Development of Androgen- and Estrogen-Responsive Bioassays, Members of a Panel of Human Cell Line-Based Highly Selective Steroid-Responsive Bioassays

Article (PDF Available)inToxicological Sciences 83(1):136-48 · February 2005with58 Reads
DOI: 10.1093/toxsci/kfi005 · Source: PubMed
Abstract
We have established highly sensitive and specific androgen and estrogen reporter cell lines which we have named AR (androgen receptor) and ERalpha (estrogen receptor alpha) CALUX (Chemically 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 bioassay contains the human androgen receptor and a luciferase reporter construct containing three androgen-responsive elements coupled to a minimal TATA promoter. This cell line was characterized 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 contaminants 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 (environmental, food, feed, and clinical) are discussed.

Figures

TOXICOLOGICAL SCIENCES 83, 136–148 (2005)
doi:10.1093/toxsci/kfi005
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,*
,1
Hendrina 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
receptor) and ERa (estrogen receptor alpha) CALUX
(Chemically
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
coupled to a minimal TATA promoter. This cell line was character-
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;
bioassay.
Steroid hormones are essential in most reproductive processes
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-
opment of straightforward screening methods, making use of the
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
only, we have developed a series of highly sensitive and specific
reporter cell lines. These bioassays form a group of the so-called
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
thereby avoids nonspecific responses. Similarly, we have devel-
oped doubly transfected cell lines expressing estrogen receptors
(ERa or ERb) as well as the 33 ERE-TATA-Luciferase con-
struct. The ERa and ERb CALUX bioassays have the advantage
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
of hormones.
1
To whom correspondence should be addressed. Fax: 131-204350757.
E-mail: edwin.sonneveld@bds.nl.
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
quantity in farm animals (Evans, 2004; Meyer, 2001). It has also
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
of androgen active compounds is important in a variety of fields,
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
made between in vivo or environmental levels of a chemical and
the effect seen in organisms (exposure and effect determina-
tions). Rapid advances in molecular biology and biotechnology
have allowed identification of mechanisms of action of toxicants
and facilitated development of simple assays based on these
signaling mechanisms. These assays have the potential to be
used as monitoring tools for chemical contaminants
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 response elements (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 5
0
-TGTTCT-3
0
within 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
that may respond to other hormonal and cellular stimuli, thereby
modulating steroid responses (Aurrekoetxea-Hernandez and
Buetti, 2004; Uchiumi et al., 1998).
Several stable reporter gene assays have been described for
androgens. However, these systems still have several drawbacks,
since they either have a low responsiveness, use slowly growing
prostatic cell lines, or are not selective in their response because
of expression of other nuclear hormone receptors of the C3 class,
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
cell line U2-OS, in which the stably introduced human androgen
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
AR CALUX cell line is a member of a panel of reporter cell lines
with the same cellular background (the U2-OS cell line),
allowing efficient and convenient measurement of not only
androgen-,but also estrogen-, progesterone-, and glucocorticoid-
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
Chemicals. Androstenedione, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloro-
ethane (HPTE), corticosterone, dehydroepiandrosterone (DHEA), dexametha-
sone, diethylstilbestrol (DES), 5a-dihydrotestosterone (DHT), 17a-estradiol,
17b-estradiol (E2), estriol, estrone, 17a-ethynyl-estradiol (EE2), flutamide,
genistein, hydrocortisone, methoxychlor (MX), methyl testosterone (MT), mife-
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,p
0
-dichlorodiphenyl tri-
chloroethane (o,p
0
DDT), and p,p
0
-dichlorodiphenyl trichloroethane ( p,p
0
DDT)
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
137
Sera. 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.
DNA constructs. A blunt-ended 3050 bp SalI fragment from pSV0-hAR
(obtained from A. Brinkmann, Rotterdam, The Netherlands) containing the
full-length human androgen receptor (AR) (Brinkmann et al., 1989) was inserted
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, resulting in 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-
GTTACAAACTGTTCTAGCTCGTTACAAACTGTTCTAAGCTCAAGC-
TT (Schule et al., 1988) upstream of the minimal adenovirus E
1
B TATA
promoter sequence (GGGTATATAAT) were inserted in the multiple cloning
site of the promoter less luciferase reporter construct pLuc (Folkers et al., 1995).
The reporter construct 33 ERE-TATA-Luc was described earlier (Legler
et al., 1999).
Cell culture. The human osteoblastic osteosarcoma cell line U2-OS
(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.
Transient transfections. For transient transfections, cells were plated in
24-well tissue culture plates. After culturing for 1 day, cells were transfected with
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
activity was corrected for transfection efficiency by measuring LacZ expression
as a result of SV2-lacZ co-transfection (Kalkhoven et al., 1994).
Establishment of stable AR and ERa CALUX cell lines. U2-OS cells
were transfected with 33 HRE-TATA-Luc and pSG5-neo-hAR, using calcium
phosphate precipitation to generate AR CALUX cells. G418-resistant clones
were tested fortheirresponse to dihydrotestosterone (DHT). Eight clones showed
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
shown).
AR and ERa CALUX bioassays. AR and ERa CALUX cells were plated
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
was refreshed, and cells were incubated with human or fetal serum (0–10% [v/v])
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
DCC-FCS. After 24 h the medium was removed, cells were lysed in 30 ml Triton-
lysis buffer and measured for luciferase activity using a luminometer (Lucy2;
Anthos Labtec Instruments, Wals, Austria) for 0.1 min/well.
Western blotting. Whole-cell extracts were prepared as described
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)
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).
Immunofluorescence. Cells were grown on coverslips and fixed on ice for
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
times with PBS. Cells were incubated with anti-hAR mouse monoclonal antibody
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
in Moviol.
Data analysis. Luciferase activity per well was measured as relative light
units (RLUs). Fold induction was calculated by dividing the mean value of light
units from exposed and nonexposed (solvent control) wells. Luciferase induction
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
represented as mean values 6 SEM from at least three independent experiments,
with each experimental point performed in triplicate. Dose-response curves were
fitted using the sigmoidal fit (y 5 a
0
1 a
1
/(1 1 exp((x a
2
)/a
3
)) in GraphPad
Prism (version 4.00 for Windows, GraphPad Software, San Diego, CA), which
determines the fitting coefficients by an iterative process minimizing the c2 merit
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 was reached using the sigmoidal fit equation. The relative transactivation
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.
RESULTS
Establishment of a Panel of Steroid-responsive
CALUX Cell Lines
Based on earlier observations (Quaedackers et al., 2001) and
transient transfections using a panel of steroid receptors, 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
not shown; Quadackers et al., 2001). We were particularly inter-
ested in the activation through possible endogenous members of
the 3C group of nuclear receptors, since not only AR but also
progesterone (PR) and glucocorticoid receptors (GR) can also
activate the HRE-containing constructs. In transient transfection
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
138
SONNEVELD ET AL.
33 HRE-TATA-Luc construct or the more conventional
pMMTV-Luc construct was used. Cotransfection of the appro-
priate receptors, however, resulted in high reporter activity upon
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
- - -
0
50
100
150
200
A
DHT
-
-
-
DHT
dex
dex
org
2058
org
2058
+ AR
+ PR
+ GR
fold induction
- - -
0
50
100
B
DHT
-
-
-
DHT
dex
dex
org
2058
org
2058
fold induction
+ AR
+ PR
+ GR
FIG. 1. U2-OS as a recipient cell line for the introduction of various
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.
TABLE 1
Performance Characteristics of U2-OS-based CALUX Bioassays for Detection of Steroidal Activity
ERa CALUX
a,b
ERb CALUX
a
AR CALUX
b
PR CALUX
c
GR CALUX
c
Reference compound E2 E2 DHT Org 2058 Dex
EC50 reference compound (nM) 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
Fold induction typical 30 80 30 30 30
Inter-assay CV (%) reference compound 25 17 22 11 21
LOD reference compound (pM) 0.8 nd 3.6 1.3 0.2
Selectivity high high high high high
Stability passages 452 nd 436 448 462
Note. Nd 5 not determined.
a
Quaedackers et al., 2001.
b
Present study.
c
Sonneveld et al., manuscripts in preparation.
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
139
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
The selected AR CALUX cell line was found to be robust and
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
induction (data not shown). The range in EC50 values measured
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-
pendent laboratories using different batches of AR CALUX cells
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).
The AR CALUX cells showed high sensitivity toward all natural
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
was remarkably selective for androgens, showing no substantial
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
values, this repression was reversed by excess DHT, confirming
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
FIG. 2. Expression of hAR in stable AR CALUX reporter cells. (A)
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).
140 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
1.0 mM), and cyproterone acetate (IC50 5 7.1 nM) clearly show
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
TABLE 2
EC50, RTA, and IC50 Values of Agonistic and Antagonistic Compounds in AR and ERa CALUX Reporter Cells
AR CALUX ERa CALUX
Compound
Agonism LogEC50
(M)
RTA (%) Antagonism LogIC50
(M)
Agonism LogEC50
(M)
RTA (%) Antagonism LogIC50
(M)
MENT 10.1 6 0.1 121 45.0 7.6 6 0.0 96 nd
R1881 9.9 6 0.0 69 45.0 6.1 6 0.2 61 nd
DHT 9.9 6 0.1 100 45.0 46. 0 55nd
Nandrolone 9.5 6 0.0 92 45.0 6.7 6 0.2 94 nd
Testosterone (T) 9.2 6 0.0 94 45.0 46.0 12 45.0
Methyl testosterone (MT) 9.1 6 0.1 108 45.0 nd nd nd
Androstenedione 8.4 6 0.3 82 nd nd nd nd
MPA 8.2 6 0.1 75 45.0 46.0 55nd
17-beta-estradiol (E2) 5.5 6 0.2 93 nd 10.8 6 0.1 100 45.0
Cyproterone acetate (CA) 5.4 6 0.1 50 8.2 6 0.3 46.0 55nd
Progesterone 45.0 36 nd 46.0 55 45.0
Dexamethasone nr 8 45.0 45.0 55 45.0
RU486 45.0 55 7.6 6 0.1 46.0 31 nd
HPTE 45.0 55 6.6 6 0.1 7.1 6 0.1 101 45.0
Vinclozolin 45.0 55 6.0 6 0.0 45.0 55 45.0
o,p
0
DDT 45.0 55 6.0 6 0.1 6.0 6 0.1 129 45.0
Flutamide 45.0 55 5.9 6 0.1 nd nd nd
ICI 164.384 45.0 55 5.8 6 0.2 45.0 55 9.3 6 0.1
Penta-BDE 45.0 55 5.7 6 0.2 45.0 55 45.0
p,p
0
DDT 45.0 55 5.6 6 0.2 45.0 50 45.0
Methoxychlor 45.0 55 5.1 6 0.3 5.1 6 0.1 85 45.0
Octa-BDE 45.0 55 45.0 45.0 55 45.0
TCDD 45.0 55 45.0 45.0 55 45.0
DHEA 45.0 11 nd nd nd nd
Hydrocortisone 45.0 9 nd nd nd nd
Bethamethasone 45.0 9 nd nd nd nd
Prednisolone 45.0 7 nd nd nd nd
Corticosterone 45.0 5 nd nd nd nd
Testosterone glucuronide 45.0 25 nd nd nd nd
17-alpha-estradiol 45.0 55nd 8.8 6 0.0 104 45.0
EE2 45.0 55nd 11.1 6 0.1 92 45.0
Pregnenolone 45.0 55nd ndnd nd
OH-pregnenolone 45.0 55nd ndnd nd
Org2058 45.0 55nd ndnd nd
Raloxifen nd nd nd 46.0 5 9.9 6 0.3
Diethylstilbestrol (DES) nd nd nd 10.4 6 0.4 98 45.0
Estriol nd nd nd 9.9 6 0.1 100 45.0
Estrone nd nd nd 9.0 6 0.1 119 45.0
Genistein nd nd nd 7.3 6 0.1 135 45.0
4OH-tamoxifen nd nd nd 45.0 5 9.6 6 0.1
Tamoxifen nd nd nd 45.0 55 7.3 6 0.0
Note. EC50 values (average 6 SD) and relative transactivation activity (RTA) of various agonistic compounds and IC50 values of various antagonistic compounds
in AR and ERa CALUX reporter cells. Nd 5 not determined. nr 5 EC50 not reached.
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
141
antagonistic properties (Fig. 4B and Table 2). This set of che-
micals consisted of the environmental pesticides o,p
0
DDT,
p,p
0
DDT, 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,p
0
DDT and p,p
0
DDT 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
0
25
50
75
100
125
A
[Compound] (logM)
Luciferase induction
(% of max. DHT
activity)
-13 -12 -11 -10 -9 -8 -7 -6
0
25
50
75
100
125
B
[Compound] (logM)
Luciferase induction
(% of max. DHT
activity)
-12 -11 -10 -9 -8 -7
0
25
50
75
100
125
C
[DHT] (lo
g
M)
Luciferase induction
(% of max. DHT activity)
FIG. 3. Dose-response curves for different androgen-receptor-activating
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.
-9 -8 -7 -6 -5
0
25
50
75
100
B
[antagonist] (log M)
Luciferase induction
(% of EC
50
DHT)
-9 -8 -7 -6 -5
0
25
50
75
100
A
[antagonist] (log M)
Luciferase induction
(% of EC
50
DHT)
FIG. 4. Repression of AR activity by AR antagonists in the AR CALUX
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,p
0
DDT (&), p,p
0
DDT (~), methoxychlor (^), HPTE (!), 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.
142 SONNEVELD ET AL.
for their reversibility by adding excess of the agonist DHT. The
antagonistic effects of all of the compounds tested were reversed
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-
city of these ligands, as assessed through inhibition of expression
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
(Paris et al., 2002a; Sohoni and Sumpter, 1998; Willemsen et al.,
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-
hERa, producing the ERa CALUX bioassay. Like AR CALUX
cells, ERa CALUX cells showed a strong response (typically
between 20- and 60-fold induction) upon E2 treatment, relative
to low background values in control cells. This response to date is
stable over 52 passages (Table 1). The range in EC50 values
measured with different ligands over time, including the positive
control E2, was small (Table 2), reflected by an interassay CV of
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
these compounds.
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,p
0
DDT (1.0 mM; 129%), methox-
ychlor (8.7 mM; 85%), and p,p
0
DDT (410 m M; 50%). Both
penta- and octa-BFR technical mixtures did not show activity
toward ERa (Fig. 5B). As in the case of the AR CALUX
-13 -12 -11 -10 -9 -8
0
25
50
75
100
A
[compound] (log M)
Luciferase induction
(% of max. E2
activity)
-9 -8 -7 -6 -5
0
25
50
75
100
125
B
[compound] (log M)
Luciferase induction
(% of max. E2 activity)
FIG. 5. Dose-response curves for various estrogens and environmental
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,p
0
DDT (&),
p,p
0
DDT (~), 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.
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
143
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
as well.
Determination of Estrogens and Androgens in Serum
Using AR and ERa CALUX Cells
In addition to testing pure compounds (steroids as well as
environmentally relevant compounds with endocrine disrupting
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
clinical application, human serum was applied directly to the AR
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-
ing the advantage of demanding only very small serum volumes
(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
AR-activating compounds.
DISCUSSION
We have developed a panel of stable human cell lines that
specifically respond with compounds interacting with human
AR, PR, GR, ERa,orERb, 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,
although convenient in their use (Sohoni and Sumpter, 1998) can
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)
and in vivo (Legler et al., 2000; Lemmen et al., 2004) models for
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;
0
10
20
30
A
% human serum
Luciferase activity (%
of max. DHT/E2
induction)
1 2 3 4 6 8 10
1 2 3 4 6 8 10
0
10
20
30
B
% foetal calf serum
Luciferase activity (%
of max. DHT/E2
induction)
1 2 3 4 6 8 10
1 2 3 4 6 8 10
FIG. 6. Androgenic and estrogenic activity in human and fetal calf serum.
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.
144 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
reporter (Wilson et al., 2002). In addition to responding to andro-
gens, this cell line responds very strongly to glucocorticoids
acting through the GR that is present endogenously, making
it unsuitable as a selective screening tool. Much better androgen
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
luciferase reporter under transcriptional control of the PB-ARE2
androgen response element (Blankvoort et al., 2001). This stable
cell line shows additional hormone class specificity, as it mainly
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
PR and GR while supporting an optimal androgen response when
the cognate receptor was transiently introduced. This led to
selection of the U2-OS cell line, which has the additional advan-
tage of being robust, genetically stable, and of fast proliferation
compared to most prostatic cell lines. Through the introduction
of a highly selective and responsive reporter gene, we generated
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.,
2003; van der Burg et al., manuscript in preparation). These data
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-
dependent AR stabilization as a result of different rates of andro-
gen dissociation and AR degradation (Zhou et al., 1995), as
shown for antiestrogens on ER stability (Gibson et al., 1991;
van den Bemd et al., 1999). Ligand-dependent AR protein forms
with different transactivation capacity as described for R1881
previously (Kuil et al., 1996) might also be an explanation for the
lower maximal induction level of R1881. On the other hand, the
synthetic androgen MENT was able to induce a supramaximal
response (RTA 5 121%). This supra-induction was also
observed for genistein and o,p
0
DDT 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
coactivators, such as TIF2 and SCR-1a as shown recently for ER
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-
pounds to the GR and their response in the GR CALUX bioassay,
with the latter showing EC50 values of 0.5 nM for dexametha-
sone, 5 nM for hydrocortisone, and 15 nM for corticosterone
(Sonneveld et al., manuscript in preparation). Accordingly, GR-
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
glucocorticoids.
While active androgens bind directly to the androgen receptor
and induce luciferase activity in AR CALUX cells, androgen
precursors need metabolic activation. Androstenedione is a very
weak binder to AR (0.1% compared to DHT; van der Burg et al.,
manuscript in preparation), but is a potent androgen in the AR
CALUX bioassay, suggesting the presence of the metabolic
enzyme 17b-HSD type 5 (with 17b-ketosteroid reductase activ-
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-
ments showed the expression of CYP17 in U2-OS cells (data not
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.
Figure 4 shows that the AR CALUX bioassay readily picks up
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
HPTE was even 30 times more potent than its parental compound
methoxychlor, making this compound one of the strongest envir-
onmental AR antagonists found so far in the AR CALUX bio-
assay. The antagonistic activity of vinclozolin (IC50 5 1 mM) is
A PANEL OF STEROID-RESPONSIVE BIOASSAYS 145
rather potent compared to another AR reporter cell line, the
MDA-kb2 cells (IC50 5 10 mM; Wilson et al., 2002). In the
latter cell line, metabolites of vinclozolin, M1 and primarily M2,
are far more potent than the parental compound, suggesting that
in the AR CALUX cells vinclozolin can be metabolized to more
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-
ing to cell death, but can also be due to more specific effects such
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
of reporter gene activity rather than overall cytotoxicity and cell
death. ‘Constitutively’ expressed reporter genes that often are
used as controls have the drawback that no bona fide constitutive
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
(hormone) receptors is a well-known mechanism of interference
and might therefore produce false-negative results. 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
(Table 2). Another receptor shown to possess squelching effects
with nuclear hormone receptors is the aryl hydrocarbon receptor
(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,p
0
-DDT,
p,p
0
-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
human serum was shown by the AR and ERa CALUX bioassays,
respectively, demonstrating that these bioassays can be used to
measure low levels of bioavailable hormones directly in very
small amounts of human serum (approximally 30 ml), as demon-
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
steroids present. However, we could not demonstrate androgenic
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
146
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.
ACKNOWLEDGMENTS
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 (Stockholm University, Sweden),
W. Schoonen (NV Organon, Oss, The Netherlands) and B. Hendriks-Stegeman
(University Medical Centre, Utrecht, The Netherlands) for kindly providing the
BFR technical mixtures, the steroids, and human serum pool, respectively.
REFERENCES
Andersen, H. R., Vinggaard, A. M., Rasmussen, T. H., Gjermandsen, I. M., and
Bonefeld-Jorgensen, E. C. (2002). Effects of currently used pesticides
in assays for estrogenicity, androgenicity, and aromatase activity in vitro.
Toxicol. Appl. Pharmacol. 179, 1–12.
Aurrekoetxea-Hernandez, K., and Buetti, E. (2004). Transforming growth factor
beta enhances the glucocorticoid response of the mouse mammary tumor virus
promoter through Smad and GA-binding proteins. J. Virol. 78, 2201–2211.
Avances, C., Georget, V., Terouanne, B., Orio, F., Cussenot, O., Mottet, N.,
Costa, P., and Sultan, C. (2001). Human prostatic cell line PNT1A, a useful tool
for studying androgen receptor transcriptional activity and its differential
subnuclear localization in the presence of androgens and antiandrogens.
Mol. Cell. Endocrinol. 184, 13–24.
Bentel, J. M., Birrell, S. N., Pickering, M. A., Holds, D. J., Horsfall, D. J., and
Tilley, W. D. (1999). Androgen receptor agonist activity of the synthetic
progestin, medroxyprogesterone acetate, in human breast cancer cells. Mol.
Cell. Endocrinol. 154, 11–20.
Blankvoort, B. M., de Groene, E. M., van Meeteren-Kreikamp, A. P., Witkamp,
R. F., Rodenburg, R. J., and Aarts, J. M. (2001). Development of an androgen
reporter gene assay (AR-LUX) utilizing a human cell line with an endogen-
ously regulated androgen receptor. Anal. Biochem. 298, 93–102.
Brinkmann, A. O., Faber, P. W., van Rooij, H. C., Kuiper, J. M., Ris, C.,
Klaassen, P., van der Korput, J. A. G. M., Voorhorst, M. M., van Laar, J. H.,
Mulder, E., et al. (1989). The human androgen receptor: Domain structure,
genomic organization and regulation of expression. J. Steroid Biochem.
34, 307–310.
Claessens, F., Verrijdt, G., Schoenmakers, E., Haelens, A., Peeters, B.,
Verhoeven, G., and Rombauts, W. (2001). Selective DNA binding by the
androgen receptor as a mechanism for hormone-specific gene regulation.
J. Steroid Biochem. Mol. Biol. 76, 23–30.
de Gooyer, M. E., Deckers, G. H., Schoonen, W. G. E. J., Verheul, H. A.,
Kloosterboer, H. J. (2003). Receptor profiling and endocrine interactions of
tibolone. Steroids 68, 21–30.
Di Croce, L., Koop, R., Venditti, P., Westphal, H. M., Nightingale, K. P.,
Corona, D. F., Becker, P. B., and Beato, M. (1999). Two-step synergism
between the progesterone receptor and the DNA-binding domain of nuclear
factor 1 on MMTV minichromosomes. Mol. Cell 4, 45–54.
Dubbink, H. J., Hersmus, R., Verma, C. S., van der Korput, H. A.,
Berrevoets, C. A., van Tol, J., Ziel-Van Der Made, A. C., Brinkmann, A. O.,
Pike, A. C., and Trapman, J. (2004). Distinct recognition modes of FXXLF and
LXXLL motifs by the androgen Receptor. Mol. Endocrinol. 18, 2132–2150.
Evans, N. A. (2004). Current concepts in anabolic-androgenic steroids. Am. J.
Sports Med. 32, 534–542.
Folkers, G. E., and van der Saag P. T. (1995). Adenovirus E1A functions as a
cofactor for retinoic acid receptor beta (RAR beta) through direct interaction
with RAR beta. Mol. Cell. Biol. 15, 5868–5878.
Gibson, M. K., Nemmers, L. A., Beckman, W. C., Davis, V. L., Curtis, S. W., and
Korach, K. S. (1991). The mechanism of ICI-164,384 antiestrogenicity
involves rapid loss of estrogen receptor in uterine tissue. Endocrinology
129, 2000–2010.
Gimeno, S., Gerritsen, A., Bowmer, T., and Komen, H. (1996). Feminization of
male carp. Nature 384, 221–222.
Glass, C. K. (1994). Differential recognition of target genes by nuclear receptor
monomers, dimers and heterodimers. Endocr. Rev. 15, 391–407.
Green, S., Walter, P., Greene, G., Krust, A., Goffin, C., Jensen, E., Scrace, G.,
Waterfield, M., and Chambon, P. (1986). Cloning of the human oestrogen
receptor cDNA. J. Steroid Biochem. 24, 77–83.
Hartig, P. C., Bobseine, K. L., Britt, B. H., Cardon, M. C., Lambright, C. R.,
Wilson, V. S., and Gray, L. E., Jr. (2002). Development of two androgen
receptor assays using adenoviral transduction of MMTV-Luc reporter and/
or hAR for endocrine screening. Toxicol. Sci. 66, 82–90.
He, B., Lee, L. W., Minges, J. T., and Wilson, E. M. (2002). Dependence of
selective gene activation on the androgen receptor NH2- and COOH-terminal
interaction. J. Biol. Chem. 277, 25631–25639.
Interagency Coordinating Committee on the Validation of Alternative Methods
(ICVAMM) (2003). ICCVAM evaluation of in vitro test methods for detection
of potential endocrine disruptors: Estrogen receptor and androgen receptor
binding and transcriptional activation assays. NIH publication no. 03–4503.
Kalkhoven, E., Kwakkenbos-Isbru
¨
cker, L., de Laat, S. W., van der Saag, P. T.,
and van der Burg, B. (1994). Synthetic progestins induce proliferation of breast
tumor cell lines via the progesterone or estrogen receptor. Mol. Cell.
Endocrinol. 102, 45–52.
Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppainen, J. A., and
Wilson, E. M. (1995). Persistent DDT metabolite p,p
0
-DDE is a potent
androgen receptor antagonist. Nature 375, 581–585.
Kelce, W. R., and Wilson, E. M. (1997). Environmental anti-androgens: Devel-
opmental effects, molecular mechanisms and clinical implications. J. Mol.
Med. 75, 198–207.
Kraus, W. L., Weis, K. E., and Katzenellenbogen, B. S. (1995). Inhibitory cross-
talk between steroid hormone receptors: Differential targeting of estrogen
receptor in the repression of transcriptional activity by agonist- and
antagonist-occupied progestin receptors. Mol. Cell. Biol. 15, 1847–1857.
Kuil, C. W., and Mulder, E. (1996). DNA-binding ability of androgen receptors in
whole cells: Implications for the action of androgens and antiandrogens.
Endocrinology 137, 1870–1877.
Legler, J., Broekhof, J. L. M., Brouwer, A., Lanser, P. H., Murk, A. J.,
van der Saag, P. T., Wester, P., Zivkovic, D., and van der Burg, B. (2000).
A novel in vivo bioassay for (xeno-) estrogens using transgenic zebrafish.
Environ. Sci. Technol. 34, 4439–4444.
Legler, J., van den Brink, C. E., Brouwer, A., Murk, A. J., van der Saag, P. T.,
Vethaak, A. D., and van der Burg, B. (1999). Development of a stably trans-
fected estrogen receptor-mediated luciferase reporter gene assay in the human
T47D breast cancer cell line. Toxicol. Sci. 48, 55–66.
Lemmen, J. G., Arends, R. J., van Boxtel, A. L., van der Saag, P. T., and van der
Burg, B. (2004). Tissue- and time-dependent estrogen receptor activation in
estrogen reporter mice. J. Mol. Endocrinol. 32, 689–701.
Lemmen, J. G., van den Brink, C. E., Legler, J., van der Saag, P. T, and
van der Burg, B. (2002). Detection of oestrogenic activity of steroids present
A PANEL OF STEROID-RESPONSIVE BIOASSAYS
147
during mammalian gestation using ERalpha and ERbeta specific in vitro
assays. J. Endocrinol. 174, 435–446.
Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G.,
Umesono, K., Blumberg, B., Kastner, P., Mark, M., Chambon, P., et al.
(1995). The nuclear receptor superfamily: The second decade. Cell 83,
835–839.
Meyer, H. H. (2001). Biochemistry and physiology of anabolic hormones used
for improvement of meat production. APMIS 109, 1–8.
McKenna, N. J., and O’Malley, B. W. (2002). Combinatorial control of gene
expression by nuclear receptors and coregulators. Cell 108, 465–474.
Nuclear Receptors Nomenclature Committee (1999). A Unified Nomenclature
System for the Nuclear Receptor Superfamily. Cell 97, 161–163.
Ouatas, T., Clare, S. E., Hartsough, M. T., De La Rosa, A., and Steeg, P. S.
(2002). MMTV-associated transcription factor binding sites increase
nm23-H1 metastasis suppressor gene expression in human breast carcinoma
cell lines. Clin. Exp. Metastasis 19, 35–42.
Paris, F., Balaguer, P., Terouanne, B., Servant, N., Lacoste, C., Cravedi, J. P.,
Nicolas, J. C., and Sultan, C. (2002a). Phenylphenols, biphenols, bisphenol-A
and 4-tert-octylphenol exhibit alpha and beta estrogen activities and antian-
drogen activity in reporter cell lines. Mol. Cell. Endocrinol. 193, 43–49.
Paris, F., Servant, N., Terouanne, B., Balaguer, P. Nicolas, J. C., and Sultan, C.
(2002b). A new recombinant cell bioassay for ultrasensitive determination
of serum estrogenic bioactivity in children. J. Clin. Endocrinol. Metab. 87,
791–797.
Paris, F., Servant, N., Terouanne, B., and Sultan, C. (2002c). Evaluation of
androgenic bioactivity in human serum by recombinant cell line: Preliminary
results. Mol. Cell. Endocrinol. 198, 123–129.
Quaedackers, M. E., van den Brink, C. E., Wissink, S., Schreurs, R. H. M.,
Gustafsson, J. A., van der Saag, P. T., and van der Burg, B. (2001). 4-
Hydroxytamoxifen Trans-represses nuclear factor-kB activity in human
osteoblastic U2-OS cells through estrogen receptor (ER) alpha, and not
through ERbeta. Endocrinology 142, 1156–1166.
Routledge, E. J., White, R., Parker, M. G., and Sumpter, J. P. (2000). Differential
effects of xenoestrogens on coactivator recruitment by estrogen receptor
(ER) a and ERb. J. Biol. Chem. 275, 35986–35993.
Schule, R., Muller, M., Kaltschmidt, C., and Renkawitz, R. (1988). Many tran-
scription factors interact synergistically with steroid receptors. Science 242,
1418–1420.
Sohoni, P., and Sumpter, J. P. (1998). Several environmental oestrogens are
also anti-androgens. J. Endocrinol. 158, 327–339.
Sonneveld, E., Jansen, H. J., Riteco, J. A. C., Brouwer, A., and van der Burg, B.
(2003). CALUX reporter cell lines as sensitive bioassays for monitoring
biological activity of various classes of steroid hormones reveal the repres-
sive effect of TCDD on the estrogen receptor. Organohal. Compounds 60,
263–266.
Sonneveld, E., van den Brink, C. E., van der Leede, B. M., Schulkes, R. K.,
Petkovich, M., van der Burg, B., and van der Saag, P. T. (1998). Human
retinoic acid (RA) 4-hydroxylase (CYP26) is highly specific for all-trans-
RA, and can be induced through retinoic acid receptors in human breast
and colon carcinoma cells. Cell Growth Differ. 9, 629–637.
Spangenberg, C., Eisfeld, K., Stunkel, W., Luger, K., Flaus, A., Richmond, T. J.,
Truss, M., and Beato, M. (1998). The mouse mammary tumour virus promoter
positioned on a tetramer of histones H3 and H4 binds nuclear factor 1 and
OTF1. J. Mol. Biol. 278, 725–739.
Sutherland, R. L., Hall, R. E., Pang, G. Y., Musgrove, E. A., and Clarke, C. L.
(1988). Genetic instability and the development of steroid hormone
insensitivity in cultured T 47D human breast cancer cells. Cancer Res. 48,
4340–4347.
Terouanne, B., Tahiri, B., Georget, V., Belon, C., Poujol, N., Avances, C.,
Orio, F., Balaguer, P., and Sultan, C. (2000). A stable prostatic biolumines-
cent cell line to investigate androgen and antiandrogen effects. Mol. Cell.
Endocrinol. 160, 39–49.
Uchiumi, F., Sato, T., and Tanuma, S. (1998). Identification and characteriza-
tion of a tannic acid-responsive negative regulatory element in the mouse
mammary tumor virus promoter. J. Biol. Chem. 273, 12499–12508.
van den Bemd, G. J. C. M., Kuiper, G. G. J. M., Pols, H. A. P., and van
Leeuwen, J. P. T. M. (1999). Distinct effects on the conformation of estrogen
receptor alpha and beta by both the antiestrogens ICI 164,384 and ICI
182,780 leading to opposite effects on receptor stability. Biochem. Biophys.
Res. Commun. 261, 1–5.
van der Burg, B., Rutteman, G. R., Blankenstein, M. A., de Laat, S. W., and
van Zoelen, E. J. (1988). Mitogenic stimulation of human breast cancer cells in
a growth factor-defined medium: Synergistic action of insulin and estrogen.
J. Cell Physiol. 134, 101–108.
Vermeer, H., Hendriks-Stegeman, B. I., van den Brink, C. E., van der Saag, P. T.,
van der Burg, B., van Buul-Offers, S. C., and Jansen, M. (2003). A novel
specific bioassay for the determination of glucocorticoid bioavailability in
human serum. Clin. Endocrinol. 59, 49–55.
Willemsen, P., Scippo, M., Kausel, G., Figueroa, J., Maghuin-Rogister, G.,
Martial, J. A., and Muller, M. (2004). Use of reporter cell lines for detec-
tion of endocrine-disrupter activity. Anal. Bioanal. Chem. 378,
655–663.
Wilson, V. S., Bobseine, K., Lambright, C. R., and Gray, L. E., Jr. (2002). A novel
cell line, MDA-kb2 that stably expresses an androgen- and glucocorticoid-
responsive reporter for the detection of hormone receptor agonists and
antagonists. Toxicol. Sci. 66, 69–81.
Zhou, Z. X., Lane, M. V., Kemppainen, J. A., French, F. S., and Wilson, E. M.
(1995). Specificity of ligand-dependent androgen receptor stabilization:
Receptor domain interactions influence ligand dissociation and receptor
stability. Mol. Endocrinol. 9, 208–218.
148 SONNEVELD ET AL.
    • "This activity is generally associated with known androgenic compounds, including natural and synthetic steroid hormones (e.g. testosterone, nandrolone and levonorgestrel), UV-filters and perfluorinated compounds, all of which have been previously found in these environments (Bjerregaard-Olesen et al., 2014; Freyberger et al., 2010; Sonneveld et al., 2005). Compared to other international studies, our results suggests that although estrogenic activity was measured in 70% of the samples in the present study, the BEQs were generally low compared to those from other studies reporting on indoor environments and outdoor air samples from smog episodes (Wenger et al., 2009; Novák et al., 2009; Novák et al., 2013; Klein et al., 2006; Kennedy et al., 2009 ). "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. Objectives: The objective of this study was to investigate and compare the overall estrogen and androgen activating potential of PM10 air samples at an urban, rural and industrial location in Flanders, using a human in vitro cell bioassay. Methods: PM10 samples were collected on glass fiber filters every six days between April 2013 and January 2014 using a high-volume sampler. Extraction was executed with a hexane/acetone mixture before analysis using a recombinant estrogen- or androgen responsive human carcinoma cell line. Results were expressed as bioanalytical equivalents (BEQs) per cubic meter of air. Results: High fluctuations in estrogenic activity were observed during the entire sampling period, with median BEQs of 32.1, 35.9 and 31.1 fg E2-Eq m(-)³ in the industrial, urban and rural background area, respectively. Estrogenic activity was measured in 70% of the samples, while no androgenic activity was observed in any of the samples. The estrogenic activity in the industrial area was positively correlated with the airborne concentration of the sum of the non-carcinogenic PAHs pyrene and fluoranthene (rho=0.48; p<0.01) and the sum of the carcinogenic PAHs (rho=0.36; p=0.05). Conclusions: This study showed that no androgenic activity was present in PM10 and that although the median estrogenic activity was rather low and comparable in the three locations, high fluctuations in estrogenic response exist over time. While atmospheric PAHs contributed to the observed estrogenic response, especially in the industrial area, the chemicals responsible for the majority of estrogenic activity remain to be identified.
    Full-text · Article · Oct 2016
    • "To avoid false negative results, only the samples classified as non-cytotoxic (cell viability >80 % according to ISO 10993-5: 2009) within the MTT assay performed according to Mosmann (1983) were applied in the CALUX tests to determine the endocrine activity. The test procedure was the same for all CALUX assays in accordance with operation procedures developed by BDS (BioDetection Systems 2009; Sonneveld et al. 2005; van der Burg et al. 2010a , b). In a 96- microwell plate, 10 × 10 4 cells/mL in assay medium (DMEM/F-12 free of phenol red supplemented with 14.5 % stripped FCS, 4.7 % gentamicin, and 3.2 % MEM-NEAA), resulting in a density of 1 × 10 4 cells per well, was seeded and incubated for 24 h (±1 h) at culture conditions. "
    [Show abstract] [Hide abstract] ABSTRACT: (Anti-)Estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: Comparison of three in vitro bioassays L. Gehrmann & H. Bielak et al., 2016 Endocrine disrupting chemicals, which are discharged into the environment mainly by wastewater treatment plants (WWTP), have been shown to induce adverse effects in aquatic life and to be of potential risk for human. Advanced treatment with ozone successfully removes organic micropollutants but, in previous studies, an increase of estrogenic effects after the ozonation of hospital wastewater was observed. In order to investigate this effect, estrogenic and androgenic as well as anti-estrogenic and anti-androgenic activities were determined during treatment of hospital wastewater using three different effect-based reporter-gene bioassays. Despite different matrix influences, sensitivities and test-specific properties, the actual assessment of the obtained results was in accordance in all systems. Estrogenic and androgenic activities were mainly reduced during the biological treatment and further removed during ozonation and sand filtration, resulting in no detectable agonistic activities in the final effluent. Antagonistic effects were removed in the biological treatment by up to 50% without further reduction in the advanced treatment. Therefore, this study showed the relevance of antagonistic activities hospital WWTPs and illustrates the need for a better understanding about antagonistic effects.
    Full-text · Article · Jul 2016
    • "The aim of this study was to develop and evaluate reduced volume procedures for the steps of exposure and chemical dilution in medium in an AR reporter gene human cell line assay (van der Burg et al., 2010a ) capable of producing results that are (i) within assay acceptance criteria, and (ii) comparable to results obtained though the higher volume procedures recommended by the method protocol, related test guidelines and previous studies. For that, the cells were exposed in 96-well plates in medium volume of around 1/3 of recommended procedures for reporter gene cell-based assays (Sonneveld et al., 2005; van der Burg et al., 2010a; OECD, 2012; Besselink et al., 2015; OECD, 2015 ). The low-volume dosing was achieved by diluting the chemicals in medium inside microvolume glass inserts, mini mizing sample losses during pipetting steps and simulating the procedure that can be done with sample extracts or fractions, often collected and stored in such vials. "
    [Show abstract] [Hide abstract] ABSTRACT: Bioactivity screening studies often face sample amount limitation with respect to the need for reliable, reproducible and quantitative results. Therefore approaches that minimize sample use are needed. Low-volume exposure and chemical dilution procedures were applied in an androgen receptor reporter gene human cell line assay to evaluate environmental contaminants and androgen receptor modulators, which were the agonist 5α-dihydrotestosterone (DHT); and the antagonists flutamide, bisphenol A, 1-hydroxypyrene and triclosan. Cells were exposed in around 1/3 of the medium volume recommended by the protocol (70μL/well). Further, chemical losses during pipetting steps were minimized by applying a low-volume method for compound dilution in medium (250μL for triplicate wells) inside microvolume glass inserts. Simultaneously, compounds were evaluated following conventional procedures (200μL/well, dilution in 24-well plates) for comparison of results. Low-volume exposure tests produced DHT EC50 (3.4-3.7×10(-10)M) and flutamide IC50 (2.2-3.3×10(-7)M) values very similar to those from regular assays (3.1-4.2×10(-10) and 2.1-3.3×10(-7)M respectively) and previous studies. Also, results were within assay acceptance criteria, supporting the relevance of the downscaling setup for agonistic and antagonistic tests. The low-volume exposure was also successful in determining IC50 values for 1-hydroxypyrene (2.1-2.8×10(-6)M), bisphenol A (2.6-3.3×10(-6)M), and triclosan (1.2-1.9×10(-6)M) in agreement with values obtained through high-volume exposure (2.3-2.8, 2.5-3.4 and 1.0-1.3×10(-6)M respectively). Finally, experiments following both low-volume dosing and exposure produced flutamide and triclosan IC50 values similar to those from regular tests. The low-volume experimental procedures provide a simple and effective solution for studies that need to minimize bioassay sample use while maintaining method reliability. The downscaling methods can be applied for the evaluation of samples, fractions or chemicals which require minimal losses during the steps of pipetting, transference to medium and exposure in bioassays.
    Full-text · Article · Jul 2016
Show more