Plastic components affect the activation of the aryl hydrocarbon and the androgen receptor.

Abstract

Phenols and plasticizers are widely used in the plastic industry, in food packaging and to impart softness and flexibility to normally rigid plastic medical devices and children's toys. The effects on the aryl hydrocarbon receptor (AhR) and the androgen receptor (AR) were assessed using luciferase reporter gene assays of the following compounds: bisphenol A (BPA), 4-n-nonylphenol (nNP), 4-tert-octylphenol (tOP), bis(2-ethylhexyl) phthalate (DEHP), di-isononyl phthalate (DINP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DNOP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), 4-chloro-3-methylphenol (CMP), 2-phenylphenol (2-PP), 2,4-dichlorophenol (DCP), resorcinol and bis(2-ethylhexyl) adipate (DEHA). Furthermore, a mixture of selected compounds was tested at the no-observed-effect concentration (NOEC), the low-observed-effect concentration (LOEC) and the half-maximum-effect/inhibitory concentration (EC50/IC50) of the single chemicals. Both receptors were affected by BPA, nNP, BBP, CMP, DCP and resorcinol whereas DEHP, DIDP and DBP affected only the AhR and tOP and 2-PP antagonised the AR activity. The mixture was composed of 6 compounds, of which one compound weakly induced the AhR but all compounds antagonized the AR activation. Using the concentration addition principle additive effects were observed at the NOEC, LOEC and EC50/IC50 for both receptors. Our in vitro data suggest that the effect of a mixture depends on the concentration, character, potency and composition of the single mixture compounds and that also the combined effects of the compounds should be taken into consideration for risk assessment of human health.

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Available online at www.sciencedirect.com
Toxicology 246 (2008) 112–123
Plastic components affect the activation of the aryl
hydrocarbon and the androgen receptor
Tanja Kr¨ uger, Manhai Long, Eva C. Bonefeld-Jørgensen∗
Unit of Cellular and Molecular Toxicology (CMT), Institute of Public Health, University of Aarhus, Denmark
Received 12 November 2007; received in revised form 20 December 2007; accepted 29 December 2007
Available online 10 January 2008
Abstract
Phenols and plasticizers are widely used in the plastic industry, in food packaging and to impart softness and flexibility to normally rigid plastic
medical devices and children’s toys. The effects on the aryl hydrocarbon receptor (AhR) and the androgen receptor (AR) were assessed using
luciferasereportergeneassaysofthefollowingcompounds:bisphenolA(BPA),4-n-nonylphenol(nNP),4-tert-octylphenol(tOP),bis(2-ethylhexyl)
phthalate (DEHP), di-isononyl phthalate (DINP), diisodecyl phthalate (DIDP), di-n-octyl phthalate (DNOP), dibutyl phthalate (DBP), benzyl butyl
phthalate (BBP), 4-chloro-3-methylphenol (CMP), 2-phenylphenol (2-PP), 2,4-dichlorophenol (DCP), resorcinol and bis(2-ethylhexyl) adipate
(DEHA). Furthermore, a mixture of selected compounds was tested at the no-observed-effect concentration (NOEC), the low-observed-effect
concentration (LOEC) and the half-maximum-effect/inhibitory concentration (EC50/IC50) of the single chemicals.
Both receptors were affected by BPA, nNP, BBP, CMP, DCP and resorcinol whereas DEHP, DIDP and DBP affected only the AhR and tOP and
2-PP antagonised the AR activity.
Themixturewascomposedof6compounds,ofwhichonecompoundweaklyinducedtheAhRbutallcompoundsantagonizedtheARactivation.
Using the concentration addition principle additive effects were observed at the NOEC, LOEC and EC50/IC50for both receptors.
Our in vitro data suggest that the effect of a mixture depends on the concentration, character, potency and composition of the single mixture
compounds and that also the combined effects of the compounds should be taken into consideration for risk assessment of human health.
© 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aryl hydrocarbon receptor; Androgen receptor; Mixtures; Phenols; Phthalates; Reporter gene assay
1. Introduction
Numerous man-made chemicals are produced and released
into the environment through different routes and a number
have been shown to disturb the endocrine system by mim-
icking, enhancing or antagonizing the endogenous hormones.
Theyarenamedendocrinedisruptingchemicals(EDCs)(Bigsby
et al., 1999; Bonefeld-Jorgensen and Ayotte, 2003). Plastic
components, such as phthalates, alkylphenols and adipates, are
synthesized chemicals widely used in industry and day-life.
The plastic components have been found in water (Brossa et
al., 2005; Kuch and Ballschmiter, 2001), foods (Petersen and
Breindahl, 2000; Sharman et al., 1994), indoor air (Fromme
et al., 2004; Rudel et al., 2003) and house dust (Bornehag
∗Correspondingauthorat:UnitofCellularandMolecularToxicology(CMT),
InstituteofPublicHealth,UniversityofAarhus,VennelystBoulevard6,Building
260, DK-8000 Aarhus C, Denmark. Tel.: +45 8942 6162; fax: +45 8942 6199.
E-mail address: ebj@mil.au.dk (E.C. Bonefeld-Jørgensen).
et al., 2005, 2004; Fromme et al., 2004; Rudel et al., 2003).
Thus, humans are exposed to these components on a daily basis
and bisphenol A (BPA) and 4-n-nonylphenol (nNP) have been
detected in human urine samples at 1.28?g/l (median) and
<0.1?g/l (median), respectively (Calafat et al., 2005). Phtha-
late metabolites are found in the body of more than 75% of
subjects sampled in the U.S. (Silva et al., 2005) and have been
detected at median values ranging from 12.7?g/l for benzyl
butyl phthalate (BBP) to 91.8?g/l for dibutyl phthalate (DBP)
inadulthumanurinesamplesandtwotofourtimeshigherlevels
in the urine of children (Koch et al., 2004, 2005). Furthermore,
inbloodofnewborninfantsaftertransfusion,theconcentrations
of bis(2-ethylhexyl) phthalate (DEHP) were found between 3.4
and 21.6?g/ml (Plonait et al., 1993).
High molecular weight phthalates like DEHP, di-isononyl
phthalate (DINP), diisodecyl phthalate (DIDP) and di-n-octyl
phthalate (DNOP) are used in the manufacture of flexible vinyl
used in floor and wall covering, food packaging, blood storage
bags, medical devices and children’s toys (reviewed in (Hauser
0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.tox.2007.12.028

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T. Kr¨ uger et al. / Toxicology 246 (2008) 112–123
113
and Calafat, 2005)). The low molecular weight phthalates like
BBPandDBPareusedinpersonal-careproducts(perfumes,cos-
metics,lotions,nailpolish)andinlacquersandvarnishes.BPAis
used in polycarbonate plastic and the BPA-based polycarbonate
is among other used as a coating in metal cans and food con-
tainers (Braunrath et al., 2005), baby bottles (Brede et al., 2003)
and dental sealants (Arenholt-Bindslev et al., 1999; Olea et al.,
1996). Alkylphenols such as nNP and 4-tert-octylphenol (tOP)
are mainly used in the production of alkylphenol polyethoxy-
lates. They are non-ionic surfactants often added to soaps,
cosmetics,paints,herbicidesandpesticideformulationsandalso
asadditivesinplastic(reviewedin(Yingetal.,2002)).4-Chloro-
3-methylphenol (CMP) and resorcinol are used as antiseptics,
2-phenylphenol (2-PP) is a fungicide used for waxing citrus
fruits and 2,4-dichlorophenol (DCP) is primarily used to pro-
duce the herbicide 2,4-dichloropheoxyacetic acid (Kim et al.,
2005). Finally, bis(2-ethylhexyl) adipate (DEHA) is an alter-
native compound for phthalates in flexible polyvinyl chloride
(PVC) products.
The aryl hydrocarbon receptor (AhR) is a ligand-dependent
transcription factor present in almost every tissue of animal
and humans. It mediates many of the toxic and biologic effects
of halogenated aromatic hydrocarbons (HAHs) such as poly-
chlorinated dibenzo-p-dioxins/furans (PCDDs/PCDFs), non-
or mono-ortho polychlorinated biphenyls (PCBs), as well as
numerouspolycyclicaromatichydrocarbons(PAHs)andrelated
chemicals (Denison and Nagy, 2003). However, recent stud-
ies have revealed that the AhR can be activated by numerous
chemicals with little structural similarity and physiochemical
characteristics distinct from classical AhR agonists (Denison
and Nagy, 2003; Long et al., 2003). Moreover, cross-talk exist
between the estrogen receptor (ER), androgen receptor (AR),
AhR and other nuclear receptors (Pocar et al., 2005). To our
knowledge, little is known about the effects of plastic compo-
nents on the AhR function.
The AR is the key regulatory element of the androgen cell
signalling and essential for the male reproductive function and
development, including spermatogenesis. In vitro effects on the
AR for several plastic components have been reported (Araki
et al., 2005; Bonefeld-Jørgensen et al., 2007; Lee et al., 2003;
Roy et al., 2004; Sohoni and Sumpter, 1998; Xu et al., 2005;
Stroheker et al., 2005; Takeuchi et al., 2005) but phenols such
as 2-PP, CMP and resorcinol as well as the plasticizers DNOP
and DEHA was not previously investigated.
The present study was a part of the EU supported research
project ENDOMET with the objectives to determine effects of
plasticcomponentson:alterationinsteroidhormoneproduction
(Mlynarcikovaetal.,2005);thesulphatesupplyenzymes(Turan
etal.,2005);estrogensulphotransferase(Harrisetal.,2005);the
thyroid hormone (Ghisari and Bonefeld-Jorgensen, 2005); tran-
scriptional activity of the sodium/iodide symporter (Breous et
al., 2005) as well as on the activity of the ER, AR, AhR and the
aromatase enzyme. The ENDOMET data is expected to provide
a range of biomarker tests to predict the potential of EDCs in
humans. Studies of some estrogenic and anti-androgenic chem-
icals in vitro as well as in vivo (Birkhoj et al., 2004; Nellemann
et al., 2003; Payne et al., 2000, 2001; Rajapakse et al., 2002)
have shown that the combined effects of single compounds of
low potency cannot be ignored. Therefore, in the ENDOMET
research project selected compounds, showing effects on
the majority of the test systems, were chosen for mixture
analysis.
TheaimofthisstudywastodeterminetheeffectofBPA,nNP,
tOP, DEHP, DINP, DIDP, DNOP, DBP, BBP, CMP, 2-PP, DCP,
resorcinol and DEHA, alone or in mixtures of selected com-
pounds,ontheAhRandARfunctionusingchemicallyactivated
luciferase gene expression (CALUX) bioassay in recombinant
mouse Hepa1.12cR cells (AhR-CALUX) and in transient trans-
fected Chinese Hamster Ovary (CHO-K1) cells (AR-CALUX).
2. Materials and methods
2.1. Chemicals
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, 98%) was obtained from
Cambridge by Bie and Berntsen and used as dose-response control in the
AhR-CALUX assay. The synthetic AR agonist methyltrienolone (R1881)
(PerkinElmer, Hvidover, DK) and the AR antagonist hydroxyflutamide (HF)
(Mikromol Gmbh, Luckenwalde, Germany) were used as dose–response con-
trolsineachAR-CALUXassay.Allthetestedplasticcomponents(Table1)were
provided by the coordinator of ENDOMET, Dr. Rosemary H. Waring (Birming-
ham, England), to all partners of the ENDOMET project to guarantee identical
batches. The purity of test compounds was above 97%. Most compounds were
harmful and appropriate personal protective methods and materials were used
throughout all experiments and all compounds were handled avoiding of light.
2.2. AhR-CALUX assay
The mouse hepatoma Hepa1.12cR cells stably transfected with the
PAH/HAH-inducibleluciferaseexpressionvectorpGudLuc1.1(kindlyprovided
byM.S.Denison(UniversityofCalifornia,Davis,CA,USA))respondinatime-
, dose-, and AhR-dependent manner (Garrison et al., 1996) and were used for
determination of effects on the AhR activity as described (Long et al., 2006).
For each compound, a stock solution of 1×10−2M dissolved in dimethyl
sulfoxide (DMSO) (BDH Laboratory Supplies, pool, UK) was prepared. The
same stock solution was used for all the assays and tested in the concentration
range from 1×10−10to 1×10−4M. The plastic components were tested alone
or in co-treatment with 60pM TCDD (TCDD−EC50). The solvent (DMSO)
did not exceed 1% (v/v) and did not affect cell viability.
The average intra coefficient of variation (CV) and inter CV of the solvent
controls (1% DMSO±60pM TCDD) or the positive controls (TCDD−EC50)
were below 8% and 21%, respectively.
2.3. AR-CALUX assay
The AR transactivity was determined in the CHO-K1 cells by transient
co-transfection with the MMTV-LUC reporter vector (kindly provided by Dr.
Ronald M. Evans, Howard Huges Medical Institute, CA) and the AR expression
plasmidpSVAR0(kindlyprovidedbyDr.A.O.Brinkmann,ErasmusUniversity,
Rotterdam,NL).Thereportergeneassaywasperformedasdescribed(Andersen
et al., 2002) with minor modifications. The transfection was carried out with
150ng DNA, with a pSVAR0:MMTV-LUC ratio of 1:100, and the test com-
pounds were added to the cells in triplicate without removal of the transfection
reagent.
Stock solutions of each compound were prepared in DMSO at a concen-
tration of 5×10−2M, stored at room temperature and diluted in culture media
immediately before use. For determination of the agonistic/antagonistic effects
of the test compounds a concentration range from 1×10−10to 1×10−4M
with or without 25pM R1881 (R1881−EC50) was prepared. The final DMSO
concentrations in the culture medium did not exceed 0.2%, not affecting cell
viability.

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T. Kr¨ uger et al. / Toxicology 246 (2008) 112–123
Table 1
Information of the tested compounds
PhenolsStructure CAS. no. CompanyPlasticizersStructure CAS. no. Company
Bisphenol A (BPA)
80-05-7 Sigma–Aldrich
(UK)
Dibutyl phthalate
(DBP)
84-74-2 Aldrich
(Germany)
4-n-Nonyl-phenol
(nNP)
104-40-5 Lancaster Synthesis
Ltd (UK)
Benzyl butyl
phthalate (BBP)
85-68-7Aldrich
(Germany)
4-tert-Octyl phenol
(tOP)
140-66-9Aldrich (Germany) Bis (2-ethylhexyl)
phthalate (DEHP)
117-81-7Lancaster
Synthesis Ltd
(UK)
2-Phenyl-phenol
(2-PP)
90-43-7Aldrich (Germany)Diisodecyl
phthalate (DIDP)
26761-40-0Fluka
(Germany)
4-Chloro-3-methyl-
phenol
(CMP)
59-50-7Sigma–Aldirch
(Germany)
Diisononyl
phthalate (DINP)
68515-48-0Aldrich
(Germany)

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T. Kr¨ uger et al. / Toxicology 246 (2008) 112–123
115
Table 1 (Continued)
Phenols
Structure
CAS. no.
Company
Plasticizers
Structure
CAS. no.
Company
2,4-Dichloro-
phenol
(DCP)
120-83-2
Lancaster Synthesis
Ltd (UK)
Dioctyl phthalate
(DNOP)
117-84-0
Fluka
(Germany)
Resorcinol
108-46-3
Riedel-de Ha¨ en
Fine Chemicals
(Germany)
Bis (2-ethylhexyl)
adipate (DEHA)
10-23-1
Lancaster
Synthesis Ltd
(UK)
The average intra CV and inter CV of the solvent controls (0.2%
DMSO±25pM R1881) or the positive controls (R1881−EC50) were below
11% and 25%, respectively.
Togetherwithco-workerswepreviouslyreportedtheARantagonisticeffects
of BPA and nNP (Bonefeld-Jørgensen et al., 2007) using the same reporter gene
assay but carried out in another laboratory. Since BPA and nNP are included
in the mixture analyses we tested these compounds again in our laboratory to
confirm previously obtained results.
2.4. Luciferase activity measurement
The cells were lysed followed by luciferase activity and protein content
measurementandtheactivitywasexpressedasrelativelightunitspermicrogram
cell protein (RLU/?g protein) as described (Bonefeld-Jorgensen et al., 2005).
2.5. Cytotoxicity
Parallel to the AhR- and AR-CALUX assays the single compounds and
mixturesweretestedforcytotoxicityusingtheprotocoloftheCellTiter96assay
from Promega (Madison, WI) and the Cytotoxicity detection kit (LDH) from
Roche (Hvidover, DK) as described (Bonefeld-Jorgensen et al., 2005; Long et
al., 2003).
2.6. Statistical analysis
Each compound was tested in the range of concentrations in at least three
independent experiments. Within each experiment each concentration was run
in triplet or quadruplicate and if one of the values deviated more than 30%
from the other values, the mean was calculated from the other wells only. The
statistical analyses were performed on the mean values from the independent
experiment. However, BPA and nNP were tested in two independent experi-
ments to confirm previously obtained results (Bonefeld-Jørgensen et al., 2007)
andthestatisticalanalysiswasperformedontheobtainedvaluesateachconcen-
tration from the two independent experiments. For each compound, only results
obtained at non-cytotoxic concentrations were included in statistical analysis.
Due to inequality of variance and relatively few data points per concentration,
nonparametricstatisticswasused.TheKruskal–Wallistestwasusedtocompare
differencesbetweenconcentrationsandtheJonckheere–Tepstratest(twotailed)
was used to analyze for a linear trend between concentration and response. If
one or both tests showed a significant difference (p<0.05), the Mann–Whitney
test was used to compare each concentration with the control.
2.7. EC50/IC50calculations
In AhR-CALUX, the EC50was calculated by fitting dose-response data to
a three- parameter sigmodal Hill curve using Sigma Plot (SPSS, Chicago, IL,
USA) as described (Long et al., 2006; Long et al., 2003).
In AR-CALUX, concentration–response relationships were plotted using
Sigma Plot and fitted to the sigmoidal Chapman, 4 parameters equation which
was found to yield the best fit for the data analysis.
2.8. Methods for evaluation of mixture effects
SinceBPA,nNP,BBP,CMP,resorcinolandtOPshowedeffectsonthemajor-
ity of the test parameters in the ENDOMET project they were recommended by
the ENDOMET team as the components of mixture analyses. They were mixed
at their respective no-observed-effect concentration (NOEC), low-observed-
effectconcentration(LOEC)andhalf-maximum-effect/inhibitoryconcentration
(EC50/IC50), respectively.
By applying the principle of concentration addition (Payne et al., 2001), the
concentration of the compounds in a mixture, at an observed mixture effect, can
be predicted using the concentration–response data for each compound alone.
Knowledge of the ratio of the compounds in the mixture is a prerequisite for
using this method.

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The following expression is given for every effect level, Emix, assuming
additive mixture effects:
??Ci-mix
where Ci-mixis the concentration of the compound i in the mixture causing the
effect Emix. Ciis the concentration of i alone causing the same Emix. For exam-
ple a mixture of the compounds at their NOEC (CBPA-NOEC-mix, CnNP-NOEC-mix,
CBBP-NOEC-mix, CCMP-NOEC-mix, CRes-NOEC-mix and CtOP-NOEC-mix) gives the
effect ENOEC-mix. From the dose response curves for the single compounds the
concentration of the single compounds at this ENOEC-mixis determined, i.e. the
concentration of BPA at ENOEC-mixis determined to be CBPA.
Underassumptionofadditivity,concentration–responserelationshipsforthe
mixture can be predicted using knowledge from concentration-response curves
of the individual compounds and the observed effects of the mixture. The pre-
dicted mixture effect concentration was calculated by redefining Eq. (1) and
replacing Ci-mix=pi×Cmixto obtain:
???pi
where picorresponds to the ratio of compound i present in the mixture, Cmix,
that is required to produce Emix. For example, in the NOEC mixture the ratio of
BPA is:
Ci
?
= 1 (1)
Cmix=
Ci
??−1
(2)
pi= pBPA-NOEC=
CBPA-NOEC-mix
[CBPA-NOEC-mix+ CnNP-NOEC-mix+ CBBP-NOEC-mix
+CCMP-NOEC-mix+ CRes-NOEC-mix+ CtOP-NOEC-mix]
.
Thus, the mixture of the compounds at their NOEC in Eq. (2) gives:
??pBPA-NOEC
+
CCMP
CNOEC-mix=
CBPA
?
+
?
?pnNP-NOEC
+
CnNP
?
+
?pBBP-NOEC
+
CBBP
?
??−1
?pCMP-NOEC
?pRes-NOEC
CRes
??ptOP-NOEC
CtOP
Using the dose–response of each single compound the Ciwas determined
at the observed mixture effects ENOEC-mix, ELOEC-mixand EEC50/IC50-mix. These
Ciconcentrations together with the ratios for each compound are used to cal-
culate the predicted mixture effect concentrations, CNOEC-mix, CLOEC-mixand
CEC50/IC50-mix, respectively, using Eq. (2). Then the predicted mixture effect
concentrations were compared to the observed mixture effect concentrations. If
the predicted mixture effect concentrations overlap with the observed mixture
effect concentrations, an additive effect is suggested. If the predicted concentra-
tionishigherthantheobservedconcentration,asynergisticeffectofthemixture
components is indicated because a lower concentration of the mixture is actu-
ally required to produce the observed effect. On the contrary, a lower predicted
concentration than the observed concentration suggests less than additivity of
the mixture components.
3. Results
3.1. AhR-CALUX
Most of the compounds as well as the mixtures were not
toxic to the Hepa1.12cR cells in the tested concentration range
except BBP, DCP, 2-PP and tOP at concentrations higher than
1×10−5M. The results given below refer only to effects
observed at concentrations not being toxic.
3.1.1. AhR agonistic responses
DBP elicited significantly dose-dependently agonistic
response in the range of 1×10−5M to 1×10−4M, being 5.49-
fold higher than the solvent control (Table 2A and Fig. 1).
Weakly induced AhR activities were observed for DEHP and
Fig. 1. Concentration–response curves for TCDD and compounds inducing the
AhR activity. Data represents mean from 3 independent assays and the curves
werefittedusingathree-parametersigmodalHillcurveinSigmaPlot.Thecurve
for nNP was previously reported (Bonefeld-Jørgensen et al., 2007), but shown
here for information of mixture analyses.
DIDP, reaching 1.75 and 3.78-fold above the solvent control,
respectively (Table 2A and Fig. 1). The AhR agonistic effects
of these compounds were in the range of 5.1–16% of the TCDD
maximum response. At the highest tested concentration CMP
decreased the constitutive basal AhR luciferase activity to 40%
of the solvent control (Table 2A).
3.1.2. Competitive effects on TCDD induced AhR activity
Antagonistic or potentiating effects of the test compounds
on the TCDD induced AhR activity were evaluated by co-
treatment of Hepa1.12cR cells with the compound and 60pM
TCDD (TCDD-EC50). CMP elicited antagonistic effect on the
TCDD induced AhR activity at 1×10−4M down to 18% of
TCDD−EC50(Table 2A). Although not eliciting an agonistic
AhR response on its own, BBP and resorcinol further enhanced
the TCDD induced AhR activity in a dose-dependent manner,
and DCP further enhanced the effect of TCDD at 1×10−9M
(Table 2A). However, none of the compounds showed a normal
dose–response curve (not shown).
3.1.3. Mixture effects
Inthemixtureof6selectedcompoundsonlynNPwasapoten-
tial AhR agonist. Therefore tentative NOEC, LOEC and EC50
of BPA, BBP, CMP, resorcinol and tOP were used (Table 3 leg-
end). Significant agonistic AhR response of the mixture was
observedatNOECandLOEC,being1.23and1.63-foldoverthe
solvent control, respectively (Table 4A). In general, the mixture
inducedresponseswerehigherthanthatofthesinglecompounds
alone. At EC50the mixture did not significantly differ from the
solvent control (Table 4A). The predicted concentration of the
mixturewasonlyslightlyhigherthantheobservedconcentration
(Fig. 2A).
Upon co-treatment with the mixture and TCDD-EC50 a
dose-dependent antagonistic effect on TCDD induced AhR
transactivitywasobserved(Table4AandFig.2B).Theobserved
mixture effects antagonized the TCDD induced AhR response
(set to 1) to 0.96 at ENOEC-mix, 0.75 at ELOEC-mix, and 0.48

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T. Kr¨ uger et al. / Toxicology 246 (2008) 112–123
117
Table 2
The agonistic/competitive CALUX responses induced by tested compounds
Agonistic response (compound alone)Competitive response (compound in the presence of TCDD)
LOEC (M) MOEC (M)% of SC at MOECa
EC50(M)LOEC (M)MOEC (M)% of SC at MOECa
EC50(M)
(A) AhR-CALUX
TCDD
BPAb
nNPb
CMP
DCP
Resorcinol
DBP
BBP
DEHP
DIDP
2×10−12
5×10−5
5×10−8
1×10−4
–
–
1×10−5
–
1×10−4
1×10−4
1×10−9
1×10−4
5×10−5
1×10−4
–
–
1×10−4
–
1×10−4
1×10−4
34456×10−11
nd
2.4×10−5
nd
–
–
nd
–
nd
nd
2×10−12
1×10−4
2.5×10−5
1×10−4
1×10−9
1×10−8
–
1×10−8
–
–
1×10−9
1×10−4
5×10−5/1×10−4
1×10−4
1×10−9
1×10−7
–
1×10−5
–
–
100
44
146/61
18
138
175
–
165
–
–
6×10−11
–
1.0×10−4
–
–
7.6×10−5
–
8.4×10−5
–
–
46
799
40
–
–
549
–
175
378
Competitive response (compound in the presence of R1881)
LOEC (M)MOEC (M)% of SC at MOECa
IC50(M)
(B) AR-CALUX
HF
BPA
nNP
tOPc
2-PP
DCP
CMP
Resorcinol
BBPc
1×10−8
1×10−6
1×10−6
1×10−5
1×10−5
1×10−5
1×10−6
–d
1×10−5
51.0×10−7
1×10−5
1×10−5
1×10−5
1×10−4
1×10−4
1×10−4
1×10−4
1×10−5
34
23
71
30
18
15
18
53
52
5.8×10−8
3.2×10−6
2.2×10−5
1.2×10−6
6.3×10−6
1.2×10−5
1.9×10−5
2.9×10−5
1.3×10−5
LOEC: the lowest tested concentration at which a significant effect (p<0.05) was detected. MOEC: the lowest tested non-cytotoxic concentration causing the
maximum effect. AhR-CALUX: No significant effects were observed for tOP, 2-PP, DNOP, DINP and DEHA. AR-CALUX: No significant effects were observed
for DBP, DNOP, DIDP, DINP, DEHP and DEHA.
aPercent of response obtained from compound compared to the controls at MOEC.
bPreviously reported (Bonefeld-Jørgensen et al., 2007), but shown here for information of mixture analyses.
cBecause of high standard deviations and cytotoxicity at the highest tested concentration, the LOEC and MOEC were the same but a dose response curve could
be created and the IC50determined.
dBecause of high standard deviations none of the tested concentrations were significantly different from the SC and LOEC could not be determined although a
dose response curve could be created.
nd: not determined.
at EEC50-mix (Table 4A and Fig. 2B), which equals to an
inhibition of 4%, 25% and 52% inhibition, respectively. The
predictedconcentrationsofthemixtures(CNOEC-mix,CLOEC-mix
and CEC50-mix) were calculated from Eq. (2) (Section 2.8)
and the effects were plotted into the graph (Fig. 2B). The
predicted PNOEC-mixand PLOEC-mixwere within the 95% con-
fidence interval of the observed dose–response curve (Fig. 2B),
whereas the concentration of the observed EEC50-mixwas only
slightlyhigherthantheconcentrationofthepredicted CEC50-mix
(Fig. 2B).
3.2. AR-CALUX
The compounds BPA, nNP, tOP and BBP were toxic at
≥1×10−4M. The other tested compounds as well as the mix-
tures were not toxic to the cells at the test concentrations. The
results given below refer only to effects observed at concentra-
tions not being toxic to the cells.
None of the 14 tested chemicals reacted as agonists in the
androgen reporter gene assay – neither alone nor in the mixture
(data not shown).
3.2.1. Competitive effects on R188 induced AR activity
In co-treatment with 25pM R1881 (R1881−EC50) all the
phenols and one plasticizer elicited AR antagonistic effects
althoughresorcinolwasnotsignificant(Table2BandFig.3).At
the MOEC the compounds antagonized the response of 25pM
R1881 in the range of 71–15%, with DCP being the most potent
anti-androgeniccompoundwithanIC50of1.2×10−5(Table2B
and Fig. 3).
3.2.2. Mixture effects
A dose-dependent antagonistic effect of the mixture on
R1881 induced AR activity was observed (Table 4B and
Fig. 4). In contrast to the compounds alone, the R1881 induced
AR activity was significantly antagonized by the mixture at
the NOEC (Table 4B), whereas at LOEC, the single com-
pounds and their mixture antagonized the R1881 induced
activity. Compared to the single compounds the LOEC mix-
ture significantly further antagonized the AR activity except for
BBP (Table 4B). Also at the IC50of the single compounds,
the mixture further antagonized the R1881 induced activity
(Table 4B).

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T. Kr¨ uger et al. / Toxicology 246 (2008) 112–123
Table 3
Ratios and concentrations of the compounds used in the mixture assay
BPAnNP tOP CMPResorcinolBBP
A. AhR
pi(equation 2, Section 2.8) in the tested mixtures
NOEC
LOEC
EC50
0.199
0.1998
0.194
0.005
0.001
0.030
0.199
0.1998
0.194
0.199
0.1998
0.194
0.199
0.1998
0.194
0.199
0.1998
0.194
Ci(equation 2, Section 2.8) giving the observed effects given in ?M
123 (ENOEC-mix)a
–b
163 (ELOEC-mix)a
–b
99 (EEC50-mix)a
–b
1.90×10−7
4.40×10−7
–b
–b
–b
–b
–b
–b
–b
–b
–b
–b
–b
–b
–b
B. AhR+60 pM TCDD
pi(equation 2, Section 2.8) in the tested mixtures
NOEC
LOEC
EC50
0.199
0.1998
0.194
0.005
0.001
0.030
0.199
0.1998
0.194
0.199
0.1998
0.194
0.199
0.1998
0.194
0.199
0.1998
0.194
Ci(equation 2, Section 2.8) giving the observed effects
96 (ENOEC-mix)a
75 (ELOEC-mix)a
48 (EEC50-mix)a
5.28×10−5
6.89×10−5
9.26×10−5
9.52×10−5
9.82×10−5
1.02×10−4
7.81×10−5
8.43×10−5
9.33×10−5
2.39×10−6
8.81×10−6
2.81×10−5
6.35×10−5
7.90×10−5
1.06×10−4
6.35×10−5
7.9×10−5
–b
C. AR+25 pM R1881
pi(equation 2, Section 2.8) in the tested mixtures
NOEC
LOEC
IC50
0.107
0.026
0.015
0.107
0.026
0.216
0.036
0.043
0.051
0.357
0.431
0.247
0.357
0.431
0.448
0.036
0.043
0.022
Ci(equation 2, Section 2.8) giving the observed effects
72 (ENOEC-mix)a
45 (ELOEC-mix)a
27 (EIC50-mix)a
6.44×10−7
4.52×10−6
7.83×10−6
4.61×10−6
3.29×10−5
5.11×10−5
5.53×10−7
1.44×10−6
2.10×10−6
1.16×10−5
2.21×10−5
2.96×10−5
30.9×10−6
–b
–b
1.28×10−6
2.13×10−5
4.06×10−5
The predicted mixture effect concentration was calculated using the equation Cmix=[?(pi/Ci)]−1(see Section 2.8): piis the ratio of the concentrations of compound i
presentinthemixture;Ciistheconcentrationofcompoundiattheobservedeffectofthemixture,determinedfromthedose–responsecurvesofthesinglecompounds.
In the AhR–CALUX assay for single compounds, BPA showed a borderline effect on AhR activity at 10−5M. This concentration was defined as tentative LOEC
while tentative NOEC (10−6M) was the concentration below LOEC and tentative EC50(2.5×10−5M) was that above LOEC. For other compounds not showing
any effect on AhR activation, the same tentative NOEC, LOEC and EC50as those of BPA were used. These tentative concentrations were used to calculate pi.
a% response of the solvent control±TCDD/R1881.
bNo effect concentration could be estimated due to no induced effect.
TheobservedmixtureeffectsantagonizedtheR1881induced
AR response (set to 1) to 0.72 at ENOEC-mix, 0.45 at ELOEC-mix,
and 0.27 at EIC50-mix (Table 4B and Fig. 4). The predicted
PNOEC-mixwasequaltotheobservedeffect(Fig.4)andthetenta-
tivepredictedPLOEC-mixwaswithinthe95%confidenceinterval
of the observed dose–response-curve (Fig. 4). The observed
EIC50-mixconcentration was only slightly higher than the pre-
dicted CIC50-mixconcentration (Fig. 4).
4. Discussion
In the present study we demonstrated the potential of four
phthalates (DBP, DEHP, DIDP and BBP) and three phenols
(CMP,DCPandresorcinol)toaffecttheAhRfunction,andseven
of the tested plastic components elicited significant AR antago-
nistic effects in a concentration-dependent manner, whereas no
AR agonistic effects were observed. For the mixture consisting
of six selected compounds additive effects were observed for
both the AhR and AR.
The phthalates DEHP, DIDP and DBP elicited weak agonis-
tic AhR activity. To our knowledge, we report for the first time
the dose-dependent induction of the AhR transactivity of DBP.
Previous in vitro studies showed that DBP inhibited estradiol
(E2):ER mediated gene expression (Zacharewski et al., 1998)
and E2 induced proliferation of human breast cancer MCF-7
cells (Okubo et al., 2003). Thus, DBP may elicit antiestrogenic
effects via the AhR pathway. We observed effects of DEHP and
DIDP at the highest concentration tested (100?M). However,
the metabolite of DEHP, mono-(2-ethylhexyl) phthalate, was
previously reported to induced the gene expression of AhR in
granulose cells at 50?M (Lovekamp-Swan et al., 2003), sug-
gesting DEHP may activate AhR via its metabolite.
BBP alone did not show significant agonistic AhR effect
whereas the compound further enhanced the TCDD induced
AhR activity in a dose-dependent manner. Our observation sup-
portsthereportthatBBPexposureoffemaleratswasassociated
with a significant increase in liver EROD activity (Singletary
et al., 1997). Also DCP and resorcinol further enhanced the

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119
Table 4
CALUX responses induced by the single compounds and mixtures
Agonistic response (compound alone) Competitive response (compound in the presence of TCDD)
NOEC LOECEC50
NOECLOEC EC50
A. AhR-CALUX
Solvent control
60pM TCDD
1.0 ± 0.03
15.2 ± 3.26a
1.0 ± 0.03
15.2 ± 3.26a
1.0 ± 0.05
15.2 ± 3.26a
–––
1.0 ± 0.07 1.0 ± 0.071.0 ± 0.15
Single compounds
BPA
nNP
BBP
CMP
Resorcinol
tOP
1.15 ± 0.30
1.08 ± 0.19
0.99 ± 0.12b
1.01 ± 0.20
0.86 ± 0.06b
1.05 ± 0.08b
1.23 ± 0.07a
1.40 ± 0.24
1.22 ± 0.16a
1.18 ± 0.12
0.92 ± 0.18b
0.85 ± 0.16b
1.09 ± 0.28
1.63 ± 0.29a
1.49 ± 0.38
1.85 ± 0.13a
1.28 ± 0.18
0.81 ± 0.08
0.86 ± 0.09
1.09 ± 0.14
0.99 ± 0.26
1.11 ± 0.14
1.04 ± 0.16
1.24 ± 0.31
0.99 ± 0.22
1.29 ± 0.29
1.15 ± 0.28
0.96 ± 0.13
1.01 ± 0.18b
1.05 ± 0.18b
1.50 ± 0.36a,b
0.73 ± 0.08
1.21 ± 0.40b
1.01 ± 0.29
0.75 ± 0.09a
0.74 ± 0.45
1.12 ± 0.04b
0.26 ± 0.14a
0.21 ± 0.06a
0.25 ± 0.12a
0.21 ± 0.11a
0.48 ± 0.11a
Mixture
Competitive response (compound in the presence of R1881)
NOEC LOEC IC50
B. AR-CALUX
25pM R1881 1.0 ± 0.091.0 ± 0.06 1.0 ± 0.04
Single compounds
BPA
nNP
BBP
CMP
Resorcinol
tOP
1.11 ± 0.08
1.21 ± 0.13
1.23 ± 0.14
1.05 ± 0.16
1.12 ± 0.17
1.18 ± 0.19
0.72 ± 0.05a
0.77 ± 0.10a,b
0.75 ± 0.15a,b
0.65 ± 0.15a
0.65 ± 0.12a,b
0.75 ± 0.10a,b
0.65 ± 0.06a,b
0.45 ± 0.02a
0.62 ± 0.20a,b
0.58 ± 0.16a,b
0.62 ± 0.13a,b
0.44 ± 0.09a,b
0.67 ± 0.13a,b
0.42 ± 0.07a,b
0.27 ± 0.05a
Mixture
Values represent means±S.D. of triplicate independent determinations.
aSignificantly different (p<0.05) from the solvent control or 60pM TCDD/25pM R1881.
bSignificantly different (p<0.05) from mixture.
TCDD induced AhR activity. The mechanism behind the fur-
ther enhancing TCDD effect of BBP, DCP and resorcinol is not
yet known. Based on the mechanistic studies reported (Ikuta et
al.,1998;Pollenz,2002),wespeculatethatBBP,DCPandresor-
cinol may inhibit AhR degradation and subsequently result in
increased levels of ligand mediated gene induction.
CMP was previously reported to express weak estrogenic
activity via ER in MCF-7 cells (Korner et al., 1998). In the
present study, CMP was shown to antagonize AhR transactivity
both in the absence and in the presence of TCDD, supporting
the inhibitory cross talk between AhR and ER (Pocar et al.,
2005).
To our knowledge we here report for the very first time
that 2-PP, DCP, CMP and resorcinol antagonize the AR func-
tion. Together with co-workers we previously reported the AR
antagonisticeffectsofBPAandnNP(Bonefeld-Jørgensenetal.,
2007). The same reporter gene assay was used but carried out
in another laboratory and similar IC50values were found. In
the present study we observed anti-androgenic effects of tOP
being consistent with the effects reported in stably transfected
AR-EcoScreencells(derivedfromChinesehamsterovaryCHO
cells)(Arakietal.,2005)andinstabletransfectedPC-3prostate
cells called PALM (Paris et al., 2002). In contrast, tOP did not
affect the AR in stable transfected CHO-K1 cells (Roy et al.,
2004).
For the phthalates no effect of DNOP and DEHA on the
AR activity was observed. To our knowledge in vitro effects
of these compounds were not previously reported. BBP showed
anti-androgenic activity as previously reported for transiently
transfected CHO-K1 cells (Takeuchi et al., 2005). In contrast,
BBP showed no effect in the stable transfected CHO-K1 cells
(Roy et al., 2004) or in the PALM cells (Sultan et al., 2001).
Neither in our transiently transfected CHO-K1 system nor in
stable transfected CHO-K1 cells (Roy et al., 2004) any effect
of DBP was observed, whereas an anti-AR effect of DBP was
reportedforAR-EcoScreencells(Arakietal.,2005),andintran-
siently transfected CHO-K1 cells using a pIND-ARE reporter
vector and the AR expression plasmid pZeoSV2AR (Takeuchi
et al., 2005). The discrepancy between our data and the data
of Takeuchi et al. (2005) could be a result of the use of differ-
ent AR response elements in the two reporter gene assays. The
phthalates DIDP, DINP and DEHP did not affect the AR in our
in vitro assay being consistent with other reports (Araki et al.,
2005; Roy et al., 2004; Takeuchi et al., 2005).
ThediscrepancybetweenthereportedAReffectsoftheabove
mentioned compounds gives evidences to cell line-dependent
effects which might be due to different design and/or sensitivi-
ties of the various assays as well as co-factor differences in the
different cell lines (Sommer and Haendler, 2003; Verrijdt et al.,
2003).

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Fig. 2. The observed and the predicted effects of the mixture on the agonistic
AhR function. Calculations were described in Section 2.8. (A) Only nNP con-
tribute to the predicted effects since no concentration (Ci, Table 3) could be
determined for the other compounds. No effect could be predicted at EC50. A:
arbitraryNOECforthemixture.(B)PEC50-mixistentative,sincenoconcentration
(Ci, Table 3) for BBP could be determined at EEC50-mix.
The compounds BBP, DCP and resorcinol were found to
further enhance the TCDD effect on the AhR whereas these
compounds antagonized the AR. Jana et al. (1999) reported
inhibitoryAhR-ARcrosstalkinLNCaPcells.Testosteronetreat-
ment dose-dependently inhibited the TCDD-induced CYP1A1
mRNA accumulation and enzyme activity (Jana et al., 1999).
Furthermore, TCDD dose-dependently inhibited testosterone-
dependent transcriptional activity (Jana et al., 1999). Inhibitory
AhR-ARcrosstalkwasinvestigatedusingtworelatedandrogen-
responsiveconstructsoftheratprobasingenepromoter,pPBand
the more androgen-responsive pARR3(Morrow et al., 2004).
Dihydrotestosterone (DHT) significantly inhibited the TCDD
induced activity in cells transfected with pPB but not pARR3,
demonstratingthatinhibitorycrosstalkispromoterspecific.Fur-
thermore, DHT induced an increased in AR protein levels in
LNCaPcells,whichcouldbeblockedbyAhRagonists(Morrow
et al., 2004).
The phthalates DEHP, DIDP and DBP showed no effects on
the AR in our assay, whereas they elicited weak agonistic AhR
activity. In utero exposure to these phthalates have been shown
to disrupt differentiation of androgen-dependent tissues in male
rat offspring (Gray et al., 2000, 1999; Parks et al., 2000). The
Fig. 3. Concentration–response curves for the inhibitory control HF and com-
poundsantagonizingtheR1881inducedARactivity.Datarepresentsmeanfrom
2 (BPA and nNP) or 3 (tOP, 2-PP, DCP, CMP, resorcinol and BBP) independent
assays and the curves were fitted using Chapman, 4 parameters equation in
Sigma Plot. “C”: BPA, nNP, tOP and BBP were found to be cytotoxic at that
concentration. (A) Compounds used in the mixture assay. (B) Compounds not
used in the mixture assay.
Fig. 4. The observed and the predicted antagonistic effects of the mixture. Cal-
culations were described in Section 2.8. A: arbitrary NOEC for the mixture.
PLOEC-mixand PIC50-mixare tentative, since no concentration (Ci, Table 3) for
Resorcinol could be determined at ELOEC-mixand EIC50-mix.

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121
reproductivetractmalformationsintheandrogen-dependenttis-
sues are similar but not equal to effects of antiandrogens such
as vinclozolin and flutamide and it was hypothesized that the
phthalates alter reproductive development through a mecha-
nism not involving binding to the AR (Gray et al., 2000, 1999;
Parks et al., 2000). The phthalates DEHP and DBP have been
showntoreducetesticulartestosteronelevelsinfetalandneona-
tal male rats (Mylchreest et al., 2002; Parks et al., 2000), and for
DBP the decreased testosterone production has been associated
with down-regulation of genes involved in the cholesterol trans-
portandtestosteronesynthesispathwayincludingsteroidogenic
acute regulatory protein, P450 side-chain cleavage enzyme,
3?-hydroxysteroid dehydrogenase, 17?-hydroxysteroid dehy-
drogenase,andscavengerreceptorclassB-1(Barlowetal.,2003;
Ryu et al., 2007). Also exposure to TCDD has been reported
to result in the reduction of testosterone (Choi et al., 2007),
and TCDD modulated the level of cytochrome P450 enzymes
involved in the steroid biosynthetic cascade possibly through
an AhR interaction with dioxin-responsive elements present in
the genes encoding these enzymes (Dasmahapatra et al., 2000).
Thus,weproposethattheweakagonisticAhRactivityofDEHP,
DIDP and DBP could be involved in the altered reproductive
development observed in male rat offspring after administration
of these phthalates.
Humans are exposed to a complex mixture of chemicals.
Therefore,assessmentofthecombinedeffectofmixturesisvery
important. The method based on the principle of concentration
addition (CA) has been shown to be a valid tool for assessing
mixture effects of similarly and dissimilarly acting xenobiotics
in vitro (Birkhoj et al., 2004; Payne et al., 2000). In this study
we performed preliminary analyses to evaluate the effect of
mixtures composed of selected compounds.
The mixture composed of six compounds of which only
nNP had a weak agonistic AhR potential, weakly induced the
AhR activity at NOEC and LOEC compared to the compounds
alone. This suggests that the non-AhR active compounds can
act together with a weak AhR agonist to increase the AhR trans-
activity. Thus, further research of risk assessment of mixtures
consisting of low effect concentrations is needed. As previously
shown for xeno-estrogens (Rajapakse et al., 2002), we observed
for xeno-androgens a significantly antagonized effect of R1881
inducedARactivity,whenthecellswereexposedtothemixture
at NOEC.
For the TCDD or R1881 induced AhR and AR activities, the
observed ENOEC-mixand ELOEC-mixdid not differ from the pre-
dicted values, whereas the concentration at EEC50/IC50-mixwas
slightly higher than the predicted value. However, tentative and
highEEC50/IC50valueswereusedforthepredictionandthus,the
concentration addition principle holds at all levels.
Insummary,toourknowledge,wereportforthefirsttimethe
potential of seven plastic components to affect the AhR func-
tion as well as AR antagonistic effects of 2-PP, DCP, CMP and
resorcinol. The effects were observed at relatively high concen-
trations compared to levels found in humans. However, humans
are exposed to several EDCs simultaneously and in support to
earlier reports our mixture analyses suggested that the com-
bined effect of all the compounds present in the human body
must be taken into consideration for risk assessment. Further-
more,asreportedfromtheotherpartsoftheENDOMETproject
(Bonefeld-Jørgensen et al., 2007; Breous et al., 2005; Ghisari
and Bonefeld-Jorgensen, 2005; Turan et al., 2005), the plastic
componentshavetheabilitytoactviamorethanonemechanism
and this might enhance the biological effect in the intact organ-
ism, since the final response is likely to be determined by the
interactionofallpathwaysimplicated.However,sincetheAhR-
and AR-CALUX are tools for initial screening of compounds,
in vivo studies are needed to further elucidate the effects of the
suspected compounds.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
We thank the CMT group: Inger Sørensen and Birgitte S.
Andersen for technical assistance; Mandana Ghisari and Philip
S. Hjelmborg for scientific support.
This study is part of the ENDOMET Project supported by
The European Commission (Contract no. QLRT-2001-02637).
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