Endocrine Disruption
Estrogen Receptor
Transcriptional Activation
(Human Cell Line HeLa-9903)
Background Information
‘The interaction of estrogens
with ERs can affect transcription
of estrogen-controlled
genes, which can lead to the
induction or inhibition of cellular
processes, including those
necessary for cell proliferation,
normal fetal development, and
reproductive function.’
OECD TG 455: The Stably
Transfected Human Estrogen
Receptor-alpha Transcriptional
Activation Assay for Detection
of Estrogenic Agonist-Activity of
Chemicals
2
• Estrogen agonists which activate the
transcription of estrogen responsive genes
are considered to be a key mechanism of
endocrine disruption1. The mechanism by
which this occurs is that ligands bind to the
estrogen receptor (ER) in the cell resulting
in dimerisation. This ligand-bound estrogen
receptor dimer complex can then interact
with and activate specific DNA sequences
called estrogen responsive elements (ERE)
which regulate the transcription of estrogen
responsive genes.
• The in vitro estrogen receptor
transcriptional activation (ERTA) assay
identifies chemicals that bind to and
activate the estrogen receptor (ER) (i.e.,
ER agonists). In addition to an introduced
ERα construct, the human cell line HeLa9903 has been engineered with a second
DNA construct which contains an ERE
promoter linked to a luciferase reporter
gene.
• Cyprotex follow the EPA OPPTS 890.1300
guideline1 and the OECD test guideline
4552 for estrogen receptor transcriptional
activation using human HeLa-9903 cells
and the assay can be performed under
GLP or non-GLP conditions.
Protocol
Guidance Protocols
EPA OPPTS 890.1300
OECD Test Guideline 455
Test System
hERα-HeLa 9903
Test Article Exposure Concentrations
8 concentrations ranging from 10-3 to 10-11M or up
to limit of solubility and/or cytotoxicity.
(initial range finding study is performed)
Exposure Time
24 hours
Number of Replicates
6 replicates per exposure concentration
2 replicates co-exposed with antagonist
ICI 182,780 per exposure concentration
Typical Vehicle Controls
DMSO, water, ethanol
Reference Controls
Negative control: Corticosterone
Positive controls: Strong agonist:17ß-Estradiol (E2)
Agonist: 17α-Estradiol
Weak agonist:
17α-Methyltestosterone
Test Article Requirements
Solid, water and liquids
Alternative formulations available on request
Analysis Method
Solubility by nephelometry (light scatter) and visual
assessment
Viability by 2-read propidium iodide assay
Transactivation by luminescence assay
Report
Final Report
Completed Data Entry Spreadsheet (DEST)
Completed Data Evaluation Report (DER) upon
request
OSRI assistance upon request
Proficiency Data Report
Available upon request
To find out more contact enquiries@cyprotex.com
‘Perturbation of normal estrogenic systems may have the potential to trigger adverse effects
on normal development (ontogenesis), reproductive health and the integrity of the reproductive
system.2’
Figure 1
Representative graphs showing data from the Estrogen Receptor Transcriptional Activation assay using hERα-HeLa 9903 cells.
B.
160
17ß-estradiol
140
17ß-estradiol + ICI 182,780
120
% Maximal Induction Control
% Maximal Induction Control
A.
120
100
80
60
40
20
0
Text Article X
Test Article X + ICI 182,780
100
80
60
40
20
0
-20
-15
-13
-11
-9
-11
-9
-7
Concentration (log M)
Concentration (log M)
The data in Figure 1A represent the potent positive reference
control, 17β-estradiol (dark blue circles), and 17β-estradiol coexposed with the hERα-HeLa 9903 antagonist, ICI 182,780 (light
blue circles). As expected, in the presence of ICI 182,780, inhibition
of the hERα-HeLa 9903 mediated induction of the luciferase gene
is observed confirming 17β-estradiol as a true hERα-HeLa 9903
agonist.
The data in Figure 1B represent a false positive test article X
(dark blue circles) where non-specific (i.e., non-hERα-HeLa
9903-mediated) induction of the luciferase gene is occurring which
is not inhibited by the potent hERα-HeLa 9903 antagonist, ICI
182,780 (light blue circles). For this reason, co-exposure with ICI
182,780 is essential for confirming a true hERα-HeLa 9903 agonist.
Cyprotex and its partners are able to offer the full range of EDSP series 890 protocols. A number of general endocrine disruption screening
services are also available.
Related Services
EDSP Series 890
OPPTS 890.1150: Androgen receptor binding (rat prostate cytosol)
OPPTS 890.1200: Aromatase (human recombinant)
Screening Services
Estrogen Receptor Transactivation (ER-TA)
Androgen Receptor Transactivation (AR-TA)
Androgen Receptor (AR) Modulation
OPPTS 890.1250: Estrogen receptor binding assay using rat uterine
cytosol
Estrogen Receptor alpha (ER-α) binding
*OPPTS 890.1550: Steroidogenesis using human cell line H295R
Androgen Receptor (AR) Binding
Estrogen Receptor beta (ER-β) binding
Steroidogenesis
*OECD guideline available on request
References
1
2
-5
OPPTS 890.1300: Estrogen Receptor Transcriptional Activation (Human Cell Line (HeLa-9903))
OECD TG 455: The Stably Transfected Human Estrogen Receptor-alpha Transcriptional Activation
Assay for Detection of Estrogenic Agonist-Activity of Chemicals