PROPOSED CLASSIFICATION SYSTEM FOR TIER-1 SCREENING OFESTROGENIC EDCs USING THE LUMI-CELL ER BIOASSAYGordon, J.D.1 , Denison, M.S.2 , Chu, A.C.1 , Chu, M.D.2 , Matherly, C.1 , Kayama, F.4 and Clark, G.C.1
1Xenobiotic Detection Systems Inc., 1601 E. Geer St. Suite S, Durham, NC 27704 Phone: (919) 688-4804; FAX:(919) 688-4404; 2 Department of Environmental Toxicology, Meyer Hall, Univ. of California, Davis, CA 95616.
3Alta Analytical Perspectives, 2714 Exchange Drive, Wilmington, NC 28405; Jichi Medical School, 329-0498
Japan.
Introduction
Concern worldwide has risen over the past several years due to the strong association between the exposure andbioaccumulation of endocrine disruptor chemicals (EDCs) and their adverse effects on human and wild life
populations 1,2 . Some examples of the effects of EDCs are: decreased reproductive success and feminization ofmales in several wildlife species; increased hypospadias along with reductions in sperm counts in men; increase in
the incidence of human breast and prostate cancers; and endometriosis 3-5 . Due to the detrimental effects ofenvironmental exposure to EDCs, there is an obvious need to develop a classification system for these compounds.
A tier-1 screening classification system for EDCs would allow researchers and regulatory officials to determine
which chemicals are more likely to result in detrimental effects due to exposure and prioritize research efforts. In
this study, 50 compounds, which were on ICCVAM’s list of compounds to validate an estrogen receptor
transcriptional activation (ER TA) assay, were analyzed using the LUMI-CELL ER bioassay for estrogen agonists.
Twenty-five of these compounds were described as positive for ER agonistic effects across all Mammalian Cell
Reporter Gene (MCRG) studies and the other 25 were described as negative for ER agonistic effects across all
MCRG studies9,10 . Dose response data on all of the compounds were graphed to determine if a logical classification
scheme could be developed. Factors such as the association between estrogenic response and logical breaks in dose
response of compounds, and potential of pharmacological concentrations being achieved in exposure scenarios weretaken into account when the classification system was developed.
Methods and Materials
Compound Preparation and Analysis Procedure: A 10 mg/ml solution of each compound was prepared in DMSO.
A range finding assay was preformed on each compound using six log dilutions. A serial dilution of the compound
was analyzed in triplicate in the activity range as determined by the LUMI-CELL ER bioassay range finding
studies.
LUMI-CELL ER Bioassay. The BG1Luc4E2 cell line was constructed as previously described by Rogers andDenison (2000). Briefly, BG1 cells were transfected with an estrogen-responsive luciferase reporter gene plasmid(pGudLuc7ere) and stable transfectants selected using G418 resistance5 . For analysis, the BG1Luc4E2 cell clone
resulting from this selection was grown in RPMI 1640 medium. The cells were transferred into flasks containing
phenol red-free DMEM media (supplemented with 5% carbon stripped fetal calf serum and G418 sulfate solution),and incubated for four days before harvesting for BG1Luc4E2 bioassay plating. The cells were then plated in 96
well plates and incubated at 37o C for 24-48 hours prior to dosing. The media solution in each well was removed andtwo hundred micro liters of phenol red-free DMEM containing the indicated concentration of the desired chemical
to be tested was added to each well. The plate was then incubated for 20 hours before analysis of luciferase activity.
Measurement of Luciferease Activity. After lysing the cells (Promega lysis buffer), the luciferase activity wasmeasured in a Berthold Orion Microplate Luminometer, with automatic injection of 50 micro liters of luciferase
enzyme reagent (Promega) into each well. The relative light units (RLUs) measured were compared to that induced

by the 17beta-estradiol (E2) standard after subtraction of the background activity. Each compound was tes ted atleast three times on three different sets of plates and the EC50 value in Molar concentration was determined using
the Microsoft Excel Forecast function.
Results and Discussion
There is a growing need for a reliable tier-1 screening classification system for estrogenic EDCs. The concern for
EDCs arises from the detrimental effects on human and wildlife populations resulting from its bioaccumulation in
the food chain. A classification system would help researchers and regulators determine which compounds pose thegreatest potential threat to human and wildlife populations. Here we report a possible method for classification of
xenoestrogenic EDCs based on dose response criteria using the LUMI-CELL ER bioassay.
After observing the all of the data from the 50 compounds tested for this study, which are depicted in Figures 1 and2, there were several observations made which lead to the first part of the classification scheme. The firstobservation was that there were two major groupings of data, where the estrogen responsive element (ERE) wasbeing activated. The first was a grouping of compounds being activated around the 10-5 M to10-6 M concentrations,and the second was a grouping around the 10-9 M to 10-11 M concentration. There was also a region from 10-7 M to
10-9 M showing little or no compounds with activity at these concentrations. This same spread in activity has been
observed by other researchers such as; Sonneveld et. al. 2005, Kojima et. al., 2003, and Legler et. al., 2002. Theseobservations lead to the first set of criteria in the classification of estrogenic EDCs. This rule classifies anycompound with an EC50 of less than 1x10-8 M as a “Strong” inducer of estrogenic responses and any compound with
an EC50 of greater than 1x10-8 M as a “Weak” inducer of estrogenic responses.
The second part of the classification scheme deals with the midpoint Relative Light Unit (RLU) value for 17βestradiol (E2). E2 is the standard used in the LUMI-CELL ER agonist bioassay. The second part of theclassification scheme states that any compound with a V-Max greater than or equal to the midpoint RLU for E2would be classified as a Class I compound. Any compound with a V-Max above background, but less than themidpoint RLU for E2 would be classified as a Class II Compound. V-Max is being defined as the maximal RLU
response of the compound.
The Third part of the classification scheme states that any compound with a V-Max RLU less than the 3 times thestandard deviation of the background solvent (DMSO in this case) would be classified as a Class III compound.
Figure 1 depicts 25 compounds tested using the LUMI-CELL ER bioassay. These compounds were described as
positive for ER agonistic effects across all Mammalian Cell Reporter Gene (MCRG) studies9,10 . Figure 2 depicts
another 25 compounds, which were described as negative for ER agonistic effects across all MCRG studies9,10 . Theboxes represent the classification of the compounds (i.e. Strong, Weak, or Class I, Class II or Class III), or thecriteria for each of these classifications. We suggest this classification system as a means of priority setting forpotential estrogenic chemical entities that should be investigated for their ability to alter hormonal function inanimal models. This system of in vitro classification should help limit the number of animals that are used foranalysis of endocrine disrupting activity and provide initial dose response data on concentrations to test in animalmodels therefore limiting the number of animals necessary for further scientific investigations. The LUMI-CELL®
ER in vitro testing method may actually be useful in replacing the use of animal models, in some instances, since it
has been shown that the response in this system correlates highly with classical estrogen mediated responses such as
uterine weight and cellular proliferation in uterine tissues2 .
Acknowledgements: This work was supported in part by grants from the National Institutes of Environmental
Health Small Business Initiated Research program (ES10533-03) and a Superfund Basic Research Grant ES04699.

Figure 1. Classification scheme for Tier-1 In Vitro Screening of Estrogenic EDCs compounds previously classified
as Positive for ER agonistic activity across all MCRG studies9,10 .

Defining Estrogenic Activity

StrongAdjusted RLU above background

16000

Weak

1x10 -8 M
cut-off

14000

E2 RLU
midpoint

12000
10000

3 x std. Dev.
DMSObackground

8000
6000

Class I

4000

Class II2000
0-20001.00E-14

1.00E-13

1.00E-12

1.00E-11

1.00E-10

1.00E-09

1.00E-08

1.00E-07

1.00E-06

1.00E-05

1.00E-04

Class III
1.00E-03

1.00E-02

Molar
E2 EC50DMSO

BPA

butylbenzyl phthate

Corticosterone

Dexamethasone

pp' DDEpp' DDTdibenzo(a,h)anthracene

dibutyl phthalate

DES

a-Estradiol

ß-Estradiol

ethynyl estadiol

EstroneEthyl Parben

Fenarimol

FlavoneKaemoferol

KeponeMetoxychlor

Nonylphenol

Norethynedrel

Octyphenol

Tamoxifin

2,4,5-TCA

zearlenone

Figure 2. Classification scheme for Tier-1 In Vitro Screening of Estrogenic EDCs compounds previously classified
as Negative for ER agonistic activity across all MCRG studies9,10 .

Defining Estrogenic Activity

StrongAdjusted RLU above background

12000

Weak

1x10-8 M
cut-off

10000

6000
4000

Class IE2 RLU
midpoint

8000

3 x std. Dev.
DMSO
background

Class II

2000
0

Class III-2000-4000
1.00E-14 1.00E-13

1.00E-12

1.00E-11 1.00E-10

1.00E-09

1.00E-08 1.00E-07 1.00E-06

Molar

1.00E-05

1.00E-04 1.00E-03 1.00E-02

E2 EC50
Ammonium perchlorate

DMSO
4-Androstenedione

ß-Estradiol
2-sec-ButylphenolActinomycin D
Corticosterone

Cycloheximide
Haloperidol
Mifepristone
Pimazide

Cyproterone acetate
Ketoconazole
Morin
Procymidon

Diethylhexyl Phthalate
LinuronNilutamide
Progesterone

Flutamide
Medroxyprogestrone Acetate
Phenolphthlin
Propylthiouracil

Reserpine

Sprionolactone

L-Thyroxine

VinclozolinReferences
1. Jarry H, Christoffel J, Rimoldi G, Koch L, Wuttke W. (2004). Toxicology. 205(1-2):87-93.
2. Jefferson W.N., Padilla-Banks E., Clark G., Newbold R.R. (2002). J Chromatogr B Analyt Technol Biomed
Life Sci. 777:179-189.
3. Markey C.M., Coombs M.A., Sonnenschein C., 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 Vitro Mol Toxicol. 13:67-82.
6. Sonneveld E., Riteco J., Jansen H., Pieterse B., Brouwer A., Schoonen W., Burg B. (2005). Toxicol Sci.
89:173-187.
7. Kojima H., Iida M., Katsura, E., Kanetoshi A., Hori Y., Kobayashi K. (2003). EHP. 111:497-502.
8. Legler J., Zeinstra L., Schuitemaker F., Lanser P., Bogerd J., Brouwer A., Vethaak A., Voogt P., Murk A., Burg
B., (2002). Env. Sci. and Tech. 36:4410-4415.
9. Current Evaluation of In Vitro Test Methods for Detecting Potential Endocrine Disruptors (May 2003).
ICCVAM, NTP, NICEATM, NIEHS. NIH Publication No. 03-4503.
10. Current Status of Test Methods for Detecting Endocrine Disruptors: In Vitro Estrogen Receptor Transcriptional
Activation. http://iccvam.niehs.nih.gov/docs/docs.htm#endocrine andhttp://iccvam.niehs.nih.gov/methods/endodocs/final/erta_brd/erta_all.pdf.