## 3 Results  
Impact of Estrogens and of the Pollutant Mixture on the Expression of the Estrogen Receptors in Various Intestinal Segments  
To better picture the consequences of ovariectomy on the gut, we first quantified the expression levels of the estrogen receptors in the jejunum, ileum, and colon as previously performed in the liver and adipose tissues of the same groups of female mice [19]. We observed that the expression levels of Esr1 and Esr2 genes encoding the nuclear estrogen receptor (ER) \(\alpha\) and \(\beta\), respectively, and Gper1 encoding the G-protein-coupled estrogen receptor 1 (GPR30) differ depending on the intestinal regions, with the highest level of expression of all three receptors in the colon, then the ileum and jejunum (Figure 1). Ovarietomy significantly affected Esr1 mRNA levels only in the colon (positively), while E2 replacement resulted in reduced mRNA levels in the jejunum and ileum of both Esr1 and Gper1 as compared to Ovx mice. Esr2 mRNA levels did not change between groups, regardless of the gut segment (Figure 1). Exposure to pollutants had an inhibitory effect on mRNA levels of Esr1 and Gper1 mRNA levels in the ileum (Figure 1). An upward trend in Esr1 mRNA levels (\(p\) = 0.06) was observed in the colon of Ovx mice exposed to pollutants compared to Ovx mice (Figure 1). Thus, exposure to pollutants triggered effects similar to those resulting from E2 replacement, suggesting that the mixture might have estrogen-mimetic activities in the jejunum and ileum. Impact of Estrogen Withdrawal/Replacement and Pollutant Mixture on the Expression of Xenobiotic Processing Genes  
Preceding reports indicated that the pollutants of the used mixture can interact with xenobiotic receptors [27; 28]. We focused on the expression levels of genes encoding the aryl hydrocarbon receptor (Ahr), the constitutive androstane receptor CAR (Nr1i3), and the pregnane xenobiotic receptor PXR (Nr1i2). We observed that Ahr mRNA levels did not vary between groups regardless of the intestinal segment (Figure 2 and Figure S1). Regarding the expression of the Nr1i3 gene, the jejunum responded to estrogen withdrawal with a significant increase not detected in ovariectomized mice given to E2 implants. Ovarietomy did not significantly affect Nr1i2 mRNA levels, which were significantly decreased in E2-replaced Ovx mice compared to Ovx mice (Figure 2). No inter-group variation was detected in the ileum and colon, with respect to Nr1i2 and Nr1i3 mRNA levels (Figure S1). CAR and PXR immediate target genes, which encode the cytochrome P450 enzymes CYPB10 and CYP3A11, respectively [29], were also surveyed. With the exception of Cyp3a11 mRNA levels in the colon, which were in the same range in Sham and Ovx mice, we observed upregulation of both Cyps in response to estrogen withdrawal which was avoided in E2-replaced Ovx mice in the jejunum, ileum, and colon (a trend in the ileum) (Figure 2). As observed with xenobiotic receptors, exposure to pollutants did not cause any change in the expression levels of Cyps (Figure 2). To complete the study, we compared these results in the gut with data from the liver Contrasting the jejunum, we found that estrogen withdrawal caused downregulation of Ahr mRNA levels in the liver, an effect not evidenced in E2-replaced Ovx mice compared to Ovx mice. However, Nr1i2 and Nr1i3 mRNA levels did not fluctuate in the liver between groups. In addition, the expression of both hepatic Cyps was downregulated in Ovx mice (a trend for Cyp2b10) with further reduction in E2-replaced Ovx mice as compared to Ovx mice (Figure S2). Adipose tissues, which also express AHR but not PXR and CAR (Ref. in [5]) were investigated and Ahr mRNA levels fluctuated negatively with estrogens in the  
Figure 1: Effect of ovariectomy alone (Ovx) or with 17ß-estradiol (E2) supplementation (Ovx+E2) or with exposure to pollutants (Ovx + Poll) on the expression of genes encoding the estrogen receptors ER\(\alpha\), ER\(\beta\), GPR30 in various segments of the gut: The jejunum (**A**), the ileum (**B**), and the colon (**C**).