Certain chemicals in the environment are estrogenic. The low potencies of these compounds, when studied singly, suggest that they may have little effect on biological systems. The estrogenic potencies of combinations of such chemicals were screened in a simple yeast estrogen system (YES) containing human estrogen receptor (hER). Combinations of two weak environmental estrogens, such as dieldrin, endosulfan, or toxaphene, were 1000 times as potent in hER-mediated transactivation as any chemical alone. Hydroxylated polychlorinated biphenyls shown previously to synergistically alter sexual development in turtles also synergized in the YES. The synergistic interaction of chemical mixtures with the estrogen receptor may have profound environmental implications. These results may represent a previously uncharacterized level of regulation of estrogen-associated responses.

In the uterus insulin-like growth factor-1 (IGF-1) signaling can be initiated by estradiol acting through its nuclear receptor (estrogen receptor (ER)) to stimulate the local synthesis of IGF-1. Conversely, in vitro studies have demonstrated that estradiol-independent ER transcriptional activity can be induced by IGF-1 signaling, providing evidence for a cross-talk mechanism between IGF-1 and ER. To investigate whether ER␣ is required for uterine responses to IGF-1 in vivo, both wild-type (WT) and ER␣ knockout (␣ERKO) mice were administered IGF-1, and various uterine responses to IGF-1 were compared. In both WT and ␣ERKO mice, IGF-1 treatment resulted in phosphorylation of uterine IGF-1 receptor (IGF-1R) and formation of an IGF-1R/insulin receptor substrate-1/ phosphatidylinositol 3-kinase signaling complex. In addition, IGF-1 stimulated phosphorylation of uterine Akt and MAPK in both WT and ␣ERKO mice. However, IGF-1 treatment stimulated BrdUrd incorporation and proliferating cell nuclear antigen expression in WT uteri only. To determine whether ER␣ can be activated in vivo by IGF-1 signaling, transgenic mice carrying a luciferase gene driven by two estrogen response elements (ERE-luciferase mice) were utilized. Treatment of ovariectomized ERE-luciferase mice with IGF-1 resulted in an increase in uterine luciferase activity that was attenuated in the presence of the ER antagonist ICI 182,780. Together these data demonstrate that 1) functional signaling proximal to IGF-1R is maintained in the ␣ERKO mouse uterus, 2) ER␣ is necessary for IGF-1 induction of uterine nuclear proliferative responses, and 3) cross-talk between IGF-1R and ER signaling pathways exists in vivo.Epithelial cells of the mammalian uterus undergo a wave of DNA synthesis followed by mitosis in response to 17␤-estradiol (E 2 ), 1 which regulates the transcription of numerous target genes by binding to and activating the nuclear estrogen receptor (ER). Among the genes identified as targets for regulation by the E 2 /ER complex in the uterus is that encoding insulinlike growth factor-1 (IGF-1). Studies have demonstrated that rodent uterine IGF-1 mRNA levels increase after exposure to E 2 (1, 2). Furthermore, presumably through increasing local production of IGF-1, E 2 has been shown to stimulate uterine IGF-1 receptor (IGF-1R) signaling as measured by tyrosine phosphorylation of IGF-1R and the formation of a signaling complex composed of IGF-1R, insulin receptor substrate-1 (IRS-1), and p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (3, 4). These studies suggested that IGF-1 signaling is involved in E 2 -induced uterine growth, and in support of this mechanism, other studies have shown that, like E 2 , IGF-1 can induce DNA synthesis in cells of the rodent uterus (5). A more recent study further demonstrated a role for IGF-1 in E 2 -induced uterine proliferation by demonstrating that IGF-1 is required for E 2 -induced uterine epithelial cell mitosis (6). In that study, DNA synthesis occurred in IGF-1 knockout (IGF-1KO) m...

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.ImagesFigure 1.Figure 2. AFigure 2. BFigure 3.Figure 4.Figure 5.