Organohalo gen Compounds, Dioxin 2003, Volume 65, p.78-81.

VALIDATION OF THE LUMI-CELL ER RECOMBINANT BIOASSAY
FOR RAPID EVALUATION OF CHEMICALS FOR POTENTIALESTROGENIC ACTIVITY
John D. Gordon1 , Andrew C. Chu1 , Michael D. Chu2 , Charlotte L. Taylor1 , Michael S. Denison3
and George C. Clark1
1. Xenobiotic Detection Systems Inc., 1601 E. Geer St. Suite S, Durham, NC 27704
Phone: (919) 688-4804; FAX: (919) 688-4404
2. Alta Analytical Perspectives, 2714 Exchange Drive, Wilmington, NC 28405
3. Department of Environmental Toxicology, Meyer Hall, Univ. of California, Davis, CA 95616.

Introduction
The association between the exposure and bioaccumulation of endocrine disruptor chemicals(EDCs) and their adverse effects on human and wild life populations has raised concern
worldwide1,2 . Due to the detrimental effects of environmental exposure to EDCs, there is an
obvious need to develop a relevant bioassay, which can both detect these chemicals, as well as
provide a relevant estimate of their endocrine disrupting potency. Some examples of the effects ofEDCs are: decreased reproductive success and feminization of males in several wildlife species;
increased hypospadias along with reductions in sperm counts in men; increase in the incidence ofhuman breast and prostate cancers; and endometriosis 3-5 . Because these chemicals are ubiquitous,
highly lipophilic, and often chlorinated, ensures their persistent presence in the environment
resulting in their bioaccumulation in the food chain.
In fact, these concerns over the effects of environmental EDCs, lead to the passage ofCongressional legislation (the Food Quality Act of August 1996 and the Safe Water
Reauthorization Act and Amendments of 1996), which mandated the EPA to investigate theeffects of exposure to environmental EDCs 6,7 . Based on this mandate, the EPA established theEndocrine Disruptor Steering and Testing Advisory Committee (EDSTAC). The EPA alsoestablished the Endocrine Disruptor Screening Program (EDSP) within the agency. Therefore,
there is a growing need for a fast reliable high-throughput system for the screening of known andpotential environmental contaminants, which act to disrupt normal endocrine homeostasis.
In order to detect EDCs using a high-throughput bioassay system, Xenobiotics Detection System(XDS) Inc. in collaboration with Dr. Michael S. Denison (Univ. California -Davis), havedeveloped the LUMI-CELL ER bioassay. BG-1 cells were stably transfected with an estrogenresponsive luciferase reporter gene plasmid (pGudLuc7ere)5 .
The resulting cell line
(BG1Luc4E2 ), responds to estrogenic chemicals in a time-, dose dependent- and chemical-specificmanner with the induction of luciferase gene expression5 . In initial studies, BG1Luc4E2 cells,
using the LUMI-CELL ER bioassay, were able to detect as little as 0.1pM of 17β-estradiol5 .
In May of 2002 XDS submitted preliminary data to ICCVAM for review as a validated regulatory
method in response to the Federal Register Notice (Vol. 66, No. 57/Friday, March 23, 2001) as a78

Organohalo gen Compounds, Dioxin 2003, Volume 65, p.78-81.

HTPS method for estrogen active compounds8 . Here we describe studies in which XDS’s LUMICELL ER estrogenic cell bioassay system was used for high throughput screening (HTPS)
analysis of the ICCVAM EDWG proposed 78 substances for validation of estrogen receptor (ER)and androgen receptor (AR) binding and transcriptional activation assays 8 . Our results
demonstrate the utility of XDS’s BG1Luc4E2 LUMI-CELL ER bioassay HTPS system forscreening chemicals for estrogenic/antiestrogenic activity.
Methods and Materials
Chemicals were purchased from the Aldrich Chemical Co. Sigma Chemical Corporation, andChem Service Inc.
LUMI-CELL ER Bioassay. The BG1Luc4E2 cell line was constructed as previously describedby Rodgers and Denison (2000). Briefly, BG1 cells were stably transfected with an estrogenresponsive luciferase reporter gene plasmid (pGudLuc7ere) and selected for using G418
resistance5 .
Cell Culture and Bioassay Plates. BG1Luc4E2 cells were grown in RPMI 1640. The cells weretransferred into flasks containing DMEM media (supplemented with 5% carbon stripped fetal calf
serum and G418 sulfate solution), and incubated for four days before harvesting for BG1Luc4E2
bioassay plates. The cells were then plated in 96 well plates and incubated at 37o C for 24-48
hours prior to dosing.
Bioassay Dosing Process. Once the assay plate completed its incubation, the media solution ineach well was removed and two hundred microliters of DMEM containing the indicatedconcentration of the desired chemical to be tested was added to each well. The plate was thenincubated for 24 hours before analysis of luciferase activity.
Bioassay Analysis by Berthold Luminometer. After lysing the cells (Promega lysis buffer), theluciferase activity was measured in a Berthold Orion Microplate Luminometer, with automatic
injection of 50 microliters of luciferase enzyme reagent (Promega) to each well. The relative lightunits (RLUs) measured were compared to that induced by the 17beta-estradiol standard after
subtraction of the background activity. Each compound was tested at least three times on threedifferent sets of plates and the EC50 value in mmol/ml was determined using the Microsoft Excel
Forecast function.
Results and Discussion
There is a growing concern for a need for a system fast, reliable, inexpensive method to detect
EDCs in the environment. This concern arises from the detrimental effects of EDCs on humanand wildlife populations resulting from its bioaccumulation in the food chain. Here we report afast, reliable, relatively inexpensive high throughput cell based recombinant bioassay screening
method (LUMI-CELL ER bioassay) for xenoestrogenic EDCs.
Seventy-Eight chemicals were tested using XDS’s LUMI-CELL ER bioassay system. Twentyfour of these chemicals were recommended by ICCVAM for validation of ER binding and79

1.00E-13

-5000

0

5000

10000

15000

20000

25000

1.00E-12

1.00E-07

Coumestrol

Medroxyprogestrone Acetate
Kaempferol

Genistein

Progesterone
Corticosterone

1.00E-06

Keponemmole/ml

1.00E-08

a-Zearalenol

1.00E-09

DDD

1.00E-10ß-estradiol

1.00E-11

Estrogenic Compounds1.00E-04

1.00E-03

Dexamethasone

5B-Androstane3A-OL-17-One

Daidzein

17a-Ethilnyl Estradiol

chlordane

1.00E-05

1.00E-02

Organohalo gen Compounds, Dioxin 2003, Volume 65, p.78-81.

Figure 1. Selected compounds tested to demonstrate dose dependent response relationships using
XDS’s BG1Luc4E2 LUMI-CELL ER HTPS bioassay system for known and potential estrogenic
EDCs.

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Adjusted RLU above backgroundOrganohalo gen Compounds, Dioxin 2003, Volume 65, p.78-81.

transcriptional activation assays and are known to posses estrogenic activity, thereby making thempotential endocrine disruptors8 . Fifty-four of these chemicals are environmental contaminants,
and were tested to determine their estrogenic activity as unknowns and their potential to be
endocrine disruptors. Five other compounds on the ICCVAM list of chemicals to test for ER andAR validation, which have previously been shown to not posses’ estrogenic activity, and are
therefore not potential endocrine disruptors, were also tested8 .
Of the Seventh-Eight chemicals tested by XDS’s LUMI-CELL ER bioassay system, 61demonstrated estrogenic activity, while 17 showed no activity. All of the 24 chemicals tested,which were recommended by ICCVAM for validation, with the exception of Progesterone, which
has both agonist and antagonist activity, demonstrated estrogenic activity. Of the Fifty-fourchemicals tested, which were not included in the ICCVAM requirements for validation, 27 werefound to have estrogenic activity and 17 showed no activity. Five of the 17 chemicals notshowing any activity, had been previously shown to not have estrogenic activity8 . 17β-Estradiolproved to have the highest estrogenic activity, having an EC50 detection level of 1.43x10-11mmol/ml.
Figure 1 shows the results of an analysis of 15 of the chemicals tested in this study via the LUMICELL ER bioassay. Eleven of these significantly induced luciferase reporter gene activity at orbelow the 1 µM level and 4 were essentially inactive. Of the eleven chemicals showing activity,
nine were on the list of chemicals suggested by ICCVAM for validation of ER binding andtranscriptional activation assays and two (DDD and Chlordane) had not been previously shown toposses estrogenic activity8 . Three of the four chemicals showing no activity were on theICCVAM list for validation and according to their report had no estrogenic activity8 . The fourthchemical (5B-Androstane3A-OL-17-One) was tested as an unknown. This data clearly
demonstrates that XDS’s LUMI-CELL ER high-throughput bioassay system is a fast, reliable,
and relatively inexpensive method for detection of environmental EDCs, meeting the requirements
mandated by the EPA and ICCVAMs Tier I (screening) requirements for EDC detection assays.
Acknowledgements
This research was supported generously by NIEHS SBIR grant ES10533-03 and a Superfund
Basic Research Grant ES04699.
References
1. Jefferson W.N., Padilla-Banks E., Clark G., and Newbold R.R. (2002). J Chromatogr B
Analyt Technol Biomed Life Sci. 777:179-189.
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Pregnancy. Thieme-Stratton. New Yo rk.
3. Markey C.M., Coombs M.A., Sonnenschein C., and Soto A.M. (2003). Evol Dev. 5:67-75.
4. Safe, S.H. (2002). Health Perspect. 110: 925-929.
5. Rogers J.M, and Denison M.S. (2000). In Vitr Mol Toxicol. 13:67-82.
6. Food Quality Protection Act of 1996. (1996a). Public Law 104-170.
7. Safe Drinking Water Act Amendments of 1996. (1996b). Public Law 104-182.
8. Current Status of Test Methods for Detecting Endocrine Disruptors: In Vitro Estrogen
Receptor Transcriptional Activation. http://iccvam.niehs.nih.gov/docs/docs.htm#endocrineand http://iccvam.niehs.nih.gov/methods/endodocs/final/erta_brd/erta_all.pdf.

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