Analysis of the anti-estrogenic activity of TCDD in human ovarian carcinoma (BG-1) cells

In an attempt to detect compounds that can disrupt estrogen homeostasis and to analyze the mechanism(s) by which they may exert their actions, I have developed, in Chapter 2 of this dissertation, a recombinant human ovarian cell line (BG1Luc4E₂) that is capable of detecting estrogenic chemicals with the induction of luciferase. This sensitive bioassay is specific for estrogens and has a minimal detection limit of 0.1–1 pM and an ED ₅₀ of 10 pM estradiol. By growing these cells in estrogen stripped media (ESM), prior to treatment, we can remove 99% of background luciferase activity. In Chapter 3 I have demonstrated the aryl hydrocarbon receptor (AhR) dependent inhibition of estrogen signaling (30%–60%) by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild type BG-1 and BG1Luc4E₂ cells. I have also analyzed individual steps in the pathway of estrogen signaling to determine which are disrupted by TCDD. Pre-treatment of BG1Luc4E₂ cells with TCDD failed to increase the inhibitory effect and excess estradiol did not eliminate the effects of TCDD, thereby suggesting that metabolism of estradiol is not responsible for the antiestrogenic effect. TCDD also decreased estrogen receptor (ER) levels by 30%–60% under standard media conditions. However, growth in ESM decreased ER by about 7-fold without altering either the AhR level or function, the absolute maximum luciferase activity or the degree of inhibition by TCDD. I demonstrate further that TCDD does not decrease ER levels when cells are grown in ESM, that the AhR does not bind, in vitro, to the estrogen responsive DNA binding site, and that there is no decrease in ER-DNA binding from nuclear extracts of BG1Luc4E₂ cells treated with TCDD. Since inhibition of protein synthesis by cyclohexamide eliminated the inhibition of estradiol signaling by TCDD, we conclude that the effect is mediated by a secondary, TCDD-induced protein. Taken together, our data suggests that TCDD activates the AhR resulting in the induction of a secondary protein. This unknown protein most likely acts at a step following ER/DNA binding, either at the transcriptional or post-transcriptional level, to inhibit estrogen signaling in BG1Luc4E ₂ cells.