Structural essentials of xenoestrogen dialkyl phthalates to bind to the estrogen receptors

Article abstract of DOI:10.1016/S0378-4274(00)00253-8

Xenoestrogen dialkyl phthalates, C₆H₄(COOC_nH_m)₂, lack the phenolic hydroxyl group that is an essential structural component of the steroid A ring of 17β-estradiol. In order to examine whether dialkyl phthalates imitate the steroid structure, we have synthesized a series of 4-hydroxyl derivatives of dialkyl phthalates. The compounds were examined for their ability to displace [³H]17β-estradiol from the recombinant human estrogen receptor, which was expressed on Sf9 cells using the vaculovirus expression system. Dialkyl 4-hydroxyl phthalates were found to exhibit several-fold higher binding affinities compared to phthalates without the 4-hydroxyl group. From the analyses of receptor binding modes of dialkyl phthalates with and without the 4-hydroxyl group, it was deduced that the phthalic benzene ring mimics the steroid A ring. A biphasic binding curve observed for dicyclohexyl phthalate was also depicted by its 4-hydroxyl derivative, but it increased binding affinity only at the high affinity binding site. These data suggest that the phthalate benzene moiety recognizes the core of the estrogen receptor binding site and the hydrophobic interaction of the dialkyl moiety substantiates the binding characteristics of the phthalates. The present data indicate that even chemicals with slight structural analogy and weak receptor affinity can perturb the endocrine system when administered in high concentrations.

Full text of DOI:10.1016/S0378-4274(00)00253-8

Toxicology Letters 118 (2000) 1–8
www.elsevier.com/locate/toxlet
Structural essentials of xenoestrogen dialkyl phthalates to
bind to the estrogen receptors
Daisuke Asai a, Yoshiko Tahara a, Makoto Nakai b, Yoshikuni Yakabe b,
Mineo Takatsuki b, Takeru Nose a, Teruo Shinmyozu c,
Yasuyuki Shimohigashi a,*
a Laboratory of Structure–Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences,
Kyushu Uni6ersity, Fukuoka 812-8581, Japan
b Chemicals Assessment Center, Chemicals E6aluation and Research Institute, Kurume 830-0023, Japan
c Institute for Fundamental Research of Organic Chemistry (IFROC), Fukuoka 812-8581, Japan
Received 15 May 2000; received in revised form 31 July 2000; accepted 1 August 2000
Abstract
Xenoestrogen dialkyl phthalates, C6H4(COOCnHm)2, lack the phenolic hydroxyl group that is an essential
structural component of the steroid A ring of 17b-estradiol. In order to examine whether dialkyl phthalates imitate
the steroid structure, we have synthesized a series of 4-hydroxyl derivatives of dialkyl phthalates. The compounds
were examined for their ability to displace [3H]17b-estradiol from the recombinant human estrogen receptor, which
was expressed on Sf9 cells using the vaculovirus expression system. Dialkyl 4-hydroxyl phthalates were found to
exhibit several-fold higher binding affinities compared to phthalates without the 4-hydroxyl group. From the analyses
of receptor binding modes of dialkyl phthalates with and without the 4-hydroxyl group, it was deduced that the
phthalic benzene ring mimics the steroid A ring. A biphasic binding curve observed for dicyclohexyl phthalate was
also depicted by its 4-hydroxyl derivative, but it increased binding affinity only at the high affinity binding site. These
data suggest that the phthalate benzene moiety recognizes the core of the estrogen receptor binding site and the
hydrophobic interaction of the dialkyl moiety substantiates the binding characteristics of the phthalates. The present
data indicate that even chemicals with slight structural analogy and weak receptor affinity can perturb the endocrine
system when administered in high concentrations. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Dialkyl phthalates; Estrogen receptor; Receptor binding; Xenoestrogens; Endocrine disruptors
1. Introduction
Dialkyl phthalates C6H4(COOCnHm)2 have
* Corresponding author. Tel.: +81-92-6422584; fax: +81-
been described as potential xenoestrogens, the in
92-6422584.
vitro estrogenic behavior of which has been evi-
denced, for example, in the estrogen receptor
E-mail address: shimoscc@mbox.nc.kyushu-u.ac.jp (Y. Shi-
mohigashi).
0378-4274/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0378-4274(00)00253-8

2
D. Asai et al. / Toxicology Letters 118 (2000) 1–8
binding assay and in the recombinant yeast screen
(Sharpe et al., 1995; Harris et al., 1997). Dialkyl
phthalates are thought to disrupt the steroidal
regulation of normal development and the func-
tion of the male and female reproductive tracts
(Colborn et al., 1993; Auger et al., 1995; Danzo,
1997). Dialkyl phthalates are utilized widely as
plasticizers, since they provide flexibility and
workability to the plastic intermediates and end
products. However, their usage as plasticizers is
just as an additive and, thus, they are not bound
covalently to the products. This allows the phtha-
lates to leach from plastic wares into the environ-
found to be engaged in hydrogen bondings with
the receptor residues. If the phthalic benzene ring
mimics the steroid A ring, the 4-hydroxyl deriva-
tive of dialkyl phthalates would increase the bind-
ing affinity of non-hydroxyl dialkyl phthalates due
to additional hydrogen bonding(s) just as observed
for the phenolic hydroxyl group of 17b-estradiol.
In the present study, in order to demonstrate
that the phthalic benzene ring mimics the steroid
A ring, we have synthesized a series of 4-hydroxyl
derivatives of dialkyl phthalates (Fig. 1) and ex-
amined their binding characteristics for estrogen
receptor. Although such 4-hydroxyl derivatives are
neither industrial products nor physiologically rel-
evant metabolites of phthalates, this kind of struc-
ture–activity study is now extremely important for
understanding the structural basis of chemicals in
the assessment of their potentials, that is, whether
they are to be an endocrine disruptor. In particu-
lar, for analysis of the quantitative structure–ac-
tivity relationships (QSAR) between the chemicals
and the nuclear receptors, such structural informa-
tion is crucially important for evaluation of the
extent to which a certain chemical structure is
engaged in the receptor interaction (Fig. 1).
mental
medium,
causing
pollution
and
contamination. Also, our direct exposure to such
phthalates through plastic wares and fatty foods
has been noted as possible pathways to concen-
trate xenoestrogens in the body (Jobling et al.,
1995).
Since dialkyl phthalates and the native estrogen,
17b-estradiol, share only the benzene ring as a
core structure, it is hard to understand the struc-
tural basis which determines why dialkyl phtha-
lates bind to the estrogen receptor. Several
structure-activity studies on phthalates have re-
cently been reported (Harris et al., 1997; Waller et
al., 1996), and we examined a series of alkyl-chain
and ring isomers of dialkyl phthalates to explore
the structural essentials for binding to the estrogen
receptor (Nakai et al., 1999). It was found that
phthalates having an alkyl chain of more than C3
(n=3) exhibit a distinct full receptor binding in a
dose-dependent manner and that the interaction is
optimized with dibutyl phthalates of C4. These
structure–activity studies, however, gave no direct
evidence regarding the structural resemblance be-
tween dialkyl phthalates and 17b-estradiol.
In our evaluation, even the most potent dialkyl
phthalate, di-n-butyl phtalate, was found to be
much less potent than 17b-estradiol in the receptor
binding assay (Nakai et al., 1999). This diminished
activity has been tentatively explained by the lack
of a hydroxyl group corresponding to the estra-
diol–phenolic group. The A ring moiety of 17b-
estradiol is phenol, and dialkyl phthalates are
devoid of a phenolic hydroxyl group. From the
crystallographic study of the 17b-estradiol/recep-
tor complex, the phenolic hydroxyl group was
Fig. 1. Chemical structures of: (A) di-n-alkyl phthalates
C6H4(COOCnH2n+1)2; and (B) their 4-hydroxyl derivatives.

D. Asai et al. / Toxicology Letters 118 (2000) 1–8
3
2. Materials and methods
(DMSO-d6) d 1.25 (m, 6H, CH3), 4.23 (m, 4H,
CH2CH3), 6.90–6.97 and 7.68 (m, 3H, ArH),
10.60 (br, 1H, ArOH). Elemental analysis: C,
60.50; H, 5.92% (Calculated values for C12H14O5:
C, 60.50; H, 5.92%).
2.1. Materials
17b-Estradiol was purchased from RBI Re-
search Biochemicals International (Natick, MA).
A series of dialkyl phthalates were from Wako
(Osaka), and 4-hydroxyphthalic acid from Acros
(Geel, Belgium). All other chemicals were of the
best grade available.
2.2.3. Di-n-propyl 4-hydroxyphthalate
HOꢀC6H3(COOC3H7)2
This was prepared from 4-hydroxyphthalic acid
(911 mg, 5 mmol) and 1-propanol (7.5 ml, 100
mmol): yield, 1.17 g (88%); m.p. 67–68°C; Rf
1
2.2. Synthesis of dialkyl 4-hydroxyphthalates
0.72; H-NMR (DMSO-d6) d 0.91 (m, 6H, CH3),
1.65 (m, 4H, CH2CH2CH3), 4.13 (m, 4H,
CH2CH2CH3), 6.91–6.98 and 7.69 (m, 3H, ArH),
10.60 (br, 1H, ArOH). Elemental analysis: C,
63.14; H, 6.83% (Calculated values for C14H18O5:
C, 63.14; H, 6.81%).
Alcohols were purified by distillation just be-
fore esterification reaction. Desiccants used are
magnesium for MeOH and EtOH and calcium
hydride for 1-propanol, 1-butanol, and cyclohex-
anol. High-performance thin-layer chromatogra-
phy was carried out on silica gel 60 (Merck,
Frankfurt) with the solvent system of CHCl3–
MeOH (5:1, v/v) and the mobility is shown by Rf.
Melting points were not corrected.
2.2.4. Di-n-butyl 4-hydroxyphthalate
HOꢀC6H3(COOC4H9)2
This was prepared from 4-hydroxyphthalic acid
(911 mg, 5 mmol) and 1-butanol (9.2 ml, 100
mmol): yield of an oil, 1.30 g (89%); Rf 0.73;
1H-NMR (DMSO-d6) d 0.91 (m, 6H, CH3), 1.36
(m, 4H, CH2CH2CH2CH3), 1.62 (m, 4H,
2.2.1. Dimethyl 4-hydroxyphthalate
HO-C6H3(COOCH3)2
The reaction mixture of 4-hydroxyphthalic acid
(911 mg, 5 mmol) and concentrated H2SO4 (0.1
ml) in MeOH (4.1 ml, 100 mmol) was refluxed for
5 h, and then evaporated in vacuo to leave an oil.
The residue was treated with 5% Na2CO3 and
CHCl3, and the aqueous layer was further ex-
tracted three times with CHCl3. A combined solu-
tion of organic extracts was washed with water,
4% NaHCO3, and saturated NaCl, and dried over
Na2SO4. After evaporation, the residue was crys-
tallized from CHCl3-ether: yield, 786 mg (85%);
CH2CH2CH2CH3),
4.18
(m,
4H,
CH2CH2CH2CH3), 6.91–6.98 and 7.68 (m, 3H,
ArH), 10.59 (br, 1H, ArOH). Elemental analysis:
C, 65.25; H, 7.57%. (Calculated values for
C16H22O5: C, 65.29; H, 7.53%).
2.2.5. Dicyclohexyl 4-hydroxyphthalate
HOꢀC6H3(COOC6H11)2
This compound was prepared from 4-hydrox-
yphthalic acid (911 mg, 5 mmol) and cyclohexanol
(10 ml, 100 mmol): yield, 1.21 g (83%); m.p.
1
1
m.p. 110–111°C; Rf 0.64; H-NMR (DMSO-d6) d
127–128°C; Rf 0.76; H-NMR (DMSO-d6) d 1.2–
3.77 (m, 6H, CH3), 6.91–6.97 and 7.70 (m, 3H,
ArH), 10.67 (br, 1H, ArOH). Elemental analysis:
C, 56.95; H, 4.76% (Calculated values for
C10H10O5: C, 57.14; H, 4.80%).
1.9 and 4.84 (m, 22H, cyclohexyl H), 6.89–6.95
and 7.66 (m, 3H, ArH), 10.55 (br, 1H, ArOH).
Elemental analysis: C, 69.28; H, 7.53% (Calcu-
lated values for C20H26O5: C, 69.34; H, 7.56%).
2.2.2. Diethyl 4-hydroxyphthalate
2.3. Estrogen receptor binding assay
HOꢀC6H3(COOC2H5)2
The title compound was prepared from 4-hy-
droxyphthalic acid (911 mg, 5 mmol) and EtOH
(5.8 ml, 100 mmol) as described above. Yield, 1.07
The binding assay for estrogen receptors to
evaluate the inhibition of radiolabeled estrogen
was carried out essentially as reported previously
(Nakai et al., 1999). Briefly, a solution (10 ml) of
g
(90%); m.p. 67–68°C; Rf 0.69; 1H-NMR

4
D. Asai et al. / Toxicology Letters 118 (2000) 1–8
recombinant human estrogen receptor, which was
expressed in Sf9 cells using the vaculovirus expres-
sion system (a kind gift from Sumitomo Chemi-
cal), at a concentration of ꢀ0.1 nM was added to
the buffer of Tris-HCl (pH 7.4, 70 ml) containing
1 mM EDTA, 1 mM EGTA, 1 mM NaVO4, 10%
glycerol, 10 mg/ml g-globulin, 0.5 mM phenyl-
methylsulfonyl fluoride, and 0.2 mM leupeptin.
Samples were dissolved in DMSO at 0.1 M initia-
tion concentrations in DMSO (30% DMSO for
dicyclohexyl 4-hydroxyphthalate), then diluted by
the same Tris-HCl buffer (pH 7.4) to prepare
sample solutions of B3% DMSO. After the sam-
ple solution (10 ml) and 0.5 nM [2,4,6,7,16,17-
3H]17b-estradiol (10 ml; 128 Ci/mmol, Amersham,
Buckinghamshire, UK) were added, the assay so-
lution was incubated for 1 h at 25°C. Free radioli-
gand was removed by incubation with 0.2%
activated charcoal (Wako) and 0.02% dextran
(Sigma, St. Louis, MO) in PBS (pH 7.4) for 10
min at 4°C and centrifugation for 10 min at
15 000 rpm. Binding assays were carried out by
three independent experiments, and the data was
analyzed by the computer program ALLFIT (De
Lean et al., 1978) to depict the binding curves and
to estimate the IC50 value, the dose which pro-
duces a 50% inhibition of the binding of [3H]17b-
estradiol.
2.6. Ab initio molecular orbital calculations
Ab initio molecular orbital (MO) calculations
were carried out using the program of GAUS-
SIAN94 (Frisch et al., 1995) on a computer Sili-
con Graphics Octane. The structures were
optimized at the RHF/6-31+G* level of theory.
3. Results and discussion
3.1. Synthesis of 4-hydroxyl deri6ati6es of dialkyl
phthalates
The synthesized derivatives of dialkyl phtha-
lates in this study are a series of dialkyl 4-hydrox-
yphthalates, HO-C6H3(COOCnHm)2, where CnHm
are CH3 (n=1), CH2CH3 (n=2), CH2CH2CH3
(n=3),
CH2CH2CH2CH3
(n=4),
and
cyclo(CH2CH2CH2CH2CH2CH2) (n=6)) (Fig. 1).
These were prepared from 4-hydroxyphthalic acid
HO-C6H3(COOH)2 (5 mmol) by simultaneous es-
terification of dicarboxylic acids with alcohols
(100 mmol) of methanol, ethanol, 1-propanol,
1-butanol, and cyclohexanol, respectively, in the
presence of concentrated H2SO4 (2.5 mmol) as a
catalyst. Eventually, dimethyl, diethyl, di-n-pro-
pyl, and dicyclohexyl 4-hydroxyphthalates were
obtained as crystalline compounds (mean yield,
87%), whereas di-n-butyl 4-hydroxyphthalate was
obtained as an oil (89% yield).
2.4. High-performance liquid chromatography.
Analytical HPLC was performed on a Hitachi
655A-11 liquid chromatograph using an L-
column ODS reversed-phase column (4.6×250
mm) (Chemicals Evaluation and Research Insti-
tute, Tokyo, Japan) in 0.1% trifluoroacetic acid
with a linear gradient of acetonitrile (30–95%) for
65 min with a flow rate of 1 ml/min.
The purity was verified by high-performance
thin-layer chromatography and elemental analy-
sis. All compounds purified were eluted as a single
peak on reversed-phase HPLC and their mass
numbers were confirmed by mass spectroscopy
1
(data not shown). In the H-NMR measurements,
a typical splitting pattern of proton signals was
observed for the aromatic envelope of 4-hydroxyl
derivatives of dialkyl phthalates.
1
2.5. H-NMR measurements
Samples were prepared by dissolving 5 mg of
the compound in 0.5 ml of DMSO-d6 (99.6% d,
CDN Isotopes, Quebec, Canada). All H-NMR
3.2. Receptor binding affinity of dialkyl
4-hydroxyphthalates
1
spectra were recorded on a JEOL JNM-EX270
FT NMR System spectrometer operating at 270
MHz H frequency at 25°C. The chemical shifts
were determined using tetramethylsilane as an
internal standard.
Binding assay for the estrogen receptor was
carried out using tritium-labeled natural estrogen,
[2,4,6,7,16,17-3H]17b-estradiol for recombinant
human estrogen receptor expressed on Sf9 cells
1

D. Asai et al. / Toxicology Letters 118 (2000) 1–8
5
essentially as described previously (Nakai et al.,
1999). A series of phthalates were first dissolved in
neat DMSO at the 0.1 M concentration, then
diluted stepwise with the aqueous buffer. For
dicyclohexyl 4-hydroxyphthalate, 1 M solution in
DMSO was first diluted ten times to make a
solution of 0.1 M concentration in 30% DMSO.
This dilution method makes the DMSO concen-
tration to be B3% in the assay solutions. Even at
the highest concentration of 3% DMSO, the bind-
ing characteristics of compounds did not change
at all. The solubility of aromatic organic com-
pounds to the aqueous solution is usually ex-
tremely low, though the receptor binding assays
require high concentrations owing to weak bind-
ing affinities. We found that the method to trans-
fer the DMSO solution is an effective way to
make dilution. It should be noted, however, that
DMSO affects the ligand binding significantly
when its concentration becomes more than 5%.
Among the synthesized dialkyl 4-hydroxyph-
thalates, diethyl 4-hydroxyphthalate (n=2), di-n-
propyl 4-hydroxyphthalate (n=3), and di-n-butyl
4-hydroxyphthalate (n=4) were found to bind to
the estrogen receptor. Their IC50 values were cal-
culated to be 256918, 82.8912 and 19.691.1
mM, respectively. Di-n-butyl 4-hydroxyphthalate
was the most potent among these three active
compounds. Dimethyl 4-hydroxyphthalate did not
bind to the receptor at all.
Fig. 2. Concentration-dependent curves of 17b-estradiol (ꢀ),
di-n-butyl phthalate (ꢁ), and its 4-hydroxyl derivative ( ) in
the receptor binding assay to measure the ability of the
compounds to displace [3H]17b-estradiol in the recombinant
human estrogen receptor expressed on Sf9 cells. These show
representative dose-dependent binding curves, which give the
IC50 value closest to the mean IC50 from three assays. IC50
values showed a between-experiment coefficient of variation of
6–14%.
times more potent than the parent compound in
regards to receptor binding. The parallelism was
also observed between these compounds and 17b-
estradiol (Fig. 2). Similar results regarding the
binding curve parallelism and activity enhance-
ment were found for a set of di-n-propyl phtha-
lates with and without a 4-hydroxyl group (data
not shown).
3.3. Phthalic benzene ring mimics the steroid A
ring
When the activity profiles were compared be-
tween dialkyl phthalates and their 4-hydroxyl
derivatives, several characteristic features became
prominent. First, a remarkable difference has
emerged for diethyl ester derivatives. Diethyl ph-
thalate with no 4-hydroxyl group was unable to
bind to the estrogen receptor. However, its 4-hy-
droxyl derivative exhibited a distinct receptor
binding in a dose-dependent manner. This is cer-
tainly due to the existence of the hydroxyl group
at the position 4 of the phthalate benzene ring.
Another important finding was confirmed when
di-n-butyl phthalates with and without a 4-hy-
droxyl group were compared. These two phtha-
lates exhibited the dose-dependent binding curves
parallel to each other, indicating that their bind-
ing modes are exactly the same (Fig. 2). In this
parallelism, the 4-hydroxyl derivative was several
The estrogen receptor, a member of a super-
family of transcription factors (Mangelsdorf et al.,
1995), has three different functional domains, and
has a relatively large ligand binding cavity (450
3
,
A , approximately twice the molecular volume of
estradiol). The most important receptor interac-
tions of 17b-estradiol are the hydrogen bondings
between the 17b-estradiol phenolic-hydroxyl
group and receptor Glu353 and Arg394 (Brzo-
zowski et al., 1997). The molecular recognition of
17b-estradiol by the estrogen receptor is accom-
plished by the non-polar hydrophobic interaction
of the rest of the estradiol structure in addition to
these specific hydrogen bondings.
The results obtained from the binding assay
indicated that the placement of the 4-hydroxyl
group reinforces the receptor binding affinity sev-
eral times owing to the formation of hydrogen

6
D. Asai et al. / Toxicology Letters 118 (2000) 1–8
bonding between the 4-hydroxyl group and the
receptor residues, presumably Glu353 and Arg394
as described above. The parallelism of dose-de-
pendent binding curves further indicated that di-
alkyl phthalates bind to the receptor in a manner
similar to those of 4-hydroxyl dialkyl phthalates
and 17b-estradiol. These data provide evidence
that the phthalate-benzene ring of dialkyl phtha-
lates imitates the steroid A ring despite its lack of
a phenolic hydroxyl group. This was confirmed in
the present study by the computer-assisted ab
initio molecular orbital (MO) calculations carried
out using the program of GAUSSIAN (Frisch et
al., 1995). When the phthalate-benzene ring was
superimposed on the steroid A ring, it was found
that dialkyl phthalates are able to locate the first
3ꢀ4 methylenes of ester-alkyl groups just at the
place corresponding to the steroid interface (data
not shown). The alkyl moiety larger than the
butyl would not fit the receptor binding site be-
cause of their steric hindrances.
preservation suggests that the low affinity binding
site possesses no site complementary to the phe-
nolic hydroxyl group. The estrogen receptor ag-
gregates in a dimeric form by the binding of
17b-estradiol, and this dimerization is responsible
for positive cooperativity in the binding to DNA
(Anolik et al., 1995). The present result clearly
shows that [3H]17b-estradiol binds to both high
and low binding sites, and only dicyclohexyl ph-
thalates are able to distinguish these two sites with
very weak affinities. It is not clear yet at this
moment what is the second binding site in the
estrogen receptor, since this second site appears
not to require the phenolic hydroxyl group for
specific interaction.
3.5. Structural basis of dialkyl phthalate toxicity
Chemicals which act as endocrine disruptors
usually carry little structural resemblance to natu-
ral estrogens. Indeed, dialkyl phthalates have little
structural analogy to 17b-estradiol. The present
results suggest that the phthalate benzene moiety
recognizes the core of the estrogen receptor bind-
ing site just as like the phenolic A ring of 17b-
estradiol to substantiate its binding to the
receptor. The important point is that we are able
to use this kind of structural information for
3.4. Di6alent binding of dicyclohexyl phthalates
Although the binding assays were performed
utilizing a single type of receptor, namely, a-estro-
gen receptor, dicyclohexyl phthalate was found to
bind to the estrogen receptor with a biphasic
binding curve. Apparently, this biphasic curve of
dicyclohexyl phthalate is not due to the existence
of two different receptor populations. In the
present study, dicyclohexyl 4-hydroxyphthalate
also depicted a biphasic binding curve. As shown
in Fig. 3, both dicyclohexyl phthalates exhibited
biphasic binding curves, indicating the presence of
the two distinct binding sites with high and low
affinities. It should be noted that the binding
affinity of 4-hydroxyl derivative was increased
several times only at the high affinity binding site
as compared with the non-hydroxyl derivative.
There was no increase at the low affinity binding
site (Fig. 3).
Affinity enhancement at the high affinity bind-
ing site was judged to be also due to the hydrogen
bonding to the receptor residue meditated though
the 4-hydroxyl group. The placement of the hy-
droxyl group does not affect the affinity of phtha-
lates at the low affinity binding site. The affinity
Fig. 3. Representative concentration-dependent curves of dicy-
clohexyl phthalate (ꢁ) and its 4-hydroxyl derivative ( ) in the
receptor binding assay to measure the ability of the com-
pounds to displace [3H]17b-estradiol in the recombinant hu-
man estrogen receptor expressed on Sf9 cells. Three
independent assays depicted almost the same binding curves
for each compound.

D. Asai et al. / Toxicology Letters 118 (2000) 1–8
7
prediction of the ability of various chemicals in
binding to the receptors. The establishment of the
structure–activity relationships between chemicals
and receptors is now a keen requirement espe-
cially to assess the toxicity of chemicals.
estrogenic effect, and some phthalates are indeed
known to be inactive as estrogen. Also, in many
cases, phthalates are known to be rather easily
degraded into mono esters and alcohols. For pre-
diction whether the phthalate is estrogenic or not,
the structures of dialkyl and monoalkyl phthalates
should be evaluated also for their alkyl ester
moieties, especially examining whether they mimic
the hydrophobic ring conformation of 17b-estra-
diol. It appears now that such structure–activity
studies are necessary for any chemicals having the
benzene ring.
In spite of a structural imitation of the steroid
A ring by the phthalate aromatic ring, the fact is
that the binding affinities of dialkyl phthalates are
extremely weak as compared to the native estro-
gen, 17b-estradiol. However, the possibility of
these chemicals to elicit equivocal hormonal ac-
tions might be warranted by their inevitable high
concentrations. The physicochemical characteris-
tics of dialkyl phthalates such as the lipophilic
nature and structural stability may allow them to
accumulate and remain longer in fatty tissues of
the body, thus increasing their concentration and
bioavailability (Danzo, 1997; Hooper et al., 1990).
This possibility would become a serious problem
of utmost importance when we take phthalates
directly, for example, through daily meals. Most
recently in Japan, a very high concentration of
di(2-ethylhexyl) phthalate (DEHP) was detected
in boxed meals, and this DEHP contamination
was found to be due to the leach from polyvinyl
chloride (PVC) gloves worn by kitchen staff for
preventing germ contamination when cooking and
packing the food. DEHP is a plasticizer of PVC
and melts easily in an oil used for fried foods.
Given that phthalates melted in oil were intro-
duced into the body, they would be easily accu-
mulated in the fatty tissues. Thus, an intake of
intact phthalates appears to be very serious. Re-
cent study by Wine et al. (1997) has shown that
the most potent phthalate in the receptor binding,
di-n-butyl phthalate, is a reproductive/develop-
mental toxicant in rats exposed directly both as
adults and during development. Phthalates are
present also in various environmental waters, but
in highly variable concentrations ranging from
nanograms to milligrams per liter, and their con-
centration through the food chains must produce
a similar accumulation in the body of animals,
including humans.
Acknowledgements
We thank Sumitomo Chemical for their kind
donation of recombinant human estrogen recep-
tor expressed on Sf9 cells.
References
Anolik, J.H., Klinge, C.M., Hilf, R., Bambara, R.A., 1995.
Cooperative binding of estrogen receptor to DNA depends
on spacing of binding sites, flanking sequence, and ligand.
Biochemistry 34, 2511–2520.
Auger, J., Kunstmann, J.M., Czyglik, F., Jouannet, P., 1995.
Decline in semen quality among fertile men in Paris during
the past 20 years. N. Engl. J. Med. 332, 281–285.
Brzozowski, A.M., Pike, A.C.W., Dauter, Z., Hubbard, R.E.,
8
Bonn, T., Engstro¨m, O., Ohman, L., Greene, G.L.,
,
Gustafsson, J-A, Carlquist, M., 1997. Molecular basis of
agonism and antagonism in the oestrogen receptor. Nature
389, 753–758.
Colborn, T., vom Saal, F.S., Soto, A.M., 1993. Developmental
effects of endocrine-disrupting chemicals in wildlife and
humans. Environ. Health Perspect. 101, 378–384.
Danzo, B.J., 1997. Environmental xenobiotics may disrupt
normal endocrine function by interfering with the binding
of physiological ligands to steroid receptors and binding
proteins. Environ. Health Perspect. 105, 294–301.
De Lean, A., Munson, P.J., Rodbard, D., 1978. Stimultaneous
analysis of families of sigmoid dose response curves. Am.
J. Physiol. 235, E97–E102.
Frisch, M.J., Trucks, G.W., Schlegel, H.B., Gill, P.M. W.,
Johnson, B.G., Robb, M.A., Cheeseman, J.R., Keith, T.,
Petersson, G.A., Montgomery, J.A., Raghavachari, K.,
Al-Laham, M.A., Zakrzewski, V.G., Ortiz, J.V., Fores-
man, J.B., Cioslowski, J., Stefanov, B.B., Nanayakkara,
A., Challacombe, M., Peng, C.Y., Ayala, P.Y., Chen, W.,
Wong, M.W., Andres, J.L., Replogle, E.S., Gomperts, R.,
Martin, R.L., Fox, D.J., Brinkley, J.S., Defrees, D.J.,
The present results have shown that the ben-
zene ring of dialkyl phthalates mimics the steroid
A ring of 17b-estradiol. However, this is not to
say that all dialkyl phthalates elicit an in vivo

8
D. Asai et al. / Toxicology Letters 118 (2000) 1–8
Nakai, M., Tabira, Y., Asai, D., Yakabe, Y., Shinmyozu, T.,
Barker, J., Stewart, J.P., Head-Gordon, M., Gonzalez, C.,
Pople, J.A., 1995. Gaussian, Pittsburgh, PA.
Harris, C.A., Henttu, P., Parker, M.G., Sumpter, J.P., 1997.
The estrogenic activity of phthalate esters in vitro. Envi-
ron. Health Perspect. 105, 802–811.
Noguchi, M., Takatsuki, M., Shimohigashi, Y., 1999.
Binding characteristics of dialkyl phthalates for the estro-
gen receptor. Biophys. Biochem. Res. Commun. 254, 311–
314.
Sharpe, R.M., Fisher, J.S., Millar, M.M., Jobling, S.,
Sumpter, J.P., 1995. Gestational and lactational exposure
of rats to xenoestrogens results in reduced testicular size
and sperm production. Environ. Health Perspect. 103,
1136–1143.
Waller, C.L., Oprea, T.I., Chae, K., Park, H.-K., Korach,
K.S., Law, S.C., Wiese, T.E., Kelce, W.R., Gray, L.E., Jr.,
1996. Ligand-based identification of environmental estro-
gens. Chem. Res. Toxicol. 9, 1240–1248.
Wine, R.N., Li, L.-H., Barns, L.H., Gulati, D.K., Chapin,
R.E., 1997. Reproductive toxicity of di-n-butylphthalate in
a continuous breeding protocol in Sprague-Dawley rats.
Environ. Health Perspect. 105, 102–107.
Hooper, S.W., Pettigrew, C.A., Sayler, G.S., 1990. Ecological
fate, effects and prospects for the elimination of environ-
mental polychlorinated-biphenyls (PCBs). Environ. Toxi-
col. Chem. 9, 655–667.
Jobling, S., Reynolds, T., White, R., Parker, M.G., Sumpter,
J.P., 1995. A variety of environmentally persistent chemi-
cals, including some phthalate plasticizers, are weakly es-
trogenic. Environ. Health Perspect. 103, 582–587.
Mangelsdorf, D.J., Thummel, C., Beato, M., Herrlish, P.,
Schu¨tz, G., Umesono, K., Blumberg, B., Kastner, P.,
Mark, M., Chambon, P., Evans, R.M., 1995. The nuclear
receptor superfamily: the second decade. Cell 83, 835–839.
.