Organohalogen Compounds, Dioxin 2004, Volume 66, p.169-174.

Detection of Estrogen Receptor Endocrine Disruptor Potency of Commonly
Used Organochlorine Pesticides Using The LUMI-CELL Bioassay
John D. Gordon1 , Andrew C. Chu1 , Michael D. Chu2 , Michael S. Denison3 and George C. Clark1,4
1.
2.
3.

Xenobiotic Detection Systems Inc., 1601 E. Geer St. Suite S, Durham, NC 27704. Phone:(919) 68-4804; FA8X: (919) 688-4404.
Alta Analytical Perspectives, 2714 Exchange Drive, Wilmington, NC 28405.
Department of Environmental Toxicology, Meyer Hall, University of California, Davis,
CA 95616.

Introduction
Organochlorine pesticides are found in many ecosystems worldwide as result of agricultural andindustrial activities and exist as complex mixtures. The use of these organochlorine pesticides has
resulted in the contamination of lakes and streams, and eventually the animal and human foodchain. Many of these pesticides, such as pp’-DDT, pp’-DDE, Kepone, Vinclozolin, andMethoxychlor (a substitute for the banned DDT), have been described as putative estrogenic
endocrine disruptors, and act by mimicking endogenous estrogen 1-3 .
Estrogenic compounds can have a significant detrimental effect on the endocrine and reproductive
systems of both human and other animal populations4 . Studies by Jefferson et al, (2002) haveshown a strong association between several EDCs (17ß-Estradiol, DES, Zeralanol, Zeralenone,
Coumestrol, Genistein, Biochanin A, Diadzein, Naringenin, Tamoxifin) and estrogenic activity via
uterotropic assay, cell height, gland number, increased lactoferrin, and a transcriptional activity
assay using BG1Luc4E2 cells (provided by Xenobiotic Detection Systems International Inc.
(XDS), Durham, NC)4 . Some other examples of the effects of these EDCs 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 of human breast andprostate cancers; and endometriosis 3-5 . Because these chemicals are ubiquitous, highly lipophilic,and often chlorinated, it ensures their persistent presence in the environment (i.e. water supply,
soil, river sediment) resulting in their bioaccumulation in the food chain 3,5,6 . Taken together, thesedata show a pattern of food chain contamination and the detrimental effects of EDCs, therefore
understanding the estrogenic potency of these and other potential EDCs, is extremely important.
The association between the exposure and bioaccumulation of endocrine (hormone) disruptor
chemicals (EDCs) and their adverse effects on human and wild life populations has raised concern
worldwide. These concerns over the effects of environmental EDCs, lead to the passage of U.S.
Congressional legislation (Food Quality Act of 1996 and Safe Water Reauthorization Act
Amendments of 1996), which mandated the EPA to investigate the exposure to environmental
EDCs 7,8 . Based on this mandate, the EPA established the Endocrine Disruptor Steering andTesting Advisory Committee (EDSTAC), a committee charged with defining the course of action
to accomplish this goal. EDSTAC submitted their report to the U.S. Congress in August of 2000.
Subsequent to this report, the EPA established the Endocrine Disruptor Screening Program(EDSP) within the agency.
This EDSTAC report proposed that the EPA pursue thestandardization and validation of Tier I (screening) and Tier II (testing) assays specific andsensitive for EDCs, which may act as agonists and/or antagonists. Therefore, there is a growing

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Organohalogen Compounds, Dioxin 2004, Volume 66, p.169-174.

need for a fast reliable high-throughput system for the screening of known and potential
environmental contaminants, which act to disrupt normal endocrine homeostasis 9 .
In order to detect the endocrine disrupting potency of organochlorine pesticides and othercompounds using a bioassay system, Xenobiotic Detection System (XDS) in collaboration with
Dr. Michael S. Denison (Univ. California-Davis), have developed the LUMI-CELL ER
bioassay. BG-1 (human ovarian carcinoma) cells were stably transfected with an estrogenresponsive luciferase reporter gene plasmid (pGudLuc7ere) containing the estrogen responsive
element (ERE) and luciferase reporter gene6 . The resulting cell line (BG1Luc4E2), used in XDS’s
LUMI-CELL ER estrogenic cell bioassay system, responds in a time-, dose dependent- andchemical-specific manner with the induction of luciferase gene expression in response to exposure
to estrogen and estrogenic chemicals in a high-throughput screening (HTPS) format6 . In initialstudies, BG1Luc4E2 cells were able to detect as little as 0.1pM of 17ß-estradiol6 . In initialstudies, treatment of BG1Luc4E2 resulted in significant induction of luciferase activity for
estrogen, diethylstilbesterol, 17ß -estradiol, o,p’-DTT, Bisphenol A, Nonylphenol, Genistein, andDiadzein. Luciferase activity was not increased by any other steroid tested (i.e. progesterone,
testosterone, all-trans retinoic acid, thyroid hormone, and dihydrotestosterone) 6 . These results are
consistent with previously published data indicating the lack of estrogenic activity of thesechemicals 10-12 , and clearly demonstrates the specificity of this system as a bioassay to detect
estrogenic chemicals.
Here we describe studies in which XDS’s LUMI-CELL ER estrogenic cell bioassay system wasused for high throughput screening (HTPS) analysis of the estrogenic disrupting potency ofseveral commonly used pesticides and organochlorines: p,p’DDT; p,p’-DDE; DDD; α-chlordane;ψ-chlordane; Kepone; Methoxychlor; Vinclozolin; Fenarimol; 2,4,5-Trichlorophenoxyacetic Acid;and Dieldrin. Our results demonstrate the utility of XDS’s LUMI-CELL ER bioassay HTPS
system for screening chemicals for estrogenic activity.
Methods and Materials
The majority of chemicals were purchase from the Aldrich Chemical Co., Sigma Chemical
Corporation, and Chem Service Inc.
LUMI-CELL ER Bioassay. The BG1Luc4E2 cell line was constructed as previously describedby Rodgers and Dennison (2000) 6 . Briefly, BG1 cells were stably transfected with an estrogenresponsive luciferase reporter gene plasmid (pGudLuc7ere) and selected for using G418
resistance.
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 indicated170Organohalogen Compounds, Dioxin 2004, Volume 66, p.169-174.

concentration of the desired chemical to be tested (dissolved in DMSO) was added to each well.
The plate was then incubated 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 17β-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.
Discussion and Results
There is a growing concern for a need for a system fast, reliable, inexpensive method to detect
estrogenic EDCs in the environment. This concern arises from the detrimental effects of EDCs on
human and wildlife populations resulting from its bioaccumulation in the food chain. Here we
report a fast, reliable, relatively inexpensive high throughput cell based recombinant bioassay
screening method (LUMI-CELL ER bioassay) for xenoestrogenic EDCs.
Thirteen organochlorine pesticides suspected of possessing xenoestrogenic endocrine disrupting
potential were tested using XDS’s LUMI-CELL ER estrogenic cell bioassay system for thisstudy. Four of these pesticides (p,p’-DDE, p.p’-DDT, Kepone, and Methoxychlor) wereTable 1: Compounds tested with XDS’s LUMI-CELLTM bioassay, CAS numbers and theirrelative EC 50 values.
Relative
Induction
to highest
Pesticide

CAS Number

EC 50 Value

Relative
Inductionto β-Estradiol

ß-Estradiol
a-Chlordane

50-28-2
12789-03-6

1.55E-11 ± 6.93E-13
1.32E-06 ± 2.01E-07

1.00E+00
1.17E-05

1.00

Kepone143-50-0

1.38E-06 ± 3.97E-07

1.13E-05

0.96

DDD
pp' DDT

72-54-8
789-02-6

1.71E-06 ± 5.10E-07
2.94E-06 ± 3.78E-07

9.06E-06
5.28E-06

0.77
0.45

Metoxychlor
ψ-Chlordane

72-43-5
57-74-9

3.46E-06 ± 6.10E-07
4.86E-06 ± 1.67E-06

4.48E-06
3.19E-06

0.38
0.27

pp' DDE

72-55-9

6.74E-06 ± 2.58E-06

2.30E-06

0.20

1.92E-06
1.29E-06

0.16
0.11

1.12E-06
1.07E-06
N/A
N/A
N/A

0.10
0.09
N/A
N/A
N/A

CompoundFenarimol
2,4,5-Trichlorophenoxyacetic Acid

60168-88-9
93-76-5

Dieldrin
Linuron
Mirex
Vinclozolin
Progesterone

60-57-1
330-55-2
2385-85-550471-44-8
57-83-0

8.11E-06 ± 1.26E-06
1.21E-05 ± 2.60E-06
1.39E-05 ± 8.51E-07
1.45E-05 ± 6.25E-06
Non-active
Non-active
Non-active

171

-

172
1.00E-13

-2000

0

2000

4000

6000

8000

10000

12000

1.00E-12

1.00E-10

1.00E-09

ß-Estradiol
pp' DDEa-Chlordane
Progesterone
Metoxychlor
DDD
2,4,5-Trichlorophenoxyacetic Acid
Mirex

1.00E-11mmole/ml

1.00E-08

1.00E-07

1.00E-05

pp' DDTy-Chlordane
KeponeFenarimol
Vinclozolin
Dieldrin
Linuron

1.00E-06

1.00E-04

Organochlorine Pesticide Estrogenic Compounds

1.00E-03

1.00E-02

Organohalogen Compounds, Dioxin 2004, Volume 66, p.169-174.

Figure 1. Organochlorine Pesticide compounds tested to demonstrate dose dependent response
relationships using XDS’s LUMI-CELL ER HTPS bioassay system for known and potential
estrogenic EDCs.
Adjusted RLU above backgroundOrganohalogen Compounds, Dioxin 2004, Volume 66, p.169-174.

recommended by ICVAM for validation of ER binding and transcriptional activation assays andare thought to posses estrogenic activity, thereby making them potential endocrine disruptors 2 . An
additional six pesticides, not included in the ICVAM requirements for validation, which are foundas environmental contaminants, were tested to determine their potential estrogenic activity3,13-18 .
In this study, all of the compounds with historical data suggesting that they may poses estrogenic
activity were shown to poses estrogenic activity (Figure 1 and Table 1). Vinclozlin and Mirex
were the only organochlorine pesticides not to demonstrate any estrogenic activity. This is not
surprising given the results of previous studies showing Vinclozlin not to possess endocrine
disruptive potency at concentration below 600 ppm19 .
When comparing the estrogenic potency of the organochlorine pesticides (Table 2), the order of
induction of activity with respect to their EC50 values is α-Chlordane > Kepone > DDD > pp'
DDT > Methoxychlor > ψ-Chlordane > pp' DDE > Fenarimol > 2,4,5-Trichlorophenoxyacetic
Acid > Dieldrin > Linuron > Mirex = Vinclozolin.
For the most part organochlorines pesticides start to show significant induction of luciferase
reporter gene activity at or below the 1 µM (or ~1ppm) (Fig. 1). This is lower than several otherreports showing affect in animal studies. Okoumassoun et al. (2002), found a significant increasein vitellogenin, the serum phospholipoglycoprotein precursor to egg yolk, in rainbow trout
hepatocytes and male rainbow trout when exposed to as little as 10 µM DDT13 . Laug et al. lookedat rat liver and found effects at doses as low as 5 ppm DDT20 . Khasawinah et al., in two separate
studies, found the same lower effect level (5 ppm) in ICR mice and Fischer 344 rat’s livers when
dosed with Chlordane21 . Davis et al., found when C3HeB/Fe mice and C3H mice wereadministered as little as 10 ppm Dieldrin there was a significant increase in benign hepatomas andhepatic carcinomas 22 . When Tomatis et al. fed CF-1 mice 250 ppm DDD for 150 weeks or 250
ppm DDE for 130 weeks, they noted a significant increase in lung tumors and the in incidence ofhepatomas, respectively 23 . A significant increase in sperm-head abnormalities was observed when
Swiss-Webster mice were administered as little as 25 ppm Trichloroacetic acid 24 . When dogs
were dosed with Vinclozo lin, the lowest significant effect was seen 600 ppm with in increase in
adrenal gland weight19 . This is consistent with our data for Vinclozolin, showing no activity atlower levels.
According to the previously published data, the average minimal effective dose for pesticides /organochlorines in animals appears to be 5 ppm or greater13,20-24 . XDS’s LUMI-CELL ER
bioassay is capable detecting pesticides and organochlorines at < 1ppm (with a lower limit ofdetection of < 1ppt). If limits are to be imposed on the food, feed and pharmaceutical industries as
to the content of estrogenic EDCs in consumable products, the limit should reflect the lower
average minimal effective dose of 5 ppm or lower. The LUMI-CELL ER bioassay has an EC50
detection of 1.99 x 10-11 for 17β-estradiol. This level of detection is far lower than any limit likely
to be imposed by any regulatory agency. This data clearly demonstrates that XDS’s LUMICELL ER high-throughput bioassay system is a fast, reliable, and relatively inexp ensive methodfor detection of environmental EDCs, meeting requirements mandated by the EPA and ICCVAMs
Tier I (screening) requirements for EDC detection assays.

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Acknowledgements
This research was supported generously by NIEHS SBIR grant ES10533-03 and a Superfund
Basic Research Grant ES04699.
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