Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals

Abstract

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.

Figure 1

A scattergram of gene expression analyzed by DNA microarray. For each gene, the relative mRNA level in the control is given on the x axis and the expression level for the same transcript in the experimental sample (estrogenic compound exposed) is plotted on the y axis. Each graph displays two lines indicating 2-fold up-regulation or down-regulation in the expression level of each individual probe set comparing treated vs. control sample. The linear line (P < 0.05) was fitted to the microarray data.

Figure 2

Venn diagram showing the number of genes induced by E2 and EDs. The altered gene profiles by E2, OP and DES (overlapped 21 genes; A) or NP, BPA and Gen (overlapped 30 genes B), respectively, were summarized. A hierarchical clustering analysis (C) was performed following treatments with E2 and other EDs in the uterus of immature rats. Two-dimensional hierarchical clustering was applied to the expression data from 7.5 k genes, which showed significant changes in the balanced differential expression. Increased expression levels are shown in red and decreased expression levels are shown in green.

Figure 3

Confirmation of gene profiles by real-time PCR analysis. Relative values of expression of the altered genes quantified by real-time PCR are shown in graphs, indicating the comparison of fold change determined by real-time PCR analysis by E2, DES, OP, NP, BPA, and Gen in the uteri of immature rats. The representative genes are CaBP-9k, oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (calcyclin). Total RNAs from the uterus following treatment with estrogenic compounds were used to quantify altered gene expression normalized by cytochrome oxidase subunits I (1A) as a control.

Figure 4

Expression of CaBP-9k, oxytocin, Acrp30, Ldha and calcyclin mRNAs in the uterus of adult rats during estrous cycle. To investigate the expression of these genes during estrous cycle, the expression levels of these genes in the uterus of adult female rats were analyzed by real-time PCR. Data were analyzed by non-parametric procedure of the Kruskal Wallis test, followed by Dunnett's test for two-pair comparisons. The values represent means ± SD. a, P < 0.05 vs. Metestrus and Diestrus.