Comparative study of the uterotrophic potency of 14 chemicals in a uterotrophic assay and their receptor-binding affinity.

Abstract

We performed an immature rat uterotrophic assay of 14 chemicals having various receptor-binding affinities in order to assess the relationship between their uterotrophic potency and receptor-binding affinity. The chemicals tested were phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, 2-ethylhexyl-p-hydroxybenzoate, 4,4'-biphenol, 4,4'-sulfonyldiphenol, 4,4'-dihydroxydiphenylmethane, 2,4-dihydroxybenzophenone, 4,4'-cyclohexylidenebisphenol, 4-t-butylpyrocatechol, clomiphene citrate, 4,4'-(1,3-phenylenediisopropylidene)bisphenol, p-t-butylphenol, and diallylterephthlate. Two of the 14 chemicals, phthalic acid di-n-propyl ester and diallylterephthlate, exhibited no receptor-binding affinity, and the receptor-binding affinity of phthalic acid di-n-hexyl ester and phthalic acid di-n-amyl ester was lower than that of the other chemicals. Ten of the chemicals showed uterotrophic potency, the exceptions being phthalic acid di-n-propyl ester, diallylterephthlate, phthalic acid di-n-hexyl ester, and phthalic acid di-n-amyl ester. The results of the present study demonstrate that the affinity of the chemicals in the receptor-binding assay correlated well with their potency in the uterotrophic assay except for a few chemicals with very low receptor-binding affinity.

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Toxicology Letters 146 (2004) 111–120
Comparative study of the uterotrophic potency of 14 chemicals
in a uterotrophic assay and their receptor-binding affinity
Kanji Yamasaki∗, Shuji Noda, Nobuya Imatanaka, Yoshikuni Yakabe
Chemicals Assessment Center, Chemicals Evaluation and Research Institute, 3-822 Ishii, Hita, Oita 877-0061, Japan
Received 23 June 2003; received in revised form 29 July 2003; accepted 29 July 2003
Abstract
We performed an immature rat uterotrophic assay of 14 chemicals having various receptor-binding affinities in order to as-
sess the relationship between their uterotrophic potency and receptor-binding affinity. The chemicals tested were phthalic acid
di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, 2-ethylhexyl-p-hydroxybenzoate, 4,4?-biphenol,
4,4?-sulfonyldiphenol,4,4?-dihydroxydiphenylmethane,2,4-dihydroxybenzophenone,4,4?-cyclohexylidenebisphenol,4-t-butyl-
pyrocatechol, clomiphene citrate, 4,4?-(1,3-phenylenediisopropylidene)bisphenol, p-t-butylphenol, and diallylterephthlate. Two
of the 14 chemicals, phthalic acid di-n-propyl ester and diallylterephthlate, exhibited no receptor-binding affinity, and the
receptor-binding affinity of phthalic acid di-n-hexyl ester and phthalic acid di-n-amyl ester was lower than that of the other
chemicals. Ten of the chemicals showed uterotrophic potency, the exceptions being phthalic acid di-n-propyl ester, diallyl-
terephthlate, phthalic acid di-n-hexyl ester, and phthalic acid di-n-amyl ester. The results of the present study demonstrate that
the affinity of the chemicals in the receptor-binding assay correlated well with their potency in the uterotrophic assay except for
a few chemicals with very low receptor-binding affinity.
© 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Agonist; Antagonist; Endocrine; Estrogen effect; Rat; Receptor-binding assay; Uterotrophic assay
1. Introduction
There is concern that certain chemicals may have
the potential to interfere with normal sexual differ-
entiation and development in animals and humans
(McLachlan, 1993; McLachlan and Korach, 1995),
and the Organisation for Economic Cooperation and
Development (OECD) has proposed the uterotrophic
assay as an in vivo screening assay to detect the estro-
∗Corresponding author. Tel.: +81-973-24-7211;
fax: +81-973-23-9800.
E-mail address: yamasaki-kanji@ceri.jp (K. Yamasaki).
genic properties of potentially endocrine-disrupting
chemicals (OECD, 1998, 2000, 2003b). The rodent
uterus and its rapid and dramatic growth in response
to estrogen during the natural estrous cycle are ba-
sis for the uterotrophic assay. The receptor-binding
assay has been developed as an in vitro prescreen-
ing assay to select chemicals that have estrogenic
properties before subjecting them to an in vivo assay
(OECD, 2003a). In our previous study, the results of
the receptor-binding assay correlated well with those
of the uterotrophic assay (Yamasaki et al., 2003), but
it was impossible to clearly define the relationship
between receptor-binding affinity and uterotrophic
0378-4274/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2003.07.003

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K. Yamasaki et al. / Toxicology Letters 146 (2004) 111–120
potency. Accordingly, in the present study we per-
formed the uterotrophic assay on fourteen chemicals
having various receptor-binding affinities in order to
better define the relationship between the results of
these two tests.
2. Materials and methods
The uterotrophic assay was performed in accor-
dance with Good Laboratory Practice guidelines.
2.1. Chemicals
The chemicals tested are listed in Table 1, and their
chemical structure is shown in Fig. 1. All chemicals
were dissolved in olive oil (Fujimi Pharmaceutical,
Company, Osaka, Japan) before use.
2.2. Animals
Crj:CD (SD) rat dams and their female 10-day-old
pups were purchased from Charles River Japan Inc.
(Shiga, Japan), and the dams and pups were housed
in polycarbonate pens until weaning. All pups were
weaned at 19 days of age and then individually
housed in stainless steel wire-mesh cages throughout
the study. The immature female rats were weighed,
weight-ranked, and assigned randomly to each of
the experimental and control groups. Body weight
and clinical signs were recorded daily throughout the
Table 1
Chemicals tested in this study
ChemicalsCAS no.Purity (%)Source
Phthalic acid di-n-hexyl ester
Phthalic acid di-n-amyl ester
Phthalic acid di-n-propyl ester
2-Ethylhexyl-p-hydroxybenzoate
4,4?-Biphenol
4,4?-Sulfonyldiphenol
4,4?-Dihydroxydiphenylmethane
2,4-Dihydroxybenzophenone
4,4?-Cyclohexylidenebisphenol
4-t-Butylpyrocatecho1
Clomiphene citrate
4,4?-(1,3-Phenylenediisopropylidene)bisphenol
p-t-Butylphenol
Diallylterephthlate
84-75-3
131-18-0
131-16-8
5153-25-3
92-88-6
80-09-1
620-92-8
131-56-6
843-55-0
98-29-3
50-41-9
13595-25-0
98-54-4
1026-92-2
99.3
99.5
98.4
99.9
99.9
99.4
99.9
99.8
99.9
99.6
Unknown
99.9
100.0
99.6
Tokyo Kasei Kogyo Co.
Tokyo Kasei Kogyo Co.
Tokyo Kasei Kogyo Co.
Wako Pure Chemicals
Tokyo Kasei Kogyo Co.
Tokyo Kasei Kogyo Co.
Tokyo Kasei Kogyo Co.
Tokyo Kasei Kogyo Co.
Tokyo Kasei Kogyo Co.
Wako Pure Chemicals
ICN
Aldrich Co.
Wako Pure Chemicals
Tokyo Kasei Kogyo Co.
study. Rats were given ad libitum access to tap water
and a commercial diet (CRF-1, Oriental Yeast Co.,
Tokyo, Japan) before weaning and ad libitum access
to water from an automatic dispenser and a com-
mercial diet (MF, Oriental Yeast Co.) after weaning.
The animal room was maintained at a temperature of
23 ± 2◦C and a relative humidity of 55 ± 5%, and
it was artificially illuminated with fluorescent light
on a 12h light/dark cycle (6:00–18:00h). All animals
were cared for according to the principles outlined in
the guide for animal experimentation prepared by the
Japanese Association for Laboratory Animal Science.
2.3. Study design
Each chemical was subcutaneously injected into the
back of 20-day-old female rats on three consecutive
days.ThedosesofeachchemicalareshowninTable2.
Ethynyl estradiol (EE, CAS no. 57-63-6, 98% purity,
Sigma) in olive oil was also subcutaneously injected
into the back of some rats in a dose of 0.6?g/kg
per day on three consecutive days after administra-
tion of the chemicals at the same doses. A vehicle
control group was injected with olive oil alone, and
a positive control group was injected with EE after
administration of olive oil. A group injected with the
estrogen-antagonist chemical tamoxifen in a dose of
1mg/kg per day plus EE was also established to con-
firm the reliability of this study. Each group consisted
of six rats. The doses of each chemical were based on
the results of a preliminary study, and the no observed

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K. Yamasaki et al. / Toxicology Letters 146 (2004) 111–120
113
Fig. 1. Chemical structures of test compounds.

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K. Yamasaki et al. / Toxicology Letters 146 (2004) 111–120
adverse effect level in the preliminary study was se-
lected as the high dose in the present study. The vol-
ume of the olive oil solution containing the chemical
or EE for subcutaneous injections was 4ml/kg. The
animals were killed by bleeding from the abdominal
vein under deep ether anaesthesia approximately 24h
after the final dose. At necropsy, the uteri were care-
fully dissected free of adhering fat and mesentery and
weighed.
2.4. Receptor-binding assay
The stock solutions (10mM) of the test substance
and [2,4,6,7,16,17-3H] 17?-estradiol (Amersham Bio-
sciences K.K., radiochemical purity; 99.9%, specific
activity; 154Ci/mmol) were prepared with DMSO
and diluted to ten fold higher concentration of the
final one with Tris–HCl (pH 7.4) containing 1mM
EDTA, 1mM EGTA, 1mM NaVO3, 10% glycerol,
10mg/ml ?-globulin, 0.5mM phenylmethylsulfonyl
fluoride, and 0.2mM leupeptin (binding buffer). A
solution (10?l) dissolved in the binding buffer of
approximately 10nM of recombinant human estro-
gen receptor ligand binding domain fused with glu-
tathione S-transferase and expressed in E. coli was
dissolved in the binding buffer. After adding the sam-
ple solution (10?l) of each chemical and 5nM of
[2,4,6,7,16,17-3H] 17?-estradiol (10?l), the solution
was incubated for 1h at 25◦C. Free radioligand was
removed by incubation with 100?l of dextran-coated
charcoal (DCC, 0.2% activated charcoal and 0.02%
dextran in PBS (pH 7.4)) for 10min at 4◦C. After
filtration, radioactivity in the filtrate was measured
using liquid scintillation counter. Chemicals were
tested over the 10−11to 10−4M concentration range.
The percent ratio (B/B0 (%)) of standard ligand
([3H] 17?-estradiol) bound to the receptor was calcu-
lated from the radioactivities of the solutions with and
without the test substance, subtracting the radioactiv-
ity due to nonspecifically bound standard ligand to the
receptor.
The B/B0values as a function of the concentration
were fit to the logistic equation and IC50 value of
each chemical was calculated by least-squares method
using the computer program GraphPad Prism®. The
binding abilities of test chemicals to the receptor were
evaluated by relative binding affinity (RBA), ratio of
IC50values to l7?-estradiol.
2.5. Statistical analysis
Differences in body weight and organ weight be-
tween the vehicle group and each of the chemical
groups and between the vehicle-plus-EE group and
each of the chemical plus EE groups were assessed
for statistical significance by the two-tailed Student’s
t-test.
3. Results
3.1. Clinical signs and body weight
Body weights are shown in Table 2. No clinical
abnormalities were observed in any of the groups, and
body weight increased normally in all groups.
3.2. Uterine weight
Uterine weights are shown in Table 2. Watery
uterine fluid was grossly detected in rats given EE,
200mg/kg 2-ethylhexyl p-hydroxybenzoate, 600mg/
kg 4,4?-biphenol, 1000mg/kg 4,4?-dihydroxydiphenyl-
methane and 2,4-dihydroxybenzophenone, and 150
mg/kg 4,4?-cyclohexylidenebisphenol.
The uterine weight of the rats given EE was higher
thanthatoftheratsgivenvehiclealone,andtheuterine
weight of the rats given tamoxifen plus EE was lower
thanthatoftheratsgivenEE,confirmingthereliability
of this study.
Uterine weight was significantly higher in the
200mg/kg 2-ethylhexyl p-hydroxybenzoate group, the
60, 200, and 600mg/kg 4,4?-biphenol groups, the 20
and 500mg/kg 4,4?-sulfonyldiphenol groups, the 100,
300, and 1000mg/kg 4,4?-dihydroxydiphenylmethane
and 2,4-dihydroxybenzophenone groups, the 30 and
150mg/kg 4,4?-cyclohexylidenebisphenol groups, the
1000mg/kg 4-t-butylpyrocatechol group, the 2, 10,
and 50mg/kg clomiphene citrate groups, the 50mg/kg
4,4?-(1,3-phenylenediisopropylidene)bisphenol group,
and the 100, 300, and 1000mg/kg p-t-butylphenol
groups. By contrast, uterine weight was significantly
lower in the 1000mg/kg phthalic acid di-n-amyl ester
group, and it was significantly higher in the 300mg/kg
phthalic acid di-n-propyl ester group, but without any
dose–effect relationship.
Uterine weight was significantly lower in the
200mg/kg 2-ethylhexyl p-hydroxybenzoate plus EE

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115
Table 2
Summary of receptor-binding (RAB) and uterotrophic assays
Chemicals RAB
(% of E2)
Dosages
(mg/kg per day)
Body weight (g)Uterine wet weight Uterine blotted weight
Absolute
(mg)
40.5 ± 8.9
39.9 ± 6.7
38.4 ± 6.1
36.3 ± 4.5
143.6 ± 23.8
154.7 ± 24.5
166.3 ± 19.0
149.1 ± 25.3
85.6 ± 8.3d
44.4 ± 6.1
37.9 ± 8.1
40.5 ± 8.0
34.0 ± 4.1b
167.5 ± 22.5
156.3 ± 26.3
150.6 ± 43.9
158.1 ± 31.0
87.3 ± 1.8d
33.5 ± 5.0
39.0 ± 5.6
42.8 ± 9.2
41.1 ± 8.7
152.0 ± 48.0
169.3 ± 20.8
144.5 ± 11.3
181.6 ± 23.2
95.7 ± 5.2c
37.7 ± 4.3
41.6 ± 7.2
43.5 ± 4.9
88.2 ± 11.3b
156.3 ± 20.1
162.9 ± 34.6
157.2 ± 9.8
105.1 ± 15.6d
89.8 ± 3.0d
Relative
(mg/100g)
68.3 ± 10.2
63.7 ± 10.5
60.7 ± 9.3
60.7 ± 7.7
237.4 ± 46.1
258.2 ± 28.7
270.8 ± 23.9
238.8 ± 43.1
141.4 ± 14.9d
73.0 ± 8.2
61.4 ± 10.6
66.9 ± 11.4
57.4 ± 7.7b
274.9 ± 35.2
261.0 ± 52.5
248.5 ± 66.8
266.0 ± 48.9
145.3 ± 5.5d
52.4 ± 5.3
60.6 ± 7.8
65.7 ± 11.5a
64.2 ± 13.1
232.4 ± 58.3
270.2 ± 41.1
243.9 ± 12.0
293.6 ± 48.0
153.8 ± 16.9c
62.5 ± 9.2
68.4 ± 10.2
73.0 ± 10.3
148.8 ± 22.1b
255.5 ± 37.2
281.3 ± 53.1
255.8 ± 13.1
172.7 ± 19.4d
153.8 ± 9.1d
Absolute
(mg)
39.6 ± 8.7
39.0 ± 6.7
37.5 ± 6.2
35.4 ± 4.3
122.7 ± 15.7
121.2 ± 13.9
130.4 ± 6.8
121.9 ± 13.3
84.0 ± 8.2d
43.0 ± 5.9
36.9 ± 7.8
39.4 ± 7.7
33.0 ± 4.0b
126.2 ± 14.0
121.4 ± 9.5
114.5 ± 24.7
124.4 ± 12.8
85.2 ± 1.4d
33.2 ± 4.9
38.6 ± 5.6
42.3 ± 9.3
40.6 ± 8.5
119.8 ± 23.5
130.8 ± 5.8
127.1 ± 15.2
139.9 ± 10.4
93.9 ± 5.1c
37.2 ± 4.2
41.2 ± 7.2
42.9 ± 4.9
86.1 ± 10.8b
124.4 ± 8.2
125.3 ± 19.3
131.9 ± 6.9
101.6 ± 15.1d
89.0 ± 2.8d
Relative
(mg/100g)
66.8 ± 10.2
62.3 ± 10.4
59.3 ± 9.5
59.2 ± 7.4
202.2 ± 28.5
203.3 ± 22.6
213.1 ± 17.1
195.3 ± 23.3
138.8 ± 14.8d
70.6 ± 7.8
59.8 ± 10.1
65.0 ± 10.9
55.7 ± 7.4b
207.5 ± 26.3
202.2 ± 20.9
189.5 ± 37.1
209.6 ± 19.7
141.8 ± 5.5d
51.9 ± 5.3
59.9 ± 7.8
65.0 ± 11.7a
63.5 ± 12.8
184.8 ± 27.2
208.5 ± 17.7
210.5 ± 14.8
225.4 ± 19.6c
150.9 ± 16.4c
61.6 ± 9.2
67.7 ± 10.1
72.0 ± 10.3
145.2 ± 20.5b
203.2 ± 17.2
217.3 ± 33.0
214.7 ± 12.5
167.0 ± 18.4d
152.4 ± 8.7d
Phthalic acid di-n-hexyl ester0.000918 Vehicle control
100
300
1000
Vehicle + EE
100 + EE
300 + EE
1000 + EE
TMX + EE
Vehicle control
100
300
1000
Vehicle + EE
100 + EE
300 + EE
1000 + EE
TMX + EE
Vehicle control
100
300
1000
Vehicle + EE
100 + EE
300 + EE
1000 + EE
TMX + EE
Vehicle control
8
40
200
Vehicle + EE
8 + EE
40 + EE
200 + EE
TMX + EE
58.8 ± 5.6
62.8 ± 4.2
63.4 ± 4.9
59.8 ± 2.3
60.9 ± 4.0
59.8 ± 4.9
61.4 ± 4.0
62.7 ± 5.7
60.6 ± 2.3
60.9 ± 3.8
61.4 ± 3.4
60.5 ± 4.2
59.5 ± 4.3
61.1 ± 5.4
60.2 ± 3.0
60.3 ± 3.6
59.4 ± 3.6
60.2 ± 2.6
63.8 ± 4.2
64.2 ± 1.2
64.7 ± 3.2
63.9 ± 3.2
64.5 ± 5.6
63.0 ± 3.6
60.2 ± 3.2
62.3 ± 5.3
62.5 ± 3.9
60.7 ± 3.4
60.7 ± 2.8
59.8 ± 2.4
59.5 ± 3.5
61.3 ± 2.0
58.2 ± 8.2
61.5 ± 1.9
60.7 ± 3.8
58.5 ± 2.0
Phthalic acid di-n-amyl ester 0.00165
Phthalic acid di-n-propyl esterNot binding
2-Ethylhexyl-p-hydroxy-benzoate 0.0519