparent in adulthood only in female offspring. As a consequence, adult females perinatally exposed to BPA have been shown to develop severe inflammatory responses in a rat model of inflammatory bowel disease, demonstrating enhanced expression and production of T-Helper 1 cytokines in inflamed areas. In mice, a mother-to-infant transport of maternal BPA is consistent with the ability of the chemical to reach the fetus through the placental barrier: BPA is present in the amniotic fluid and accumulates in the maturing gut. Although BPA, once absorbed by maternal gut, is rapidly deactivated by first pass conjugation in the liver, recent studies emphasize that BPA at low, environmentally relevant levels can transfer across the human placenta, mainly in an estrogen-active, unconjugated form. It is now thought that BPA ingested by dams has repercussions on the education of the immune system by reducing intestinal permeability from fetal stages and promotes severe inflammatory response later in life.

Bifidobacterium longum and Lactobacillus helveticus Synergistically Suppress Stress-related Visceral Hypersensitivity Through Hypothalamic-Pituitary-Adrenal Axis Modulation

Background/Aims Visceral pain and hypothalamic-pituitary-adrenal axis (HPA) dysregulation is a common characteristic in irritable bowel syndrome (IBS) patients. Previously, we reported that a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) prevents chronic stress-mediated brain function abnormalities by attenuating the HPA axis response. Here, we compared the effect between different probiotic treatments on the perception of visceral pain during colorectal distension (CRD) following a chronic stress and the consequences to the activity of the HPA axis. Methods After a 2-week treatment with a combined probiotic formulation, or L. helveticus or B. longum alone in stressed mice, the visceral pain in response to CRD was recorded. The expression of glucocorticoid receptors was determined in the different brain areas involved in the stress response (hypothalamus, hippocampus, and prefrontal cortex). The plasma levels of stress hormones were also measured. Results A pretreatment using the combination of probiotic formulation significantly reduces the chronic stress-induced visceral hypersensitivity respectively at 0.06, 0.08, and 0.10 mL CRD volume. However, a single probiotic (B. longum or L. helveticus) administration is less effective in reducing visceral pain in stressed mice. Moreover, the expression of the glucocorticoid receptor mRNA was consistently up-regulated in several brain areas after pretreatment with a combined probiotic, which correlated with the normalization of stress response compared to the inconsistent effects of a single probiotic. Conclusion The combination of L. helveticus and B. longum is more effective in regulating glucocorticoid negative feedback on the HPA axis than probiotic alone and subsequently in treating stress-induced visceral pain.

140 Protective Effect of a Phytoestrogen-Enriched Diet on the Increase in Visceral Sensitivity and Intestinal Permeability Induced by Acute Stress in Female Rats

Background: Studies have suggested obesity as a risk factor for colorectal adenoma recurrence. It is unknown if weight change affects the risk of adenoma recurrence. Aim: We sought to examine whether weight change over a four year period is associated with subsequent colorectal adenoma recurrence. Methods: A total of 1,826 participants with a history of colorectal adenoma had body weight measured at baseline and 4 years later in the Polyp Prevention Trial. Adenoma recurrence was determined by end of trial colonoscopy. We used multinomial regression models to estimate adjusted relative risk ratios (RRR) and 95% confidence intervals (CI) to evaluate body mass index (BMI), weight change over 4 years and the risk of incident non-advanced and advanced adenoma recurrence. Results: A total of 605 (33.1%) participants had non-advanced adenoma recurrence while 118 (6.5%) had advanced adenoma recurrence. Although relative risks in this data set did not quite reach statistical significance, the analytic results suggest that baseline adiposity, especially BMI ≥ 30 kg/m2, is positively associated with adenoma recurrence. When compared with participants with BMI < 25 kg/m2 (n = 466) at baseline, there was an elevated risk of nonadvanced adenoma and advanced adenoma recurrence among participants with BMI = 25 29 kg/m2 (n = 868) (RRR = 1.23; 95%CI: 0.95-1.59 and RRR = 1.02; 95%CI: 0.61-1.68, respectively) and among those with BMI ≥ 30 kg/m2 (n = 492) (RRR = 1.27; 95%CI: 0.951.70 and RRR = 1.63; 95%CI: 0.96-2.78, respectively). When compared with those with relatively stable weight (less than 5 pound weight change) over the 4-year trial, weight gain or loss was not associated with adenoma recurrence (Table); this was consistent regardless of the baseline BMI. Conclusions: We observed a suggestive positive association between baseline BMI and adenoma recurrence; however, weight loss or gain over 4 years does not affect adenoma recurrence irrespective of baseline weight. Adjusted for age, sex, non-steroidal anti-inflammatory drug use, smoking and family history of colorectal cancer

Mo1659 Perinatal Exposure to Low Doses of the Food Endocrine Disruptor Bisphenol A Impairs Oral Tolerance and Sensitization in Offspring Rats at Adulthood

Background: The food contaminant Bisphenol A (BPA) is the building plastic monomer widely used in food packaging and epoxy resins lining metal cans. BPA is a xenoestrogen known to disrupt endocrine function, liver metabolism and intestinal barrier function. We have previously shown that BPA exposure in utero and through lactation at the No Observed Adverse Effect Level (NOAEL) (5mg/kg/j) decreased colonic paracellular permeability and enhanced the TNBS-induced colitis severity in adult female offspring. Herein we aimed to address the consequences of perinatal exposure to 1000 time lower than NOAEL dose of BPA (5μg/kg/day) on the upper intestinal immune homeostasis at adulthood. Material and methods: Gravid and breast feeding rats were treated per os from the 15th day of gravidity to pups weaning (D21) with or without BPA [5μg/kg/d]. Paracellular (4kDa dextran-FITC) and transcellular (Horse Radish Peroxidase) jejunal permeability were addressed by Ussing chamber on female adult offspring aged of 45 days. In parallel, effects of BPA perinatal exposure on oral tolerance and systemic immunisation to ovalbumin (OVA) in adult offspring were tested. Anti-OVA IgG titers were measured by ELISA as well as IFNγ secretion after In Vitro stimulation of either spleen ormesenteric lymph nodes cells culture.Results: Perinatal treatment with BPA induced a decrease of jejunal transcellular permeability compared to controls (0.03±0.0045x10-7 vs 0.09±0.02 x10-7 cm/s; p<0.05) but did not affect jejunal paracellular permeability. Furthermore, BPA perinatally treated adult rats exhibited higher anti-OVA IgG titers following either an oral tolerance protocol (162±88.7x103 compared to 6.2±1.9 x103 in controls; p<0.03) or a sensitization with OVA (783±127x103 compared to 433±100 x103 in controls; p<0.03). Enhanced humoral response in perinatally BPA treated adult rats was associated with a higher production of IFNγ by spleen in sensitized rats (628.2±392 compared to 114.9±72 pg/ml in controls) and by mesenteric lymph nodes in tolerized rats (707±325 compared to 54.6±43 pg/ml in controls). Conclusion: Perinatal exposure to low doses of BPA decreased jejunal transcellular permeability and impaired oral tolerance and sensitization to dietary antigen protein in adult offsprings. These results suggest that the perinatal period is a critical window for BPA exposure that may trigger food intolerance in later life.

M1689 The Endocrine Disruptor Bisphenol a (BPA) Decreases Paracellular Permeability in the Female Rat Colon At Environmentally Relevant Doses and Increases Tight Junction Protein Expression in CACO-2 Cells

Early epithelial restitution is an important repair modality in the gut mucosa and occurs as a consequence of epithelial cell migration. Our previous studies have shown the canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca2+ channel in intestinal epithelial cells (IECs) and regulates early mucosal restitution. Increased TRPC1 channel activity increases store-operated Ca2+ entry (SOCE) and enhances IEC migration during restitution. However, the exact upstream signals initiating TRPC1 activation after mucosal injury remain elusive. Recently, stromal interaction molecule 1 (STIM1) has been identified as a store Ca2+ sensor and can rapidly translocate to the plasma membrane (PM) where it interacts with and modulates Ca2+ channels. We hypothesized that STIM1 is crucial for stimulation of IEC migration after wounding by activating TRPC1 channel activity.Methods: Studies were conducted in IEC-Cdx2L1 cells, which represent differentiated IECs. STIM1 subcellular distribution was analyzed by surface biotinylation assays and immunohistochemical staining, while its functions were investigated by specific siRNA (siSTIM1) and ectopic overexpression of constitutively active STIM1 EF-hand mutants. [Ca]cyt was measured by fluorescence digital imaging analysis. Cell migration was measured in a model that mimics cell division-independent epithelial restitution. Results: IECs highly expressed STIM1 in cultured cells and in the intestinal mucosa of the rat. STIM1 translocation to the PM increased significantly within 10 min after wounding, peaked between 30 to 60 min, and then gradually returned to normal. Maximum increases in levels of STIM1 at PM were ~six times the prewounding control, which was associated with a dramatic increase in SOCE. There were no changes in total STIM1 protein levels after wounding. STIM1 directly interacted with TRPC1 in the PM as measured by immunoprecipitation assays. Ectopic expression of constitutively active STIM1 EF-hand mutants increased SOCE (by ~60%) and also stimulated IECmigration (by ~35%) after wounding. In contrast, STIM1 silencing by siSTIM1 decreased SOCE, inhibited IEC migration, and delayed epithelial restitution in cells overexpressing TRPC1. Levels of Ca2+ influx due to SOCE were decreased by ~70% in STIM1-silenced populations, whereas cell migration was inhibited by ~45%. Conclusions: These findings indicate that 1) wounding increases STIM1 translocation to the PM in IECs and 2) STIM1 at the PM interacts with and activates TRPC1, thus stimulating epithelial restitution as a result of increase in SOCE.