## 4 Discussion
Such hormonal complexity probably explained why their regulation following ovariectomy and E2 replacement differed between the liver and gut (as observed in the present study) since these organs are distinctly regulated by hormones, not to mention estrogens [41; 62; 63]. Maintenance of the gut barrier function is essential for metabolic and immune health. It requires tightly regulated processes to cope with the transport, digestion, and absorption of nutrients that take place throughout the intestine from the duodenum to the colon and ensure intestinal immunity, food tolerance, and limited inflammation [64; 65]. While ovariectomy induced obesity and triggered dysbiosis [66], we observed little changes in the mRNA expression levels of genes involved in epithelial cell differentiation and permeability throughout the gut segments with the exception of fatty acid transporters (Cd36 and Got2), Mmp7 in the jejunum, Muc2 in the ileum, and Tjp1 and Atoh1 in the colon. It has been demonstrated that transcription factors controlling intestinal differentiation [34] and intestinal permeability were deregulated in mice fed a high fat diet [65]. Worthy of note, temporal and regional changes in the in vivo intestinal permeability have recently been reported following loss of estrogen [33]. These authors demonstrated that while ovariectomy resulted in disturbed permeability accompanied with changes in tight-junction gene expression early after surgery, a normalization of the effects occurred over a period of 4 to 8 weeks that the authors suggested to be due to complex compensatory mechanisms. Since in the present study, the analysis was performed 13 weeks after surgery, compensatory mechanisms may also explain the absence of changes in the expression levels of genes involved in gut permeability. Determination of protein levels and immunohistochemical analysis at tight junctions would clarify whether or not there are changes in intestinal permeability under the different conditions studied. Based on the findings that the Ovx mice given E2 supplementation exhibited an alteration in cholesterol metabolism possibly related to the over-expression of Esr1, Cyp7a1, and Hmgcr and the reduced expression of Nr1h4 (FXR) in the liver [19], we explored bile acid biosynthesis in both the liver and the gut. Indeed, overexpression of ER\(\alpha\) in the liver triggers a loss in the fine tuning of negative feedback regulation of cholesterol synthesis [67] and HMG-CoA reductase and CYP7A1 are the rate-limiting enzymes of cholesterol metabolism and degradation in bile acids, respectively. On the panel of genes studied, transporters of bile acids were the most highly regulated both in liver and in ileum with profiles consistent with estrogens increasing efflux through BSEP from the liver but decreasing BA uptake in both the ileum (IBAT) and the liver (NTCP). In addition, E2 replacement caused a decrease of Fgf15 mRNA expression but no change in Nr1h4 (FXR) in the ileum, all highlighting deregulation of the BA biosynthesis process. Importantly, the mixture of pollutants produced effects similar to the E2 replacement on Slc10a2 and Fgf15 (although not significant), and on Nr1i1 (a trend) and Nr1c1 in the ileum. Since VDR and PPAR\(\alpha\) contribute to BA biosynthesis [39; 68], further studies will have to be undertaken to determine whether pollutants act on VDR and/or PPAR\(\alpha\) to mediate their effects. The relevance of our results must be compared with the dosage of the mixture in the range of the TDIs, thus not expected individually to generate a metabolic effect [16]. Consequently, the protocol set up here by demonstrating that several genes were affected (at least at the mRNA level) by the mixture of pollutants extended the concept of the cocktail effect. This concept states that the effect resulting from a mixture cannot be derived from the expected effect of a chemical analyzed individually, as reviewed [46; 69].