ENDOCRINEANALYSIS O F T H E ANTI-ESTROGENIC E F F E C T O F TCDD IN
HUMAN OVARIAN CARCINOMA (BG-1) C E L L S
Jane M. Rogers and Michael S. Denison
Department of Environmental Toxicology, University of Califomia, Davis, CA 95616 USA

Introduction
Concems have been raised over the last decade regarding the prevalence in the environment ofcompounds that are able to disrupt the estrogen homeostasis of an organism. Although effects onhuman health remain controversial, the reproductive success of several wildlife populations has
been defrimentally affected by such compounds''^. Often, these compounds act as esfrogenagonists by interacting directly with the estrogen receptor (ER). However, the halogenatedaromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-/7-dioxin (TCDD), is known to exhibit an arrayof anti-estrogen effects in vivo and in cells in culture, yet the mechanism(s) for this inhibition has
not been elucidated. Numerous investigators have focused on determining the mechanism for theanti-estrogenic action ofTCDD. Several hypotheses have been proposed, including the increasedmetabolism and clearance of estradiol by TCDD induced enzymes' and down-regulation of ER
mRNA and protein'' . It has also been proposed that the aryl hydrocarbon receptor (AhR)
interacts with an inhibitory dioxin responsive element (iDRE) located adjacent to, or overlapping,
the ER binding site in the upstream regions of the cathepsin-D and pS2 genes, thereby blocking
ER-DNA binding via steric hinderance''*. However, this mechanism does not seem to be
responsible for the TCDD induced repression of other estrogen induced genes'.
In previous work we have demonsfrated the AhR-dependent inhibition of estrogen signaling byTCDD in wild type human ovarian carcinoma (BG-1) cells and in recombinant BG-1 cells(BGILUC4E2) that have been stably transfected with an estrogen-responsive luciferase reporter
gene'**. We have also examined individual steps in the pathway of estrogen signaling in order todetermine those that are affected by TCDD. We have previously demonstrated that pre-treatmentof BGILuc4E2 cells with TCDD failed to increase the inhibitory effect and that 1,000-fold excessestradiol did not eliminate the effect of TCDD'**. TCDD also decreased ER protein and mRNAlevels under standard media conditions. However, growth in estrogen-stripped media (ESM)(which is used to decrease background luciferase levels) decreased ER protein and mRNA levels
by about 7-fold without altering either the AhR level or function'". This decrease in ER but notAhR results in a significant (~ 7-fold) increase in the AhR/ER ratio in cells grown in ESM
compared to those grown in standard media, and yet no change in maximal luciferase induction or
the degree of inhibition by TCDD was observed. We also demonstrated that TCDD does notdecrease ER protein levels when cells are grown in ESM'". Taken together, these resuhs from ourprevious studies suggest that the TCDD-dependent inhibition of estrogen signaling in BGlLuc4E2
cells is not mediated by a decrease in either estradiol or ER levels. In the present study we haveextended this work by examining additional steps in the pathway of estrogen signal transduction in
an attempt to determine the mechanism by which TCDD inhibits estrogen signaling in BG-1 andBGlLuc4E2 cells.
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Materials and Methods
Luciferase Expression Assav for BGlLuc4E2 Cells. Cells were grown for 6 days in estrogen
stripped media (ESM) (phenol red free MEM with 5% dextran-coated charcoal stripped FBS),
with daily media changes. Cells were exposed to the indicated chemical for increasing amounts oftime, followed by lysis and measurement of luciferase activity as described in detail".
Northem Blot Analvsis. RNA from treated cells was isolated using the RNeasy Mini Kit from
Qiagen. Northem blot analysis was carried out on total RNA (20 fig) as previously described'".
The resulting blots were visualized by autoradiography and quantified using a Molecular
Dynamics Phosphorlmager Sl, and pS2 values were normalized to those of GAPDH.
Results and Discussion
To determine how rapidly TCDD inhibits esfrogen-induced luciferase activity we analyzed thetime-course ofTCDD inhibition in BGlLuc4E2 cells grown in ESM. In general, TCDD-dependent
inhibition occurred between 7 hours and 18 hours after treatment (fig. 1). These results suggestthat the effect of TCDD on estrogen-induced luciferase activity was not a direct, primary event,which would have been observed much earlier, but was the result of an indirect, secondary
response. To further examine whether the anti-estrogenic action ofTCDD in BGlLuc4E2 cells ismediated directly by the AhR or indirectly by a TCDD-induced protein, BGlLuc4E2 cells weregrown in standard media and the effect ofthe protein synthesis inhibitor cyclohexamide (CHX) onthe TCDD-dependent inhibition of the endogenous estrogen-dependent pS2 gene expression wasexamined. TCDD reduced pS2 mRNA levels to 60% - 70% of that of DMSO treated cells,
however, in the presence of cyclohexamide, TCDD did not significantly reduce pS2 mRNA levels
as compared to the DMSO control (fig. 2). These data strongly support a role for a TCDDinduced protein as being responsible for mediating the inhibition of estrogen signaling in BG-1cells.
A critical step in the pathway of esfrogen signaling is the binding ofthe ER complex to DNA. To
examine the effect of TCDD on ER-DNA binding we first determined whether the AhR could
directly bind the estrogen responsive element (ERE) which was responsible for estrogen signalingin our reporter system. Although we observed that reticulocyte lysate expressed TCDD:AhR:Amtcomplex could bind to a ["P] DRE oligonucleotide, no TCDD-inducible protein-DNA complex
was formed using a ['^P]-labelled oligonucleotide containing the ERE from the chicken
vitellogenin A2 gene. We also examined the ability of nuclear proteins from conlrol and TCDD
treated BGlLuc4E2 cells, grown in standard media, to bind these oligonucleotides. As expected, asmall TCDD induced protein-DNA band was formed with the ['"P] DRE oligonucleotide, however
no TCDD-inducible AhR-DNA complex was formed with the chicken vitellogenin ERE. Theseresults clearly indicate that the AhR does not bind, at least in vitro, to the ERE we have used in oursystem. The ER:ERE complex that formed was unaffected by TCDD treatment, indicating that, invitro, TCDD does not alter binding of the ER to DNA, even though total cellular ER levels are
decreased by approximately 40%'". We therefore propose a scenario for the inhibition of estrogensignaling by TCDD in BGlLuc4E2 cells which resuhs from a TCDD- and AhR-dependent
induction of an as yet, unknown "repressor" protein. Our data suggests that this TCDD-induced
protein acts at a step that follows ER/DNA binding and most likely occurs at the transcriptional or
post-transcriptional level. A model of the proposed mechanism of the anti-estrogenic action ofTCDD in BGlLuc4E2 cells is shown in figure 3.

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Acknowledgments
This work is supported by the National Institutes of Health (ES07685, ES04699 and ES05707)(MSD) and a UC Toxic Substances Research and Teaching Program fellowship (JMR). We wish
to thank Dr. George Clark (Xenobiotic Detection Systems) for the BG-1 cells. Dr. Larry
14•

Control•

1 n M Estradiol

12
A I n.M TCDO + I nM Estradiol

10 > u'I 2 8 <a.
6 -J —

020

15

102S

30

Time (hours)

Hjelmeland (UC Davis) for GAPDH and Dr. William Helferich (University of Illinois) for pS2.
Figure 1. Time course of inhibition of estrogen-induced luciferase activity by TCDD in
BGlLuc4E2 cells. Cells were grown for 6 days in ESM and then treated with the indicatedchemical for increasing amounts of time. Luciferase activity was determined as described. Values
represent mean ± SD of four determinations.

EthanolcTCHXT

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Figure 2. Cyclohexamide blocks TCDD-dependent inhibition of estrogen-induced pS2 mRNA
expression in BGlLuc4E2 cells. Ceils were grown in standard media and treated for 24 hours withDMSO (C) or 10 nM TCDD (T) with or without 10 pM cyclohexamide (CHX). Isolation of RNAand Northem analysis was cartied out as described in Materials and Methods. This experiment
was performed in triplicate with similar results, and a representative blot is shown.
Figure 3. Proposed mechanism for the TCDD-dependent decrease in estrogen signaling in
BGlLuc4E2 cells. Abbreviations: BTM (basal transcriptional machinery), Hsp's (heat shockproteins), DREs (dioxin responsive elements), EREs (estrogen responsive elements), AhR (arylhydrocarbon receptor), ARNT (AhR nuclear translocator), XAP2 (hepatitis B vims X-associatedprotein 2).
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