ENDOCRINE DISRUPTORSAPPLICATION OF A HUMAN OVARIAN CELL BIOASSAY FOR THE
DETECTION AND ANALYSIS OF ESTROGEN-TOXICANT
INTERACTIONS
Jane M. Rogers and Michael S. Denison
Departinent of Environmental Toxicology, University of Califomia, Davis, CA 95616 USA

Introduction
Environmental contaminants which can dismpt the hormonal homeostasis of an organism havegained widespread attention in recent years, and their defrimental effects on the reproductive
success of many wildlife populations have been well documented''^. Although many of thesecompounds can act as esfrogen agonists, the dioxin-like halogenated aromatic hydrocarbons(HAHs) are a class of chemicals which have been shown to interact in an inhibitory manner withthe esfrogen system, and yet the mechanism for these interactions has still not been resolved.
There appear to be several mechanisms by which 2,3,7,8-tefrachlorodibenzo-/7-dioxin (TCDD),
tae most potent HAH and aryl hydrocarbon receptor (AhR) ligand, may exert its anti-esfrogenicaction. It has been reported that inhibition of induction of some esfrogen dependent genes such ascathepsin-D and pS2 is due to binding of thefransformedTCDD:AhR complex to an imperfectdioxin responsive element which overlaps the esfrogen responsive element (ERE) and prevents
DNA binding of the esfrogen receptor (ER)'"*. However, this mechanism does not appear to be
responsible for the TCDD induced repression of other esfrogen dependent genes'. Additional
mechanisms, which involve changes in ER phosphotylation, down regulation of ER gene
expression and increased metabolism and clearance of esfradiol, have also been proposed,
however, tae role of these events remains to befirmlyestablished*''. The majority ofthe detailed
mechanistic work on the anti-esfrogenic effect of TCDD has focused on examining its effect on
the promoter and upsfream regulatory elements of a specific esfrogen responsive gene'".
However, tae presence of overlapping and/or adjacent DNA binding sites for transcription factors
and hormone receptors can complicate assessment as to whether TCDD can directly affect the ER
dependent signaling mechanism. To minimize these potential interference's we have developed arecombinant, human ovarian, cell line (BGlLuc4E2), which has been stably fransfected wita aluciferase reporter plasmid that is under the esfrogen inducible confrol of four EREs'. In addition
to its use as a detection tool for the identification of esfrogenic and anti-estrogenic compounds inenvfronmental samples, the development of this recombinant cell line will also provide us with aconfrolled system with which to stady the mechanism by which TCDD and related HAHs dismpt
tae esfrogen signaling pathway.
Materials and Methods
Luciferase Expression Assay for BGlLuc4E, Cells: Cells were grown in either regular media(DMEM wita 10% fetal bovine semm (FBS)) or for 7 days in esfrogen sfripped media (ESM)(phenol red free MEM with 5% dexfran-coated charcoal sfripped FBS). Unless otherwise statedthe cells were exposed to the indicated chemicals for 24 hours, followed by lysis and measurementof luciferase activity as described in detail*.
Westem Blot Analysis: Whole cell exfracts, cytosolic protein or nuclear proiein (5pg), wasseparated by SDS PAGE, followed by elecfroblotting to nifrocellulose membranes. Blots wereprobed with ERa antibody (Santa Cmz Biotechnology, Inc.) as previously described'.
ER Ligand Binding Analysis: Whole cell extracts (0.5 mg) were incubated with 2 nM 'Hestradiol for 2 hr at 4°C. Exfracts were then incubated with a charcoal pellet for 15 min. at 4°Cand taen cenfrifiiged at 3500 rpm for 15 min. The supematant was then added to scintillation

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cocktail and counted in a liquid scintillation counter. Non-specific binding was determined byaddifion of 100 fold excess ofthe synthetic esfrogen diethylsfilbesterol.
Results and Discussion
The development ofthis recombinant human ovarian cell line provides us with a novel bioassay
for detecting esfrogenic and anti-estrogenic compounds. We have optimized growth and assay
conditions so as to essentially eliminate background luciferase activity attributed to endogenous
esfrogenic chemicals'. BGlLuc4E2 cells have previously been shown to respond to esfrogen in atime-, dose-, and chemical-specific manner, with a detection limit of as low as 0.1 - 1 pM
esfradioP.
BGlLuc4E2 cells are cunently being utilized to determine which step(s) in the esfrogen signaling
pathway is dismpted by TCDD. Growth of these cells in regular media, containing phenol red and
10% FBS, results in high constitative luciferase activity (due to esfrogens within the media) which
is not further increased by esfradiol but is decreased in a dose-dependant manner by TCDD. To
show a more direct effect of TCDD on esfrogen signaling the cells were grown in ESM for several
days to reduce background luciferase activity. Co-treatment of these cells with 1 nM esfradiol and
10 nM TCDD results in a 30% - 60% decrease in esfrogen induced luciferase. Pre-freatment ofthe cells with TCDD for 24 h or 48 h fails to fiirther reduce the esfrogen response, compared tosimultaneous freatment (Figure IA), and an excess of estradiol (1000 fold) does not eliminate theeffect of TCDD (Figure 1B). Taken together these data suggest that the anti-esfrogenic effect of
TCDD in this system is not due to enhanced esfrogen metabolism by TCDD induced enzymes.
We next examined the effect of TCDD on the amount of ER in this cell line. Radioactive ligandbinding analysis of whole cell exfracts (WCE) from freated cells grown in regular media showsthat TCDD reduces the amount of ER able to bind 'H esti'adiol by about 30% - 60%, a similar
amount of inhibition as seen with the luciferase assay (Figure 2A). In addition, both WCE andnuclear and cytosolic protein from cells grown in regular media and treated for 24 h with 10 nM
TCDD show a decrease in the amount of ERa compared wita DMSO freated cells, as seen byWestem blot (Figure 2B). However, both ligand-binding and Westem blot analyses show asubstantial decrease in the amount of ER in cells grown in ESM for several days compared withregular media. A time course of days grown in ESM shows a conelation between the decrease in
ERa and an increase in esfrogen inducibility of luciferase which implies the presence of spare
receptors that are not required for the response of these cells to esfrogen, or for inhibition of that
response by TCDD. Although we delect only low levels of ERa from cells grown in ESM taeselevels do not appear to be decreased fiirther by TCDD freatment. These data indicate taatalthough TCDD is able to reduce the amount of ER in cells grown under conditions which resultin maximal levels of ER, only a fractional amount of that ER is apparently required for anesfrogen response in this system. Therefore, the observed decrease in ERa does not appear to be
responsible for the inhibition of esfrogen induced luciferase activity seen in BGILuc4E2cells.
Acknowledgments
This work is supported by tae National Institutes of Environmental Healta Sciences (ES07685 anda Superfiind Basic Research Grant ES04699) and a UC Toxic Substances Research and Teaching
Program fellowship.
References
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Nimrod, A.C. and Benson, W. H. (1996). Crit. Rev. Toxicol. 26(3): 335-364.
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Giesy, J.P., Ludwig, J.P. and Tillitt, D.E. (1994) Environ. Sci. Technol. 28, 128A135A.
3.
Krishnan, V., Porter, W., Santostefano, M., Wang, X. and Safe, S. (1995) Molec. CeU.
Biol. 15,6710-6718.

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4.
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8.

Gillesby, B.E., Santostefano, M., Porter, W., Safe, S., Wu, Z.F. and Zacharewski, T.R.
(1997) Biochem. 36, 6080-6089.
Nodland, K.L, Wonnke, M. and Safe, S. (1997) Arch. Biochem. Biophys. 338, 67-72.
Spink, D.C, Hayes, C.L., Young, N.R., Christou, M., Sutter, T.R., Jefcoate, CR. andGiertiiy, J.F. (1994) J. Steroid Biochem. Molec. BioL 51,251-258.
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Cellular Biochem. 60:173-184.
Rogers, J.M. and Denison, M.S. (2000) In Vitro and Molec. Tox. (In Press)

Figure 1. A. Effect of TCDD co-freatment and 24 hr and 48 hr pre-freatment on the esfrogen
induced luciferase activity in BGlLuc4E2 cells. B. Effect of 1000 fold excess of estradiol (1 pM)
on the anti-esfrogenic effect of TCDD in BG I Luc4E2 cells. Cells were grown for one week inesfrogen stripped media and exposed for 24 hrs (unless otaerwise indicated) to the mdicatedcompounds. Values represent mean relative light units/mg protein ± SD of at least triplicate
determinations.

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10 nM TCDDDMSOBMW(Kda)217—•*^^»*w*ss«^^.
'^**!^?T^'**'i^iW»ri~ ^*126—•
1 iW*W-**»¥4f} ^ .:73 — •ERa

43.5

T
CytosolicProtein

CI

NuclearProteinFigure 2. A. Specific binding of 'H esfradiol in whole cell extt'acts of BGlLuc4E2 cells freatedfor 24 hrs witfi DMSO or 10 nM TCDD. B. Westem blot showing tfie decrease in ERa in botii
cytosolic and nuclear fractions of BG1Luc4E2 cells freated for 24 hrs wita DMSO (C) or 10 nM
TCDD (T).

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