Yumi Akahoria,b,*, Makoto Nakaib, Kanji Yamasakic, Mineo Takatsukib, Yasuyuki Shimohigashid, Masahiro Ohtakia 

Department of Human Environmental Science, Ochanomizu University, 2-1-1, Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Japan (CERI) 1600, Shimo-Takano, Sugito-machi, Kitakatsushika-gun, Saimata 345-0043, Japan
Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Japan (CERI) 3-822 Ishii-machi, Hita, Oita 877-0061, Japan
Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 
* Corresponding author. Address: Department of Human Environmen-tal Science, Ochanomizu University, 2-1-1, Otsuka, Bunkyo-ku, Tokyo112-8610, Japan. Tel.: +81 480 37 2601; fax: +81 480 37 2861. E-mail address: akahori-yumi@ceri.jp(Y. Akahori).

Received 2 May 2007; accepted 8 August 2007
Available online 23 August 2007 

Abstract

For screening chemicals possessing endocrine disrupting potencies, the uterotrophic assay has been placed in a higher level in the OECD testing framework than the ER binding assay to detect ER-mediated activities. However, there are no studies that can demonstrate a clear relationship between these assays. In order to clarify the relationship between the in vitro ER binding and in vivo utero-trophic assays and to determine meaningful binding potency from the ER binding assay, we compared the results from these assaysfor 65 chemicals spanning a variety of chemicals classes. Under the quantitative comparison between logRBAs (relative binding affinities)and logLEDs (lowest effective doses), the log RBA was well correlated with both logLEDs of estrogenic and anti-estrogenic compounds at r2= 0.67 (n= 28) and 0.79 (n= 23), respectively. The RBA of 0.00233% was found to be the lowest ER binding potency to elicit estrogenic or anti-estrogenic activities in the uterotrophic assay, accordingly this value is considered as the detection limit of estrogenic oranti-estrogenic activities in the uterotrophic assay. The usage of this value as cutoff provided the best concordance rate (82%). Thesefindings are useful in a tiered approach for identifying chemicals that have potential to induce ER-mediated effects in vivo.



1. Introduction 

After a variety of studies showed that certain chemicalsmight disrupt the sex hormonal systems of wildlife andhumans, the Organization for Economic Cooperationand Development (OECD), European countries, the United States and Japan initiated efforts to develop and validate assays and screen chemicals for their potential todisrupt the endocrine system of human and wildlife (CEC, 2004; EPA, 1998; Gelbke et al., 2004). Under theOECD activity, ‘‘Conceptual Framework for the Testingand Assessment of Endocrine Disrupting Chemicals’’ wasdeveloped. This framework has five components or levelseach corresponding to a different level of biological complexity (OECD, 2002). The in vitro assays such as receptorbinding assay are placed in level 2 in this framework toprovide mechanistic information and serve for screeningpurposes. As a tool to detect hormonal effects mediated thorough specific hormone receptors, especially for estrogen receptors (ERs), the application of the ER bindingassay has been investigated as a quick and cost-effective method.  Furthermore,  the  rodent uterotrophic assay, which has been placed in level 3 to provide data about single endocrine mechanism and effects, has also been recognized as an in vivo assay to detect estrogen receptormediated effects (Kanno et al., 2001; Owens and Koeter, 2003). 

Since the uterotrophic assay and ER binding assay bothdetect ER-mediated activities, a clear relationship betweenthe results from both two assays is expected. However, there are no studies that can demonstrate a clear relationship between two assays on a variety of chemical structures, although studies comparing at limited number ofchemical structures has been available (Takemura et al.,2005; Hong et al., 2005; Legler et al., 2002; Yamasakiet al., 2004). Since the receptor binding assay is consideredas one of the screening assays to determine if a chemicalhas potential to trigger receptor-mediated endocrine disruption such as ER and androgen receptor (AR). Theresults of receptor binding studies need to be related to biological relevance. The purpose of this study is to clarify therelationships between the results obtained in the in vitro ER binding assay and in vivo uterotrophic assay, and to determine the meaningful cutoff value from the ER bindingassay. For this purpose, we compared the results of receptor binding assay using human ERα(hERα) with immaturerat uterotrophic assay for 65 selected chemicals.

2. Materials and methods

2.1. Chemicals

The chemical names, CAS numbers and sources of 65 test chemicals subjected in this study are listed in Table 1. Parabenes, benzophenones, biphenyls, diphenylmethanes, diphenylethylenes, phthalates, phenols and other chemicalstructure classes were included. All test chemicals used inthis study had more than 95% purity. Tris(hydroxy-methyl)aminomethane (Tris), phenylmethylsulfonyl fluo-ride (PMSF) and 17β-Estradiol were purchased from Sigma-Aldrich. Dimethylsulfoxide(DMSO), leupeptinhemisulfate monohydrate, sodium metavanadate, (NaVO3)dithiothreitol (DTT), glycerol and bovine serum albumin(BSA) were obtained from Wako Pure Chemical Industries,Ltd. and disodium salt dihydrate, ethylenediaminetetraace-tic acid (EDTA) and ethyleneglycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA) were from Dojindo Laboratories Inc.

2.2. In vitro ER binding assay

The receptor binding assay using recombinant humanestrogen receptor α(hERα) was conducted by the methodpreviously reported(Nakai et al., 1999; Yamasaki et al.,2004). Briefly, a recombinant hERα ligand binding domain(hERα-LBD) fused with glutathione-S-transferase (GST) was expressed in E. coli and purified using affinity chroma-tography technique. After the addition of sample solution(10μL) of varied concentration (1·10-11 to 1·10-4M as final concentrations) and [2,4,6,7,16,17-3H] 17β-estradiol([3H]-E2, 10μL; final conc. 0.5 nM, 155 Ci/mmol, Amar-sham Biosciences Corp.) in Tris-HCl (pH 7.4, 50μL) con-taining 1 mM EDTA, 1 mM EGTA, 1 mM NaVO3,1mMDTT, 10% glycerol, 10 mg/mL BSA, 0.5 mM PMSF, and 0.2 mM leupeptin, a solution of recombinant hERα-LBD(30μL; final conc. 0.2 nM) was gently mixed. This mixture solution was incubated for 1 h at 25 C. Free radiolabelled ligands were removed by incubation with 0.4% dextran-coated charcoal (Sigma) (100μL) for 10 min at 4 C followed by filtration. The radioactivity of residual radiolabelled ligands bound to receptors in filtrate were measured by liquid scintillation counting. The assay was repeated more than three times for each test chemical.

2.3. Immature rat uterotrophic assay

The chemicals listed in Table 1 were tested in the immature rat uterotrophic assay according to the OECD drafttest guideline as previously reported (Yamasaki et al.,2004) in compliance with good laboratory practice (GLP).

Crj:CD (SD) IGS female rat pups (10-day old) purchased from Charles River Japan Inc. (Shiga, Japan) were weaned with their dams and individually housed until 19-days old. These immature female rats were weighted and weight-ranked to assign to each of the treated and control groups (6 rats/group). Three doses were used for each testchemical and the highest dose was set at the maximum tol-erance dose based on the results of dose-range finding test. The limit dose was set at 1000 mg/kg/day. Each group of six immature, 20-days old female rats received subcutaneous injections of a test chemical into their back for three consecutive days (4 mL/kg/day) for evaluation of estro-genic activity. The vehicle control group treated with oliveoil (s.c.) and the positive control group for estrogenic activity (s.c. 0.6μg/kg/day of 17α-ethynylestradiol (EE, Sigma)) were concurrently run for each test chemical. To evaluate anti-estrogenic activity in another set of treatment groups,EE (0.6μg/kg/day) was co-administered with the testchemical. The positive control group for anti-estrogenicactivity received subcutaneous co-administration of 1 mg/kg/day of tamoxifen (TAM, Sigma) and 0.6μg/kg/day of EE. The animals were sacrificed by bleeding from the abdominal vein under deep ether anesthesia 24 h after the final administration, and body weight and uterine weight of each animal were recorded.

2.4. Data analysis

The resulting data from the ER binding assay were analyzed using GraphPad Prism®, Version 4 (GraphPad Soft-ware, Inc.), and the IC50 value for each test chemical wasobtained by logistic equation. The relative binding affinity (RBA) of each test chemical was calculated using the following equation:
RBA = (IC50 for E2) / (IC50 for test chemical) x 100

When IC50 was not calculated and the maximum displacement of radiolabelled ligand was between 20% and 50%, the binding potency of test chemical was shown as ‘‘N.D. (not determined)’’. When the maximum displacement of radiolabelled ligand was below 20%, the bindingpotency of test chemical was shown as ‘‘N.B. (not bound)’’.

The Dunnet test was used to analyze the data from theuterotrophic assay. When the significant increase of uterine weights in agonism assay or significant decrease in antago-nism assay were observed, the test chemical was evaluatedas estrogenic or anti-estrogenic, respectively.

The lowest effective dose (LED,µmol/kg/day), the low-est dose showing a statistically significant effect in thisassay, was employed as a quantitative parameter in thiscomparison study with logRBAs from the in vitro ER binding assay, and the correlation coefficients (r2) and its P values were calculated by GraphPad Prism® version 4 (GraphPad Software, Inc.). Also, contingency analyseswere performed to calculate concordance (the rate agree-ment of the results among assays), false-negative (the rateof negatives in the ER binding assay identified as positivein the uterotrophic assay) and false-negative rates (the rateof positives in the ER binding assay identified as negativesin the uterotrophic assay).

Table 1. List of test chemicals and summary results of ER binding and uterotrophic assays

Chemical Name CAS No. Sourcea logRBA logLED (µmol/kg/day)
Estrogenic Anti-estrogenic
17β-Estradiol 50-28-2 NA 2.00 <-2.43b N.A.
4-n-Amylphenol 14938-35-3 TCI -2.49 3.69
p-Dodecyl-phenol 104-43-8 Kanto Chem. -0.62 2.18
p-tert-Butylphenol 98-54-4 Wako -2.63 2.82 3.30
p-(tert-Pentyl) phenol = p-(tert-Amyl) phenol 80-46-6 Wako -1.76 3.09 3.09
4-Cyclohexylphenol 1131-60-8 TCI -1.40 3.05
4-(1-Adamantyl)phenol 29799-07-3 Aldrich 0.23 1.54 -
2,4-Di-tert-butylphenol 96-76-4 TCI -2.81
Pentachlorophenol 87-86-5 Wako N.B.
2-Naphthol 135-19-3 Wako -2.98
p-Hydroxybenzoic acid 99-96-7 Wako N.B.
Ethyl-p-hydroxybenzoate 120-47-8 Wako N.D. -
2-Ethylhexyl-4-hydroxybenzoate 5153-25-3 Wako -1.28 2.90 2.90
4,4'-Dimethoxybenzophenone 90-96-0 TCI N.B.
4-Hydroxybenzophenone 1137-42-4 Sigma -1.97 3.00 3.00
4,4'-Dihydroxybenzophenone 611-99-4 Wako -1.77 2.97 2.97
2,4-Dihydroxybenzophenone 131-56-6 TCI -1.86 2.67 3.15
2,4,4'-Trihydroxybenzophenone 1470-79-7 Aldrich -1.13 2.24 2.24
2,2',4,4'  -Tetrahydroxybenzophenone 131-55-5 Wako -1.03 2.91 2.21
4-Fluoro-4'-hydroxybenzophenone 25913-05-7 TCI -2.50 2.67
2,3,4-Trihydroxybenzophenone 1143-72-2 Wako -2.05 3.11 3.64
2,2-Bis(4-hydroxyphenyl)-4-methyl-n-pentane 6807-17-6 Wako 0.45 0.87 0.87
4,4'-Cyclohexylidenebisphenol 843-55-0 TCI -0.67 2.05 2.05
4,4'-(Octahydro-4,7-methano-5H-inden-5-ylidene) bisphenol 1943-97-1 Acros 0.34 0.80 0.80
4,4'-(Hexafluoroisopropylidene)diphenol 1478-61-1 Aldrich -0.11 1.08 1.08
4-(Phenylmethyl)-phenol 101-53-1 TCI -1.65 3.04 3.04
4,4'-Dihydroxydiphenylmethane 620-92-8 TCI -1.14 2.70 2.70
4,4'-Sulfonyldiphenol 80-09-1 TCI -2.26 1.90 3.30
4,4'-Thiobis-phenol 2664-63-3 TCI -0.61 0.96 1.66
Clomiphene citrate (cis and trans mixture) 50-41-9 ICN 1.57 0.52 0.52
4,4'-Dimethoxytriphenylmethane 7500-76-7 KKC N.D.
3,3'-Dichlorobenzidine dihydrochloride 612-83-9 SIGMA -3.36
4,4'-Biphenol 92-88-6 TCI -1.05 2.51 2.51
4'-Hydroxy-4-biphenylcarbonitrile 19812-93-2 Wako -2.84
3,3',5,5' -Tetramethyl-(1,1'-bisphenyl)-4,4' -diol 2417-04-1 Aldrich -2.39 -
Diethylphthalate 84-66-2 Kanto Chem. N.B. -
Di-n-propyl phthalate 131-16-8 TCI N.D.
Di-n-pentyl phthalate 131-18-0 TCI -2.78
Di-n-hexyl phthalate 84-75-3 TCI -3.04
Diheptyl phthalate 3648-21-3 Aldrich -2.95
Diisononyl phthalate 28553-12-0 Wako -3.49
Diisodecyl phthalate 26761-40-0 TCI -3.46
Di(2-ethylhexyl) phthalate 117-81-7 Wako -1.15
Diallyl tetephthlate 1026-92-2 TCI N.B.
Testosterone enanthate 315-37-7 Wako N.B. 1.40 -
Methyltestosterone = 17α-Methyltestosterone 58-18-4 Wako N.D. 1.52
N-Cyclohexyl-2-benzothiazolesulfenamide 95-33-0 TCI -2.33
2,2'-Dibenzothiazolyl disulfide = 2,2'-Dithiobis[benzothiazole] 120-78-5 Wako -1.89
2-Benzothiazolethiol = 2-Mercaptobenzothiazole 149-30-4 Wako -2.78
4-tert-Butylpyrocatechol = 4-tert-Butylcatechol 98-29-3 Wako -1.72 3.78 3.26
p-Dichlorobenzene 106-46-7 TCI N.B.
Benzanthrone 82-05-3 Wako N.B. 3.64
Flutamide 13311-84-7 SIGMA N.B.
3-Amino-1,2,4-triazole 61-82-5 TCI N.B.
Benomyl 17804-35-2 SIGMA N.B.
Hexachlorocyclopentadiene 77-47-4 Wako -1.97 -
Captafol;1,2,3,6-Tetrahydro-N-(1,1,2,2-tetrachloroethylthio)phthalimide 2425-06-1 Wako -1.34
Di (2-ethylhexyl) adipate= Bis(2-ethylhexyl)adipate 103-23-1 Wako N.B. -
Disulfiram 97-77-8 Wako -1.34 - 2.53
4,4'-(1,3-Phenylendiisopropylidene)bisphenol 13595-25-0 Aldrich -0.76 2.16 0.76
1,1,3-Tris(2-methyl-4-hydroxy-5-tert-butylphenyl)butane 1843-03-4 Wako -1.67
3,3,3',3'-Tetramethyl-1,1'-spirobisindane-5, 5',6,6'-tetrol 77-08-7 TCI -1.00 3.37
Diphenyl-p-phenylenediamine 74-31-7 Wako -1.87 2.58
Atrazine 1912-24-9 TCI N.B. 2.97
4-Hydroxyazobenzene 1689-82-3 Wako -1.13 2.30
4-Diethylaminobenzaldehyde 120-21-8 Wako N.B. 3.05
N.A.: not available, N.B. (not bound): the maximum displacement of radiolabelled ligand was below 20%, N.D. (not determined): IC50 was not calculatedand the maximum displacement of radiolabelled ligand was between 20–50%.
–: Significant increase or decrease of uterine weight was not observed in estrogenic or anti-estrogenic assay systems, respectively.
aTCI; Tokyo Chemical Industry Co., Ltd., Kanto Chem.; Kanto Chemical Co., Inc., Wako; Wako Pure Chemical Industries, Ltd., Acros; AcrosOrganics, ICN; KKC; Kankyo Kagaku Center Inc.
bThe agonistic logLED of E2 was determined by the subcutaneous injection for three consecutive days to immature rat (from 23-days old, SD rat)conducted byPadilla-Banks et al., 2001). This value was not used for the quantitative and qualitative analyses in the study.



3. Results

3.1. In vitro ER binding assay

The results of the in vitro binding assay to hERα for 65 chemicals are shown in Table 1.

RBA values were obtained for 47 of 65 chemicals. The highest and lowest logRBAs were 1.57 of clomiphene citrate and 3.49 of diisononyl phthalate, respectively.

The remaining 18 chemicals were regarded as negatives (non-binders) in the concentration tested. Among them, although 4 chemicals, i.e., ethyl p-hydroxybenzoate, di(n-propyl)phthalate, 17α-methyltestosterone and 4,4’-dim-ethoxytriphenylmethane showed 20–50% displacementand they were regarded as negatives in this study.

3.2. Immature rat uterotrophic assay

Sixty-five chemicals were tested by immature rat utero-trophic assay in both estrogenic and anti-estrogenic assaysystems. The test chemical was evaluated as estrogenic or anti-estrogenic if the uterine weights were significantlyincreased in the estrogenic assay or decreased in the anti-estrogenic assay. In such cases, the log lowest effectivedoses (logLED,µmol/kg/day) were shown in Table 1.

Based on this evaluation, 31 and 25 chemicals were identified as estrogenic and anti-estrogenic in immature ratuterotrophic assay, respectively. Twenty-one, including alldiphenylmethanes tested, exhibited both estrogenic andanti-estrogenic responses. On the other hand, none of thephthalates tested in this study have either estrogenic noranti-estrogenic.

3.3. In vitro ER binding assay vs. in vivo uterotrophic assay

3.3.1. Comparison between logRBA and logLED values

The logRBAs obtained from the in vitro ER bindingassay were compared with logLEDs from the uterotrophicassay. As shown in Fig. 1, the logRBAs were well correlated with both logLEDs in estrogenic and anti-estrogenicassay systems r2= 0.67 (n= 28, P< 0.0001) and 0.79 (n= 23, P< 0.0001), respectively. The lowest logRBA that can detect estrogenic or anti-estrogenic response in the uterotrophic assay was 2.63 (RBA = 0.00233%) of p-tert-butylphenol, respectively.


3.3.2. Consistency between ER binding and uterotrophic assays for detecting estrogenicity/anti-estrogenicity

The results of ER binding and uterotrophic assays basedon the evaluation of the ER related response as detectable (positive) or not (negative) are compared in Table 2-1. Therates of concordance, false negative and false positive for all chemicals tested was 66%, 14% and 57%, respectively.


Fig. 1. Relationships between logLED and logRBA values. a, Relation-ship between estrogenic logLEDs and logRBAs b, Relationship between anti-estrogenic logLEDs and logRBAs


Fig. 2. Changes of indexes by contingency analysis depending on cutoff of RBA. The rates of concordance, false negative and false positive areshown as open circle with solid line, closed triangle with solid line andopen triangle with dash line, respectively.


Seventeen chemicals found to bind to the ER were neitherestrogenic nor anti-estrogenic in the uterotrophic assay.Among these chemicals, 3 benzothiazoles and 6 phthalateswere included, and the logRBAs of the remaining 8 chemicals were relatively weak ranging from –3.36 to –1.34. Fivechemicals were classified as non-binders that showed estro-genic or anti-estrogenic responses in the uterotrophic assay. All 3 non-ER binder chemicals that showed estrogenicity inthe uterotrophic assay were androgens (testosterone enan-thate and 17α-methyltestosterone) and p-diethylamino-benzaldehyde. The other 2 non-ER binders that showedanti-estrogenic effects were atrazine and benzanthrone.

Based on the comparison above, the ER binding assayseemed to have higher sensitivity than immature rat utero-trophic assay. In order to determine the lowest biologicallyeffective binding potency in the ER binding assay, the relationship between RBA cutoff value and the rates of concordance, false negative and false positive from contingencyanalysis were investigated (Fig. 2). As mentioned above,the lowest RBA that showed estrogenic or anti-estrogenicresponses in the uterotrophic assay was 2.63 (RBA = 0.00233) of p-tert-butylphenol in this study. Whenthis value is used as a cutoff value, the rates of concordanceand false positive rate ratios were refined at 82% and 23%,respectively without increasing the false-negative rate (Table 2-2). This cutoff achieved the best concordance andlowest false-negative ratios as shown in Fig. 2.

Table 2-1. Contingency table between ER binding and uterotrophic assays without applying cutoff

 

ER-binding assay

Total

Index

Rate(%)

P

N

 

 

 

Uterotrophic assay

P/P

21

0

21

Concordance

66

Estrogenic

P/N

7

3

10

 

 

/Anti-estrogenic

N/P

2

2

4

False negative

14

activities

N/N

17

13

30

 

 

Total

 

47

18

65

False positive

57

P: positives, N: negatives, P/N: positives in estrogenic and negatives in anti-estrogenic activities, P/P: positives in both estrogenic and anti-estrogenicactivities, N/P: negatives in estrogenic and positives in anti estrogenic activities and N/N: negatives in both estrogenic and anti-estrogenic activities.

Table 2-2. Contingency table based on the RBA giving a maximum concordance

 

ER-binding assay

Total

Index

Rate(%)

P

N

 

 

 

Uterotrophic assay

P/P

21

0

21

Concordance

82

Estrogenic

P/N

7

3

10

 

 

/Anti-estrogenic

N/P

2

2

4

False negative

14

activities

N/N

7

23

30

 

 

Total

 

37

28

65

False positive

23

P: positives, N: negatives, P/N: positives in estrogenic and negatives in anti-estrogenic activities, P/P: positives in both estrogenic and anti-estrogenicactivities, N/P: negatives in estrogenic and positives in anti estrogenic activities and N/N: negatives in both estrogenic and anti-estrogenic activities.

4. Discussion

After the potential of chemicals to disrupt the endocrinebecame apparent, numerous efforts have been made to testand assess chemicals for their endocrine disrupting potential. To detect ER mediated effects, the application of the in vitro ER binding assay and in vivo rodent uterotrophicassay have long been investigated since ER mediation hasbeen considered as a major mechanism of endocrine disruption of exogenous chemicals.

In order to understand the relationship between the in vitro ER binding and in vivo uterotrophic assays andto investigate the biologically meaningful binding potencyfrom an in vitro assay, we compared the results obtainedfrom a receptor binding assay using hERa and the immature rat uterotrophic assay for 65 chemicals spanning avariety of chemicals classes.

For a quantitative comparison of logRBAs and logL-EDs, the log RBA was found to be well correlated withboth log LEDs of estrogenic and anti-estrogenic assayresults r2= 0.67 and 0.79, respectively (Fig. 1). Theseresults strongly suggest that there was a positive relationship between the two assays and that both assays detectsame biological mechanism, i.e., ER mediated biologicalresponses. It also suggests that the result from the utero-trophic assay can be predicted, in some instances, fromthe results of the ER binding assay. However, care mustbe taken to extrapolate in vitro data because some important factors, such as the interaction of the ER with otherendocrine related systems and metabolism of the test chemical in in vivo situation cannot be negligible.

The contingency table analysis of the results from the in vitro ER binding and the in vivo uterotrophic assaysfor all 65 chemicals revealed a relatively good concordanceratio (66%). In this comparison, androgens, phthalates andother classes of chemicals were identified as presenting conflicting results in the two assays under the test conditions.Two androgens, testosterone enanthate and 17α-methyltes-tosterone, were identified as non-ER binders that wereestrogenic in the uterotrophic assay. The potential ofandrogens to stimulate uterine growth in immature female rat is known (Armstrongm et al., 1976).Armstrongm et al.(1976) investigated the effect of testosterone on uterineweight of immature female rat by subcutaneous adminis-tration, and clearly demonstrated the increase of uterineweight and the potential of aromatization to convert testosterone to E2. And the enzymatic activity of aromatase inimmature female rat has been also observed (el-Maasaranyet al., 1991). Testosterone enanthate could be converted totestosterone, i.e. the precursors of estrogens, by hydrolysisin the body. The aromatization of 17α-methyltestosteroneto 17α-methylestradiol has been confirmed in in vitro assayusing human aromatase (de Gooyer et al., 2003). Thus,both testosterone enanthate and 17α-methyltestosteronecan be precursors of estrogens and can elevate the estrogenlevels caused by aromatization of these administratedandrogens, and this would be expected to result in anincrease of the uterine weight. At this moment, the metabolic fate of test chemicals in the immature rat uterotrophicassay cannot be estimated precisely and therefore theimpact of metabolic system on the inconsistency betweenthese assays cannot be fully explained. Accordingly, themetabolic issue on their assay systems should be extensively explored in the future. p-Diethylaminobenzaldehydethat showed the same discrepancy as androgens has beenreported as androgen receptor antagonist in the transcriptional activation assay (Araki et al., 2005). But its anti-androgenic effect on the uterotrophic assay is not knownand the further investigation may be necessary. There wereseventeen chemicals that had ER binding potency but neither estrogenic nor anti-estrogenic activities in the utero-trophic assay. Three benzothiazoles and six phthalateswere included among these chemicals. Benzothiazolesseems to be readily metabolized and at least two benzo-thiazoles that had more than 0.002% of RBA would bemetabolized to 2-mercaptobenzothiazole having 0.00165%of RBA (el Dareer et al., 1989; Elfarra and Hwang, 1990;Fukuoka and Tanaka, 1987). In this study, 9 phthalateswere tested and 6 of them had ER binding affinity rangingfrom -3.49 to -1.15 as logRBA. However, none of phthalates elicited estrogenic or anti-estrogenic responses in theuterotrophic assay in this study. Some phthalates showed ER-mediated activities in in vitro assays but no estrogenicresponse in in vivo model as shown in this study (Honget al., 2005; Zacharewski et al., 1998). These discrepancies between in vitro and in vivo assays in phthalates are probably caused by the deactivation of phthalates to monoalkyl phthalates (Harris et al., 1997; Picard et al., 2001;Zacharewski et al., 1998). The other chemicals with inconsistent response outliers between the in vitro and in vivo assay comparison had relatively weak ER bindingpotencies.

The quantitative comparison found that the 0.00233% of RBA of p-tert-butylphenol was the lowest ER bindingpotency detected in the ER binding assay that elicitedestrogenic or anti-estrogenic activities in the immature ratuterotrophic assay and this RBA is considered as the detection limit of estrogenic or anti-estrogenic activitiesobserved in the uterotrophic assay. The use of this cutoffvalue considerably improved the concordance betweenthe two assays without increasing the false negative rateby excluding the weak ER binders for which estrogenicor anti-estrogenic activities cannot be detected in the in vivo assay.

Our studies revealed that the quantitative relationship between the ER receptor binding assay and uterotrophic assay, and the application of cutoff based on meaningful ER binding affinity can provide the best concordance between two assays. These findings are useful in a tiered approach for identifying chemicals that have potential toinduce ER-mediated effects in in vivo, though it is necessary to consider the metabolic capacity in in vivo situation.

Acknowledgement 

This study was financially supported by Ministry of Economic, Trade and Industry, Japan (METI).


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