Christine Belloir

Dietary xenoestrogens differentially impair 3T3-L1 preadipocyte differentiation and persistently affect leptin synthesis

Recent observations have highlighted adipogenesis alterations under exposure to several xenoestrogens at critical stages, and pointed at their possible involvement in the pathogenesis of obesity. However, it remains unclear whether these effects are mediated by classical estrogen receptor (ER) binding and subsequent transcriptional modulation. The aim of this study was to determine the (anti-)adipogenic impact of apigenin, bisphenol A, genistein and 17beta-estradiol at the onset of adipose cell maturation, and to correlate it to their estrogenic potential. In steroid-free conditions, 3T3-L1 preadipocytes were induced to differentiate in the presence of xenoestrogens for 2 days. DNA and triglyceride levels, leptin secretion and expression of Pref-1, C/EBPbeta, PPARgamma2, FAS, leptin and ERs were measured on days 0, 3 and 8 of differentiation. Genistein potently blocked mitotic clonal expansion and all markers of maturation. Bisphenol A and estradiol did not modify triglyceride accumulation but increased the expression of differentiation genes. Apigenin caused a weak but reversible delay in adipogenesis although it unexpectedly enhanced leptin synthesis. However, the expression of steroid hormone receptors was not associated with these differential effects. In conclusion, we could not put a clear estrogen-dependent mechanism forward, but early exposure to xenoestrogens persistently disrupted adipocyte gene expression and leptin synthesis.

Expression and differential localization of xenobiotic transporters in the rat olfactory neuro-epithelium

Transporters, such as multidrug resistance P-glycoproteins (MDR), multidrug resistance-related proteins (MRP) and organic anion transporters (OATs), are involved in xenobiotic metabolism, particularly the cellular uptake or efflux of xenobiotics (and endobiotics) or their metabolites. The olfactory epithelium is exposed to both inhaled xenobiotics and those coming from systemic circulation. This tissue has been described as a pathway for xenobiotics to the brain via olfactory perineural space. Thereby, olfactory transporters and xenobiotic metabolizing enzymes, dedicated to the inactivation and the elimination of xenobiotics, have been involved in the toxicological protection of the brain, the olfactory epithelium itself and the whole body. These proteins could also have a role in the preservation of the olfactory sensitivity by inactivation and clearance of the excess of odorant molecules from the perireceptor space. The goal of the present study was to increase our understanding of the expression and the localization of transporters in this tissue. For most of the studied transporters, we observed an opposite mRNA expression pattern (RT-PCR) in the olfactory epithelium compared to the liver, which is considered to be the main metabolic organ. Olfactory epithelium mainly expressed efflux transporters (MRP, MDR). However, a similar pattern was observed between the olfactory epithelium and the olfactory bulb. We also demonstrate distinct cellular immunolocalization of the transporters in the olfactory epithelium. As previously reported, Mrp1 was mainly found in the supranuclear portions of supporting cells. In addition, Mrp3 and Mrp5 proteins, which were detected for the first time in olfactory epithelium, were localized to the olfactory neuron layer, while Mdr1 was localized to the capillary endothelium of lymphatic vessels in the subepithelial region. The pattern of expression and the distinct localization of the olfactory transporters showed in this work may highlight on their specific function in the whole olfactory epithelium.

Inhibition of Carcinogen-Induced DNA Damage in Rat Liver and Colon by Garlic Powders With Varying Alliin Content

Abstract: The present study was designed to investigate the protective efficiency of three garlic powders, obtained from bulbs grown in soils with different levels of sulfur fertilization, against DNA damage. Increasing fertilization of soil resulted in an increased alliin content of the powders. Garlic powders were administered to rats for 2 weeks (5% of the diet) and their antigenotoxic effects were examined in the liver and the colon using the comet assay. Consumption of the different garlic powders induced a 35–60% reduction in DNA damage induced by N-nitrosodimethylamine (NDMA) in rat liver. Increased alliin content of the garlic powder was associated strongly with a proportional decrease in NDMA induced DNA alteration. DNA damage induced by aflatoxin B1 in the liver or by 1,2-dimethylhydrazine in the colon were also decreased strongly by the three garlic powders but these decreases were not correlated to the alliin content of the garlic powders. Feeding garlic powders did not modify the genotoxic activity of the direct-acting carcinogen methylnitrosourea in the colon. Part of our results supports evidence that fertilization can have an impact on the protective capacity of garlic bulbs.

Efficient Production and Characterization of the Sweet-Tasting Brazzein Secreted by the Yeast Pichia pastoris

Brazzein is a small, heat-, and pH-stable sweet protein present in the fruits of the West African plant Pentadiplandra brazzeana Baillon. It exists in two forms differing in sweetness intensity. The major form, called pyrE-bra, contains a pyroglutamic acid at its N-terminus, while the minor form, called des-pyrE-bra, lacks this residue. Here we describe the heterologous expression in the methylotrophic yeast Pichia pastoris of two natural forms of brazzein, pyrE-bra and des-pyrE-bra, and an additional form, called Q1-bra, which is not naturally occurring in the fruit. Q1-bra differs from pyrE-bra in having a glutamine residue instead of pyrE at its N-terminus. Over an expression period of 6 days, we obtained approximately 90, 30, and 90 mg/L of purified recombinant pyrE-bra, Q1-bra, and des-pyrE-bra brazzein forms, respectively. Recombinant proteins were purified and submitted to mass spectrometry and (1)H NMR spectroscopy. The data indicate that the recombinant brazzein forms were properly folded. Moreover, they activated the human sweet receptor in vitro and evoked sweetness in vivo with properties similar to those of the two natural brazzein forms.

Identification of Odorant-Receptor Interactions by Global Mapping of the Human Odorome

The human olfactory system recognizes a broad spectrum of odorants using approximately 400 different olfactory receptors (hORs). Although significant improvements of heterologous expression systems used to study interactions between ORs and odorant molecules have been made, screening the olfactory repertoire of hORs remains a tremendous challenge. We therefore developed a chemical systems level approach based on protein-protein association network to investigate novel hOR-odorant relationships. Using this new approach, we proposed and validated new bioactivities for odorant molecules and OR2W1, OR51E1 and OR5P3. As it remains largely unknown how human perception of odorants influence or prevent diseases, we also developed an odorant-protein matrix to explore global relationships between chemicals, biological targets and disease susceptibilities. We successfully experimentally demonstrated interactions between odorants and the cannabinoid receptor 1 (CB1) and the peroxisome proliferator-activated receptor gamma (PPARγ). Overall, these results illustrate the potential of integrative systems chemical biology to explore the impact of odorant molecules on human health, i.e. human odorome.

The membrane-associated MUC1 improves adhesion of salivary MUC5B on buccal cells. Application to development of an in vitro cellular model of oral epithelium.

OBJECTIVES The mucosal pellicle is a thin layer of salivary proteins, mostly MUC5B mucins, anchored to epithelial oral cells. This pellicle is involved in protection of oral mucosae against abrasion, pathogenic microorganisms or chemical xenobiotics. The present study aimed at studying the involvement of MUC1 in mucosal pellicle formation and more specifically in salivary MUC5B binding using a cell-based model of oral epithelium. DESIGN MUC1 mRNAs were not detected in TR146 cells, and therefore a stable cell line named TR146/MUC1 expressing this protein was developed by transfection. TR146 and TR146/MUC1 were incubated with human saliva in order to evaluate retention of MUC5B by epithelial cells. RESULTS The cell surface of both TR146 and TR146/MUC1 was typical of a squamous non-keratinized epithelium, with the presence of numerous microplicae. After incubation for 2h with saliva diluted in culture medium (1:1) and two washes with PBS, saliva deposits on cells appeared as a loose filamentous thin network. MUC5B fluorescent immunostaining evidenced a heterogeneous lining of confluent cell cultures by this salivary mucin but with higher fluorescence on TR146/MUC1 cells. Semi-quantification of MUC5B bound to cells confirmed a better retention by TR146/MUC1, evaluated by Dot Blot (+34.1%, p<0.05) or by immunocytochemistry (+44%, p<0.001). CONCLUSION The membrane-bound mucin MUC1 is a factor enhancing the formation of the mucosal pellicle by increasing the binding of salivary MUC5B to oral epithelial cells. An in vitro model suitable to study specifically the function and properties of the mucosal pellicle is proposed.

Sweeteners and sweetness enhancers

Purpose of review The current review summarizes and discusses current knowledge on sweeteners and sweetness enhancers. Recent findings The perception of sweet taste is mediated by the type 1 taste receptor 2 (T1R2)/type 1 taste receptor 3 (T1R3) receptor, which is expressed in the oral cavity, where it provides input on the caloric and macronutrient contents of ingested food. This receptor recognizes all the compounds (natural or artificial) perceived as sweet by people. Sweeteners are highly chemically diverse including natural sugars, sugar alcohols, natural and synthetic sweeteners, and sweet-tasting proteins. This single receptor is also the target for developing novel sweet enhancers. Importantly, the expression of a functional T1R2/T1R3 receptor is described in numerous extraoral tissues. In this review, the physiological impact of sweeteners is discussed. Summary Sweeteners and sweetness enhancers are perceived through the T1R2/T1R3 taste receptor present both in mouth and numerous extraoral tissues. The accumulated knowledge on sugar substitutes raises the issue of potential health effects.

Highly sensitive olfactory biosensors for the detection of volatile organic compounds by surface plasmon resonance imaging

Nowadays, monitoring of volatile organic compounds (VOCs) is very important in various domains. In this work, we aimed to develop sensitive olfactory biosensors using odorant binding proteins (OBPs) as sensing materials. Three rat OBP3 derivatives with customized binding properties were designed and immobilized on the same chip for the detection of VOCs in solution by surface plasmon resonance imaging (SPRi). We demonstrated that the proteins kept their binding properties after the immobilization under optimized conditions. The obtained olfactory biosensors exhibited very low limits of detection in both concentration (200 pM of β-ionone) and in molecular weight of VOCs (100 g/mol for hexanal). Such a performance obtained with SPRi in solution is especially remarkable. We hypothesized that the binding of VOCs to the active sites of OBPs induced a local conformational change in the proteins. This change would give rise to a variation of refractive index, to which SPRi is extremely sensitive. In addition, the olfactory biosensors showed a high selectivity especially at relatively low VOC concentrations. With optimized regeneration procedures, they also showed very good repeatability not only from measurement to measurement but also from chip to chip with a lifespan up to almost two months. These olfactory biosensors are particularly interesting for trace detection of VOCs in solution.

Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli

Umami taste perception is mediated by the heterodimeric G-protein coupled receptors (GPCRs), formed by the assembly of T1R1 and T1R3 subunits. T1R1 and T1R3 subunits are class C GPCRs whose members share common structural homologies including a long N-terminal domain (NTD) linked to a seven transmembrane domain by a short cysteine-rich region. The NTD of the T1R1 subunit contains the primary binding site for umami stimuli, such as L-glutamate (L-Glu) for humans. Inosine-5’-monophosphate (IMP) binds at a location close to the opening of the T1R1-NTD “flytrap”, thus creating the observed synergistic response between L-Glu and IMP. T1R1/T1R3 binding studies have revealed species-dependent differences. While human T1R1/T1R3 is activated specifically by L-Glu, the T1R1/T1R3 in other species is a broadly tuned receptor, sensitive to a range of L-amino acids. Because domestic cats are obligate carnivores, they display strong preferences for some specific amino acids. To better understand the structural basis of umami stimuli recognition by non-human taste receptors, we measured the binding of selected amino acids to cat T1R1/T1R3 (cT1R1/cT1R3) umami taste receptor. For this purpose, we expressed cT1R1-NTD in bacteria as inclusion bodies. After purification, refolding of the protein was achieved. Circular dichroism spectroscopic studies revealed that cT1R1-NTD was well renatured with evidence of secondary structures. Using size-exclusion chromatography coupled to light scattering, we found that the cT1R1-NTD behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that cT1R1-NTD is capable of binding L-amino acids with Kd values in the micromolar range. We demonstrated that IMP potentiates L-amino acid binding onto renatured cT1R1-NTD. Interestingly, our results revealed that IMP binds the extracellular domain in the absence of L-amino acids. Thus, this study demonstrates that the feasibility to produce milligram quantities of cT1R1-NTD for functional and structural studies.

P259 Identification de nouveaux antagonistes des récepteurs aux endocannabinoïdes à action périphérique susceptibles d’améliorer les paramètres glucido-lipidiques chez la souris obèse

Introduction Le systeme endocannabinoide (SEC) est une cible therapeutique potentielle pour lutter contre l’obesite. Ainsi, le Rimonabant®, un antagoniste des recepteurs aux endocannabinoides 1 (CB1R) diminue la masse corporelle et ameliore les parametres glucido-lipidiques de patients obeses. Neanmoins, ce medicament a ete retire du marche suite a des effets centraux induisant des troubles neuropsychiatriques. L’objectif de cette etude est d’identifier des analogues du Rimonabant® a action peripherique susceptibles d’avoir des effets benefiques sur le metabolisme glucido-lipidique de la souris. Materiels et methodes Des analogues du Rimonabant® ne passant pas la barriere hemato-encephalique ont ete selectionnes dans une chimiotheque a l’aide d’un modele predictif QSAR. La capacite des molecules candidates a moduler l’activite du SEC et a exercer des effets sur le metabolisme glucido-lipidique a ete testee chez la souris par des approches in vivo et in vitro. Resultats Sur huit molecules selectionnees (M1-M8), trois molecules (M2, M5 et M6) ont exerce un effet sur l’expression du gene CB1R dans des explants de foie de souris obese. L’administration intra-peritoneale aigue de M2, M5 et M6 a induit une amelioration de la tolerance au glucose (OGTT) chez des souris controles. Les tests repetes chez la souris CB1R-/- ont suggere que seule M6 a une action dependante du CB1R. Parallelement, l’etude des interactions ligandrecepteur basee sur la mesure des variations intracellulaires d’AMPc dans des cellules transfectees par CB1R a montre que M6 exerce un effet agoniste inverse sur le recepteur. Enfin, l’administration chronique de M6 a des souris obeses a induit une amelioration de la tolerance au glucose et de la sensibilite a l’insuline independamment d’une diminution de la prise alimentaire et du poids corporel. Conclusion En conclusion, nos resultats montrent des effets favorables de l’analogue du Rimonabant® M6 (JM00266) sur le metabolisme glucidolipidique de souris obeses et confortent l’hypothese selon laquelle l’inactivation ciblee du SEC peripherique constitue une strategie therapeutique contre les desordres lies a l’obesite.