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Previous research from our laboratory has determined the transcript profiles for developing fetal rat female and male reproductive tracts following transplacental exposure to estrogens. Prenatal exposure to bisphenol A (BPA) or 17-α-ethynyl estradiol (EE) significantly affects steroidogenic acute regulatory (StAR) protein transcript levels in the developing male rat reproductive tract. The purpose of this study was to establish the intratesticular distribution and temporal expression pattern of StAR, a key gene involved in steroidogenesis. Beginning on gestation day (GD) 11, pregnant Sprague-Dawley rats were exposed daily to 10μg/kg/day EE and fetal testes were harvested at GD16, 18, or 20. Quantitative reverse transcriptase PCR (QRT-PCR) demonstrated no significant difference in StAR transcript levels present at GD16. However, at GD18, StAR transcripts were significantly decreased following exposure. Immunohistochemistry demonstrated similar StAR protein levels in interstitial region of GD16 testes and an obvious decrease in StAR protein levels in the interstitial region of GD18 testes. Moreover, starting at GD11 additional dams were dosed with 0.001 or 0.1 μg/kg/day EE or 0.02, 0.5, 400 mg/kg/day BPA via subcutaneous injections. QRT-PCR validated previous microarray dose-related decreases in StAR transcripts at GD20, whereas immunohistochemistry results demonstrated decreases in StAR protein levels in the interstitial region at the highest EE and BPA doses only. Neither EE nor BPA exposure caused morphological changes in the developing seminiferous cords, Sertoli cells, gonocytes, or the interstitial region or Leydig cells at GD16-20. High levels of estrogens decrease StAR expression in the fetal rat testis during late gestation., (© 2012 Wiley Periodicals, Inc.)

A reliable in vitro model to determine the potential estrogenic activity of chemicals of interest is still unavailable. To further investigate the usefulness of a human-derived cell line, we determined the transcriptional changes induced by bisphenol A (BPA) in Ishikawa cells at various doses (1 nM, 100 nM, 10 microM, and 100 microM) and time points (8, 24 and 48 h) by comparing the response of approximately 38,500 human genes and ESTs between treatment groups and controls (vehicle-treated). By trend analysis, we determined that the expression of 2794 genes was modified by BPA in a dose- and time-dependent manner (p< or =0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest doses of BPA evaluated (10-100 microM), while the genomic response of the cells exposed to low doses of BPA was essentially negligible. By comparing the Ishikawa cells' response to BPA vs.17 alpha-ethynyl estradiol we determined that the change in the expression of 307 genes was identical in the direction of the change, although the magnitude of the change for some genes was different. Further, the response of Ishikawa cells to high doses of BPA shared similarities to the estrogenic response of the rat uterus, specifically, 362 genes were regulated in a similar manner in vivo as well as in vitro. Gene ontology analysis indicated that BPA results in changes to multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response after exposure to chemicals with varied estrogenic activity, and offer an in vitro model to assess this mode of action., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 alpha-ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001-10 microg EE/kg/day, 0.001-100 mg Ges/kg/day, or 0.002-400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.

Exposure to some compounds with estrogenic activity, during fetal development, has been shown to alter development of reproductive organs, leading to abnormal function and disease either after birth or during adulthood. In order to understand the molecular events associated with the estrogenicity of different chemicals and to determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have used microarray technology to determine the transcriptional program influenced by exposure to this class of compounds during organogenesis and development. Changes in patterns of gene expression were determined in the developing uterus and ovaries of Sprague-Dawley rats on GD 20, exposed to graded dosages (sc) of 17alpha-ethynyl estradiol (EE), genistein, or bisphenol A (BPA) from GD 11 to GD 20. Dose levels were roughly equipotent in estrogenic activity. We compared the transcript profiles between treatment groups and controls, using oligonucleotide arrays to determine the expression level of approximately 7000 rat genes and over 1000 expressed squence tags (ESTs). At the highest tested doses of EE, BPA, or genistein, we determined that less than 2% of the mRNA detected by the array showed a 2-fold or greater change in their expression level (increase or decrease). A dose-dependent analysis of the transcript profile revealed a common set of genes whose expression is significantly and reproducibly modified in the same way by each of the 3 chemicals tested. Additionally, each compound induces changes in the expression of other transcripts that are not in common with the others, which indicated not all compounds with estrogenic activity act alike. The results of this study demonstrate that transplacental exposure to chemicals with estrogenic activity changes the gene expression profile of estrogen-sensitive tissues, and that the analysis of the transcript profile of these tissues could be a valuable approach to determining the estrogenicity of different compounds.