Differential effects of phthalate esters on transcriptional activities via human estrogen receptors α and β, and androgen receptor

Toxicology 210 (2005) 223–233

Differential effects of phthalate esters on transcriptional activities via human estrogen receptors ␣ and ␤, and androgen receptor Shinji Takeuchi a , Mitsuru Iida b , Satoshi Kobayashi a , Kazuo Jin a , Tadashi Matsuda c , Hiroyuki Kojima a,∗ a

Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819, Japan EDC Analysis Center, Otsuka Pharmaceutical Company Ltd., Tokushima 771-0195, Japan Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan b

c

Received 21 January 2005; accepted 9 February 2005 Available online 19 March 2005

Abstract Some phthalates are suspected to disrupt the endocrine system, especially by mimicking estrogens. In this study, we characterized the activities of human estrogen receptor ␣ (hER␣), human estrogen receptor ␤ (hER␤), and human androgen receptor (hAR) in the presence of 22 phthalates including 3 of their metabolites using highly sensitive reporter gene assays. Of the 22 compounds tested, several phthalate diesters with alkyl chains ranging in length from C3 to C6 exhibited not only hER␣-mediated estrogenic activity, but also hER␤-mediated antiestrogenic activity in a dose-dependent manner. In addition, we found that some phthalate diesters possess hAR-mediated antiandrogenic activity. However, the phthalates having side chains with very short length (diethyl) or very long length (diheptyl), and three metabolites (monoesters) were found to have no effect on the activities of the three receptors. These results indicate that several phthalate esters simultaneously act as agonists and/or antagonists via one or more hormonal receptors, and interaction of phthalate esters with the estrogen and androgen receptors requires certain size and bulkiness with alkyl groups. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Antiandrogenic activity; Antiestrogenic activity; Estrogenic activity; Human estrogen receptor ␣; Human estrogen receptor ␤; Phthalate

1. Introduction

∗ Corresponding author. Tel.: +81 11 747 2733; fax: +81 11 736 9476. E-mail address: [email protected] (H. Kojima).

Exogenous compounds such as endocrine-disrupting chemicals (EDCs) are of special interest because they mimic, block or in some way alter the activities of endogenous hormones. Most of the EDCs are derived from agricultural, industrial, and household sources in

0300-483X/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2005.02.002

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the environment. Several studies have demonstrated that EDCs are capable of interacting with estrogen and androgen receptors (AR), suggesting that the adverse effects elicited by these substances may be mediated via hormonal receptors (Bonefeld-Jorgensen et al., 2001; Gaido et al., 1999, 2000; Kelce et al., 1995; Nishihara et al., 2000). Therefore, these chemicals pose a potential threat to human and wildlife reproduction (Colborn et al., 1993). Phthalates are commonly used in large quantities as plasticizers to impart flexibility to a variety of plastics. Because of their leachability from plastics, phthalates have been identified in several environmental sectors, polyvinyl chloride medical devices (Inoue et al., 2002), fatty foods (Sharman et al., 1994), indoor air (Rudel et al., 2003; Saito et al., 2002), and house dust (Rudel et al., 2003). There are many kinds of phthalates formed through the ester binding of various side chains to phthalic acid. Some phthalates are suspected to disrupt the endocrine system, especially by mimicking estrogens. Actually, several in vitro studies have shown that phthalates such as di-n-butyl phthalate (DBP) and butylbenzyl phthalate (BBeP) are capable of binding to estrogen receptor ␣ (ER␣), inducing ER␣mediated gene expression, and enhancing the proliferation of MCF-7 human breast cancer cells expressing abundant ER␣ (Andersen et al., 1999; Harris et al., 1997; Jobling et al., 1995; Nishihara et al., 2000; Soto et al., 1995; Zacharewski et al., 1998). Estrogenic responses are mediated via two separate estrogen receptors; ER␣ (Green et al., 1986) and ER␤ (Kuiper et al., 1996; Ogawa et al., 1998), which are members of the nuclear receptor family. ER␣ and ER␤ have a similar affinity for estrogen, but exhibit distinct tissue distribution and physiological functions (Couse and Korack, 1999). Therefore, the complete estimation of the estrogenicity of chemicals requires data concerning their effects on both ER␣ and ER␤, but there have been no reports on the ER␤-mediated estrogenic responses of various phthalates. In addition, DBP, di-(2-ethylhexyl) phthalate (DEHP) and dicyclohexyl phthalate (DcHP) were reported to bind not only to ER␣, but also weakly to the androgen receptor, which is another member of the nuclear receptor family (Satoh et al., 2001). With regard to effects of phthalates on transcriptional gene expression via AR, only one study has demonstrated that estrogenic BBeP is also antiandrogenic using in vitro yeast-based assays (Sohoni and Sumpter, 1998).

Therefore, it is still unclear whether various phthalates act as agonists or antagonists via AR. A series of in vitro assays has been developed to detect EDC activity as a first screening test. We have developed a novel screening method using Chinese hamster ovary (CHO) cells, which is highly sensitive and specific to chemicals having hER␣/␤ and hAR activities (Kojima et al., 2003, 2004), and recently screened 200 pesticides for activities of hormonal receptors, and demonstrated that various pesticides possess estrogenic activity and/or antiandrogenic activity (Kojima et al., 2004). In the present study, we aimed to elucidate the structure–activity relationship of phthalates suspected as EDCs, and determined the effects of 19 phthalate diesters having distinctive side chains, such as various kinds of aromatic rings and alkyl chains of various lengths, on ER␣/␤ and AR activities. In addition, we examined three primary metabolites of phthalates, mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBeP), and mono-(2-ethylhexyl) phthalate (MEHP), which are mainly detected in human urine (Blount et al., 2000). Consequently, we found that several phthalate diesters induce ER␣mediated transcriptional activity, but simultaneously inhibit ER␤- and AR-mediated transcriptional activities. Such an activity pattern is similar to that of the methoxychlor metabolite, 2,2-bis-(p-hydroxyphenyl)1,1,1-trichloroethane (HPTE), as reported by Gaido et al. (2000). In this paper, we provide the first evidence that various phthalates demonstrate different transcriptional activity via ER␣, ER␤, and AR depending on chemical structure, and act as agonists and/or antagonists via one or more hormonal receptors.

2. Materials and methods 2.1. Chemicals, biochemicals, and cells 17␤-Estradiol (E2 ; >97% pure), 5␣-dihydrotestosterone (DHT; 95% pure), and tamoxifen citrate (98% pure) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). The structures, sources, and purity of the 22 phthalate esters tested in the present study are listed in Fig. 1. The bisphenolic metabolite of methoxychlor, 2,2-bis-(p-hydroxyphenyl)-1,1,1trichloroethane, was synthesized following the method of Gaido et al. (1999), and was more than 99% pure

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Fig. 1. Chemical structures, source, and purity of phthalate esters used in the present study.

as determined by gas chromatography. Dimethyl sulfoxide (DMSO) was used as a vehicle and purchased from Wako Pure Chemical Industries Ltd., and all compounds used were dissolved in DMSO at a concentration of 10−2 M. Dulbecco’s modified Eagle medium plus Ham’s F-12 nutrient mixture (D-MEM/F-12) and a penicillin–streptomycin solution (antibiotics) were obtained from GIBCO-BRL (Rockville, MD, USA). Fetal bovine serum (FBS) and charcoal–dextran-treated FBS (CD-FBS) were obtained from Hyclone (Logan, UT, USA). Bovine serum albumin (BSA) and 4-methylumbelliferyl-␤-d-galactoside (4-MUG) were obtained from Sigma–Aldrich (St. Louis, MO, USA). CHO-K1 cells obtained from the Dainippon Pharmaceutical Co. (Osaka, Japan) were maintained in DMEM/F-12 supplemented with 10% FBS and antibiotics. All compounds were diluted to the desired concentrations in phenol red-free D-MEM/F-12 immediately before use. The final solvent concentration in the culture medium did not exceed 0.1%, and this concentration did not affect cell yields.

2.2. Plasmids The expression plasmids pcDNAER␣, pcDNAER␤, and pZeoSV2AR as well as the reporter plasmids pGL3-tkERE and pIND-ARE were prepared as previously described (Kojima et al., 2003, 2004). The internal control plasmid pCMV␤-Gal was purchased from Clontech (Palo Alto, CA, USA). 2.3. Transfection of plasmids to cells and luciferase activity assay The host CHO-K1 cells were plated in 96-well microtiter plates (Nalge, Nunc, Denmark) at a density of 8400 cells per well in phenol red-free D-MEM/F-12 containing 5% CD-FBS (complete medium) 1 day before transfection. For detection of hER␣ or hER␤ activity, cells were transfected with 5 ng pcDNAER␣ or 5 ng pcDNAER␤, 50 ng pGL3-tkERE, and 5 ng pCMV␤Gal per well using the FuGENE 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN, USA). For detection of hAR activity, cells were transfected

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with 2.5 ng pZeoSV2AR, 50 ng pIND-ARE, and 5 ng pCMV␤-Gal per well using the FuGENE 6 transfection reagent. After a 3-h transfection period, cells were dosed with various concentrations of test compounds or with 0.1% DMSO (vehicle control) in complete medium. To avoid cell toxicity by the phthalates, assays were performed for phthalates at concentrations less than 10−5 M. For the measurement of the antagonistic activities via hER␣, hER␤, and hAR, the test compound was added to the cell cultures together with 10−11 M E2 , 10−10 M E2 , and 10−10 M DHT, respectively. After an incubation period of 24 h, cells were rinsed with phosphate-buffered saline (pH 7.4) and lysed with passive lysis buffer (50 ␮L/well; Promega, Madison, WI, USA). We measured the firefly luciferase activity with a MiniLumat LB 9506 luminometer (Berthold, Wildbad, Germany) in one reaction tube with a 5-␮L aliquot of the cell lysate using the Luciferase Assay System (Promega), following the manufacturer’s instructions. The luciferase activity was normalized based on the ␤-galactosidase activity for each treatment. Results are expressed as means ± S.D. from at least threeindependent experiments. 2.4. ␤-Galactosidase activity assay The measurement of ␤-galactosidase activity was performed by a fluorescence method. Ninety microliters of the ␤-galactosidase substrate solution (0.2 mM 4-MUG, 1 mM MgCl2 , 100 mM NaCl, 0.1% BSA, 10 mM Na–phosphate buffer, pH 7.0) was added to 10 ␮L of the cell lysate in the second 96-well plate. After incubation at 37 ◦ C for 30 min, 100 ␮L of the stop solution (100 mM glycine–NaOH, pH 10.3) was added to a reaction mixture. The fluorescence was determined at 460 nm with a fluorescence microplate reader, fmax (Molecular Devices, Sunnyvale, CA, USA) using an excitation wavelength of 355 nm. 2.5. Evaluation of agonistic and antagonistic activities In order to estimate the potency of the receptoragonistic activity of the compounds tested, the luminescence intensity of the assay was represented in a dose–response curve. The concentration of the compound equal to 20% of the maximal response of E2

or DHT was evaluated from a dose–response curve of the luminescence intensity, and expressed as 20% relative effective concentration (REC20 ). The results for the receptor-antagonistic activities of the compound were expressed as 20% relative inhibitory concentration (RIC20 ); that is, the concentrations of the test compounds showing 20% inhibition of the activities induced by 10−11 M E2 , 10−10 M E2 , and 10−10 M DHT for ER␣, ER␤, and AR, respectively.

3. Results 3.1. Agonistic activities of the 22 phthalates via ER␣ and ER␤ Fig. 2A show the relative estrogenic activities of 19 phthalate diesters and 3 monoesters via ER␣ and ER␤ at a concentration of 10−5 M compared with 100% activity defined as the activity achieved with 10−9 M E2 . Nine of 22 compounds tested were found to induce ER␣-mediated estrogenic activity over 20% of the maximum activity with E2 . The relative potencies of their estrogenic activities descended in the order BBeP > DcHP > DiHP > DiBP, DBP, DPeP, DHP > DEHP, DiHepP. The slight activity smaller than REC20 was recognized in DiPrP, DPrP, DAP, or DHepP. On the other hand, among all the compounds tested, only BBeP showed estrogenic activity via ER␤, though its activity was smaller than that via ER␣. The three monoesters were inactive in all of the assays. In Fig. 2B–J, all of the nine selected phthalates showed enhancement of transcriptional activity in a dose-dependent manner, but these estrogenic responses at 10−5 M were completely inhibited by 10−6 M of tamoxifen, which is known as an ER antagonist. In addition, the methoxychlor metabolite, HPTE, also showed estrogenic activities via ER␣ and ER␤ at an approximately 1000-fold lower concentration than those of BBeP, which has the most potent estrogenic activity among the phthalates (Fig. 2K). 3.2. Antagonistic activities of the 22 phthalates via ER␣ and ER␤ Fig. 3A shows the antiestrogenic activities via ER␣ and ER␤ in the presence of E2 . Although none of the 22 compounds tested showed antiestrogenic ac-

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tivity via ER␣, 7 phthalates (DcHP, BBeP, DPeP, DiHP, DHP, DEHP, and DiHepP) showed antiestrogenic activities over the 20% inhibitory effect in the hER␤-transactivation assay with 10−10 M E2 . The

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three monoesters were inactive in all of the assays. Dose–response curves of antiestrogenic activity via ER␤ for the seven compounds together with HPTE are shown in Fig. 3B–H. Among the seven compounds,

Fig. 2. Estrogenic effects of 22 phthalates and dose–response curves of selected phthalates in the hER␣- and hER␤-transactivation assays. CHO cells were transiently transfected with an expression plasmid for human ER␣ or ER␤ as well as a reporter-responsive firefly luciferase plasmid and a constitutively active ␤-galactosidase expression plasmid. (A) Cells were treated with 10−5 M of 19 phthalates and their 3 metabolites. (B–L) Cells were treated with increasing concentrations of DiBP, DBP, DcHP, BBeP, DPeP, DiHP, DHP, DEHP, DiHepP, HPTE, and E2 . The results were shown as agonistic activity via ER␣ () or via ER␤ (䊉). Cells were also treated with 10−5 M of these selected phthalates in the presence of 10−6 M tamoxifen, an ER antagonist, and the results were shown as agonistic activities via ER␣ (♦) or via ER␤ (). The firefly luciferase activity was normalized based on the ␤-galactosidase activity. Values represent the means ± S.D. of three-independent experiments and are presented as the percentage of the response, compared with 100% activity defined as the activity achieved with 10−9 M E2 .

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Fig. 3. Antiestrogenic effects of 22 phthalates and dose–response curves of selected phthalates in the hER␣- and hER␤-transactivation assays. CHO cells were transiently transfected with an expression plasmid for human ER␣ or ER␤ as well as a reporter-responsive firefly luciferase plasmid and a constitutively active ␤-galactosidase expression plasmid. (A) Cells were treated with 10−5 M of 19 phthalates and their 3 metabolites in the presence of 10−11 M E2 for ER␣ or 10−10 M E2 for ER␤. (B–I) In ER␤ assay, cells were treated with increasing concentrations of DcHP, BBeP, DPeP, DiHP, DHP, DEHP, DiHepP, and HPTE to detect ER␤-antagonistic activity. The firefly luciferase activity was normalized based on the ␤-galactosidase activity. Values represent the means ± S.D. of three-independent experiments and are presented as the percentage of the response, compared with 100% activity defined as the activity achieved with 10−11 M E2 for ER␣ or 10−10 M E2 for ER␤.

DcHP exhibited the most potent inhibitory effect via ER␤. HPTE also showed antiestrogenic activity via ER␤, but not via ER␣ (Fig. 3I). 3.3. Antagonistic activities of 22 phthalates via AR In the AR assay, none of the compounds tested showed androgenic activity (data not shown). We examined 22 phthalates for their inhibitory effect on

the androgenic activity induced by DHT (10−10 M). As shown in Fig. 4A, of the 22 tested chemicals, 9 phthalates (DAP, DiBP, DBP, DcHP, BBeP, DPeP, DiHP, DHP, and DiHepP) showed antiandrogenic activities over the 20% inhibitory effect in the hAR-transactivation assay with 10−10 M DHT. Dose–response curves of antagonistic activity via hAR for the nine compounds together with HPTE are shown in Fig. 4B–K. Among the phthalates, BBeP was found

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Fig. 4. Antiandrogenic effects of 22 phthalates and dose–response curves of selected phthalates in the hAR-transactivation assays. CHO cells were transiently transfected with an expression plasmid for human AR as well as a reporter-responsive firefly luciferase plasmid and a constitutively active ␤-galactosidase expression plasmid. (A) Cells were treated with 10−5 M of 19 phthalates and their 3 metabolites in the presence of 10−10 M DHT. (B–K) Cells were treated with increasing concentrations of DAP, DiBP, DBP, DcHP, BBeP, DPeP, DiHP, DHP, DiHepP, and HPTE to detect AR antagonistic activity. The firefly luciferase activity was normalized based on the ␤-galactosidase activity. Values represent the means ± S.D. of three-independent experiments and are presented as the percentage of the response, compared with 100% activity defined as the activity achieved with 10−10 M DHT.

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Table 1 Comparison of agonistic and antagonistic activities of 19 phthalates and 3 metabolites via ER␣, ER␤, and AR Compounds

E2 DMP DEP DiPrP DPrP DAP DiBP DBP DcHP DPhP BBeP DPeP DiHP DHP DiHepP DEHP DHepP DOP DiNP DiDP MBP MBeP MEHP HPTE

Agonistic activity, REC20 a (M)

Antagonistic activity, RIC20 b (M)

ER␣

ER␤

ER␤

AR

2.5 × 10−12

5.3 × 10−12

–c – – – – 6.1 × 10−6 6.0 × 10−6 2.8 × 10−6 – 1.7 × 10−6 5.7 × 10−6 2.8 × 10−6 5.6 × 10−6 6.3 × 10−6 5.5 × 10−6 – – – – – – – 2.0 × 10−9

– – – – – – – – – 3.8 × 10−6 – – – – – – – – – – – – 2.7 × 10−9

– – – – – – – 2.5 × 10−6 – 9.4 × 10−6 5.7 × 10−6 3.2 × 10−6 4.0 × 10−6 5.3 × 10−6 3.4 × 10−6 – – – – – – – 1.2 × 10−7

– – – – 1.2 × 10−6 6.2 × 10−6 4.8 × 10−6 3.8 × 10−6 – 2.9 × 10−6 5.7 × 10−6 3.4 × 10−6 3.5 × 10−6 3.8 × 10−6 – – – – – – – – 4.5 × 10−8

20% relative effective concentration, the concentration of the test compound showing 20% of the agonistic activity of 10−9 M E2 . 20% relative inhibitory concentration, the concentration of the test compound showing 20% of the antagonistic activity of 10−10 M E2 via ER␤ or 10−10 M DHT via AR, respectively. c No effect (REC or RIC > 10−5 M). 20 20 a

b

to be most antiandrogenic and HPTE was found to be more antiandrogenic than the most active phthalate. The agonistic or antagonistic effects of 22 phthalates and HPTE via ER␣, ER␤, and AR are summarized in Table 1 as REC20 or RIC20 , respectively. None of the tested compounds in the selected dose range induced cytotoxicity or suppression of ␤-Gal activity (data not shown).

4. Discussion There have been a number of in vitro studies on the estrogenic effect of some phthalates by ER␣-binding assays as well as proliferation assays using MCF-7 cells and ER␣-dependent transcription assays (Andersen et al., 1999; Harris et al., 1997; Jobling et al., 1995; Nishihara et al., 2000; Soto et al., 1995; Zacharewski

et al., 1998); however, ER␤- and AR-dependent transcription induced by phthalates has not been fully elucidated. To examine the relationship between the chemical structures of phthalates and their endocrinedisrupting effects, we investigated the potential receptor activities of 22 phthalates, including 3 metabolites, by highly sensitive reporter gene assays using CHOK1 cells transfected with expression vectors for human ER␣, ER␤, and AR along with the appropriate receptor plasmids. As shown in Table 1, we succeeded to reveal that several phthalates exhibited not only agonistic activities via ER␣, but also antagonistic activities via ER␤ and AR, and there were structure– activity relationships of the phthalates, as described below. Among the 22 compounds tested, 9 compounds; DiBP, DBP, DcHP, BBeP, DPeP, DiHP, DHP, DEHP, and DiHepP, each of which has alkyl chains between

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C3 and C6 in length, were found to induce ER␣mediated estrogenic activity. Moreover, these estrogenic responses at 10−5 M were suppressed by the ER antagonist, tamoxifen, suggesting that the estrogenic activities of phthalates might be induced by binding to ER␣ (Fig. 2). Nakai et al. (1999) have reported that among the series of di-n-alkyl phthalates, the highest affinity for ER␣ was attained by DBP, and phthalates with alkyl groups longer than the hexyl (C6 ) and shorter than the ethyl (C2 ) were almost completely inactive in ER binding, a finding, which appears to support our results. In addition to the phthalates already reported to be estrogenic, we were able to find here that DPeP and DiHepP possess ER␣-agonistic activities. This suggests that our reporter gene assays are highly sensitive and specific. In the ER␤ assay, seven phthalates (DcHP, BBeP, DPeP, DiHP, DHP, DEHP, and DiHepP) surprisingly showed antiestrogenic activity (Fig. 3). It is worth noting that these seven phthalates also have alkyl chains ranging in length from C4 to C6 , and have both ER␣agonistic and ER␤-antagonistic activities. However, the overall structure of the ER␤-ligand binding domain is very similar to that of ER␣-ligand one, and most of the compounds demonstrate similar binding affinities and transcriptional activities with ER␣ and ER␤ (Kuiper et al., 1997). Actually, we also showed recently that a variety of pesticides have both ER␣and ER␤-agonistic activities (Kojima et al., 2004). On the other hand, some chemicals have been reported to have differing activities via the two subtypes of ER. For example, the bisphenolic metabolite of methoxychlor, 2,2-bis-(p-hydroxyphenyl)-1,1,1trichloroethane (HPTE) (Gaido et al., 1999), R,Renantiomer of tetrahydrochrysene (Sun et al., 1999), and phytochemicals such as ferutinine, a sesquiterpenoid (Ikeda et al., 2002), have been shown to have differing activities via ER␣ and ER␤ in human hepatoma (HepG2) cells, human endometrial cancer (HEC-1) cells, and human embryonic kidney carcinoma (293T) cells, respectively. In the present study, we also confirmed that HPTE acts via ER␣ as an agonist, but via ER␤ as both an agonist and antagonist in CHO cells (Figs. 2K and 3I). And therefore, the results for HPTE suggest that the differential activities of phthalates are specific not only in CHO cells, but possibly in other cells, although several papers have reported that agonistic and antagonistic responses via ER are dependent

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on cell type, and reporter plasmid specificity (Jones et al., 1999; Paech et al., 1997; Watanabe et al., 1997). There is one possibility that the helix 12 region is involved in the mechanism of the differing activities of phthalates via ER␣ and ER␤ and plays an important role. The helix 12 is present in both estrogenic receptors, and the agonist orientation of helix 12 in ER␤ has been reported to be unstable, and thus easier to be antagonistic than that in ER␣ (Gaido et al., 2000). Though the differing activities of seven phthalates via ER␣ and ER␤ were very weak in comparison with that of HPTE (Table 1), phthalates may also be able to stabilize helix 12 in the agonist orientation for ER␣ but not for ER␤. This indicates that several phthalates might act as an ER␣-selective agonist and an ER␤-selective antagonist. A recent paper has reported that BBeP had agonistic activity via ER␣, but not via ER␤ in 293T cells, and therefore BBeP acts as an ER␣-selective agonist (Fujita et al., 2003). However, in the present study using CHO cells, BBeP was found to have both estrogenic and antiestrogenic activities via hER␤ in addition to estrogenic activity via ER␣ (Table 1). The liganddependent activation of ERs requires ligand-dependent association of protein cofactors and basal transcription factors (McKenna et al., 1999), and the expression levels of these factors differ from cell to cell, which may contribute to the cell type-specific transcriptional activation induced by certain ligands (Montano et al., 1995). This suggests that the discrepancy in the estrogenicity of BBeP between 293T and CHO cells can be explained by cell context. We also found that nine phthalates (DAP, DiBP, DBP, DcHP, BBeP, DPeP, DiHP, DHP, and DiHepP) possessed antiandrogenic activity (Fig. 4), while none of the tested compounds showed androgenic activity. This result suggests that the phthalates, which have the alkyl chains ranging in length from C3 to C6 , have antiandrogenic activities via hAR. Therefore, six phthalates; DcHP, BBeP, DPeP, DiHP, DHP, and DiHepP, show simultaneously both ER␣-agonistic and ER␤- and AR-antagonistic activities. We and other researchers have already shown that a lot of environmental estrogens are also antiandrogenic (Kojima et al., 2004; Sohoni and Sumpter, 1998; Vinggaard et al., 1999). The transcriptional activities of these active phthalates via the estrogen and/or androgen receptors require certain size and bulkiness with alkyl groups.

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MEHP, MBP, and MBeP are the monoester derivatives of DEHP, DBP, and BBeP, respectively. Some metabolic studies indicated that orally administrated phthalate diesters are rapidly hydrolyzed to their corresponding monoesters by non-specific esterases in the gut and other tissues (Albro et al., 1973). Recently, Okubo et al. (2003) have reported that MBP and MBeP showed inhibitory effects on the proliferation of MCF7 human breast cancer cells at high concentrations (>10−4 M). In the present study, however, we did not observe any ER␣-antagonistic activity by the phthalate monoesters because 10−4 M of tested phthalates decreased the ␤-Gal activity of the toxicity control (data not shown). Although most of the phthalates, which have alkyl chains ranging in length from C3 to C6 , demonstrated ER␣/␤ and AR activities, only DPhP (C4 ) was inactive (Table 1, Fig. 1). This suggests that the diphenyl residue in DPhP may impede the physical interaction of DPhP with ER␣/␤ and AR, being distinct from the case of cyclohexyl residue in DcHP. Meanwhile, the phthalates with alkyl chains of C1 , C2 , C7 , C8 , or C9 in length were also inactive (Table 1). Taken together, the present study demonstrate for the first time that several phthalates currently used in large quantities as plasticizers simultaneously induce ER␣-mediated transcriptional activity, and inhibit ER␤- and AR-mediated transcriptional activity, and that agonistic and antagonistic activities of the phthalate esters via ER␣/␤, and AR are restricted to dialkyl phthalates having alkyl chains ranging in length from C3 to C6 , except for DPhP. Thus, phthalates may disrupt endocrine systems by acting as agonists and/or antagonists via one or more hormonal receptors.

Acknowledgements We thank Dr. Shinichi Kudo of Hokkaido Institute of Public Health for his help in reviewing the manuscript. This study was supported by the Hokkaido government.

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