Masking effect of anti-androgens on androgenic activity in European river sediment unveiled by effect-directed analysis.

Abstract

This study shows that the androgen receptor agonistic potency is clearly concealed by the effects of androgen receptor antagonists in a total sediment extract, demonstrating that toxicity screening of total extracts is not enough to evaluate the full in vitro endocrine disrupting potential of a complex chemical mixture, as encountered in the environment. The anti-androgenic compounds were masking the activity of androgenic compounds in the extract with relatively high anti-androgenic potency, equivalent to 200 nmol flutamide equivalents/g dry weight. A two-step serial liquid chromatography fractionation of the extract successfully separated anti-androgenic compounds from androgenic compounds, resulting in a total androgenic potency of 3,820 pmol dihydrotestosterone equivalents/g dry weight. The fractionation simplified the chemical identification analysis of the original complex sample matrix. Seventeen chemical structures were tentatively identified. Polyaromatic hydrocarbons, a technical mixture of nonylphenol and dibutyl phthalate were identified to contribute to the anti-androgenic potency observed in the river sediment sample. With the GC/MS screening method applied here, no compounds with AR agonistic disrupting potencies could be identified. Seventy-one unidentified peaks, which represent potentially new endocrine disrupters, have been added to a database for future investigation.

Full text

Page 1

PAPER IN FOREFRONT
Masking effect of anti-androgens on androgenic activity
in European river sediment unveiled by effect-directed
analysis
Jana M. Weiss & Timo Hamers & Kevin V. Thomas &
Sander van der Linden & Pim E. G. Leonards &
Marja H. Lamoree
Received: 25 February 2009 /Revised: 14 April 2009 /Accepted: 16 April 2009 /Published online: 6 May 2009
# The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract This study shows that the androgen receptor
agonistic potency is clearly concealed by the effects of
androgen receptor antagonists in a total sediment extract,
demonstrating that toxicity screening of total extracts is not
enough to evaluate the full in vitro endocrine disrupting
potential of a complex chemical mixture, as encountered in
the environment. The anti-androgenic compounds were
masking the activity of androgenic compounds in the
extract with relatively high anti-androgenic potency, equiv-
alent to 200 nmol flutamide equivalents/g dry weight. A
two-step serial liquid chromatography fractionation of the
extract successfully separated anti-androgenic compounds
from androgenic compounds, resulting in a total androgenic
potency of 3,820 pmol dihydrotestosterone equivalents/g
dry weight. The fractionation simplified the chemical
identification analysis of the original complex sample
matrix. Seventeen chemical structures were tentatively
identified. Polyaromatic hydrocarbons, a technical mixture
of nonylphenol and dibutyl phthalate were identified to
contribute to the anti-androgenic potency observed in the
river sediment sample. With the GC/MS screening method
applied here, no compounds with AR agonistic disrupting
potencies could be identified. Seventy-one unidentified
peaks, which represent potentially new endocrine disrupt-
ers, have been added to a database for future investigation.
Keywords Androgenicity.Androgenreceptor(AR).Effect-
directedanalysis(EDA).Sediment.Polycyclicaromatic
hydrocarbons(PAHs).MODELKEY
Introduction
In areas influenced by anthropogenic activity, the environ-
ment is often contaminated by a complex mixture of
substances that may have toxicological effects on organ-
isms. One arising concern has been the world-wide
observation of effects consistent with exposure to endocrine
disrupting compounds in invertebrates, fish, wildlife and
even humans [1–3]. Endocrine disrupting effects can be
profound due to the role hormones play in the development
of an organism. Androgens are one group of these crucial
hormones. Androgens stimulate and control the develop-
ment and maintenance of masculine characteristics by
binding to the androgen receptor (AR) and inducing a
cascade of hormone responses. This includes the activity of
the accessory male sex organs and development of male
secondary sex characteristics. Androgens are also the
precursor of all estrogens, the female sex hormones.
Androgen-mediated dysfunction has been reported in fish,
such as skewed sex ratio, masculinisation of females,
disturbed relative weight and histopathology [2]. Sexual
dimorphism in fish has been demonstrated to be affected by
both androgens and estrogens [4]. Also, vitellogenin
Anal Bioanal Chem (2009) 394:1385–1397
DOI 10.1007/s00216-009-2807-8
J. M. Weiss:T. Hamers:P. E. G. Leonards:M. H. Lamoree (*)
Institute for Environmental Studies (IVM),
Faculty of Earth and Life Sciences, VU University,
De Boelelaan 1087,
1081 HV Amsterdam, The Netherlands
e-mail: marja.lamoree@ivm.vu.nl
K. V. Thomas
Norwegian Institute for Water Research (NIVA),
Gaustadalléen 21,
0349 Oslo, Norway
S. van der Linden
BioDetection Systems (BDS) B.V.,
Science Park 406,
1098 XH Amsterdam, The Netherlands

Page 2

induction, normally a measure of estrogenic activity, has
been observed in both sexes probably as a result of
aromatisation of androgens. Synthetic androgens (anabolic
steroids) are used to promote muscle strength in humans
(e.g. athletes) and animals [5, 6], to increase the meat
quantity in farm animals [7], and in androgen replacement
therapies [8]. Synthetic and natural androgenic steroids
(such as methyl-testosterone and testosterone, respectively)
bind to the androgen receptor (AR), inducing an activation
of the receptor (AR agonism) leading to transcription of
genes responsible for androgenic effects. It has been found
that environmental pollutants, e.g. pesticides and industrial
chemicals, can interfere with the androgen system in both
wildlife and humans, mainly as anti-androgens [2]. Anti-
androgens (such as the anti-androgenic drug flutamide) can
bind to the AR, but are unable to activate the receptor (AR
antagonism).
To fully investigate the androgenic potential of the
sediment extract, we followed an effect-directed analysis
approach (EDA). EDA has been developed in order to
identify toxic compounds in the environment [9]. EDA
studies use bioassay-directed fractionation techniques in
order to select those fractions containing toxic compounds,
and hence perform a toxicity characterization of the sample.
In EDA, a non-destructive clean up procedure is used
which enables the inclusion of as many compounds as
possible present in a sample that exert a specific mode of
action in a bioassay, i.e. a non-selective identification of
relevant compounds that may cause a specific effect in the
environment [9]. The aim is, with the assistance of
bioassays responding to specific toxicological/biological
endpoint(s), to select active fractions of the sample for
further fractionation in order to simplify the complexity of
the extracts. The final goal is to identify—by chemical
analytical means—the effect-causing compounds in the
active fractions.
The EDA approach has successfully been applied to
evaluate endocrine potencies in several water systems, such
as wastewater treatment plants [10, 11], rivers [11–13],
harbour areas [14, 15] marine sediments [16] and biota
[17]. For example, almost 100% of estrogenic and
androgenic activity in fish bile and effluents from waste-
water treatment plants can be explained by the occurrence
of natural and synthetic steroids [11, 17]. An earlier
published review further describes a range of important
results obtained from bioassay-directed studies [9].
The study presented here focuses on a sediment sample
from the river Scheldt water basin collected close to
Antwerp (Belgium). This sample was selected after a
toxicity characterization of three European river systems
(11 samples from the rivers: Elbe in the Czech Republic
and Germany; Llobregat in Spain; Scheldt in Belgium and
The Netherlands) within the EU funded MODELKEY
project (SSPI-CT-2003-511237-2; http://www.modelkey.
ufz.de/). The bioassay used to direct the analysis is the
Chemically Activated Luciferase gene eXpression assay for
androgen detection (AR CALUX®), for both agonistic and
antagonistic responses, expressed as dihydrotestosterone
(DHT) equivalents and flutamide (FLU) equivalents,
respectively. The sediment extract chosen for further EDA
investigation in this study was selected because it had the
highest anti-androgenic (i.e. AR antagonistic) potency after
initial toxicity screening. No androgenic (i.e. AR agonistic)
potency was observed.
The aim of the work described here was to use an EDA
approach to evaluate the full androgenic potential in a
sediment sample from a European river system. Because
the presence of AR agonists may be masked by the
presence of AR antagonists in the sample, this includes
AR agonistic screening of the fractions of the original AR
antagonistic Scheldt sample. Potent compounds in the
bioassay are tentatively identified by qualitative Gas
Chromatography–Mass Spectrometry (GC/MS) screening.
Materials and methods
Sample information
The sediment sample that has been investigated is from the
river Schijn, a tributary to the river Scheldt, at the location
Eenhoorn, Wijnegem (51°13′13.77″N, 4°32′50.79″E), close
to Antwerp in Belgium (Fig. 1). Eenhoorn is situated below
the outlet of the Grote Merriebeek, which is a water-stream
canalised through an industrialised area, parallel to the
Albert Canal, and is a point discharge of polluted water into
the Schijn.
Fig. 1 Map of sediment sampling area in Belgium, close to Antwerp,
location Eenhoorn (star)
1386J.M. Weiss et al.

Page 3

Sediment (top 10–20-cm layer) was sampled in June
2006 with a hand-operated Ponar grab. The sediment
contained fine muddy particles, was rich in organic material
and was darkly coloured with light oily specks that were
observed during sampling. The dry weight of the sample as
received was 24%. The sample was stored in a climate
room (4 °C) until analysis. Before clean up, it was sieved
(125 μm) followed by freeze drying.
Pre-treatment procedure
All solvents were pro analysis quality or better and were
purchased from JT baker (The Netherlands) or Merck
(Germany) unless stated otherwise. The pre-treatment
procedure is outlined in Fig. 2.
Extraction
The extraction method used for the sediment sample (40 g
dry weight [dw]) was Accelerated Solvent Extraction
(ASE), an exhaustive extraction method [18]. Dichloro-
methane (DCM): acetone (3:1, v/v) was used as extraction
solvent. ASE extractions were performed at 50°C,
2,000 psi, with three extraction cycles [15]. Extracts were
evaporated under a gentle N2-gas stream until almost
dryness and re-dissolved in DCM (1 ml).
Gel permeation chromatography for clean up
The ASE-extract was cleaned up using Gel Permeation
Chromatography (GPC) equipped with two polystyrene-
divinylbenzene columns in series (10 μm, 50 Å, 600×
25 mm, Polymer Laboratories) and with 10 ml/min DCM as
eluent [19]. The ASE-extract was injected via a 1.5-ml
injection loop onto the GPC column. The major part of the
matrix elutes in the first fraction up to ca 18 min following
injection. Based on the results of the elution profile of test
compounds [19], the fraction 16:30—24:00 min was
collected (fraction GPC1). The fraction containing sulphur,
which is known to be cytotoxic in the bioassay, elutes
between 25 and 29 min and was discarded. Another
fraction was also collected (29–36 min) after the heart cut
of sulphur to see if late-eluting androgenic or anti-
androgenic compounds were present (fraction GPC2). The
separate GPC-extracts were split, 5% was used for direct
bioassay analysis (evaporated to dryness and re-dissolved
in 100 μl dimethylsulfoxide [DMSO, spectrophotometric
grade, 99.9%, Acros, Belgium]), and the remaining 95%
was used for high-performance liquid chromatography
(HPLC) fractionation (evaporated to dryness and re-
dissolved in methanol [MeOH]: water [H2O], 1:1v/v).
Only the first GPC fraction (GPC1, 20 g sediment/ml)
showed activity in the bioassay and was further analysed.
Due to the lack of bioassay response (both AR agonistic as
well as AR antagonistic) in GPC fraction 2, this fraction
was not included in the rest of the study.
Reversed phase fractionation
The first fractionation was performed using a reversed-
phase (RP) HPLC system at 22 °C, with a C18 semi-
preparative column (Vydac 201TP510, 5 μm, 10.0×
250 mm; mobile phase initially 50% MeOH and 50%
H2O (4.7 ml/min), changing linearly to 100% MeOH in
50 min and kept at 100% MeOH during 40 min) [17]. The
GPC-extract (GPC1) in 1 ml MeOH:H2O (1:1v/v) was
quantitatively injected and five fractions with time windows
0–9, 9–24, 24–42, 42–63 and 63–90 min, indicated as
fractions RP1–RP5, were collected. After quantitative
injection, the vial was rinsed with hexane to dissolve any
compounds possibly remaining on the vial wall that were
too non-polar to dissolve in the carrier solvent (MeOH:
H2O) prior to fractionation. This non-polar residual fraction
was evaporated and dissolved in 100 μl DMSO and tested
with AR CALUX assays without further fractionation.
RP-HPLC fractions were evaporated with N2-gas at
40 °C and split into two portions: 20% was evaporated and
dissolved in 100 μl of DMSO (80 g sediment/ml) for AR
CALUX measurements, while the remaining 80% of the
active fractions in the bioassays were further fractionated.
Active fractions
further fractionated
Sediment sample (40 gram)
Sieved and freeze dried
ASE extraction
GPC clean up
(16.5-24 min and
29-36 min fraction)
NP-HPLC fractionation
(8 fractions)
Chemical analysis
GC/MS
RP-HPLC fractionation
(5 fractions + residual)
Bioassay
5%
Active fractions
further fractionated
Bioassay
20%
Bioassay
10%
Fig. 2 An outline of the pre-treatment procedure used in the effect-
directed analysis of the sediment sample
Masking effect of anti-androgens on androgenic activity1387

Page 4

Normal phase fractionation
As the second step in the two-step serial fractionation
strategy, the reversed-phase fractions were further fraction-
ated into eight sub-fractions by Normal Phase (NP) HPLC
with a Waters μPorasil semi-preparative column (7.8×
300 mm, particle size 10 μm; mobile phase initially 100%
hexane (5 min), 1%/min of DCM for 5 min, 4%/min of
DCM for 25 min to 100% DCM (10 min) followed by
10%/min of acetonitrile (ACN) for 5 min, and then back to
100% DCM (5 min) and finally 6%/min to 100% of hexane
to re-equilibrate (10 min) as described by Fernandez and
Bayona [20]. Around 14% of the eluate of each NP fraction
(NP1–NP8) was evaporated, dissolved in 50 μl DMSO
(80 g sediment/ml) for AR CALUX measurements. The
remaining extracts (re-dissolved in hexane) were used for
chemical screening.
Bioassays
Androgenic and anti-androgenic potencies of the extract
and its fractions were determined in the AR CALUX®
bioassay (BioDetection Systems, Amsterdam, The Nether-
lands). The AR CALUX bioassay is a reporter gene assay,
which consists of a human osteoblast cell line carrying a
luciferase gene under transcriptional control of an androgen
responsive element [21]. Dihydrotestosterone (DHT,
Sigma-Aldrich, The Netherlands) and flutamide (FLU,
Sigma-Aldrich, The Netherlands) are used as androgenic
and anti-androgenic reference compounds, respectively.
The assay is performed according to the original publica-
tion by Sonneveld et al. [21] and AR agonistic and AR
antagonistic responses are expressed as relative amounts of
reference compound equivalents per g of dry weight (dw)
sediment. Triplicates of a dilution series in DMSO of the
sediment total extracts and fractions were analysed. Dose–
response curves of the reference compounds were fitted
using the sigmoidal fit with variable slope in GraphPad
Prism (version 4.01 for Windows, GraphPad Software;
Fig. 3a and b). The amount of reference compound
equivalents (eq) in the sample was determined by interpo-
lating the response of the sample into the dose–response
curve of the reference compound (Figs. 3c and d). A
minimum of 1% induction was set as limit of detection
(LOD) for androgenic effects, and 20% inhibition for
antagonistic androgenicity. The most diluted extract still
containing activity higher than the LOD was used for
quantification to avoid possible matrix effects. In order to
avoid false positives, the results from the non-diluted
fraction (x1) are not quantified (reported as + in Table 1)
if activity was not observed in the next diluted fraction (x3).
As a confirmation step of the bioassay effect and the
analytical quality (recovery) following normal phase sub-
fractionation the sub-fractions were pooled into their
original reversed-phase fractions constitution, i.e. RPXNP1
to RPXNP8 (where X=2, 3 or 4) is pooled and evaporated
into the same end volume as the original RPX fraction and
re-analysed in the bioassay [22]. This will clarify whether
the observed change in activity of the fractions are due to
loss of potent androgenic compounds during fractionation,
0.1 0.11110 10100
pMpM
1000 10000100000 1000 10000100000
00
20 20
4040
60 60
8080
100 100
120120
Induction (%) of DHT activity
b. DHT curve
100
Induction (%) of DHT activity
0.0001 0.00010.001 0.001 0.01
1/Dilution 1/Dilution
0.10.1 111010
00
2020
4040
6060
80 80
100100
120 120
LOD >1%
induction induction
d. Fraction RP3NP7
0.01
LOD >1%
0.10.1111010 100
nM
1000 10000100000 1000 10000100000
40
60
80
100
Induction (%) at 200 pM DHT
a. FLU curve
100
0
20
120
Induction (%) at 200 pM DHT
0.0001 0.0001 0.001 0.0010.01
1/Dilution1/Dilution
0.10.1111010
00
2020
40 40
6060
80 80
100 100
120 120
LOD >20%
inductioninduction
c. Fraction RP3NP2
0.01
LOD >20%
Fig. 3 Reference compounds
antagonistic flutamide (FLU)
curve (a) and agonistic dihydro-
testosterone (DHT) curve (b) in
AR CALUX, which are
expressed as % of inhibition at a
constant DHT concentration
(200 pM) and % of induction
compared to maximum DHT
concentration (10 nM), respec-
tively. Illustrated in c is the
sediment extract RP3NP2 AR
antagonistic potency and d the
sediment extract RP3NP7 AR
agonistic potency, in a dilution
series of ×1–×1,000
1388J.M. Weiss et al.

Page 5

too low bioassay sensitivity to observe activity in each
fraction or to interactions between AR agonistic and AR
antagonistic compounds.
Quality control
The reproducibility of the EC50s and IC50s of the reference
curves (n=19) are expressed as coefficients of variation
(CV) and were 47% for DHT and 19% for the FLU
between analyses. The variance is high between experi-
ments but considered acceptable since the responses are
expressed as relative effect potencies and a dose–response
curve of the reference compound is included in every
experiment.
To evaluate the inter-laboratory stability of the bioassay,
eight samples (GPC1, GPC2, RP1–RP5 and residual
reversed-phase fraction) were analysed for AR antagonistic
potency at two laboratories (IVM and BioDetection
DHT equivalentsFLU equivalents
ng/g sed pmol/g sed
μg/g sednmol/g sed
GPC fraction 1 (16.5-24 min)
GPC fraction 2 (29-36 min)
RP1
RP2
RP3
RP4
RP5
Residual fraction
Sum RP
RP2NP1
RP2NP2
RP2NP3
RP2NP4
RP2NP5
RP2NP6
RP2NP7
RP2NP8
Pooled RP2NP1–8
RP3NP1
RP3NP2
RP3NP3
RP3NP4
RP3NP5
RP3NP6
RP3NP7
RP3NP8
Pooled RP3NP1-8
RP4NP1
RP4NP2
RP4NP3
RP4NP4
RP4NP5
RP4NP6
RP4NP7
RP4NP8
Pooled RP4NP1–8
Sum individual NP
Sum pooled NP
<<
<<
<<
250
120
<<
<<
<<
370
<<
<<
<<
<<
<<
<<
120
<<
320
<<
<<
<<
<<
<<
<<
940
<<
110
<<
<<
<<
<<
<<
<<
<<
<<
+
1060
430
<<
<<
<<
890
440
<<
<<
<<
1330
<<
<<
<<
<<
<<
<<
420
<<
1170
<<
<<
<<
<<
<<
<<
3400
<<
380
<<
<<
<<
<<
<<
<<
<<
<<
+
3820
1550
55
<<
<<
+
30
6.9
<<
<<
37
<<
<<
<<
<<
<<
+
<<
<<
+
<<
6.0
+
+
2.4
4.5
<<
<<
27
<<
3.4
<<
<<
<<
<<
+
<<
9.3
16
36
200
<<
<<
+
110
25
<<
<<
130
<<
<<
<<
<<
<<
+
<<
<<
+
<<
22
+
+
8.8
16
<<
<<
98
<<
12
<<
<<
<<
<<
+
<<
34
59
130
Table 1 The agonistic (ng and
pmol of dihydrotestosterone
(DHT) equivalents/g dw) and
antagonistic (μg and nmol flu-
tamide (FLU) equivalents/g dw)
androgenicity measured in GPC
fraction 1 and 2, reversed-phase
(RP) fractions, the residual (non-
dissoluble fraction at reversed-
phase fraction), normal phase
(NP) fractions and pooled NP
fractions
<< below limit of detection
(>1% induction in the agonistic
AR CALUX, >20% inhibition
in the antagonistic AR
CALUX), + not quantified, i.e.
effect only observed in the non-
diluted fraction (×1) and not in
the diluted fractions
(×3–×1,000)
Masking effect of anti-androgens on androgenic activity1389

Page 6

Systems). The results did not differ (paired t test p>0.05)
and the CV was determined to be 10% between laboratories
(data not shown). To evaluate the inter-assay comparability,
the reversed-phase fractions (RP1–RP5) were analysed in
the yeast-based bioassay (YAS) for antagonistic androge-
nicity at the Centre for Environment, Fisheries and
Aquaculture Science (CEFAS, Essex, United Kingdom).
RP3 was successfully identified as the most potent AR
antagonistic fraction but the reported value was 300 times
higher (9000 μg/g dw) than in the AR CALUX. This
indicates the differences in response factors and the
difficulties to compare results between assays, but also
illustrates the comparability of the patterns of the observed
toxicity.
Chemical analysis
All normal phase sub-fractions were screened with Gas
Chromatography (GC, Agilent 6890) with a quadrupole
Mass Selective Detector (MS, Agilent 5973), equipped with
an SGE-BPX5 column (25 m, 0.22 mm i.d., 0.3 μm film
thickness). Two microlitres of fractions (in hexane) were
splitless injected. The column oven temperature programme
was as follows: 60°C (1 min), 7°C/min to 200°C (15 min),
5°C to 265°C (5 min) and finally 10°C/min to 275°C
(5 min). Run time, 60.00 min. Helium was used as carrier
gas (1 ml/min). The detector was operated in full scan mode
(m/z 50–650) and electron ionisation (EI) mode. For
additional analytical information on the fractions, analyses
were performed with negative chemical ionisation (NCI)
operated in full scan mode (m/z 50-650) and Selected Ion
Monitoring (SIM) for chlorine (m/z 35/37) and bromine
content (m/z 79/81) with the same column and column
temperature programme as described above.
Mass spectra were deconvoluted using the Automated
Mass Spectral Deconvolution and Identification System
(AMDIS version 2.64) and compared with reference spectra
in the National Institute of Standards and Technology
(NIST version 02) main mass spectral database (match
factor ≥80%) for tentative identification. The Kovats
Retention Indices (KRI) values were used to identify the
compounds using the Quality Peak Identification Database
(QPID) that was partly developed in the framework of the
MODELKEY project. This database stores unknowns after
attributing them a number, which are recognised peaks
according to a number of established GC/MS settings, e.g.
peak shape, purity of spectra, intensity check etc. These
unknowns will later be thoroughly evaluated within the
MODELKEYproject and comparisons on the emergence of
specific pollutants can be made between sample locations.
It is also possible to separate interesting compounds from
the trivial ones by using filters present in the QPID
software. In our study, the background contamination (i.e.
compounds present in analytical blank samples) as well as
compounds present in adjacent, non-active fractions was
subtracted from the active fraction result list. Only the
interesting compounds (i.e. present only in the active
fractions) and the number of unknowns are reported here.
Results
Anti-androgenicity
The AR antagonistic activity originally observed in the
toxicity screening of the river sediments performed within
MODELKEY was confirmed in the total extract, i.e. GPC1
(200 nmol FLU eq/g dw, Table 1). The AR antagonistic
potency following reversed phase fractionation was
110 nmol FLU eq/g dw in fraction 3 (RP3) and 25 nmol
FLU eq/g dw in fraction 4 (RP4; Fig. 4a). Summation of
activity shows that the potency was 30% lower than in the
original extract GPC1. Following normal phase sub-
fractionation, two groups of potent AR antagonistic fractions
were distinguished, i.e. normal phase sub-fractions 2 of
reversed-phase fractions 3 and 4 (RP3NP2 + RP4NP2) and
normal phase sub-fractions 5 and 6 of reversed-phase
fractions 3 (RP3NP5 + RP3NP6; Fig. 4a). A total AR
antagonistic activity of 59 nmol FLU eq/g dw was found by
summation of all normal phase sub-fractions together, i.e.
about 50% lower than in the original reversed-phase
fractions (Fig. 5a). When all normal phase sub-fractions
(NP1–8) of reversed-phase fraction RP3 were pooled into
the original reversed-phase constitution and re-tested in the
Fig. 4 Anti-androgenic (a) and androgenic (b) responses in AR
CALUX (nmol FLU eq/g dw and pmol DHT eq/g dw, respectively) in
reversed-phase fractions (RP1-5) and normal phase sub-fractions
(NP1-8)
1390J.M. Weiss et al.

Page 7

bioassay, the original AR antagonistic potency was
obtained again, i.e. 98 nmol FLU eq/g dw (Fig. 5a). The
same was observed following pooling of the normal phase
sub-fractions of reversed-phase fraction RP4 (34 nmol FLU
eq/g dw). The decrease in total AR antagonistic potency
after sub-fractionation was recovered, indicating either a
synergistic effect of all present compounds, or that the
bioassay is not sensitive enough to detect all AR antago-
nistic effects present in the separate normal phase sub-
fractions.
Androgenicity
Upon screening in the AR CALUX assay, no detectable
agonistic androgenic activity was present in the total
sediment sample extract, i.e. GPC1 (Table 1). After the
first fractionation on the reversed-phase column, however, a
significant AR agonistic activity was measured in reversed-
phase fractions 2 (RP2) and 3 (RP3), 890 and 440 pmol
DHT eq/g dw, respectively (Fig. 4b). Thus, reversed-phase
fraction 3 possesses an AR antagonistic as well as AR
agonistic potency. The observed AR agonistic potency of
RP2 was lower after normal phase sub-fractionation, and
the remaining activity was found in normal phase fraction 7
(RP2NP7, 420 pmol DHT eq/g dw). In normal phase sub-
fraction 7 of reversed-phase fraction 3 (RP3NP7), an AR
agonistic potency of 3,400 pmol DHT eq/g dw was
observed, which is almost eight times higher than in the
original fraction RP3 (Fig. 4b).
Following pooling of all normal phase sub-fractions of
reversed-phase fraction 3 into the original RP3 constitution
and re-tested in the bioassay, the original AR agonistic
potency of RP3 was interestingly obtained again (380 pmol
DHT eq/g dw) (Fig. 5b), suggesting that antagonistic
compounds present in the other normal phase sub-
fractions than the active RP3NP7 masked the agonistic
potency in RP3. The opposite effect was observed when
RP2 normal phase sub-fractions were pooled and re-tested,
the AR agonistic activity was increased to its original RP2
potency, showing that the lower potency found following
normal phase fractionation was not due to loss of
compounds. This might be due to either synergism between
compounds present in the RP2 fraction or low sensitivity of
the bioassay when analysing the normal phase sub-fractions
of RP2.
Chemical screening
In the normal phase sub-fractions active in the AR CALUX
bioassay, chemical screening revealed the tentative identity
of seventeen compounds (Table 2) with the GC/MS settings
applied in the AMDIS deconvolution procedure, and as
many as 71 unknown spectra were added to the QPID
database for further investigation (Table 3).
The two distinguished groups of sub-fractions with AR
antagonistic activity contained structurally different com-
pounds. The first group of fractions (RP3NP2 + RP4NP2),
eluting early on the normal phase column, contains non-
polar compounds. Many polycyclic aromatic hydrocarbons
(PAHs) were detected in RP3NP2 (Fig. 6a, Table 2) and
RP4NP2 (chromatogram not shown), together with 28
unknown peaks, i.e. peaks that fulfil the GC/MS setting
criteria and not present in non-active fractions or procedural
blank fractions (attributed peak-numbers in Table 3).
Moderately polar compounds are expected to be present
in the second group (RP3NP5 + RP3NP6). Apart from a
technical nonylphenol mixture, 4′-Propoxy-2-methylpropio-
phenone, 4-tert-pentylphenol, N-[4-(phenylamino)phenyl]-
benzamide, and dibutylphthalate (Table 2), 41 unknown
peaks (Table 3) were detected in the fractions RP3NP5
(Fig. 6b) and RP3NP6 (chromatogram not shown).
Tris(1-chloro-2-propyl) phosphate was the only tenta-
tively identified compound in the AR agonistic potent
fraction RP2NP7 (Table 2). Two distinct isomer clusters,
with GC-retention between 18–26 min and 40–52 min,
were detected in EI mode in both fractions RP2NP7
(Fig. 6c) and RP3NP7, but in the latter fraction, the cluster
was more intense (chromatogram not shown). The first
cluster was not detected in NCI mode but the second was.
The clusters did not contain any bromines or chlorines and
are yet of unknown identity. The AR agonistic potent
fractions contained only two unknown peaks (Table 3). The
low number of peaks may be an indication that GC/MS is
not the preferred method for analysis of the more polar
compounds, such as natural hormones.
Fig. 5 A comparison of bioassay results obtained in GPC fraction,
individual reversed-phase fractions (RP) and normal phase (NP) sub-
fractions compared to pooled NP fractions to illustrate the AR
antagonistic (a) and AR agonistic (b) potency recovery and
interactions
Masking effect of anti-androgens on androgenic activity 1391

Page 8

Discussion
Effect-directed analysis
This study shows that the application of EDA to a
contaminated sediment sample unravels the different con-
tributions to the observed biological activity, in our case in
the AR CALUX assay, and leads to a distinction of both
agonistic and antagonistic effects. This is an important step
towards the identification of potential endocrine disrupting
compounds that can interfere with the androgen hormone
system. The compounds identified here have been distin-
guished purely based on their activity in the bioassay and
not due to their presence on priority lists, production
volumes or prediction models, as is commonly the case.
EDA studies have earlier revealed that a majority of key
toxicants present in European water systems could not be
found on the list of priority pollutants [9]. EDA is therefore
a powerful tool to discover a wide range of biologically
active contaminants, including emerging pollutants as well
as ‘classical’ toxicants. The early detection of the presence
of key toxicants that may hamper the chemical and/or
ecological status enables the implementation of monitoring
campaigns for environmental pollutants that are not, or not
yet, routinely monitored.
In the sample investigated in this study, the AR agonistic
potency is clearly concealed by the effects of AR antagonists
in the total extract (GPC1), demonstrating that toxicity
screeningof totalextractsisnotenough toevaluatethe fullin
vitro endocrine disrupting potential of a complex chemical
mixture as found in the environment. The two-step serial
fractionations enable the separation of androgenic and anti-
Table 2 Tentatively identified compounds (match factor≥80% in NIST database) found in fractions with a specified activity (androgen receptor
agonistic or antagonistic), with the compound name, CAS number, Logarithm octanol–water coefficient (Log Kow)a, the structure of each
compound and the androgenic activity according to literature
receptor agonistic or antagonistic), with the compound name, CAS
Fraction AR activityNameCAS number Log Kow
Structure Literature
RP2NP7Agonistic Tris (1-chloro-2-propoyl) phosphate 13674-84-52.89
O
ClP
O
O
O
Cl
Cl
Anti-
androgenic
[38-41]
RP3NP2AntagonisticFluoranthene206-44-04.93Anti-
androgenic
[27]
RP3NP2 and
RP4NP2
AntagonisticBenz[a]anthracene56-55-3 5.52
Anti-
androgenic
[27]
RP3NP2 AntagonisticPyrene129-00-04.93 Not active
[27]
RP3NP2 AntagonisticPhenanthrene 85-01-84.35Not active
[27]
RP3NP2Antagonistic1,3-bis(1,1-Dimethylethyl)-benzene 1014-60-45.81
RP3NP2 Antagonistico-Terphenyl84-15-15.52
RP3NP2Antagonisticp-Terphenyl92-94-45.52
RP3NP2Antagonistic4-Methyl-phenanthrene832-64-4 4.89
RP3NP2Antagonistic1,4-bis(Phenylmethyl) benzene793-23-76.04
No data
available
No data
available
No data
available
No data
available
No data
available
Table 2 Tentatively identified compounds (match factor≥80% in
NIST database) found in fractions with a specified activity (androgen
number, Logarithm octanol–water coefficient (Log Kow)a, the structure
of each compound and the androgenic activity according to literature
1392J.M. Weiss et al.

Page 9

androgenic compounds into different sub-fractions. The
masking of the AR agonistic effect by antagonistic com-
pounds has been reported earlier; e.g. in sediment sample
from the river Lambro, Italy and in paper and pulp mill
effluents. In both cases, antagonistic but no agonistic AR
activity was observed in the total extracts, but AR agonistic
activity appeared following reversed phase fractionation
[13, 23]. In the latter report, it was suggested that the AR
agonistic activity could be attributed to natural compounds
originated from decaying wood [23].
Anti-androgenic activity
The anti-androgenic activity observed in the total extract
(GPC1) was detected in reversed-phase fractions RP3 and
RP4 (Table 1). From the reversed phase fractionation
procedure, it can be derived that the log octanol/water
coefficient (log Kow) of the AR antagonists present in RP3
and RP4 will be between 4–6 and 6–9 (± 0.5 according to
Schymanski et al. [24]), respectively, based on a tested
chemical mixture containing compounds with a wide range
of log Kows [19]. Normal phase sub-fractionation success-
fully separated two potent chemical groups from each other,
decreasing the complexity of the matrix and simplifying the
identification of compounds by chemical analysis. The first
anti-androgenic compound group consisted of lipophilic
aromatic compounds, such as PAHs (Table 2). PAHs are a
class of organic aromatic compounds comprising several
congeners that are ubiquitous in the environment and in
foodstuffs. They originate from the incomplete combustion
of fossil fuels, oil spills and industrial processes. PAHs are
reported at high concentrations in the marine environment
Table 3 The number of unknowns (n) in each androgenic and anti-androgenic active sub-fraction and the attributed number (#) given in the QPID
database
FractionActivityUnknowns (n) Unknown (#)
RP2NP7
RP3NP7
RP3NP2
RP4NP2
RP3NP5
RP3NP6
Total
Androgenic
Androgenic
Anti-androgenic
Anti-androgenic
Anti-androgenic
Anti-androgenic
1
1
61
2
65, 72, 74, 81, 85, 87, 95, 98, 107, 112-114, 117, 119-127
42, 50, 54, 63, 76, 84
1, 3-34
5, 14, 29, 34, 77, 97, 116, 118
22
6
33
8
71
RP3NP5Antagonistic4’Propoxy-2-methylpropiophenon 64436-60-83.65
O
O
RP3NP5Antagonistic4-tert-Pentylphenol 80-46-63.91
OH
Not active
[31]
RP3NP5AntagonisticN-[4-Phenylamino)phenyl]-benzamide Not available 4.00
N
H
N
H
O
RP3NP5 and RP3NP6 Antagonisticp-Nonylphenol (technicalmixture,8
identified peaks)
104-40-55.99
OH
Anti-
androgenic
[29]
RP3NP6 AntagonisticDibutylphthalate 84-74-24.61
O
O
O
O
Possible anti-
androgen
[30-33]
RP4NP2Antagonistic p-Dicyclohexylbenzene 1087-02-1 7.63
RP4NP2 Antagonistic 6-Phenyldodecane2719-62-27.87No data
available
No data
available
No data
available
No data
available
aEPI Suite calculated
Masking effect of anti-androgens on androgenic activity 1393

Page 10

(Σ 16 PAHs), e.g. up to 52 μg/g sed. in the Gulf of Gdansk
of the Baltic Sea [25] and 10 μg/g sed. in Lake Pilnok in the
Czech Republic [26]. Target analysis of PAHs in the
sediment sample investigated in this study resulted in
5 μg/g dw (not yet published). Some PAHs tentatively
identified in RP3NP2 and RP4NP2 are able to act as anti-
androgens (e.g. benzo[a]anthracene, and fluoranthene),
whereas others (e.g. pyrene and phenanthrene) are not able
to inhibit androgen receptor transactivation [27]. Fluoran-
thene was the most dominant PAH congener in the target
analysis, followed by pyrene. In petroleum-derived PAHs,
pyrene is more abundant than fluoranthene, but at higher
combustion temperatures a predominance of fluoranthene
over pyrene is characteristic. As such, a ratio value (flu/
pyr) greater than 1 is classically related to pyrogenic
sources, e.g. coal burning, wood burning, vehicular
exhaust emission, waste crankcase oil and asphalt roofing
material [28].
In the second, more polar group of anti-androgenic sub-
fractions (RP3NP5 + RP3NP6), a technical mixture of
nonylphenol could be tentatively identified (Table 2). Non-
ylphenol has been identified in estuaries in the UK [12],
and has been reported to act as a potent AR antagonist in a
yeast-based bioassay [29].
Fig. 6 Total ion chromatogram
(TIC) GC/MS chromatogram
with electron ionisation (EI) of
the anti-androgenic potent frac-
tion RP3NP2 (a), the anti-
androgenic potent fraction
RP3NP5 (b) and the androgenic
potent fraction RP2NP7 (c)
1394J.M. Weiss et al.

Page 11

The observations regarding anti-androgenic activity of
dibutylphthalate (DBP), tentatively identified in RP3NP6,
is not consistent in literature. Phthalates, such as DBP, have
been demonstrated to be an environmental anti-androgen in
vivo that disrupts androgen-regulated rat male sexual
differentiation via a non-receptor mediated mechanism
[30]. Although, DBP has been reported to be unable to
exhibit an anti-androgenic potency in a cell-based (CHO
K1 cells) in vitro bioassay at a maximum concentration of
10 μM [31], another study with the same cell line reported
anti-androgenic potency at 4.8 μM [32]. Additionally, an in
vitro receptor binding assay reported weak binding to the
androgen receptor with an IC50of 27.5 μM [33]. It can be
concluded that phthalates exhibit anti-androgenic activity
but with which mechanism and to which extent is still
unclear.
No information was available regarding the (anti-)
androgenic potency of the other tentatively identified
compounds.
Androgenic activity
The AR agonistic potency masked by AR antagonists in the
total extract (GPC1) was successfully revealed by the two-
step serial fractionations. The identification of active
fractions simplifies the chemical analytical identification
of potent AR agonistic compounds. The predicted log Kows
of the AR agonists present in RP2 and RP3 will be between
2–4 and 4–6, respectively. It can also be expected that
compounds eluting late in the normal phase sub-
fractionation are rather polar. Despite the well reported
potencies of steroid androgens, i.e. the natural hormone
testosterone and its natural and synthetic derivatives [11,
34], there are no data available on exogenous pollutants
being potent in vitro androgen receptor agonists. Out of 202
natural, synthetic and environmental chemicals evaluated in
an extensive in vitro androgen receptor binding study, only
14 of the tested compounds exhibited strong binding to the
AR, all of which were steroids [33]. The log Kows of these
steroids fit the expected log Kowof the AR agonistic potent
fractions (log Kow2-6).
A large amount of anabolic steroid hormones are
synthesised and sold to be potent androgens in human and
animal treatments [5–8]. The estimated yearly excretion of
androgens by farm animals was 7 tons in the European
Union in the year 2000 [35]. The environmental fate of
steroids originating from livestock excreta seems to be
strongly influenced by storage conditions and also by the
soil type of the fields where the dung is spread [35].
Synthetic and natural androgens from human activity have
been reported to be stable enough to be present in the
effluent of waste water treatment plants although the
majority of them are successfully removed [11, 36, 37].
DHT, androstenedione, androstanedione, 5β-androstane-
3α,11β-diol-17-one, androsterone and epi-androsterone
were identified in sewage treatment works effluents in the
UK and held responsible for 99% of the in vitro androgenic
activity determined [11]. In the present study, the only
tentatively identified compound in the androgenic fraction
was an organophosphate used as flame retardant (Table 2),
but organophosphates have been reported to be strong
androgenic antagonists [38–41]. More interesting are the
two unidentified clusters of non-halogen (chlorine or
bromine) compounds (Fig. 6c). The intensity of the clusters
increases from RP2NP7 to RP3NP7, corresponding with
the higher androgenic potency of the latter fraction
(Table 1), assuming that the ionisation efficiency of the
individual compounds is the same.
According to the fractionation scheme published by
Houtman et al. [15], estrogens are expected to elute in
reversed-phase fraction 2. Many of the known-to-be-present
estrogens can act as AR antagonists [42], although in this
study, no anti-androgenic activity was detected in RP2, only
agonistic androgenicity. Androgens and estrogens are
similar in structure and expected to have comparable log
Kow, and hence elute in the same fractions. It may therefore
be possible that estrogens present in the AR agonistic
fraction RP2NP7 still mask its full in vitro androgenic
potency. GC/MS techniques are not optimal for the analysis
of estrogens and androgens due to their high polarity,
although it is possible after derivatisation of the fractions,
and a careful choice of analytical GC column [17].
Interactions between AR agonists and antagonists
The AR antagonistic potency was lower after fractionation,
from GPC1 (200 nmol FLU/g dw, to RP3 and RP4
(130 nmol FLU/g dw together) and finally following
normal phase fractionation in RP3NP2, RP3NP5, RP3NP6
and RP4NP2 (59 nmol FLU/g dw together; Fig. 5a). Due to
the additional confirmation step where the normal phase
fractions were pooled and re-tested in the bioassay
(130 nmol FLU/g dw) it can be concluded that the
analytical recovery of the active compounds following the
two-step serial fractionations is at least 65% (from total
activity 200 nmol FLU/g dw to 130 nmol FLU/g dw). Also
the agonistic androgenic activity in reversed-phase fraction
(1,330 pmol DHT/g dw) was recovered by pooling the
normal phase sub-fractions (1,550 pmol DHT/g dw),
illustrating that no compounds were lost during normal
phase sub-fractionation (Fig. 5b). Unfortunately, it was not
possible to pool all reversed-phase fractions into their
original total extract constitution (GPC1) and to re-test it in
the bioassay, due to the lack of any remaining extract.
Reversed-phase fraction RP3 was able to both induce and
inhibittheARactivity.Theobservedeffectcouldbeexplained
Masking effect of anti-androgens on androgenic activity1395

Page 12

by the knowledge of the so-called partial agonists. These
compounds (e.g. alkylphenols) have an anti-androgenic effect
despite the fact that they can bind and activate the AR. The
partial AR agonists, however, cause a much lower maximum
response than true AR agonistic compounds, even when all
receptors are occupied.Inthe presenceoftrueagonists, partial
agonists therefore inhibit the effect of the true agonists and
thus act as anti-androgens [21, 43]. The separation of partial
AR agonists present in fraction RP3NP7 from AR antago-
nistic fractions (RP3NP2, RP3NP5, RP3NP6) can be the
explanation of the lower total AR antagonistic potency of the
normal phase sub-fractions (ΣRP3NP1–847 nmol FLU/g dw,
Table 1) compared to the original AR antagonistic potency of
the reversed-phase fraction RP3 (110 nmol FLU/g dw,
Fig. 5a and b).
Concluding remarks
Through the use of EDA fractionation strategies, it was
demonstrated that androgenic and anti-androgenic com-
pounds present in a sediment sample influence each other’s
interaction with the androgen receptor. The AR agonistic
effect appeared following both reversed and normal phase
sub-fractionation, but the potency was masked again after
pooling the normal phase sub-fractions.
As literature has indicated, it is likely that the PAHs,
nonylphenol and possibly DBP are contributing to the anti-
androgenic potency observed in the river sediment sample
analysed in this study. With the GC/MS screening method
applied here, no compounds with AR agonistic disrupting
potenciescouldbe(tentatively)identified,suggestingthatLC/
MSmaybeabetterchoiceforidentificationofthecompounds
responsible for the observed AR agonistic activity.
The power of EDA lies in its suitability to reveal the
emergence of novel environmental pollutants, including the
unravelling of agonistic and antagonistic effects in the same
sample, but there are several aspects inherent to the strategy
that need to be addressed.
Generally, in EDA studies, no internal standards or
reference compounds are added to the sample because of
the possibility that these may disturb the bioassay results.
Hence, it is not possible to accurately calculate concen-
trations of tentatively identified compounds in the active
fractions. Instead, it is advisable to carry out target analysis
of the identified compounds in remaining sample(s) from
the same locations.
In EDA studies, exhaustive extraction is usually applied to
the sediment samples in order to obtain information on all the
compounds present, ignoring the fact that under normal
conditions certain compounds may be more bioavailable than
others. In the case of the presence of both agonistic and
antagonistic compounds (in a given assay), the assessment of
the biological availability of the identified compounds in the
original sample is an important aspect.
There is a need to fully investigate the environmental
behaviour (bioavailability, persistency, distribution, metab-
olism, etc.) of the identified potent compounds, so that the
assessment of the environmental risk is firmly based on
relevant data. As a first step, the environmental behaviour
of the identified compounds can to some extent be derived
from the fractionation schemes, where the distribution/
elution of compounds depends on the physico-chemical
properties, e.g. molecular weight, lipophilicity and polarity.
Future efforts will be put into identification of the
unknown active compounds (n=71, Table 3) in these
fractions. Other sophisticated analytical techniques (e.g.
accurate mass LC/MS) will be used to target complemen-
tary groups of compounds, i.e. less volatile and more polar
compounds such as the natural hormones and pharmaceut-
icals. A summary of all toxicological and chemical analyses
on the river samples will be published within the project
MODELKEY (www.modelkey.org).
Acknowledgement
The Netherlands), Eric de Deckere (University of Antwerp, Belgium),
Sander van Vliet (National Institute for Coastal and Marine Manage-
ment, The Netherlands) and Chris van Liefferinge (University of
Antwerp, Belgium) for collecting the samples, Peter Cenijn (IVM, VU
University, The Netherlands) for technical assistance and Jan Balaam
(CEFAS,UnitedKingdom)foradditional analyses.Thisworkwas done
in the framework of the EU-supported programme MODELKEY
(Contract-No. 511237 (GOCE)) and the Marie Curie Research Training
Network KEYBIOEFFECTS (MRTN-CT-2006-035695).
WethankBertvan Hattum (IVM, VUUniversity,
Open Access
tive Commons Attribution Noncommercial License which permits any
noncommercial use, distribution, and reproduction in any medium,
provided the original author(s) and source are credited.
This article is distributed under the terms of the Crea-
References
1. Hotchkiss AK, Rider CV, Blystone CR, Wilson VS, Hartig PC,
Ankley GT, Foster PM, Gray CL, Gray LE (2008) Toxicol. Sci
105:235–259
2. WHO (2002) Global assessment of the state-of-the-science of
endocrine disruptors. International Programme on Chemical
Safety, Geneva, Switzerland
3. Mills LJ, Chichester C (2005) Sci Total Environ 343:1–34
4. Ankley G, Mihaich E, Stahl R, Tillit D, Colborn T, McMaster S,
Miller R, Bantle J, Cambell P, Denslow N, Dickerson R, Folmar
L, Fry M, Giesy J, Gray LE, Guiney P, Hutchinson T, Kennedy S,
Kramer V, LeBlanc G, Mayes M, Nimrod A, Patino R, Peterson
R, Purdy R, Ringer R, Thomas P, Touart L, van der Kraak G,
Zacharewski T (1998) Environ Toxicol Chem 17:68–87
5. Yu NH, Ho ENM, Leung DKK, Wan TSM (2005) J. Pharm.
Biomed. Anal 37:1031–1038
6. Wood RI (2008) Front. Neuroendocrinol 29:490–506
7. Meyer HHD (2001) Acta Pathol. Microbiol. Immunol. Scand
109:1–8
8. Gooren LJG, Bunck MCM (2004) Drugs 64:1861–1891
9. Brack W (2003) Anal Bioanal Chem 377:397–407
1396J.M. Weiss et al.

Page 13

10. Grung M, Lichtenthaler R, Ahel M, Tollefsen K-E, Langford K,
Thomas KV (2007) Chemosphere 67:108–120
11. Thomas KV, Hurst MR, Matthiessen P, McHugh M, Smith A,
Waldock MJ (2002) Environ Toxicol Chem 21:1456–1461
12. Thomas KV, Thain JE, Waldock MJ (1999) Environ Sci Technol
18:401–411
13. Urbatzka R, Van Cauwenberge A, Maggioni S, Viganò L,
Mandich A, Benfanati E, Lutz I, Kloas W (2007) Chemosphere
67:1080–1087
14. Houtman CJ, Cenijn PH, Hamers T, Lamoree MH, Legler J, Murk
AJ, Brouwer A (2004) Environ Toxicol Chem 23:32–40
15. Houtman CJ, Booij P, Jover E, del Rio Pascual D, Swart K, van
Velzen M, Vreuls R, Legler J, Brouwer A, Lamoree MH (2006)
Chemosphere 65:2244–2252
16. Grote M, Brack W, Altenburger R (2005) Environ Toxicol
20:475–486
17. Houtman CJ, van Oostveen AM, Brouwer A, Lamoree MH,
Legler J (2004) Environ Sci Technol 38:6415–6423
18. Seiler T-B, Schulze T, Hollert H (2008) Anal Bioanal Chem
390:1975–1985
19. Houtman CJ. Tracing endocrine disruptors—identification and
effects of endocrine disrupting compounds in the aquatic
environment. Thesis. 2007. Institute for Environmental Studies
(IVM), VU University, Amsterdam, The Netherlands.
20. Fernandez P, Bayona JM (1992) J Chromatogr 625:141–149
21. Sonneveld E, Jansen HJ, Riteco JAC, Brouwer A, Van der Burg B
(2005) Toxicol Sci 83:136–148
22. Brack W, Schmitt-Jansen M, Machala M, Brix R, Barceló D,
Schymanski E, Streck G, Schulze T (2008) Anal Bioanal Chem
390:1959–1973
23. Svenson A, Allard A-S (2004) Environ Toxicol 19:510–517
24. Schymanski EL, Meinert C, Meringer M, Brack W ((2008) Anal
Chim Acta 615:136–147
25. Falandysz J, Albanis T, Bachmann J, Bettinetti R, Bochentin I,
Boti V, Bristeau S, Daehne B, Dagnac T, Galassi S, Jeannot R,
Oehlmann J, Orlikowska A, Sakkas V, Szczerski R, Valsamaki V,
Schulte-Oehlmann U (2006) J Environ Sci Health Part A
41:2127–2162
26. Mazurová E, Hilscherová K, Jálová V, Köhler HR, Triebskorn R,
Giesy JP, Bláha L (2008) Aquat Toxicol 89:172–179
27. Vinggaard AM, Hnida C, Larsen JC (2000) Toxicol 145:173–183
28. Sanders M, Sivertsen S, Scott G (2002) Arch Environ Contam
Toxicol 43:438–448
29. Lee HJ, Chattopadhyay S, Gong E-Y, Ahn RS, Lee K (2003)
Toxicol Sci 75:40–46
30. Mylchreest E, Sar M, Cattley RC, Foster PMD (1999) Toxicol
Appl Pharmacol 156:81–95
31. Roy P, Salminen H, Koskimies P, Simola J, Smeds A, Saukko P,
Huhtaniemi IT (2004) J Steroid Biochem Mol Biol 88:157–166
32. Takeuchi S, Iida M, Kobayashi S, Jin K, Matsuda T, Kojima H
(2005) Toxicol 210:223–233
33. Fang H, Tong W, Branham WS, Moland CL, Dial SL, Hong H,
Xie Q, Perkins R, Owens W, Sheehan DM (2003) Chem Res
Toxicol 16:1338–1358
34. Sonneveld E, Riteco JAC, Jansen HJ, Pieterse B, Brouwer A,
Schoonen WG, Van der Burg B (2006) Toxicol Sci 89:173–187
35. Lange IG, Daxenberger A, Schiffer B, Witters H, Ibaretta D,
Meyer HHD (2002) Anal Chim Acta 473:27–37
36. Kolodziej EP, Gray JL, Sedlak DL (2003) Environ Toxicol Chem
22:2622–2629
37. Kirk LA, Tyler CR, Lye CM, Sumpter JP (2002) Environ Toxicol
Chem 21:972–979
38. Andersen HR, Vinggaard AM, Rasmussen TH, Gjermandsen IM,
Bonefeld-Jørgensen EC (2002) Toxicol Appl Pharmacol 179:1–12
39. Kitamura S, Suzuki T, Ohta S, Fujimoto N (2003) Environ Health
Perspect 111:503–508
40. Tamura H, Maness SC, Reischmann K, Dorman DC, Gray E,
Gaido KW (2001) Toxicol Sci 60:56–62
41. Xu L-C, Liu L, Ren X-M, Zhang M-R, Cong N, Xu A-Q, Shao J-
H (2008) Toxicol 243:59–65
42. Sohoni P, Sumpter JP (1998) J Endocrin 158:327–339
43. Singh SM, Gauthier S, Labrie F (2000) Curr Med Chem 7:211–247
Masking effect of anti-androgens on androgenic activity 1397