TOXICOLOGICAL SCIENCES 83, 136–148 (2005)
Advance Access publication October 13, 2004
Development of Androgen- and Estrogen-Responsive Bioassays,
Members of a Panel of Human Cell Line-Based Highly
Selective Steroid-Responsive Bioassays
Edwin Sonneveld,*,1Hendrina J. Jansen,* Jacoba A. C. Riteco,* Abraham Brouwer,*,† and Bart van der Burg*
*BioDetection Systems B.V., Badhuisweg 3, 1031 CM Amsterdam, The Netherlands, and †Institute for Environmental Studies,
Vrije Universiteit Amsterdam, De Boelelaan 1115, 1081 HV Amsterdam, The Netherlands
Received August 8, 2004; accepted October 4, 2004
We have established highly sensitive and specific androgen and
estrogen reporter cell lines which we have named AR (androgen
Activated LUciferase eXpression), respectively. Both bioassays are
member of a panel of CALUX reporter cell lines derived from the
human U2-OS osteosarcoma cell line, all using highly selective
reporter constructs based with a basal promoter element linked to
multimerized response elements, allowing efficient and specific
measurement of compounds interfering with androgen, estrogen,
progesterone, and glucocorticoid receptors. The AR CALUX bio-
assay contains the human androgen receptor and a luciferase
reporter construct containing three androgen-responsive elements
coupledtoaminimal TATApromoter. Thiscell linewascharacter-
ized by its stable expression of AR protein, its highly selective
response to low levels of different natural and synthetic androgens,
and its insignificant response to other nuclear hormone receptor
ligands such as estrogens, progestins, and glucocorticoids. The
EC50 of dihydrotestosterone (DHT) was found to be 0.13 nM,
consistent with the high affinity of this ligand to the human AR.
Flutamide, cyproterone acetate, and the environmental contami-
nants vinclozolin, DDT, methoxychlor, its metabolite HPTE, and
penta-BFR showed clear antagonistic activity in the AR CALUX
bioassay, competitively inhibiting DHT-mediated transactivation.
The established AR CALUX bioassay proved to excel in terms
of easy cell line maintenance, high fold induction range (typical
30 times over solvent control), low minimal detection limit
(3.6 pM), and high androgen selectivity. Potential applications
such as testing the androgenic or estrogenic activity of pure
chemicals and pharmaceuticals and complex mixtures (environ-
mental, food, feed, and clinical) are discussed.
Key Words: androgen; estrogen; receptor; CALUX; luciferase;
and can influence many other physiological processes as well.
Due to the relatively simple chemical structure and lipophilic
nature of steroids, their regulatory pathways can be readily
modified by pharmacological, environmental, and dietary
agents. The mechanism of action of steroids allows the devel-
fact that steroid receptors are transcription factors that induce
transcription of target genes after binding to specific DNA
sequences in their promoter. When these DNA sequences are
linked to the gene of a readily measurable protein (the so-called
reporter gene; e.g., firefly luciferase) and introduced into a
suitable cell line, a steroid-responsive reporter cell line can be
generated. By fusing multiple copies of a hormone response
element to a minimal promoter containing the TATA box
CALUX (Chemically Activated LUciferase eXpression) bio-
assays. These systems are exemplified by the estrogen receptor
ER CALUX bioassay consisting of the human T-47D breast
tumor cell line expressing estrogen receptors (ER) endogen-
ously together with an ER-specific 33 ERE-TATA-Luciferase
construct (Legler et al., 1999). This approach, using a minimal
reporter construct circumvents that signaling pathways other
than the signaling pathway of the steroid receptor of interest
regulate promoter activity, and luciferase expression, and
(ERa or ERb) as well as the 33 ERE-TATA-Luciferase con-
of even more selective responses toward ER interacting ligands
(Lemmen et al., 2002; Quaedackers et al., 2001).
Because of the many possible applications of steroid bio-
assays, we were interested in developing a panel of assays
using the same cellular background, in which the activity
of all major classes of steroid hormones can be determined
specifically and sensitively. In particular we were interested
in generating a selective and sensitive bioassay for androgens,
in response to the recent interest in environmental androgens
and anti-androgens (ICCVAM, 2003; Kelce et al., 1995),
together with the paucity of good assay systems for this class
1To whom correspondence should be addressed. Fax: 131-204350757.
Toxicological Sciences vol. 83 no. 1#Society of Toxicology 2005; all rights reserved.
Androgens are a major class of steroid hormones that
have critical roles in the development and maintenance of the
male reproductive system and other physiological targets,
predominantly in males. Through their anabolic effects, andro-
gens are used to promote muscle strength in athletes and meat
been found that environmental chemicals can interfere with
androgen action, thereby possibly contributing to disruption
of the endocrine system in wildlife and humans (Andersen
et al., 2002; Kelce and Wilson, 1997). Therefore, identification
ranging from pharmacological and clinical screening, food
and feed manufacturing, to toxicological monitoring and risk
assessment. Traditionally, monitoring strategies focus on two
extremes: (1) sophisticated, detailed chemical analysis and (2)
determination of biological effects using whole-animal assays
and epidemiology. With these two methods a correlation can be
the effect seen in organisms (exposure and effect determina-
tions). Rapid advances in molecular biology and biotechnology
and facilitated development of simple assays based on these
signaling mechanisms. These assays have the potential to be
used as monitoringtools
interfering with these signaling mechanisms, but also have
the potential to replace, in part, animal experimentation for
effect determination by offering prescreens to identify chemi-
cals that impact major toxicological endpoints. In the case of
androgens, the main mode of signaling is well established.
The effects of androgens in target cells are mediated by the
androgen receptor (AR), a member of the nuclear hormone
receptor superfamily that also includes receptors for other
steroid hormones like progestins and glucocorticoids, retinoids,
and thyroid hormones (Mangelsdorf et al., 1995; McKenna and
O’Malley, 2002). AR is a ligand-dependent transcription factor
that regulates specific gene expression by binding to specific
hormone responseelements(HREs) within the regulatory DNA
sequences of androgen-responsive genes (Claessens et al.,
2001). The enhancer region of the mouse mammary tumor
viral long terminal repeat (MMTV-LTR) promoter is the
most widely used enhancer to study AR function, although it
was originally isolated as a progesterone and glucocorticoid-
responsive enhancer (Di Croce et al., 1999). This can be
explained by the fact that the four inverted repeats of the core
sequence 50-TGTTCT-30within the MMTV-LTR enhancer are
recognized by: AR, glucocorticoid receptor (GR), progesterone
receptor (PR), and mineralocorticoid receptor (MR; Glass,
1994), now classified as the members of the 3C group within
the nuclear receptor family (Nuclear Receptors Nomenclature
Committee; 1999). The MMTV promoter also contains several
enhancer regions that can be addressed by transcription factors
modulating steroid responses (Aurrekoetxea-Hernandez and
Buetti, 2004; Uchiumi et al., 1998).
Several stable reporter gene assays have been described for
since they either have a low responsiveness, use slowly growing
prostatic cell lines, or are not selective in their response because
activating the transfected reporter gene through non-AR-
mediated mechanisms (Blankvoort et al., 2001; de Gooyer
et al., 2003; Paris et al., 2002a; Terouanne et al., 2000; Wilson
et al., 2002). We decided to generate a new androgen reporter
cell line that combines high specificity, sensitivity, and ease of
handling. To attain this we selected a cell line, the human bone
receptor was highly active, while expression of other C3 class
receptors is insignificant. In this line we cotransfected a highly
specific reporter construct, containing three HREs and a mini-
mal promoter linked to luciferase, and selected a stable highly
responsive clone. The AR CALUX cell line combines rapid
growth and levels of high specificity and inducibility so far
unreported. We studied its basal response characteristics, as
well as its potential to serve in a variety of applications. The
with the same cellular background (the U2-OS cell line),
allowing efficient and convenient measurement of not only
receptor interacting compounds (Quaedackers et al., 2001;
Sonneveld et al., manuscripts in preparation). Besides
describing the characteristics and applications of the AR
CALUX cell line, additional data are provided for the ERa
CALUX cell line as a complimentary bioassay in the group
of CALUX reporter cell lines.
MATERIALS AND METHODS
ethane (HPTE), corticosterone, dehydroepiandrosterone (DHEA), dexametha-
sone, diethylstilbestrol (DES), 5a-dihydrotestosterone (DHT), 17a-estradiol,
17b-estradiol (E2), estriol, estrone, 17a-ethynyl-estradiol (EE2), flutamide,
pristone (RU486), prednisolone, progesterone, testosterone, 4-OH-tamoxifen,
and tamoxifen citrate were obtained from Sigma-Aldrich (Zwijndrecht, The
Netherlands). The synthetic androgen receptor agonist methyltrienolone
(R1881) was obtained from Packard (Packard BioScience, Groningen, The
Netherlands). Bethamethasone, cyproterone acetate (CA), medroxyprogester-
one acetate (MPA), 19-nor-testosterone (nandrolone), pregnenolone, 17-
hydroxy-pregnenolone (17OH-pregnenolone), and testosterone glucuronide
were obtained from Steraloids Inc. (Newport, RI). ICI 164.384, 19-Nor-7a-
methyl-testosterone (MENT), Org 2058 and raloxifen (RAL) were kind gifts
from W. Schoonen (N.V. Organon, Oss, The Netherlands). Vinclozolin was
purchased from Riedel-de Hae ¨n (The Netherlands). o,p0-dichlorodiphenyl tri-
chloroethane (o,p0DDT), and p,p0-dichlorodiphenyl trichloroethane (p,p0DDT)
were kindly provided by J. Legler (Institute for Environmental Studies, VU,
Amsterdam, The Netherlands). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
was purchased from Cambridge Isotope Laboratories (Andover, MA). Octab-
romodiphenyl ether (Octa-LM) and pentabromodiphenyl ether (DE-71) techni-
cal mixtures were gifts from A. Bergman (Stockholm University, Sweden).
All chemicals were diluted in either ethanol or dimethylsulphoxide (DMSO;
Acros, Geel, Belgium) and stored at ?20?C. Neomycin (G418) was purchased
from Life Technologies (Breda, The Netherlands).
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
Fetal calf serum was obtained from Invitrogen (Breda, The
Netherlands). A pooled human serum batch was a gift from B. Hendriks-
Stegeman (University Medical Centre, Utrecht, The Netherlands). In short,
blood from 15 healthy adult volunteers (male/female ratio 8:7) was collected
in silica-coated tubes (Capiject, Terumo Medical Corp.). After centrifugation
serum was removed, all collected sera were pooled and stored at ?20?C.
A blunt-ended 3050 bp SalI fragment from pSV0-hAR
(obtained from A. Brinkmann, Rotterdam, The Netherlands) containing the
into the blunt-ended XhoI fragment from pSG5-neo (Sonneveld et al., 1998)
containing the neomycin resistance gene, resulting in the expression plasmid
pSG5-neo-hAR. An 1800 bp EcoRI fragment from pSG5-hERa (HEGO)
(obtained from P. Chambon, Strassbourg, France) containing the full-length
human estrogen receptor alpha (Green et al., 1986) was inserted in the EcoRI
fragment from pSG5-neo, resultingin the expression plasmid pSG5-neo-hERa.
The reporter construct pMMTVluc was described earlier (Hartig et al., 2002).
The reporter construct 33 HRE-TATA-Luc was constructed as follows: three
tandem repeats of ARE oligos AAGCTTAGAACAGTTTGTAACGAGCTC-
TT (Schule et al., 1988) upstream of the minimal adenovirus E1B TATA
promoter sequence (GGGTATATAAT) were inserted in the multiple cloning
The reporter construct 33 ERE-TATA-Luc was described earlier (Legler
et al., 1999).
(ATCC) was cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium
and Ham’s F12 medium (DF, Gibco) supplemented with 7.5% fetal calf serum.
AR and ERa CALUX cells were cultured in DF medium supplemented with
7.5% FCS and 200 mg/ml G418.
The human osteoblastic osteosarcoma cell line U2-OS
1 mg reporter plasmid (33 ERE-TATA-Luc, pMMTVluc, or 33 HRE-TATA-
Luc), 200 ng SV2-lacZ, and 200 ng expression plasmid (pSG5-neo-hERa,
pSG5-neo-hPR, pSG5-neo-hGR, or pSG5-neo-hAR) or empty vector DNA
(pSG5-neo), using the calcium phosphate coprecipitation method. Luciferase
activitywascorrectedfortransfection efficiency by measuringLacZexpression
as a result of SV2-lacZ co-transfection (Kalkhoven et al., 1994).
For transient transfections, cells were plated in
Establishment of stable AR and ERa CALUX cell lines.
were transfected with 33 HRE-TATA-Luc and pSG5-neo-hAR, using calcium
phosphate precipitation to generate AR CALUX cells. G418-resistant clones
consequent high response. One of these (clone 568) responding to the lowest
concentration of DHT (10 pM) was selected for further investigation. ERa
CALUX cells (Quaedackers et al., 2001) transfected with 33 ERE-TATA-
Luc and pSG5-neo-hERa were regenerated in our laboratories, since the
original clones showed bell-shaped dose-response curves and relatively high
backgrounds, making them less suitable for routine applications (data not
AR and ERa CALUX bioassays.
in 96-well plates (6000 cells/well) with phenol red-free DF medium supple-
mented with 5% dextran-coated charcoal-stripped FCS (DCC-FCS; van der
Burg et al., 1988) at a volume of 200 ml per well. Two days later, the medium
or the compounds to be tested (dissolved in ethanol or DMSO) in triplicate at
a 1:1000 dilution. In case of serum incubation, final serum concentration was
10% (v/v), and lower percentages of the tested sera were supplemented with
lysis buffer and measured for luciferase activity using a luminometer (Lucy2;
Anthos Labtec Instruments, Wals, Austria) for 0.1 min/well.
AR and ERa CALUX cells were plated
previously (Sonneveld et al., 1998). 20 mg of protein was run on an 8% (w/v)
SDS–polyacrylamide gel and transferred electrophoretically to nitrocellulose
sheets. Membranes were treated with blocking buffer containing 4% (w/v)
Whole-cell extracts were prepared as described
nonfat powdered milk in TBST (10 mM Tris–HCl pH 8.0, 150 mM NaCl,
0.2% (v/v) Tween-20) and then incubated for 2 h with anti-hAR mouse mono-
clonal antibody Ab-1 (clone AR441) (NeoMarkers, Fremont, CA), diluted at
1:200 in TBST buffer. After washing with TBST, the membranes were
immunostained using the ECL Western blotting system (Amersham).
15 min with 3.6% (v/v) formaldehyde in ethanol. Subsequently, the cells were
washed three times with phosphate buffered saline (PBS), permeabilized with
0.1% Triton X-100/PBS, incubated with 1% (w/v) BSA/PBS, and washed three
Ab-1 (1:80) in 10% (v/v) normal goat serum/PBS for 1 h, washed three times
with PBS, incubated with GAM-Cy3 (second) antibody (1:250) in 10% (v/v)
normal goat serum/PBS for 1 h, washed three times with PBS, and mounted
Cells were grown on coverslips and fixed on ice for
units (RLUs). Fold inductionwas calculatedby dividingthe mean value of light
as a percentage of maximal DHT (AR CALUX) or E2 (ERa CALUX) activity
was calculated by setting the highest fold induction of DHT (AR CALUX) or
E2 (ERa CALUX) at 100%. When assessing for anti-androgenic effects, the
fold induction at the EC50 concentration of DHT was set at 100%. Data are
fittedusingthesigmoidalfit(y5a01a1/(11exp(?(x?a2)/ a3))in GraphPad
Prism (version 4.00 for Windows, GraphPad Software, San Diego, CA), which
function (least squares criterion). The EC50 and 100-times EC50 values were
calculated by determining the concentration by which 50 or 100% of maximum
activity (RTA) of each compound tested was calculated as the ratio of maximal
luciferase reporter gene induction values of each compound and the maximal
luciferase reporter gene induction value of reference compound DHT
(AR CALUX) or E2 (ERa CALUX). The transactivation activity of DHT or
E2 was arbitrarily set at 100.
Luciferase activity per well was measured as relative light
Establishment of a Panel of Steroid-responsive
CALUX Cell Lines
Based on earlier observations (Quaedackers et al., 2001) and
transienttransfections using apanelofsteroidreceptors,steroid
reporter plasmids, and different cell lines (HEK293, T-47D,
U2-OS, HeLa, CHO), the osteoblastic osteosarcoma U2-OS
cell line was selected as the best candidate to serve as the
basis of androgen-, estrogen-, glucocorticoid-, and progestin-
responsive reporter cell lines. This selection was mainly based
on the observation that the U2-OS cell line showed little or no
endogenous receptor activity using reporter plasmids only,
while it supported strong hormone-mediated responses when
cognate receptors were transiently introduced (Fig. 1 and data
the 3C group of nuclear receptors, since not only AR but also
progesterone (PR) and glucocorticoid receptors (GR) can also
assays, no evidence for significant endogenous activity of AR,
PR, or GR upon ligand stimulation (DHT, Org 2058, and
dexamethasone, respectively) was found when the selective
SONNEVELD ET AL.
33 HRE-TATA-Luc construct or the more conventional
pMMTV-Luc construct was used. Cotransfection of the appro-
ligand treatment (Fig. 1). Stable transfectants were selected
from U2-OS cells transfected with the hAR (this study), hPR
and hGR (Sonneveld et al., manuscripts in preparation) and the
33 HRE-TATA-Luc reporter construct, or with the hERa in
combination with the 33 ERE-TATA-Luc reporter construct
(this study; Quadackers et al., 2001). In this manner, distinct
steroid reporter cell lines with the same cellular back-
ground (U2-OS) and comparable minimal promoter reporter
constructs (multimerized response elements coupled to the
TATA box and the luciferase reporter gene) were generated.
Table 1 shows a summary of the basic properties of these
lines, characterized by high levels of induction (fold induc-
tion ranging between 30 and 80), high stability (usually more
than 40 passages), high sensitivity (picomolar to nanomolar
range), and high selectivity. We next determined the char-
acteristics of the U2-OS cells stably transfected with hAR
and 33 ARE-TATA-Luc, which we have named the AR
CALUX cell line.
Characterization of AR CALUX Cells
Figure 2 shows the expression of human AR as determined
by Western analysis (Fig. 2A) and whole-cell immunofluor-
escence in parental U2-OS cells (Fig. 2C), and U2-OS-derived
AR CALUX cells (Figs. 2D and 2E). As shown by both
assays, maternal U2-OS cells do express small amounts of
AR, while the stable AR CALUX clone clearly expresses AR.
The human breast cancer cell line T-47D (Sutherland et al.,
1988) was used as a positive control, expressing AR endo-
genously at moderate levels (Figs. 2A and 2B; Blankvoort
et al., 2001). Both cytoplasmic and nuclear expression of AR
in AR CALUX cells was observed using immunofluorescence
(Fig. 2D). Since ligated steroid receptors have a nuclear
steroid receptors and their corresponding reporter constructs. U2-OS cells
were transiently transfected with MMTV-luc (A) or 33 HRE-TATA-Luc (B)
reporter constructs, with (black bars) or without (white bars) AR, PR, or GR
expression vectors. Fold induction indicates reporter activity in cells treated
with DHT (10 nM), Org 2058 (10 nM), or dexamethasone (10 nM) over
solvent treated cells (?). Each bar represents the mean of three independent
experiments 6 SEM.
U2-OS as a recipient cell line for the introduction of various
Performance Characteristics of U2-OS-based CALUX Bioassays for Detection of Steroidal Activity
EC50 reference compound (nM)
Fold induction typical
Inter-assay CV (%) reference compound
LOD reference compound (pM)
0.02 6 0.004
0.06 6 0.01
0.13 6 0.02
0.09 6 0.01
0.37 6 0.08
Note. Nd 5 not determined.
aQuaedackers et al., 2001.
cSonneveld et al., manuscripts in preparation.
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
localization we also treated cells with DHT (1 nM) (Fig. 2E).
The observed shift to nuclear staining confirmed the specifi-
city of the signal in the immunofluorescence and the normal
cellular distribution of the AR expressed in AR CALUX cells
(Avances et al., 2001). Furthermore, the stable homogeneous
expression of AR is demonstrated as well by immunofluor-
escence, as 100% of the AR CALUX cells stained positive
(Figs. 2D and 2E).
Maintenance, Responsiveness, and Stability of
AR CALUX Cells
easily maintainable in standard culture media with a population
doubling time of 24 h, comparable with U2-OS parental cells
(data not shown). Freeze–thaw procedures had no significant
influence on the viability of the cells, again showing the robust-
ness of the selected cell line. The cells showed a remarkably
strong response (typically between 15- and 50-fold induction)
upon DHT treatment, relative to low background values in con-
trol cells treated with solvent alone. This response has been
shown to be stable over 36 passages conducted to date
(Table 1). The range of the fold inductions was fairly wide
(15 to 50 fold), mainly due to relatively small changes in the
low background activity having a large influence on the fold
with different ligands over time, including the positive control
DHT, however, was small (Table 2), showing that changes in
fold induction did not influence quantification of the potency of
the ligands. The interassay CV was determined using EC50
values (n 5 12) obtained by various persons within two inde-
and was 22% (Table 1).
Sensitivity and Selectivity of the AR CALUX Bioassay
The sensitivity of the AR CALUX cells was assessed by
measuring the luciferase activity induced by a series of natural
steroids and precursor molecules compared to solvent control.
The most potent androgen was dihydrotestosterone, activating
these cells with an EC50 of 0.13 nM (Fig. 3A and Table 2).
androgens tested, with the following range of potencies (EC50
values in nM): DHT (0.13), testosterone (0.66), and androste-
nedione (4.5) (Fig. 3A and Table 2). The AR CALUX cell line
agonistic response to the (androgen) precursors DHEA (no
EC50 reached; 11% relative transactivation activity [RTA] to
DHT) and pregnenolone (no response; Table 2), the PR ligand
progesterone (no EC50 reached; RTA 5 36%), the ER ligand
17b-estradiol (EC50 5 3090 nM; RTA 5 93%; Table 2), and
the GR ligand hydrocortisone (no EC50 reached; RTA 5 9%).
Figure 3B shows that the selectivity for androgens was con-
firmed using a panel of synthetic ligands. Only ligands with
affinity for the AR, such as MENT (19-nor-7-alpha-methyl-
testosterone), nandrolone, the synthetic progestin MPA
(reported to possess androgenic activity as well; Bentel et al.,
1999), and R1881, strongly induced luciferase activity in AR
CALUX cells, with MENT acting as an even more potent AR
activator than DHT (EC50 5 78 pM; RTA 5 121%; Table 2).
The strong synthetic GR-activating ligand dexamethasone
did induce a response at high concentrations (0.1 mM) that
was 8% of the maximum of that reached by DHT. Other GR-
activating ligands such as prednisolone, corticosterone, and
bethamethasone showed similar responses at even higher con-
centrations (10 mM; Table 2), consistent with a minor GR-
mediated effect at high levels of ligand. No cross-reactivity
was observed with the specific PR-agonist Org 2058 (Fig. 3B)
and the ER-agonist ethynyl estradiol (EE2; Table 2), showing
the absence of PR- and ER-mediated responses in these cells,
respectively. The known AR antagonist flutamide (Fig. 3C)
repressed DHT-induced reporter gene activity. While flutamide
at 10 mM effectively repressed transactivation by DHT at EC50
the competitive nature of the antagonistic effect.
Determination of (Anti-) Androgenicity of Pure Compounds
Using AR CALUX Cells
It has been found that a variety of environmental chemicals
mimic androgens or interfere in an antagonistic fashion with
Western blotting analysis of AR protein expression in T-47D cells, parental
U2-OS cells, and U2-OS-derived AR CALUX cells. (B–E). Immunofluor-
escence staining for AR in the same cell lines (B: T-47D; C: U2-OS; D: AR
CALUX cells). In AR CALUX cells treated with DHT, translocation of AR to
the nucleus is visible (E).
Expression of hAR in stable AR CALUX reporter cells. (A)
SONNEVELD ET AL.
androgen action, thereby possibly contributing to negative
health effects in humans and wildlife (Andersen et al., 2002;
Kelce and Wilson, 1997). Particularly for the latter purpose,
screening of large numbers of chemicals is planned to be
undertaken (ICCVAM, 2003). This necessitates the use of
cost-effective screening of pure chemicals, preferably with
in vitro assays. While only a small number of environmental
compounds are currently known AR agonists, some of them are
quite potent antagonists. Figure 4 shows that the AR CALUX
bioassay readily classifies chemicals according to their anti-
androgenic properties when tested in the presence of EC50
concentrations of DHT. The well-known and widely used AR
antagonists flutamide (IC50 5 1.3 mM), vinclozolin (IC50 5
antagonistic properties, with the latter being a partial agonist
also, showing agonism for the AR at relatively high concentra-
tions (EC50 5 4.0 mM) (Fig. 4A and Table 2). In addition, we
tested a set of environmental chemicals for their agonistic and
EC50, RTA, and IC50 Values of Agonistic and Antagonistic Compounds in AR and ERa CALUX Reporter Cells
AR CALUXERa CALUX
RTA (%) Antagonism LogIC50
RTA (%) Antagonism LogIC50
Methyl testosterone (MT)
Cyproterone acetate (CA)
?10.1 6 0.1
?9.9 6 0.0
?9.9 6 0.1
?9.5 6 0.0
?9.2 6 0.0
?9.1 6 0.1
?8.4 6 0.3
?8.2 6 0.1
?5.5 6 0.2
?5.4 6 0.1
?8.2 6 0.3
?7.6 6 0.1
?6.6 6 0.1
?6.0 6 0.0
?6.0 6 0.1
?5.9 6 0.1
?5.8 6 0.2
?5.7 6 0.2
?5.6 6 0.2
?5.1 6 0.3
?7.6 6 0.0
?6.1 6 0.2
?6.7 6 0.2
?10.8 6 0.1
?7.1 6 0.1
?6.0 6 0.1
?5.1 6 0.1
?8.8 6 0.0
?11.1 6 0.1
?10.4 6 0.4
?9.9 6 0.1
?9.0 6 0.1
?7.3 6 0.1
?9.3 6 0.1
?9.9 6 0.3
?9.6 6 0.1
?7.3 6 0.0
in AR and ERa CALUX reporter cells. Nd 5 not determined. nr 5 EC50 not reached.
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
antagonistic properties (Fig. 4B and Table 2). This set of che-
micals consisted of the environmental pesticides o,p0DDT,
p,p0DDT, methoxychlor, and HPTE, as well as the penta and
octa technical mixtures of brominated flame retardants (penta-
BFR and octa-BFR). None of the above-mentioned compounds
showed agonistic properties (Table 2). As shown in Figure 4B,
the compounds o,p0DDT and p,p0DDT were able to completely
antagonize DHT-mediated AR activity with IC50 values of
1.1 mM and 2.8 mM, respectively (Table 2). The DDT-related
pesticide methoxychlor (IC50 5 8.5 mM) was a less potent
antagonist than DDT, but its metabolite HPTE was a 30-
times more potent antagonist (IC50 5 0.3 mM), being the stron-
gest environmental AR antagonist found so far in this bioassay.
The penta-BFR mixture showed antagonistic activity to the AR
(IC50 5 2.1 mM), although complete antagonism was not
reached (60% inhibition) (Fig. 4B). The octa-BFR mixture
was not able to antagonize the AR (Fig. 4B). Another environ-
mental contaminant, dioxin (TCDD), did not show agonistic or
antagonistic activity toward AR (Table 2). As a control for
nonspecific inhibition of reporter gene activity, putative antag-
onistic effects of the compounds shown in Figure 4 were tested
-13 -12 -11 -10-9 -8-7-6 -5
(% of max. DHT activity)
-10 -9-8 -7-6
(% of max. DHT activity)
-12-11 -10-9 -8-7
(% of max. DHT activity)
compounds in the AR CALUX bioassay. AR CALUX cells were plated in
96-well plates and treated with (A) the androgen progenitors progesterone (*),
DHEA (!), androstenedione (~), the natural glucocorticoid hydrocortisone
(&), and the natural androgens DHT (&) and testosterone (*), or (B) the
synthetic androgens MENT (&), R1881 (~), and nandrolone (*), the synthetic
progestins MPA (*) and Org 2058 (!), and the synthetic glucocorticoid
dexamethasone (&) for 24 h using DF medium containing 5% DCC-FCS. (C)
Luciferase induction by DHT (&) and repression of this induction by flutamide
(&) (10 mM). Each point represents the mean of at least three independent
experiments 6 SEM.
Dose-response curves for different androgen-receptor-activating
-9 -8 -7-6-5
[antagonist] (log M)
(% of EC50 DHT)
-9 -8-7 -6 -5
[antagonist] (log M)
(% of EC50 DHT)
bioassay. AR CALUX cells were plated in 96-well plates and treated with
0.13 nM DHT (EC50) and (A) the standard AR antagonists flutamide (&),
vinclozolin (*), and cyproterone acetate (!), or (B) the environmental
compounds o,p0DDT (&), p,p0DDT (~), methoxychlor (^), HPTE (!), penta-
BFR (~), and octa-BFR (*) for 24 h using DF medium containing 5% DCC-
FCS.Eachpointrepresentsthe meanof threeindependent experiments6SEM.
Repression of AR activity by AR antagonists in the AR CALUX
SONNEVELD ET AL.
for their reversibility by adding excess of the agonist DHT. The
by coincubation with excess DHT (100 times the EC50 value),
showing the specificity of the response (data not shown). In
contrast, the inhibitory effects of high levels of a number of
individual BFR congeners (not present in the mixtures used
in this study) could not be reversed by excess DHT (Hamers
et al., manuscript in preparation). This coincided with cytotoxi-
of a constitutively expressed reporter gene and a positive
response in the MTT assay (Hamers et al., manuscript in pre-
paration). The AR CALUX line is a clearly efficient tool to
screen for agonistic and antagonistic effects of compounds
toward the androgen receptor.
Determination of Estrogenicity of Pure Compounds
Using ERa CALUX Cells
Since several environmental chemicals with anti-androgenic
activity have been shown to possess estrogenic activity as well
2004), we decided to test the panel of pesticides and BFR-
mixtures in the estrogen-specific ERa CALUX bioassay. In a
similar manner as for (anti-) androgens in the AR CALUX cell
line, the ERa cell line is an effective tool to screen for agonistic
and antagonistic effects of compounds acting at the ERa. ERa
CALUX cells are also U2-OS based with the same basal char-
acteristics as other CALUX bioassays (ERb, AR, PR, and GR
CALUX), being robust, easily maintainable, highly stable,
highly responsive, and highly selective to estrogens (Tables 1
and 2). U2-OS cells transfected with 33 ERE-TATA-Luc and
hERa were described earlier (Quaedackers et al., 2001), but
bell-shaped dose-response curves and relatively high back-
grounds made this original cell line less suitable for routine
applications (data not shown). For this reason we stably trans-
fected U2-OS cells with 33 ERE-TATA-Luc and pSG5-neo-
cells, ERa CALUX cells showed a strong response (typically
between 20- and 60-fold induction) upon E2 treatment, relative
stable over 52 passages (Table 1). The range in EC50 values
25% for E2 (n 5 15; Table 1).
Typical dose-response curves for several natural as well as
synthetic estrogens are shown in Figure 5A. The ERa CALUX
cells showed high sensitivity toward all estrogens tested, with
the following range of potencies (EC50 values): EE2 (8.5 pM),
E2 (16 pM), DES (37 pM), estriol (120 pM), estrone (1.0 nM),
and 17-alpha-estradiol (1.4 nM) (Table 2). Furthermore, the
ERa CALUX cells showed high selectivity toward estrogens,
since representative steroids for other hormone receptors
(testosterone, progesterone, and dexamethasone) showed no
substantial agonistic response (Table 2). Antagonists like
raloxifen (IC50 5 0.1 nM), hydroxytamoxifen (IC50 5
0.3 nM), tamoxifen (IC50 5 55 nM), and ICI 164.384
(IC50 5 0.5 nM) repressed E2-induced reporter gene activity
(Table 2) consistent with the known anti-estrogenic nature of
To determine the (anti-) estrogenic potential of the above-
mentioned panel of environmental pesticides and BFRs, these
compounds were tested in the ERa CALUX bioassay
(Fig. 5B). None of the compounds tested showed antagonistic
activity toward ERa (data not shown). However, all of the
pesticides tested were able to transactivate ERa with the
following range of potencies (EC50 values and RTAs):
HPTE (81.3 nM; 101%), o,p0DDT (1.0 mM; 129%), methox-
ychlor (8.7 mM; 85%), and p,p0DDT (410 mM; 50%). Both
penta- and octa-BFR technical mixtures did not show activity
toward ERa (Fig. 5B). As in the case of the AR CALUX
[compound] (log M)
-11-10 -9 -8
(% of max. E2 activity)
[compound] (log M)
(% of max. E2 activity)
compounds with estrogenic activity in the ERa CALUX bioassay. ERa
CALUX cells were plated in 96-well plates and treated with (A) the natural
estrogens E2 (*), estriol (!), estrone (&), and 17a-estradiol (*) and the
synthetic estrogens EE2 (&) and DES (~), or (B) the pesticides o,p0DDT (&),
p,p0DDT (~), methoxychlor (^), and HPTE (!), and the brominated flame
retardants penta-BFR (~) and octa-BFR (*) for 24 h using DF medium
containing 5% DCC-FCS. Each point represents the mean of three
independent experiments 6 SEM.
Dose-response curves for various estrogens and environmental
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
bioassay, dioxin did not show agonistic or antagonistic activ-
ity toward ERa (Table 2; Sonneveld et al., 2003). These
experiments clearly show that certain environmental pesti-
cides with anti-androgenic activity possess estrogenic activity
Determination of Estrogens and Androgens in Serum
Using AR and ERa CALUX Cells
In addition to testing pure compounds (steroids as well as
potency) for estrogenic and androgenic activity, the need to
determine steroid bioactivity status in a wide range of pediatric
as well as adult clinical conditions is indicated. As a potential
and ERa CALUX bioassays (Fig. 6). Increasing amounts of
human serum resulted in increasing luciferase activity (Fig.
6A) in both AR and ERa CALUX cell types, indeed showing
the presence of androgenic as well as estrogenic compounds in
human serum comparable with plasma levels found in humans,
as shown recently by other ER and AR bioassays (Paris et al.,
2002b,c). The results show that these CALUX bioassays can
potentially be used in a clinical setting, thereby potentially hav-
(maximally 30 ml), making them applicable for pediatric
purposes as well. In addition to human serum we also tested
fetal calf serum for estrogenic and androgenic activities. As
shown in Figure 6B, FCS showed estrogenic activity, but no
We have developed a panel of stable human cell lines that
specifically respond with compounds interacting with human
AR, PR, GR, ERa, or ERb, allowing efficient screening of
hormonal activity of chemicals alone or in complex mixtures.
Of particular note is the AR CALUX cell line expressing an
androgen-responsive luciferase reporter gene and an androgen
receptor expression construct. The AR CALUX cells com-
bine rapid growth, high stability, high selectivity, and high
inducibility, which is, in our experience, extraordinary for an
androgen-responsive line. With its unique properties, this cell
line is potentially suitable for a wide variety of applications,
some of which we have illustrated here.
To generate an androgen reporter line superior to ones cur-
rently available, we chose not to use yeast cells, but rather
mammalian cells with an origin close to the organism of
main concern in the field of endocrine disruption (i.e., fish
and mammals, including humans). Yeast-based reporter cells,
have notably different quantitative and qualitative response to
hormonally active substances, mainly due to poor transport
across the yeast cell membrane, and are therefore not recom-
mended as screening models for endocrine disruptors
(ICCVAM, 2003). Our objective was therefore to construct a
mammalian, preferably human, reporter cell line with charac-
teristics superior to the ones available. To avoid interference of
signal transduction pathways other than AR-mediated signals,
we choose to use a minimal AR-responsive promoter element
coupled to a very minimal promoter containing a TATA box
only. This approach has been shown to be successful in genera-
tion of both in vitro (Legler et al., 1999; Lemmen et al., 2002)
selective measurement of estrogen effects. We show here that
this approach can also be successfully used to generate a highly
selective androgen reporter cell line in U2-OS cells, the AR
CALUX cell line.
Previously, the full length MMTV promoter has been used to
generate a number of androgen-responsive reporter cell lines.
Although this promoter is quite selective to AR, PR, and GR, it
also contains a number of regulatory sites that can be targeted
by different agents other than steroids (Ouatas et al., 2002;
% human serum
Luciferase activity (%
of max. DHT/E2
1 2 3 4 6 8 10
1 2 3 4 6 8 10
% foetal calf serum
Luciferase activity (%
of max. DHT/E2
1 2 3 4 6 8 10
1 2 3 4 6 8 10
AR and ERa CALUX cells were plated in 96-well plates and treated with
increasing concentrations of human (A) or fetal calf serum (B) for 24 h using
DF medium containing DCC-FCS. Black bars: AR CALUX bioassay; white
bars: ERa CALUX bioassay. Each point represents the mean of three
independent experiments 6 SEM.
Androgenic and estrogenic activity in human and fetal calf serum.
SONNEVELD ET AL.
Spangenberg et al., 1998; Uchiumi et al., 1998). MDA-kb2 is a
derivative of a human breast cancer cell line named MDA-MB-
453, containing such a stably integrated MMTV-luciferase
gens, this cell line responds very strongly to glucocorticoids
acting through the GR that is present endogenously, making
specificity was obtained by stable transfection of human pro-
static PC-3 cells with hAR and the MMTV-luciferase reporter,
named PALM cells (Terouanne et al., 2000), CHO-hAR-
MMTVluc cells (de Gooyer et al., 2003), and COS-hAR-
MMTVluc cells (Paris et al., 2002c). So far, the only cell line
that uses a simpler reporter construct, thereby avoiding influ-
ences by nonsteroidal regulatory pathways is derived from the
human breast cancer cell line T-47D, stably transfected with a
responds to progestins, due to the known over-expression of PR
in T-47D cells, and relatively low endogenous AR levels (this
study, Sonneveld et al., unpublished results; Sutherland et al.,
1988), making it less suitable as a selective screening tool.
Due to the known problems of transcriptional interference
between C3 group nuclear receptors, we choose to systemati-
cally select a line with an extremely low background activity of
the cognate receptor was transiently introduced. This led to
tage of being robust, genetically stable, and of fast proliferation
compared to most prostatic cell lines. Through the introduction
the AR CALUX cell line.
Our results with the AR CALUX cell line show that it readily
classifies the activities of pure chemicals, including natural and
synthetic steroids. The EC50 values obtained with these com-
pounds (partly listed in Table 2) correlate very well with corre-
sponding EC50 values obtained with another established AR
reporter cell line, the CHO-hAR-MMTVluc (de Gooyer et al.,
are also consistent with binding affinities to the AR of these
chemicals and the in vivo Hershberger assay (van der Burg
et al., manuscript in preparation). Not all tested androgens
reached the maximal induction level of DHT (Table 2). For
example, R1881 only reached a relative transactivation activity
of 69% compared to DHT. The reason for this lower maximal
response is not clear, but could be due to differences in ligand-
gen dissociation and AR degradation (Zhou et al., 1995), as
shown for antiestrogens on ER stability (Gibson et al., 1991;
with different transactivation capacity as described for R1881
synthetic androgen MENT was able to induce a supramaximal
response (RTA 5 121%). This supra-induction was also
observed for genistein and o,p0DDT on ERa (this study; Legler
et al., 1999) and an explanation for this phenomenon could be
ligand-dependent differences in the ability of receptor to bind
by xenoestrogens (Routledge et al., 2000).
Weak activation of reporter gene activity was obtained at
high concentrations of the strongest synthetic glucocorticoid
dexamethasone only, while other high-affinity GR ligands
such as hydrocortisone and corticosterone had little or no effect
(Table 2). This data correlates with the affinity of these com-
with the latter showing EC50 values of 0.5 nM for dexametha-
sone, 5 nM for hydrocortisone, and 15 nM for corticosterone
mediated activity is insignificant in the AR CALUX bioassay,
since only weak effects can be observed with high concentra-
tions of the strongest glucocorticoids. Such activities are
very unlikely to be present in chemicals not designed to be
and induce luciferase activity in AR CALUX cells, androgen
manuscript in preparation), but is a potent androgen in the AR
CALUX bioassay, suggesting the presence of the metabolic
enzyme 17b-HSDtype 5(with 17b-ketosteroid reductaseactiv-
ity), converting androstenedione to testosterone in AR CALUX
cells. DHEA is a very weak transactivator of AR (EC50 4
10 mM; RTA 5 11%), indicating absence or low activity of the
3b-HSD enzyme responsible for the conversion of DHEA to
androstenedione. Preliminary PCR data show that 17b-HSD
(type 5), but not 3b-HSD (type 1 and 2) is expressed in U2-
OS cells (data not shown). On the other hand, the precursor
progesterone shows induction of luciferase in AR CALUX
cells (EC50 5 8.7 mM; RTA 5 36%). This could mean that
the enzyme CYP17 is present in AR CALUX cells, converting
progesterone via OH-progesterone (17a-hydroxylase activity)
to androstenedione (17, 20 lyase activity). Indeed, PCR experi-
shown). Alternatively, since progesterone can bind AR (2%
compared to DHT), this possibly results in direct AR trans-
activation (van der Burg et al., manuscript in preparation).
Cross-talk with PR is not an issue in the bioassay, since the
PR specific synthetic ligand Org 2058 did not show activity
in the AR CALUX bioassay.
antagonistic effects of known environmental anti-androgens.
Pesticides from the DDT family clearly can antagonize the
AR with IC50 values around 1 mM, while the metabolite
onmental AR antagonists found so far in the AR CALUX bio-
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
rather potent compared to another AR reporter cell line, the
MDA-kb2 cells (IC50 5 10 mM; Wilson et al., 2002). In the
are far morepotentthan the parentalcompound, suggestingthat
active AR antagonistic compounds like M1 and M2.
Compounds, particularly at mM levels or higher, can occa-
sionally nonspecifically repress responses in reporter gene
assays. This can be due to overall cytotoxicity, ultimately lead-
as inhibition of protein synthesis or mRNA transcription. In our
experience the latter effects precede the more general cytotoxic
effects, with overt cell death as the least sensitive parameter.
Therefore, controls should be assessing nonspecific repression
death. ‘Constitutively’ expressed reporter genes that often are
promoters have been identified so far; therefore the use of these
controls should be avoided. In the case of steroid-receptor-
mediated responses, the best control for nonspecific inhibition
is considered the determination of the effect of the test com-
pound on the reporter gene activation by an excess of high-
affinity agonist. This approach worked well with the ligands
tested, and all inhibitory responses were reversed by co-
incubation with excess DHT, demonstrating the specificity of
the response. Squelching of common cofactors by other nuclear
and might thereforeproducefalse-negativeresults.An example
for this type of mechanism is the interference between PR and
ER (Kraus et al., 1995). However, squelching seems not to be
prominent in U2-OS derived CALUX bioassays, since they do
not express high levels of steroid receptors other than the stably
introduced receptor of interest. This is shown by the fact that
progestins and glucocorticoids do not interfere with DHT- or
E2-induced luciferase activity in the AR and ERa CALUX
bioassays, respectively, while androgens do not show reduced
E2-induced luciferase activity in the ERa CALUX bioassay
(AhR). Interference of the AhR ligand TCDD on ER signaling
was demonstrated in T-47D cells (ER CALUX bioassay)
expressing functional AhR (Legler et al., 1999; Sonneveld
et al., 2003), but not in U2-OS cells (ERa CALUX bioassay)
not expressing AhR (Sonneveld et al., 2003).
Several environmental contaminants have been shown to
activate the estrogen receptor, and if their effects are additive,
these may contribute to environmental and human health
impacts, most notably feminization of male fish (Gimeno
et al., 1996). Remarkably, in contrast to the estrogen receptor
that is mostly activated by environmental pollutants, the
androgen receptor seems to be prone to antagonism rather
than agonism (Paris et al., 2002a; Sohoni and Sumpter,
1998; Willemsen et al., 2004). This point is emphasized by
the fact that the pesticides tested in our study (o,p0-DDT,
p,p0-DDT, methoxychlor, HPTE) with ERa agonistic activity
were found to be also AR antagonists. This phenomenon may
contribute to the observed feminization of male fish. The
exact reason why the androgen receptor seems to be more
readily inhibited rather than activated is unclear. Possibly, a
more complex mechanism of activation with interaction
between C- and N-terminus and interactions with specific
coactivators (Dubbink et al., 2004; He et al., 2002) may
be involved in the difficulty of AR-binding pollutants leading
to activation of this receptor.
The high sensitivity and high selectivity of the AR and ERa
CALUX bioassays allowed direct measurements in nonex-
tracted biological samples. As a potential clinical application,
very low volumes of human serum were applied directly to the
CALUX bioassays. The presence of androgens and estrogens in
respectively, demonstrating that these bioassays can be used to
measure low levels of bioavailable hormones directly in very
strated recently for glucocorticoids (Sonneveld et al., manu-
script in preparation; Vermeer et al., 2003). The use of such
low volumes makes usage of these CALUX bioassays for
human serum even potentially applicable to infants, where
low-volume sample taking is desired. In fetal calf serum, high
estrogenic activity was measured. For this reason, charcoal-
stripped serum is used in the CALUX bioassays to remove all
activity in fetal calf serum, suggesting either the presence of
low levels of active androgens in fetal calf serum, or the pres-
ence of inactive precursors in the fetal serum which may be
converted to active androgens in target tissues.
The established U2-OS-based CALUX systems for estro-
gens, glucocorticoids, and progestins provide complementary
screening systems to the described AR CALUX system (see
Table 1 for an overview). Use of these CALUX bioassays will
allow the determination of full steroidal activity profiles of
compounds using the same cellular background. This has the
advantage that maintenance of the cells can be standardized,
but also provides several advantages in terms of screening
efficacy. Since compounds have different effects on the var-
ious steroid receptors ‘effect profiles’ can be derived, that
potentially give more information on the biological risk or
benefit, the specificity of the response, and the nature of
the biological active components in mixture, as compared
with measurement of a single endpoint. In particular, we
expect that profiles of compounds generated by CALUX
bioassays, either alone or in conjunction with additional end-
points, may be an important step in the prescreening of che-
micals, allowing risk ranking and toxicological prioritization
and thereby reducing the number of animal tests to be under-
taken. Chemical profiles may also be important in the first
steps of identification of chemical pollutants in complex mix-
tures such as food, feed, and environmental matrices. The
discriminative power between compounds in an ‘‘effect
SONNEVELD ET AL.
profiling’’ system will greatly improve by expanding the num-
ber of cell lines used. This will, however, lead to increased
handling and accordant additional costs. Auto-motion of the
handling will therefore be an important future step in an effi-
cient ‘‘effect profiling’’ system. With this in mind, the use of a
single robust parent cell line, such as the U2-OS cells, with
identical culture and handling conditions greatly facilitates the
possibilities for automation.
The authors wish to thank Stieneke van den Brink (Hubrecht Laboratory,
Utrecht, The Netherlands) for her excellent technical assistance and Justin
Mason (BioDetection Systems B.V.) for critically reading of the manuscript.
We also would like to thank Drs. A. Bergman (StockholmUniversity, Sweden),
W. Schoonen (NV Organon, Oss, The Netherlands) and B. Hendriks-Stegeman
(University Medical Centre, Utrecht, The Netherlands) for kindly providing the
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