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Comparative Study of the Endocrine-Disrupting Activity of Bisphenol A and 19 Related Compounds

Comparative Study of the Endocrine-Disrupting Activity of Bisphenol A and 19 Related Compounds Abstract The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17β-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5α-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3′-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities. estrogenic activity, anti-androgenic activity, thyroid hormonal activity, bisphenol A, bisphenol derivative, human breast cancer cell line MCF-7, rat pituitary cell line GH3 Bisphenol A (2,2-bis-(4-hydroxyphenyl)propane; BPA) is an industrial raw material for polycarbonate and epoxy resins, and contaminates the end products. It can be detected in liquid from canned vegetables and in the saliva of patients treated with dental sealants (Brotons et al., 1995; Hashimoto et al., 2001; Olea et al., 1996). BPA has the ability to bind DNA after metabolic activation (Atkinson and Roy, 1995). BPA also shows estrogenic activity towards cell lines such as estrogen-responsive breast cancer cell line MCF-7 cells, and endocrine-disrupting effects in vivo (Ashby and Tinwell, 1998; Ashby et al., 2000; Gaido et al., 1997; Kim et al., 2001; Krishnan et al., 1993; Matthews et al., 2001; Tinwell et al., 2000). Bisphenol B (2,2-bis-(4-hydroxyphenyl)butane; BPB), bisphenol F (4,4′-dihydroxydiphenylmethane; BPF), bisphenol AD (ethylidenebisphenol; BPAD), bisphenol AF (1,3-trifluoro-2,2-bis-(4-hydroxyphenyl)propane; BPAF), tetramethylbisphenol A (2,2-bis-(3,5-dimethyl-4-hydroxyphenyl)propane; TMBPA), 3,3′-dimethylbisphenol A (DMBPA), and bisphenol S (bis-(4-hydroxyphenyl)sulfone; BPS) are also used as materials for polycarbonate resin. Tetrabromobisphenol A (2,2-bis-(3,5-dibromo-4-hydroxyphenyl)propane; TBBPA), a halogenated derivative of BPA, is also widely used throughout the world as a flame retardant in numerous products. TBBPA was developed as a relatively nontoxic flame retardant (Helleday et al., 1999; Sellström and Jansson, 1995; Sjödin et al., 2001; Thomsen et al., 2001; Watanabe et al., 1983). Tetrachlorobisphenol A (2,2-bis-(3,5-dichloro-4-hydroxyphenyl)propane; TCBPA) has been found in the effluent from waste-paper recycling plants (Fukazawa et al., 2001). Kuruto-Niwa et al. (2002) reported that estrogenic polychlorinated BPAs were not easily biodegraded. However, the endocrine-disrupting activity of these halogenated BPA has not been reported in detail. Endocrine-active chemicals arise from many different sources, including pesticides, industrial chemicals, pharmaceuticals, and phytochemicals. These chemicals are widely distributed in the environment, and are able to mimic or antagonize the biological functions of natural hormones. Chlorinated insecticides, such as kepone, o,p′-DDT, dieldrin and methoxychlor, and compounds used in the plastics and detergent industries, such as alkylphenols and BPA, are known to have estrogenic activity (Andersen et al., 1999). p,p′-DDE, a metabolite of p,p′-DDT, vinclozolin, an antifungal agent, and chlornitrofen, fenitrothion and fenthion, insecticides, have anti-androgenic activity (Gray et al., 1999; Kelce et al., 1995; Kitamura et al., 2003a; Kojima et al., 2003; Tamura et al., 2001). Some hydroxy-PCBs such as 4,4′-dihydroxy-3,3′,5,5′-tetrachlorobiphenyl are reported to show anti-thyroid hormonal activity in addition to estrogenic activity (Cheek et al., 1999; Connor et al., 1997; Korach et al., 1988; Lans et al., 1994). Interactions of estrogenic and anti-androgenic compounds with the respective hormone receptors have been demonstrated to account for most of the endocrine-disrupting actions, and these chemicals can alter reproductive development in mammals. It is also necessary to consider the activity of the metabolites of these chemicals. In the metabolism of BPA, the 3-hydroxyl metabolite (BPA catechol) was formed by human and rat liver microsomes and exhibited estrogenic activity (Elsby et al., 2001). The glucuronide metabolite proved to have no estrogenic activity (Matthews et al., 2001; Pottenger et al., 2000). However, the relationship between the structure and activity of BPA derivatives, including metabolites, remains to be fully understood. In this report, endocrine-disrupting activity, i.e., estrogenic, anti-estrogenic, androgenic, anti-androgenic, thyroid hormonal, and anti-thyroid hormonal activities of BPA and related compounds were examined using hormone-responsive reporter assays: the human breast cancer cell-line MCF-7 for estrogenic activity, the mouse fibroblast cell line NIH3T3 for androgenic activity, and the pituitary cell line GH3 for thyroid hormonal activity. Twenty BPA derivatives were tested in this study (Fig. 1). We found that BPA and some of its derivatives exhibited estrogenic as well as anti-androgenic activity. TBBPA, TCBPA, TMBPA, and DMBPA showed significant thyroid hormonal activity. The structure-activity relationship of BPA derivatives is discussed. FIG. 1. View largeDownload slide Structures of bisphenol A and related compounds tested in this study. FIG. 1. View largeDownload slide Structures of bisphenol A and related compounds tested in this study. MATERIALS AND METHODS Chemicals. TBBPA, TCBPA, BPA, BPAD, BPB, BPF, BPAF, BPS, diphenylmethane (DPM), 4-hydroxydiphenylmethane (HDM), DMBPA, TMBPA, p-isopropylphenol (IPP), 1,1-bis-(4-hydroxyphenyl)cyclohexane (BPCH), α,α′-bis-(4-hydroxyphenyl)-1,4-diisopropylbenzene (BPDB), dihydrotestosterone (DHT), and flutamide were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), 2-(4-hydroxyphenyl)-2-phenylpropane (HPP) from Nacalai Tesque, Inc. (Kyoto, Japan), 2-(3,4-dihydroxyphenyl)-2-phenylpropane (BPA catechol) from Wako Pure Chemical Co. Ltd., (Osaka, Japan), 2,2-diphenylpropane (DPP) from Aldrich Chemical Co. (Milwaukee, WI), and L-3,5,3′-triiodothyronine (T3) and 17-β-estradiol (E2) from Sigma Chemical Co. (St. Louis, MO). 2,2-bis-(4-Hydroxyphenyl)-1-propanol (BPA ol) and 2,2-bis-(4-hydroxyphenyl)-1-propionic acid (BPA carboxylic acid) were synthesized by the methods of Spivack et al. (1994). Cell culture. Human breast cancer cell-line MCF-7 cells were maintained in DMEM (Sigma Chemical Co.) containing penicillin and streptomycin with 5% fetal bovine serum (FBS; Life Technologies, Rockville, MD). Rat pituitary cancer cell-line GH3 cells were maintained in DMEM/F12 mixed medium (Sigma Chemical Co.) containing penicillin and streptomycin with 8% horse serum (Life Technologies) and 2% FBS. Mouse fibroblast cell-line NIH3T3 cells were maintained in DMEM (Sigma Chemical Co.) containing penicillin and streptomycin with 5% calf serum (Life Technologies). Assay of estrogenic activity of BPA and related compounds. ERE-luciferase reporter assay using MCF-7 cells was performed according to the previously reported method (Kitamura et al., 2003a). Briefly, transient transfections in MCF-7 cells were performed using Transfast (Promega Co., Madison, WI), according to the manufacturer's protocol. Transfections were done in 48-well plates at 2 × 104 cells/well with 0.3 μg of p(ERE)3-SV40-luc and 2 ng of phRL-CMV (Promega Co.) as an internal standard (Sugihara et al., 2000). Twenty-four hours after addition of the sample (final concentration, 10−4 – 10−9 M), the assay was performed with a Dual Luciferase assay kit (Promega Co.). Firefly luciferase reporter activity was normalized to renilla luciferase activity from phRL-CMV, to control for the cytotoxic effects of compounds, as well as differences in transfection efficiency between culture wells. For the assay of anti-estrogens, the inhibitory effect of BPA and related compounds on the estrogenic activity of E2 at the concentration of 1 × 10−10 M was examined. Assay of androgenic activity of BPA and related compounds. Assay of androgenic activity was performed by means of ARE-luciferase reporter assay using NIH3T3 cells without expressing AR. Cells were maintained in phenol red-free DMEM (Sigma Chemical Co.) containing penicillin, streptomycin, and dextran-charcoal-treated calf serum for 2–3 days. Transient transfections in NIH3T3 cells were performed using Transfast according to the manufacturer's protocol. Transfections were done in 48-well plates at 2 × 104 cells/well with 0.3 μg of p(ARE)2-luc, 0.05 μg of pSG5-hAR, and 2 ng of phRL-CMV as an internal standard (Kitamura et al., 2003c). Twenty-four hours after addition of the sample (final concentration, 10−4 – 10−8 M) dissolved in 10 μl of ethanol, the assay was performed with a Dual Luciferase assay kit according to the manufacturer's protocol. Firefly luciferase reporter activity was normalized to renilla luciferase activity from phRL-CMV. For the assay of anti-androgenic activity, the inhibitory effect of BPA and related compounds on the androgenic activity of 1 × 10−10 or 1 × 10−11 M DHT was examined. Assay of thyroid hormonal activity of BPA and related compounds. Assay of thyroid hormonal activity was performed by measuring the induction of growth hormone production in GH3 cells as previously reported (Kitamura et al., 2002). Briefly, the cells were seeded in 24-well plates at 1 × 104 cells/well and chemicals were added the next day. Two days later, growth hormone in the culture medium was measured. For the assay of anti-thyroid hormonal activity, the inhibitory effect of BPA and related compounds on the activity of 1 × 10−7 or 1 × 10−8 M T3 was examined. Assay of estrogenic activity in vivo (uterotrophic assay). B6C3F1 female mice obtained from Charles River Co. (Kanagawa, Japan) were used. They were surgically ovariectomized at four weeks old. At the age of eight weeks, they were divided into 14 groups each consisting of five animals. The mice were treated once a day for three days with ip doses of 0.2 ml of vehicle (Panacete 810, Nippon Oils and Fats Co., Ltd., Tokyo, Japan), E2 (50 μg/kg/day), TCBPA, TBBPA, or BPA (20, 100, 300, or 500 mg/kg/day). Animals were sacrificed under anesthesia and the uterus was dissected and weighed. Data analysis. Multiple comparisons were made by ANOVA followed by Scheffe's test. EC50 values and IC50 values were calculated by fitting data to the logistic equation. RESULTS Estrogenic Activity of BPA and Related Compounds Estrogenic activity of BPA and related compounds was examined using ERE-luciferase reporter assay in MCF-7 cells. BPA, BPB, BPF, BPA ol, BPA carboxylic acid, HPP, HDM, TMBPA, BPA catechol, DDM, TBBPA, DMBPA, and TCBPA all exhibited estrogenic activity in the estrogen screening assay, but the activities varied markedly from compound to compound. TCBPA, BPAF, BPB, and HPP showed significant estrogenic activity in the concentration range of 1 × 10−7 (1 × 10−8 in the case of TCBPA) − 1 × 10−4 M. BPA and HDM also showed estrogenic activity at higher concentrations. However, DPP and DPM, which lack a hydroxyl group, were inactive (Fig. 2). DMBPA, TMBPA, BPF, BPAD, BPA catechol, BPA ol, BPA carboxylic acid, and TBBPA also showed estrogenic activity at 1 × 10−6 − 1 × 10−4 M. When ICI 182,780, a pure estrogen receptor antagonist, was added at the concentration of 1 × 10−8 M, the estrogenic activities of these compounds were markedly inhibited (data not shown). The EC50 values of estrogenic activity of the positive compounds are shown in Table 1. TCBPA showed the highest activity, followed by BPAF, BPB, HPP, BPCH, HDM, DMBPA, BPA, TMBPA, BPAD, and BPF. When the propane bridge of BPA was substituted with a hydrophilic group, the estrogenic activities of these compounds were markedly inhibited, showing the specific nature of this response (compare BPA ol and BPA carboxylic acid with BPA). On the contrary, the activity was increased by substitution with a hydrophobic group (compare BPA, BPAF and BPB with BPF). BPA catechol, 3-hydroxyl derivative of BPA, exhibited little estrogenic activity. DPP, the dehydroxylated derivative of BPA, and DPM were negative in the estrogen screening assay. IPP, lacking the B-phenyl ring, showed no activity. Thus, at least one 4-hydroxyl group of BPA derivatives is essential for estrogenic activity. The second phenyl group attached the 2-position of propane is also necessary. FIG. 2. View largeDownload slide Estrogenic activity of bisphenol A (BPA) and related compounds in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Estrogenic activity was expressed as a relative activity with respect to the control using MCF-7 cells. BPB; bisphenol B, BPAF; bisphenol AF, TCBPA; tetrachlorobisphenol A, 4-HDM; 4-hydroxydiphenylmethane, HPP; 2-(4-hydroxyphenyl)-2-phenylpropane, DPM; diphenylmethane, DPP; 2,2-diphenylpropane, E2; 17-β-estradiol. *p < 0.05, **p < 0.01 compared with control. FIG. 2. View largeDownload slide Estrogenic activity of bisphenol A (BPA) and related compounds in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Estrogenic activity was expressed as a relative activity with respect to the control using MCF-7 cells. BPB; bisphenol B, BPAF; bisphenol AF, TCBPA; tetrachlorobisphenol A, 4-HDM; 4-hydroxydiphenylmethane, HPP; 2-(4-hydroxyphenyl)-2-phenylpropane, DPM; diphenylmethane, DPP; 2,2-diphenylpropane, E2; 17-β-estradiol. *p < 0.05, **p < 0.01 compared with control. TABLE 1 EC50 Values of Bisphenol Derivatives in MCF-7 Estrogen Luciferase Reporter Assay   EC50 (μM)   TCBPA  0.02  BPAF  0.05  BPB  0.07  HPP  0.15  BPCH  0.21  HDM  0.32  DMBPA  0.42  BPA  0.63  TMBPA  0.73  BPAD  0.91  BPF  1.0  BPS  1.1  BPA acid  1.1  BPA catecol  1.8  BPA ol  11  TBBPA  19  IPP  36  DPP  >1000  DPM  >1000  BPD  >1000  E2   8.6 × 10−6     EC50 (μM)   TCBPA  0.02  BPAF  0.05  BPB  0.07  HPP  0.15  BPCH  0.21  HDM  0.32  DMBPA  0.42  BPA  0.63  TMBPA  0.73  BPAD  0.91  BPF  1.0  BPS  1.1  BPA acid  1.1  BPA catecol  1.8  BPA ol  11  TBBPA  19  IPP  36  DPP  >1000  DPM  >1000  BPD  >1000  E2   8.6 × 10−6   View Large Anti-estrogenic Activity of BPA and Related Compounds Anti-estrogenic activity of these BPA derivatives was also examined by the addition of these compounds to the E2 assay system in MCF-7 cells. Inhibitory effects of TMBPA and TBBPA on the estrogenic activity of 1 × 10−10 and 1 × 10−11 M E2 were observed at the concentration of 1 × 10−5 M. However, little effect on the estrogenic activity of E2 was observed with BPA, BPB, DPP, TCBPA, and BPF in the concentration range of 1 × 10−7 − 1 × 10−5 M (Fig. 3). Other BPA derivatives did not inhibit the estrogenic activity of E2 (data not shown). Thus, BPA and related compounds, except TMBPA and TBBPA, lack significant anti-estrogenic activity, at least in the MCF-7 reporter assay. FIG. 3. View largeDownload slide Anti-estrogenic activity of bisphenol A (BPA) and related compounds against estrogenic activity of 17-β-estradiol (E2) in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Anti-estrogenic activity was expressed as a relative activity with respect to the E2 activity using MCF-7 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 9.1- and 32-fold induction (see Fig. 2). BPB; bisphenol B, BPF; bisphenol F, TMBPA; tetramethylbisphenol A, TBBPA; tetrabromobisphenol A, TCBPA; tetrachlorobisphenol A, DPP; 2,2-diphenylpropane, TAM; tamoxifen. *p < 0.05, **p < 0.01 compared with control. FIG. 3. View largeDownload slide Anti-estrogenic activity of bisphenol A (BPA) and related compounds against estrogenic activity of 17-β-estradiol (E2) in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Anti-estrogenic activity was expressed as a relative activity with respect to the E2 activity using MCF-7 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 9.1- and 32-fold induction (see Fig. 2). BPB; bisphenol B, BPF; bisphenol F, TMBPA; tetramethylbisphenol A, TBBPA; tetrabromobisphenol A, TCBPA; tetrachlorobisphenol A, DPP; 2,2-diphenylpropane, TAM; tamoxifen. *p < 0.05, **p < 0.01 compared with control. Estrogenic Activity of BPA Derivatives in Vivo Estrogenic potential of TCBPA, TBBPA, and BPA in vivo was further investigated by use of the uterotrophic assay with ovariectomized mice. The body weight of rats administered TCBPA, TBBPA, or BPA was not decreased compared with other groups. The uterine weight in the groups given these compounds was increased compared with the group given vehicle only. BPA was most effective, followed by TBBPA and TCBPA. BPA gave a 147% increase in uterus weight over the castration control at 20 mg/kg, while 124 and 118% increases were noted in the TBBPA and TCBPA groups, respectively. Weight increase was dependent on the administered dose between 0 and 300 mg/kg for each compound, albeit changes were moderate when compared with the response to E2 (Table 2). Thus, the estrogenic effect of these bisphenol derivatives was confirmed in vivo. TABLE 2 Estrogenic Activity of Bisphenol Derivatives in Vivo Group   Uterus/body weight (mg/kg)   % of control   Control  435 ± 30  100  E2          50 μg/kg  2157 ± 518**  496  TBBPA          20 mg/kg  538 ± 85*  124      100 mg/kg  594 ± 67**  137      300 mg/kg  716 ± 56**  164      500 mg/kg  538 ± 88*  123  TCBPA          20 mg/kg  514 ± 84*  118      100 mg/kg  582 ± 52**  134      300 mg/kg  716 ± 56**  164      500 mg/kg  713 ± 82**  164  BPA          20 mg/kg  640 ± 48**  147      100 mg/kg  705 ± 75**  162      300 mg/kg  806 ± 70**  185      500 mg/kg   765 ± 131**   176   Group   Uterus/body weight (mg/kg)   % of control   Control  435 ± 30  100  E2          50 μg/kg  2157 ± 518**  496  TBBPA          20 mg/kg  538 ± 85*  124      100 mg/kg  594 ± 67**  137      300 mg/kg  716 ± 56**  164      500 mg/kg  538 ± 88*  123  TCBPA          20 mg/kg  514 ± 84*  118      100 mg/kg  582 ± 52**  134      300 mg/kg  716 ± 56**  164      500 mg/kg  713 ± 82**  164  BPA          20 mg/kg  640 ± 48**  147      100 mg/kg  705 ± 75**  162      300 mg/kg  806 ± 70**  185      500 mg/kg   765 ± 131**   176   Note. The uterotrophic assay of bisphenol derivatives. Weights of the uteri of rats are shown. Ovariectomized female B6C3F1 mice were treated with 17β-estradiol (E2; 50 μg/kg/day), 2,2-bis(3,5-dibromo-4-hydroxyphenyl)propane (TBBPA; 20, 100, 300, 500 μg/kg/day), 2,2-bis(3,5-dichloro-4-hydroxyphenyl)propane (TCBPA; 20, 100, 300, 500 mg/kg/day), or 2,2-bis(4-hydroxyphenyl)propane (BPA; 20, 100, 300, 500 mg/kg/day) for three days. Each group consisted of five animals. Uterus/body weight (mg/kg) indicates mean ± SEM of five mice. ** p < 0.01, * p < 0.05 indicate significant differences from control (OVX mice) experiments. View Large Androgenic Activity of BPA and Related Compounds Androgenic activity of BPA and related compounds was examined using NIH3T3 cells transfected with an AR responsive luciferase reporter gene. DHT exhibited markedly the androgenic activity toward NIH3T3 cells at 1 × 10−11−1 × 10−9 M. However, no androgenic activity of BPA, BPB, or BPF was observed in the concentration range of 10−7–10−4 M (Fig. 4). Other BPA derivatives did not show androgenic activity (data not shown). FIG. 4. View largeDownload slide Androgenic activity of bisphenol A (BPA) and related compounds in ARE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Androgenic activity was expressed as a relative activity with respect to the control using NIH3T3 cells. BPB; bisphenol B, BPF; bisphenol F, DHT; dihydrotestosterone. *p < 0.05, **p < 0.01 compared with control. FIG. 4. View largeDownload slide Androgenic activity of bisphenol A (BPA) and related compounds in ARE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Androgenic activity was expressed as a relative activity with respect to the control using NIH3T3 cells. BPB; bisphenol B, BPF; bisphenol F, DHT; dihydrotestosterone. *p < 0.05, **p < 0.01 compared with control. Anti-androgenic Activity of BPA and Related Compounds When BPA, BPF, or BPB was added to the DHT assay system in the concentration range of 1 × 10−7–1 × 10−5 M, the activity of 1 × 10−11 M DHT was inhibited concentration-dependently. The anti-androgenic effects of these compounds were also observed at 1 × 10−10 M DHT. However, no effect of TBBPA on the androgenic activity of DHT was observed (Fig. 5). These anti-androgenic activities were in the same order as that of flutamide. Anti-androgenic activities of BPS, HPP, and DPP were also observed at 1 × 10−6–1 × 10−4 M (data not shown). Table 3 summarizes the IC50 values of these compounds against the androgenic activity of 1 × 10−10 M DHT. The highest activity among the test compounds was that TMBPA, followed by BPAF, BPAD, BPB, DMBPA, HDM, and HPP. IPP, without a phenyl group at the 2-position of the propane moiety, was also active. 3-Hydroxylated BPA, BPA catechol, showed weak activity. DPP and DPM, the dehydroxylated compounds of BPA and BPF, showed little or no antiandrogenic activity, and TBBPA, BPA ol, and BPA carboxylic acid were negative in the assay. These results show that at least one 4-hydroxyl group of BPA derivatives is essential for the activity. 3,5-Substituents markedly influenced anti-androgenic activity. TMBPA showed the highest activity, but TBBPA and TCBPA did not show any significant anti-androgenic effect in the concentration range of 1 × 10−7–1 × 10−4 M. These findings indicate that some BPA derivatives are anti-androgenic as well as estrogenic. FIG. 5. View largeDownload slide Anti-androgenic activity of bisphenol A (BPA) and related compounds against androgenic activity of dihydrotestosterone (DHT) in ERE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Antiandrogenic activity was expressed as a relative activity with respect to DHT using NIH3T3 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 2.6- and 4.7-fold induction (see Fig. 4). BPB; bisphenol B, BPF; bisphenol F, TBBPA; tetrabromobisphenol A. *p < 0.05, **p < 0.01 compared with control. FIG. 5. View largeDownload slide Anti-androgenic activity of bisphenol A (BPA) and related compounds against androgenic activity of dihydrotestosterone (DHT) in ERE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Antiandrogenic activity was expressed as a relative activity with respect to DHT using NIH3T3 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 2.6- and 4.7-fold induction (see Fig. 4). BPB; bisphenol B, BPF; bisphenol F, TBBPA; tetrabromobisphenol A. *p < 0.05, **p < 0.01 compared with control. TABLE 3 IC50 Values of Bisphenol Derivatives for Androgen Activity of Dihydrotestosterone in NIH3T3 Luciferase Reporter Assay   IC50 (μM)   TMBPA  0.29  BPAF  1.3  BPAD  1.4  BPB  1.7  DMBPA  2.0  HDM  3.9  HPP  4.2  BPA  4.3  IPP  6.2  BPCH  7.9  BPD  7.9  BPF  12  BPA catechol  14  BPS  17  DPP  370  TCBPA  870  TBBPA  >1000  DPM  >1000  BPA ol  >1000  BPA acid  >1000  Flutamide   2.5     IC50 (μM)   TMBPA  0.29  BPAF  1.3  BPAD  1.4  BPB  1.7  DMBPA  2.0  HDM  3.9  HPP  4.2  BPA  4.3  IPP  6.2  BPCH  7.9  BPD  7.9  BPF  12  BPA catechol  14  BPS  17  DPP  370  TCBPA  870  TBBPA  >1000  DPM  >1000  BPA ol  >1000  BPA acid  >1000  Flutamide   2.5   Note. EC50 for DHT: 1.1 |×| 10−11. View Large Thyroid Hormonal Activity of BPA and Related Compounds Evaluated by Growth Hormone Production Assay of GH3 Cells The thyroid hormonal activities of BPA and related compounds were examined by measuring the ability of these compounds to induce the thyroid hormone-dependent production of growth hormone by GH3 cells. Growth hormone-releasing activity was observed with T3 in the concentration range of 1 × 10−12–1 × 10−9 M. An increase of growth hormone release from GH3 cells was observed after the addition of TBBPA or TCBPA in the concentration range of 1 × 10−6 to 1 × 10−4 M. TMBPA also weakly induced growth hormone release, but DMBPA was ineffective. The effects of DMBPA and TMBPA at 1 × 10−4 M could not be judged due to the cytotoxicity of these compounds (Fig. 6). BPA, BPF, BPS, BPAF, BPAD, and BPB also showed no activity (data not shown). TBBPA had the highest activity, followed by TCBPA and TMBPA. These results indicate that some BPA derivatives show thyroid hormone-like activity, and that a 4-hydroxyl group is essential for this activity, as well as for estrogenic and anti-androgenic activities. Bulky 3- and 5-substituents play an important role in the activity. However, other BPA derivatives were negative in the assay. In contrast, when the inhibitory effects of TBBPA, TCBPA, TMBPA, DMBPA, BPA, and BPB on the hormonal activity of T3 towards GH3 cells were examined, these compounds at 1 × 10−5 and 1 × 10−4 M showed no antagonistic action towards growth hormone production induced by the thyroid hormone (data not shown). These results suggest that TBBPA, TCBPA, TMBPA, and DMBPA act as thyroid hormone agonists, but not antagonists. FIG. 6. View largeDownload slide Thyroid hormonal activity of bisphenol A (BPA), tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA), and 3,3′-dimethylbisphenol A (DMBPA) in terms of GH production in GH3 cells. Each bar represents the mean ± SD of four experiments. Thyroid hormonal activity was expressed as a relative activity with respect to the control using GH3 cells. T3; L-3,5,3′-triiodothyronine. *p < 0.05, **p < 0.01 compared with control. FIG. 6. View largeDownload slide Thyroid hormonal activity of bisphenol A (BPA), tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA), and 3,3′-dimethylbisphenol A (DMBPA) in terms of GH production in GH3 cells. Each bar represents the mean ± SD of four experiments. Thyroid hormonal activity was expressed as a relative activity with respect to the control using GH3 cells. T3; L-3,5,3′-triiodothyronine. *p < 0.05, **p < 0.01 compared with control. DISCUSSION In this study, we examined the relationship between the structure of BPA derivatives and endocrine-disrupting activity, i.e., estrogenic, anti-estrogenic, anti-androgenic, and thyroid hormonal activities. Minimum structural requirements for estrogenic activity of BPA derivatives seems to be a 4-hydroxyl group on the A-phenyl ring and a hydrophobic moiety at the 2-position of the propane moiety, judging from the activity estimated in this study. However, a 2-hydroxyl or 3-hydroxyl group attached to a phenyl ring of biphenyl or benzophenone has been reported to be effective for estrogenic activity, and the order of the activity is 4-hydroxyl>3-hydroxyl>2-hydroxyl in both cases (Blair et al., 2000; Kawamura et al., 2003; Kitamura et al., 2003b; Paris et al., 2002; Soto et al., 1997). In our preliminary study using MCF-7 reporter assay, 2-hydroxydiphenylmethane and 3-hydroxydiphenylamine showed activity, with EC50 values of 32.4 μM (0.32 μM for 4-hydroxydiphenylmethane) and 4.2 μM (2.0 μM for 4-hydroxydiphenylamine), respectively. It is possible that a 2- or 3-hydroxyl group also contributes to the estrogen receptor affinity of bisphenol derivatives. Moreover, substituents at the 3,5-positions of the A-phenyl ring and at the methylene bridge markedly influence the estrogenic activity. TCBPA, TMBPA, and DMBPA showed high activity, but TBBPA had lower activity. Estrogenic activity of TCBPA has already been reported (Fukazawa et al., 2002; Kuruto-Niwa et al., 2002; Olsen et al., 2003). Hydrophobic substituents in place of the 1-methyl group of the propane moiety increased the hormonal activity, as seen in BPAF and BPB, but a hydrophilic group, such as a hydroxymethyl or carboxylic acid group, decreased the activity. Hydrophobicity at the methylene bridge of BPA derivatives is an important factor for the estrogenic activity (Fig. 7). FIG. 7. View largeDownload slide Structural requirement of bisphenol A and related compounds for endocrine-disrupting activity. FIG. 7. View largeDownload slide Structural requirement of bisphenol A and related compounds for endocrine-disrupting activity. For estrogen receptor ligand activity, xenobiotics require an unhindered hydroxyl group on an aryl ring and a hydrophobic group attached para to the hydroxyl group (Blair et al., 2000; Elsby et al., 2000; Fang et al., 2000; Hong et al., 2002; Nishihara et al., 2000). The key structural requirement for estrogenic activity of bisphenol derivatives is the phenolic hydroxyl group. It is reported that 3-hydroxyl group of E2 interacts with Glu353 and Arg394 at the binding pocket of human estrogen receptor α via hydrogen bonding, and the 17β-hydroxyl group interacts with His524, based on a crystallographic analysis of estrogen receptor α bound with E2 (Brzozowski et al., 1997; Shiau et al., 1998). Perhaps the 4-hydroxyl group of BPA also interacts with these amino acids. TCBPA and TMBPA exhibited higher estrogenic activity than did BPA. 3,5-Chloro and methyl substituents of BPA may assist tight fitting of the ligand into the ligand-binding pocket. However, a lower activity was observed in the case of TBBPA. This may be due to steric hindrance by the bulky bromo substituent. Regarding the estrogenic activity of TBBPA, Christiansen et al. (2000) reported that the vitellogenin level of male rainbow trout did not increase after ip injection of TBBPA. Olsen et al. (2003) reported that the estrogenic activity of TBBPA is lower than that of BPA. The activity of 3-hydroxy-BPA (BPA catechol) was lower than that of BPA. The reason may be steric hindrance, because the 3-hydroxyl group should be effective for estrogenic activity. The hydrophobic moiety of the protein, consisting of Met323, Ala350, Leu346, Phe404, Leu428, etc., is also important for the interaction with the hydrophobic B, C, and D rings of E2 (Brzozowski et al., 1997; Shiau et al., 1998). The hydrophobic propane and B-ring moieties of BPA may bind tightly with the hydrophobic binding site of the estrogen receptor. Attachment of hydrophilic substituents at the methylene bridge markedly decreased the estrogenic activity. This may be due to a decrease in the stability of the interaction with the hydrophobic protein site. In contrast, we observed antagonistic activity of TMBPA and TBBPA against the estrogenic activity of E2, but other BPA derivatives lacked this activity. TMBPA showed very high estrogenic activity among the test compounds. TMBPA and TBBPA acted as both agonist and antagonist at the estrogen receptor. The structural requirements for antagonistic action need further study. In the present study, MCF-7 was primarily used for examining estrogenic activity. Although this cell line has been widely used to screen estrogenic activity in environmental chemicals (Soto et al., 1995), assay data from a single cell line may contain both false negative and false positive results which are related to certain cross-talk pathways in the cell. Therefore, we also utilized other cell lines, including a rat pituitary cell line expressing a high level of ER α, MtT/E-2 and a mouse fibroblast cell line, NIH3T3, transiently transfected with ER α or β (Fujimoto et al., 2004; Maruyama et al., 1999). We generally confirmed the estrogenic activity of compounds with one of these cell lines after screening in MCF-7. It is noteworthy that ERE-dependent transcriptional activation of BPB and BPA reached more than 200% of the maximal E2 effect. Such “supramaximal” effects have been reported with genistein and other phytoestrogens, although the mechanism involved is not clear (Kuiper et al., 1998). It is reasonable that estrogenic activity was decreased by transformation to further oxidized metabolites, or conjugates with glucuronic acid or sulfuric acid at the hydroxyl group (Elsby et al., 2001; Matthews et al., 2001; Nakagawa and Suzuki, 2001; Pottenger et al., 2000; Snyder et al., 2000). In this study, BPA catechol, BPA ol, and BPA carboxylic acid, candidates for BPA metabolites formed by liver microsomal enzymes, showed decreased activity. However, Yoshihara et al. (2001, 2004) reported that when BPA was incubated with liver microsomes and cytosol together, the native estrogenic activity was enhanced. The enhanced activity might be due to a dimerized type of metabolite, 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene, which is a potent estrogen (Fig. 8). Further study is necessary to establish the effect of metabolic modification on the activity of BPA. The in vivo estrogenic activity of BPA has been reported (Ashby et al., 2000; Kim et al., 2001; Matthews et al., 2001; Tinwell et al., 2000): the weight of the ovary in ovariectomized rats dosed with BPA was increased compared to that in rats dosed with vehicle alone. The estrogenic activity of BPA in rats in vivo seems to be due to both BPA itself and its metabolites. However, there are some reports indicating that BPA does not show estrogenic activity in vivo (Coldham et al., 1997; Gould et al., 1998). In the present study, we examined the estrogenic activity of BPA, TCBPA, and TBBPA in vivo by means of uterotrophic assay in ovariectomized mice. These compounds were positive in this study. However, the activity of TCBPA, which showed the highest activity among the bisphenol derivatives tested in the reporter assay conducted in this study, was lower than that of BPA. Perhaps, this lower activity of TCBPA in vivo is due to greater metabolic inactivation as compared with BPA. Alternatively, metabolic activation as in the case of BPA may not occur with TCBPA. In contrast, TBBPA showed a significant estrogenic activity in vivo in ovariectomized mice, in spite of having little activity in in vitro assay. TBBPA might be resistant to metabolic inactivation by glucuronidation or sulfation at the 4,4′-dihydroxyl groups due to steric hindrance. It is clearly necessary to consider the activity of metabolites produced from the parent compounds in assessing the hormonal toxicity of environmental contaminants, including bisphenol derivatives. FIG. 8. View largeDownload slide Postulated metabolic pathways and the effect of metabolic modification on the estrogenic and anti-androgenic activities of bisphenol A. FIG. 8. View largeDownload slide Postulated metabolic pathways and the effect of metabolic modification on the estrogenic and anti-androgenic activities of bisphenol A. In this study, we found that BPA, BPB, and BPS are potent anti-androgens. However, BPA catechol, BPA ol, BPA carboxylic acid, DPM, and TBBPA were inactive. BPA is reported to have an inhibitory effect on the androgenic activity of DHT in a yeast-based assay (Sohoni and Sumpter, 1998). BPA also has binding affinity for androgen receptor in PALM cells (Paris et al., 2002). The hydroxyl group on the A-phenyl ring of BPA is essential for the anti-androgenic activity as well as estrogenic activity. In this study, anti-androgenic activity of bisphenol derivatives was restricted to 4-hydroxyl derivatives. However, a 2-hydroxyl or 3-hydroxyl group has been reported to be effective in phenylphenols and hydroxybenzophenones (Kitamura et al., 2004; Paris et al., 2002). The activity of phenylphenols was in the order of 3-hydroxyl>4-hydroxyl>2-hydroxyl, while that of hydroxybenzophenones was in the order of 3-hydroxyl>2-hydroxyl>4-hydroxyl. Substitution at the 3,5-positions markedly altered anti-androgenic activity. TMBPA showed the highest activity among the test compounds, but TBBPA and TCBPA showed little activity (Fig. 7). The crystal structures of DHT bound to rat androgen receptor (Sack et al., 2001) and of R1881 bound to human androgen receptor (Matias et al., 2000) showed that the 3-carbonyl group of DHT interacts with Arg752, and the 17β-hydroxyl group interacts with Asn705 and Thr877 at the ligand binding site consisting of 18 amino acids of the receptor, and the hydrophobic moiety of the protein fixes the B, C, and D rings of DHT. The 3-carbonyl and 17β-hydroxyl groups of DHT are essential for agonistic action. BPA derivatives may act as antagonistic agents by interfering with the interaction of DHT with Arg752 of the receptor owing to the presence of the 4-hydroxyl group and hydrophobic moiety of BPA. The 4′-hydroxyl group may also interfere with the binding of the 17β-hydroxyl group of DHT to the androgen receptor. Hydrophilic substituents at the methylene bridge of BPA may disturb the interaction with a hydrophobic moiety of the protein in the same way as with the estrogenic receptor. Unexpectedly, p-isopropylphenol, which lacks the B-phenyl ring of BPA, exhibited anti-androgenic activity. Clearly the B-phenyl ring is not essential for this activity, unlike estrogenic activity. Substitution by a bulky group at the 3,5-positions of the A-ring seems not to hinder the interaction with androgen receptor. We have already reported the agonistic activity of TBBPA and TCBPA on the thyroid hormonal activity of T3 (Kitamura et al., 2002). In the present study, we found that DMBPA and TMBPA, in addition to TBBPA and TCBPA, show thyroid hormonal activity, though other BPA derivatives do not. Moriyama et al. (2002) reported that BPA acts as an antagonist of the thyroid hormone action of T3. We could not observe agonistic or antagonistic action of BPA. Ishihara et al. (2003) also did not observe affinity of BPA for thyroid hormone receptors. Perhaps the thyroid hormonal potency of TBBPA, TCBPA, DMBPA, and TMBPA found in this study is due to their structural resemblance to thyroid hormones. The rat pituitary cell line GH3, isolated from a rat pituitary tumor, has been widely used as a standard pituitary cell model, in which the growth hormone secretion depends markedly on thyroid hormones, but little on estrogen (Kitagawa et al., 1987). In the present study using GH3 cells, we observed agonistic activities of TBBPA, TCBPA, DMBPA, and TMBPA toward thyroid hormone, but we could not detect anti-thyroid hormonal action of these compounds. A 4-hydroxyl group and double substitution by halogen or methyl group at 3,5-positions of the A-phenyl group were essential for thyroid hormonal activity in this study. Relatively large substituents at the 3,5-positions of the phenyl ring, besides the 4-hydroxyl group, seem to be necessary for thyroid hormonal activity (Fig. 7). Thyroid hormone receptor shows a rigid substrate specificity compared with estrogenic and androgenic receptors, because of the relatively smaller size of the active site. Wagner et al. (1995, 2001) conducted crystallographic analyses of rat thyroid hormone receptor α1 and its ligands, and reported that the 4′-hydroxyl group of thyroid hormones interacts with His381 of thyroid hormone receptor via hydrogen-bounds, and the 3′-iodo group interacts with the hydrophobic pocket formed by Gly290, Gly291, Phe215, and Met388. The thyroid hormonal activity decreased in the order of bromine, chlorine, and methyl substituents at the 3,5-positions of BPA. Weaker binding ability, due to these smaller-size substituents compared with iodine, with the hydrophobic pocket in the active site may be a reason for the decreased activity. Various polybrominated biphenyls, polybrominated diphenyl ethers or their metabolites with such structural features may compete with thyroid hormones for binding to the thyroid hormone receptor. Thyroid hormone disruption could occur through interaction with serum transport proteins, such as transthyretin and thyroid-binding globulin. Some PCBs, for instance, have been reported to bind to transthyretin with very high affinity. Although we previously found that both TCBPA and TBBPA are able to bind thyroid hormone receptor, the interactions of these compounds with serum transport proteins and other metabolic components should be examined in the future. The 3,5-substituents of BPA markedly influenced the endocrine-disrupting activity. TCBPA exhibited the highest estrogenic activity among the test compounds. In contrast, TBBPA exhibited the highest thyroid hormonal activity. Some 3,5-substituted BPAs showed combined endocrine-disrupting activity. TCBPA, TMBPA, and TBBPA showed both estrogenic and thyroid hormonal activities. TMBPA and TBBPA showed both estrogenic and anti-estrogenic activities. TMBPA and DMBPA showed higher anti-androgenic activity. However, anti-androgenic activity was not observed with TBBPA or TCBPA. As 3,5-substituted BPA derivatives are widely used as industrial materials or found as environmental contaminants, it is necessary to consider other possible toxicities. In contrast, BPA, BPB, BPAF, HPP, and BPF exhibited both high estrogenic and anti-androgenic activities. The endocrine-disrupting activity of BPA derivatives in vivo may be based on combinations of endocrine-disrupting actions, as observed in vitro. Hydroxy-PCBs were also reported to show both estrogenic and anti-thyroid hormonal activities (Cheek et al., 1999; Connor et al., 1997; Korach et al., 1988). 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Google Scholar Author notes *Graduate School of Biomedical Sciences and †Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan Toxicological Sciences vol. 84 no. 2 © The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

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Oxford University Press
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Toxicological Sciences vol. 84 no. 2 © The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
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Abstract

Abstract The endocrine-disrupting activities of bisphenol A (BPA) and 19 related compounds were comparatively examined by means of different in vitro and in vivo reporter assays. BPA and some related compounds exhibited estrogenic activity in human breast cancer cell line MCF-7, but there were remarkable differences in activity. Tetrachlorobisphenol A (TCBPA) showed the highest activity, followed by bisphenol B, BPA, and tetramethylbisphenol A (TMBPA); 2,2-bis(4-hydroxyphenyl)-1-propanol, 1,1-bis(4-hydroxyphenyl)propionic acid and 2,2-diphenylpropane showed little or no activity. Anti-estrogenic activity against 17β-estradiol was observed with TMBPA and tetrabromobisphenol A (TBBPA). TCBPA, TBBPA, and BPA gave positive responses in the in vivo uterotrophic assay using ovariectomized mice. In contrast, BPA and some related compounds showed significant inhibitory effects on the androgenic activity of 5α-dihydrotestosterone in mouse fibroblast cell line NIH3T3. TMBPA showed the highest antagonistic activity, followed by bisphenol AF, bisphenol AD, bisphenol B, and BPA. However, TBBPA, TCBPA, and 2,2-diphenylpropane were inactive. TBBPA, TCBPA, TMBPA, and 3,3′-dimethylbisphenol A exhibited significant thyroid hormonal activity towards rat pituitary cell line GH3, which releases growth hormone in a thyroid hormone-dependent manner. However, BPA and other derivatives did not show such activity. The results suggest that the 4-hydroxyl group of the A-phenyl ring and the B-phenyl ring of BPA derivatives are required for these hormonal activities, and substituents at the 3,5-positions of the phenyl rings and the bridging alkyl moiety markedly influence the activities. estrogenic activity, anti-androgenic activity, thyroid hormonal activity, bisphenol A, bisphenol derivative, human breast cancer cell line MCF-7, rat pituitary cell line GH3 Bisphenol A (2,2-bis-(4-hydroxyphenyl)propane; BPA) is an industrial raw material for polycarbonate and epoxy resins, and contaminates the end products. It can be detected in liquid from canned vegetables and in the saliva of patients treated with dental sealants (Brotons et al., 1995; Hashimoto et al., 2001; Olea et al., 1996). BPA has the ability to bind DNA after metabolic activation (Atkinson and Roy, 1995). BPA also shows estrogenic activity towards cell lines such as estrogen-responsive breast cancer cell line MCF-7 cells, and endocrine-disrupting effects in vivo (Ashby and Tinwell, 1998; Ashby et al., 2000; Gaido et al., 1997; Kim et al., 2001; Krishnan et al., 1993; Matthews et al., 2001; Tinwell et al., 2000). Bisphenol B (2,2-bis-(4-hydroxyphenyl)butane; BPB), bisphenol F (4,4′-dihydroxydiphenylmethane; BPF), bisphenol AD (ethylidenebisphenol; BPAD), bisphenol AF (1,3-trifluoro-2,2-bis-(4-hydroxyphenyl)propane; BPAF), tetramethylbisphenol A (2,2-bis-(3,5-dimethyl-4-hydroxyphenyl)propane; TMBPA), 3,3′-dimethylbisphenol A (DMBPA), and bisphenol S (bis-(4-hydroxyphenyl)sulfone; BPS) are also used as materials for polycarbonate resin. Tetrabromobisphenol A (2,2-bis-(3,5-dibromo-4-hydroxyphenyl)propane; TBBPA), a halogenated derivative of BPA, is also widely used throughout the world as a flame retardant in numerous products. TBBPA was developed as a relatively nontoxic flame retardant (Helleday et al., 1999; Sellström and Jansson, 1995; Sjödin et al., 2001; Thomsen et al., 2001; Watanabe et al., 1983). Tetrachlorobisphenol A (2,2-bis-(3,5-dichloro-4-hydroxyphenyl)propane; TCBPA) has been found in the effluent from waste-paper recycling plants (Fukazawa et al., 2001). Kuruto-Niwa et al. (2002) reported that estrogenic polychlorinated BPAs were not easily biodegraded. However, the endocrine-disrupting activity of these halogenated BPA has not been reported in detail. Endocrine-active chemicals arise from many different sources, including pesticides, industrial chemicals, pharmaceuticals, and phytochemicals. These chemicals are widely distributed in the environment, and are able to mimic or antagonize the biological functions of natural hormones. Chlorinated insecticides, such as kepone, o,p′-DDT, dieldrin and methoxychlor, and compounds used in the plastics and detergent industries, such as alkylphenols and BPA, are known to have estrogenic activity (Andersen et al., 1999). p,p′-DDE, a metabolite of p,p′-DDT, vinclozolin, an antifungal agent, and chlornitrofen, fenitrothion and fenthion, insecticides, have anti-androgenic activity (Gray et al., 1999; Kelce et al., 1995; Kitamura et al., 2003a; Kojima et al., 2003; Tamura et al., 2001). Some hydroxy-PCBs such as 4,4′-dihydroxy-3,3′,5,5′-tetrachlorobiphenyl are reported to show anti-thyroid hormonal activity in addition to estrogenic activity (Cheek et al., 1999; Connor et al., 1997; Korach et al., 1988; Lans et al., 1994). Interactions of estrogenic and anti-androgenic compounds with the respective hormone receptors have been demonstrated to account for most of the endocrine-disrupting actions, and these chemicals can alter reproductive development in mammals. It is also necessary to consider the activity of the metabolites of these chemicals. In the metabolism of BPA, the 3-hydroxyl metabolite (BPA catechol) was formed by human and rat liver microsomes and exhibited estrogenic activity (Elsby et al., 2001). The glucuronide metabolite proved to have no estrogenic activity (Matthews et al., 2001; Pottenger et al., 2000). However, the relationship between the structure and activity of BPA derivatives, including metabolites, remains to be fully understood. In this report, endocrine-disrupting activity, i.e., estrogenic, anti-estrogenic, androgenic, anti-androgenic, thyroid hormonal, and anti-thyroid hormonal activities of BPA and related compounds were examined using hormone-responsive reporter assays: the human breast cancer cell-line MCF-7 for estrogenic activity, the mouse fibroblast cell line NIH3T3 for androgenic activity, and the pituitary cell line GH3 for thyroid hormonal activity. Twenty BPA derivatives were tested in this study (Fig. 1). We found that BPA and some of its derivatives exhibited estrogenic as well as anti-androgenic activity. TBBPA, TCBPA, TMBPA, and DMBPA showed significant thyroid hormonal activity. The structure-activity relationship of BPA derivatives is discussed. FIG. 1. View largeDownload slide Structures of bisphenol A and related compounds tested in this study. FIG. 1. View largeDownload slide Structures of bisphenol A and related compounds tested in this study. MATERIALS AND METHODS Chemicals. TBBPA, TCBPA, BPA, BPAD, BPB, BPF, BPAF, BPS, diphenylmethane (DPM), 4-hydroxydiphenylmethane (HDM), DMBPA, TMBPA, p-isopropylphenol (IPP), 1,1-bis-(4-hydroxyphenyl)cyclohexane (BPCH), α,α′-bis-(4-hydroxyphenyl)-1,4-diisopropylbenzene (BPDB), dihydrotestosterone (DHT), and flutamide were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), 2-(4-hydroxyphenyl)-2-phenylpropane (HPP) from Nacalai Tesque, Inc. (Kyoto, Japan), 2-(3,4-dihydroxyphenyl)-2-phenylpropane (BPA catechol) from Wako Pure Chemical Co. Ltd., (Osaka, Japan), 2,2-diphenylpropane (DPP) from Aldrich Chemical Co. (Milwaukee, WI), and L-3,5,3′-triiodothyronine (T3) and 17-β-estradiol (E2) from Sigma Chemical Co. (St. Louis, MO). 2,2-bis-(4-Hydroxyphenyl)-1-propanol (BPA ol) and 2,2-bis-(4-hydroxyphenyl)-1-propionic acid (BPA carboxylic acid) were synthesized by the methods of Spivack et al. (1994). Cell culture. Human breast cancer cell-line MCF-7 cells were maintained in DMEM (Sigma Chemical Co.) containing penicillin and streptomycin with 5% fetal bovine serum (FBS; Life Technologies, Rockville, MD). Rat pituitary cancer cell-line GH3 cells were maintained in DMEM/F12 mixed medium (Sigma Chemical Co.) containing penicillin and streptomycin with 8% horse serum (Life Technologies) and 2% FBS. Mouse fibroblast cell-line NIH3T3 cells were maintained in DMEM (Sigma Chemical Co.) containing penicillin and streptomycin with 5% calf serum (Life Technologies). Assay of estrogenic activity of BPA and related compounds. ERE-luciferase reporter assay using MCF-7 cells was performed according to the previously reported method (Kitamura et al., 2003a). Briefly, transient transfections in MCF-7 cells were performed using Transfast (Promega Co., Madison, WI), according to the manufacturer's protocol. Transfections were done in 48-well plates at 2 × 104 cells/well with 0.3 μg of p(ERE)3-SV40-luc and 2 ng of phRL-CMV (Promega Co.) as an internal standard (Sugihara et al., 2000). Twenty-four hours after addition of the sample (final concentration, 10−4 – 10−9 M), the assay was performed with a Dual Luciferase assay kit (Promega Co.). Firefly luciferase reporter activity was normalized to renilla luciferase activity from phRL-CMV, to control for the cytotoxic effects of compounds, as well as differences in transfection efficiency between culture wells. For the assay of anti-estrogens, the inhibitory effect of BPA and related compounds on the estrogenic activity of E2 at the concentration of 1 × 10−10 M was examined. Assay of androgenic activity of BPA and related compounds. Assay of androgenic activity was performed by means of ARE-luciferase reporter assay using NIH3T3 cells without expressing AR. Cells were maintained in phenol red-free DMEM (Sigma Chemical Co.) containing penicillin, streptomycin, and dextran-charcoal-treated calf serum for 2–3 days. Transient transfections in NIH3T3 cells were performed using Transfast according to the manufacturer's protocol. Transfections were done in 48-well plates at 2 × 104 cells/well with 0.3 μg of p(ARE)2-luc, 0.05 μg of pSG5-hAR, and 2 ng of phRL-CMV as an internal standard (Kitamura et al., 2003c). Twenty-four hours after addition of the sample (final concentration, 10−4 – 10−8 M) dissolved in 10 μl of ethanol, the assay was performed with a Dual Luciferase assay kit according to the manufacturer's protocol. Firefly luciferase reporter activity was normalized to renilla luciferase activity from phRL-CMV. For the assay of anti-androgenic activity, the inhibitory effect of BPA and related compounds on the androgenic activity of 1 × 10−10 or 1 × 10−11 M DHT was examined. Assay of thyroid hormonal activity of BPA and related compounds. Assay of thyroid hormonal activity was performed by measuring the induction of growth hormone production in GH3 cells as previously reported (Kitamura et al., 2002). Briefly, the cells were seeded in 24-well plates at 1 × 104 cells/well and chemicals were added the next day. Two days later, growth hormone in the culture medium was measured. For the assay of anti-thyroid hormonal activity, the inhibitory effect of BPA and related compounds on the activity of 1 × 10−7 or 1 × 10−8 M T3 was examined. Assay of estrogenic activity in vivo (uterotrophic assay). B6C3F1 female mice obtained from Charles River Co. (Kanagawa, Japan) were used. They were surgically ovariectomized at four weeks old. At the age of eight weeks, they were divided into 14 groups each consisting of five animals. The mice were treated once a day for three days with ip doses of 0.2 ml of vehicle (Panacete 810, Nippon Oils and Fats Co., Ltd., Tokyo, Japan), E2 (50 μg/kg/day), TCBPA, TBBPA, or BPA (20, 100, 300, or 500 mg/kg/day). Animals were sacrificed under anesthesia and the uterus was dissected and weighed. Data analysis. Multiple comparisons were made by ANOVA followed by Scheffe's test. EC50 values and IC50 values were calculated by fitting data to the logistic equation. RESULTS Estrogenic Activity of BPA and Related Compounds Estrogenic activity of BPA and related compounds was examined using ERE-luciferase reporter assay in MCF-7 cells. BPA, BPB, BPF, BPA ol, BPA carboxylic acid, HPP, HDM, TMBPA, BPA catechol, DDM, TBBPA, DMBPA, and TCBPA all exhibited estrogenic activity in the estrogen screening assay, but the activities varied markedly from compound to compound. TCBPA, BPAF, BPB, and HPP showed significant estrogenic activity in the concentration range of 1 × 10−7 (1 × 10−8 in the case of TCBPA) − 1 × 10−4 M. BPA and HDM also showed estrogenic activity at higher concentrations. However, DPP and DPM, which lack a hydroxyl group, were inactive (Fig. 2). DMBPA, TMBPA, BPF, BPAD, BPA catechol, BPA ol, BPA carboxylic acid, and TBBPA also showed estrogenic activity at 1 × 10−6 − 1 × 10−4 M. When ICI 182,780, a pure estrogen receptor antagonist, was added at the concentration of 1 × 10−8 M, the estrogenic activities of these compounds were markedly inhibited (data not shown). The EC50 values of estrogenic activity of the positive compounds are shown in Table 1. TCBPA showed the highest activity, followed by BPAF, BPB, HPP, BPCH, HDM, DMBPA, BPA, TMBPA, BPAD, and BPF. When the propane bridge of BPA was substituted with a hydrophilic group, the estrogenic activities of these compounds were markedly inhibited, showing the specific nature of this response (compare BPA ol and BPA carboxylic acid with BPA). On the contrary, the activity was increased by substitution with a hydrophobic group (compare BPA, BPAF and BPB with BPF). BPA catechol, 3-hydroxyl derivative of BPA, exhibited little estrogenic activity. DPP, the dehydroxylated derivative of BPA, and DPM were negative in the estrogen screening assay. IPP, lacking the B-phenyl ring, showed no activity. Thus, at least one 4-hydroxyl group of BPA derivatives is essential for estrogenic activity. The second phenyl group attached the 2-position of propane is also necessary. FIG. 2. View largeDownload slide Estrogenic activity of bisphenol A (BPA) and related compounds in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Estrogenic activity was expressed as a relative activity with respect to the control using MCF-7 cells. BPB; bisphenol B, BPAF; bisphenol AF, TCBPA; tetrachlorobisphenol A, 4-HDM; 4-hydroxydiphenylmethane, HPP; 2-(4-hydroxyphenyl)-2-phenylpropane, DPM; diphenylmethane, DPP; 2,2-diphenylpropane, E2; 17-β-estradiol. *p < 0.05, **p < 0.01 compared with control. FIG. 2. View largeDownload slide Estrogenic activity of bisphenol A (BPA) and related compounds in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Estrogenic activity was expressed as a relative activity with respect to the control using MCF-7 cells. BPB; bisphenol B, BPAF; bisphenol AF, TCBPA; tetrachlorobisphenol A, 4-HDM; 4-hydroxydiphenylmethane, HPP; 2-(4-hydroxyphenyl)-2-phenylpropane, DPM; diphenylmethane, DPP; 2,2-diphenylpropane, E2; 17-β-estradiol. *p < 0.05, **p < 0.01 compared with control. TABLE 1 EC50 Values of Bisphenol Derivatives in MCF-7 Estrogen Luciferase Reporter Assay   EC50 (μM)   TCBPA  0.02  BPAF  0.05  BPB  0.07  HPP  0.15  BPCH  0.21  HDM  0.32  DMBPA  0.42  BPA  0.63  TMBPA  0.73  BPAD  0.91  BPF  1.0  BPS  1.1  BPA acid  1.1  BPA catecol  1.8  BPA ol  11  TBBPA  19  IPP  36  DPP  >1000  DPM  >1000  BPD  >1000  E2   8.6 × 10−6     EC50 (μM)   TCBPA  0.02  BPAF  0.05  BPB  0.07  HPP  0.15  BPCH  0.21  HDM  0.32  DMBPA  0.42  BPA  0.63  TMBPA  0.73  BPAD  0.91  BPF  1.0  BPS  1.1  BPA acid  1.1  BPA catecol  1.8  BPA ol  11  TBBPA  19  IPP  36  DPP  >1000  DPM  >1000  BPD  >1000  E2   8.6 × 10−6   View Large Anti-estrogenic Activity of BPA and Related Compounds Anti-estrogenic activity of these BPA derivatives was also examined by the addition of these compounds to the E2 assay system in MCF-7 cells. Inhibitory effects of TMBPA and TBBPA on the estrogenic activity of 1 × 10−10 and 1 × 10−11 M E2 were observed at the concentration of 1 × 10−5 M. However, little effect on the estrogenic activity of E2 was observed with BPA, BPB, DPP, TCBPA, and BPF in the concentration range of 1 × 10−7 − 1 × 10−5 M (Fig. 3). Other BPA derivatives did not inhibit the estrogenic activity of E2 (data not shown). Thus, BPA and related compounds, except TMBPA and TBBPA, lack significant anti-estrogenic activity, at least in the MCF-7 reporter assay. FIG. 3. View largeDownload slide Anti-estrogenic activity of bisphenol A (BPA) and related compounds against estrogenic activity of 17-β-estradiol (E2) in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Anti-estrogenic activity was expressed as a relative activity with respect to the E2 activity using MCF-7 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 9.1- and 32-fold induction (see Fig. 2). BPB; bisphenol B, BPF; bisphenol F, TMBPA; tetramethylbisphenol A, TBBPA; tetrabromobisphenol A, TCBPA; tetrachlorobisphenol A, DPP; 2,2-diphenylpropane, TAM; tamoxifen. *p < 0.05, **p < 0.01 compared with control. FIG. 3. View largeDownload slide Anti-estrogenic activity of bisphenol A (BPA) and related compounds against estrogenic activity of 17-β-estradiol (E2) in ERE-luciferase reporter assay with MCF-7 cells. Each bar represents the mean ± SD of four experiments. Anti-estrogenic activity was expressed as a relative activity with respect to the E2 activity using MCF-7 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 9.1- and 32-fold induction (see Fig. 2). BPB; bisphenol B, BPF; bisphenol F, TMBPA; tetramethylbisphenol A, TBBPA; tetrabromobisphenol A, TCBPA; tetrachlorobisphenol A, DPP; 2,2-diphenylpropane, TAM; tamoxifen. *p < 0.05, **p < 0.01 compared with control. Estrogenic Activity of BPA Derivatives in Vivo Estrogenic potential of TCBPA, TBBPA, and BPA in vivo was further investigated by use of the uterotrophic assay with ovariectomized mice. The body weight of rats administered TCBPA, TBBPA, or BPA was not decreased compared with other groups. The uterine weight in the groups given these compounds was increased compared with the group given vehicle only. BPA was most effective, followed by TBBPA and TCBPA. BPA gave a 147% increase in uterus weight over the castration control at 20 mg/kg, while 124 and 118% increases were noted in the TBBPA and TCBPA groups, respectively. Weight increase was dependent on the administered dose between 0 and 300 mg/kg for each compound, albeit changes were moderate when compared with the response to E2 (Table 2). Thus, the estrogenic effect of these bisphenol derivatives was confirmed in vivo. TABLE 2 Estrogenic Activity of Bisphenol Derivatives in Vivo Group   Uterus/body weight (mg/kg)   % of control   Control  435 ± 30  100  E2          50 μg/kg  2157 ± 518**  496  TBBPA          20 mg/kg  538 ± 85*  124      100 mg/kg  594 ± 67**  137      300 mg/kg  716 ± 56**  164      500 mg/kg  538 ± 88*  123  TCBPA          20 mg/kg  514 ± 84*  118      100 mg/kg  582 ± 52**  134      300 mg/kg  716 ± 56**  164      500 mg/kg  713 ± 82**  164  BPA          20 mg/kg  640 ± 48**  147      100 mg/kg  705 ± 75**  162      300 mg/kg  806 ± 70**  185      500 mg/kg   765 ± 131**   176   Group   Uterus/body weight (mg/kg)   % of control   Control  435 ± 30  100  E2          50 μg/kg  2157 ± 518**  496  TBBPA          20 mg/kg  538 ± 85*  124      100 mg/kg  594 ± 67**  137      300 mg/kg  716 ± 56**  164      500 mg/kg  538 ± 88*  123  TCBPA          20 mg/kg  514 ± 84*  118      100 mg/kg  582 ± 52**  134      300 mg/kg  716 ± 56**  164      500 mg/kg  713 ± 82**  164  BPA          20 mg/kg  640 ± 48**  147      100 mg/kg  705 ± 75**  162      300 mg/kg  806 ± 70**  185      500 mg/kg   765 ± 131**   176   Note. The uterotrophic assay of bisphenol derivatives. Weights of the uteri of rats are shown. Ovariectomized female B6C3F1 mice were treated with 17β-estradiol (E2; 50 μg/kg/day), 2,2-bis(3,5-dibromo-4-hydroxyphenyl)propane (TBBPA; 20, 100, 300, 500 μg/kg/day), 2,2-bis(3,5-dichloro-4-hydroxyphenyl)propane (TCBPA; 20, 100, 300, 500 mg/kg/day), or 2,2-bis(4-hydroxyphenyl)propane (BPA; 20, 100, 300, 500 mg/kg/day) for three days. Each group consisted of five animals. Uterus/body weight (mg/kg) indicates mean ± SEM of five mice. ** p < 0.01, * p < 0.05 indicate significant differences from control (OVX mice) experiments. View Large Androgenic Activity of BPA and Related Compounds Androgenic activity of BPA and related compounds was examined using NIH3T3 cells transfected with an AR responsive luciferase reporter gene. DHT exhibited markedly the androgenic activity toward NIH3T3 cells at 1 × 10−11−1 × 10−9 M. However, no androgenic activity of BPA, BPB, or BPF was observed in the concentration range of 10−7–10−4 M (Fig. 4). Other BPA derivatives did not show androgenic activity (data not shown). FIG. 4. View largeDownload slide Androgenic activity of bisphenol A (BPA) and related compounds in ARE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Androgenic activity was expressed as a relative activity with respect to the control using NIH3T3 cells. BPB; bisphenol B, BPF; bisphenol F, DHT; dihydrotestosterone. *p < 0.05, **p < 0.01 compared with control. FIG. 4. View largeDownload slide Androgenic activity of bisphenol A (BPA) and related compounds in ARE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Androgenic activity was expressed as a relative activity with respect to the control using NIH3T3 cells. BPB; bisphenol B, BPF; bisphenol F, DHT; dihydrotestosterone. *p < 0.05, **p < 0.01 compared with control. Anti-androgenic Activity of BPA and Related Compounds When BPA, BPF, or BPB was added to the DHT assay system in the concentration range of 1 × 10−7–1 × 10−5 M, the activity of 1 × 10−11 M DHT was inhibited concentration-dependently. The anti-androgenic effects of these compounds were also observed at 1 × 10−10 M DHT. However, no effect of TBBPA on the androgenic activity of DHT was observed (Fig. 5). These anti-androgenic activities were in the same order as that of flutamide. Anti-androgenic activities of BPS, HPP, and DPP were also observed at 1 × 10−6–1 × 10−4 M (data not shown). Table 3 summarizes the IC50 values of these compounds against the androgenic activity of 1 × 10−10 M DHT. The highest activity among the test compounds was that TMBPA, followed by BPAF, BPAD, BPB, DMBPA, HDM, and HPP. IPP, without a phenyl group at the 2-position of the propane moiety, was also active. 3-Hydroxylated BPA, BPA catechol, showed weak activity. DPP and DPM, the dehydroxylated compounds of BPA and BPF, showed little or no antiandrogenic activity, and TBBPA, BPA ol, and BPA carboxylic acid were negative in the assay. These results show that at least one 4-hydroxyl group of BPA derivatives is essential for the activity. 3,5-Substituents markedly influenced anti-androgenic activity. TMBPA showed the highest activity, but TBBPA and TCBPA did not show any significant anti-androgenic effect in the concentration range of 1 × 10−7–1 × 10−4 M. These findings indicate that some BPA derivatives are anti-androgenic as well as estrogenic. FIG. 5. View largeDownload slide Anti-androgenic activity of bisphenol A (BPA) and related compounds against androgenic activity of dihydrotestosterone (DHT) in ERE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Antiandrogenic activity was expressed as a relative activity with respect to DHT using NIH3T3 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 2.6- and 4.7-fold induction (see Fig. 4). BPB; bisphenol B, BPF; bisphenol F, TBBPA; tetrabromobisphenol A. *p < 0.05, **p < 0.01 compared with control. FIG. 5. View largeDownload slide Anti-androgenic activity of bisphenol A (BPA) and related compounds against androgenic activity of dihydrotestosterone (DHT) in ERE-luciferase reporter assay with NIH3T3 cells. Each bar represents the mean ± SD of four experiments. Antiandrogenic activity was expressed as a relative activity with respect to DHT using NIH3T3 cells. One-hundred percent at 10−11 M and 10−10 M corresponds to 2.6- and 4.7-fold induction (see Fig. 4). BPB; bisphenol B, BPF; bisphenol F, TBBPA; tetrabromobisphenol A. *p < 0.05, **p < 0.01 compared with control. TABLE 3 IC50 Values of Bisphenol Derivatives for Androgen Activity of Dihydrotestosterone in NIH3T3 Luciferase Reporter Assay   IC50 (μM)   TMBPA  0.29  BPAF  1.3  BPAD  1.4  BPB  1.7  DMBPA  2.0  HDM  3.9  HPP  4.2  BPA  4.3  IPP  6.2  BPCH  7.9  BPD  7.9  BPF  12  BPA catechol  14  BPS  17  DPP  370  TCBPA  870  TBBPA  >1000  DPM  >1000  BPA ol  >1000  BPA acid  >1000  Flutamide   2.5     IC50 (μM)   TMBPA  0.29  BPAF  1.3  BPAD  1.4  BPB  1.7  DMBPA  2.0  HDM  3.9  HPP  4.2  BPA  4.3  IPP  6.2  BPCH  7.9  BPD  7.9  BPF  12  BPA catechol  14  BPS  17  DPP  370  TCBPA  870  TBBPA  >1000  DPM  >1000  BPA ol  >1000  BPA acid  >1000  Flutamide   2.5   Note. EC50 for DHT: 1.1 |×| 10−11. View Large Thyroid Hormonal Activity of BPA and Related Compounds Evaluated by Growth Hormone Production Assay of GH3 Cells The thyroid hormonal activities of BPA and related compounds were examined by measuring the ability of these compounds to induce the thyroid hormone-dependent production of growth hormone by GH3 cells. Growth hormone-releasing activity was observed with T3 in the concentration range of 1 × 10−12–1 × 10−9 M. An increase of growth hormone release from GH3 cells was observed after the addition of TBBPA or TCBPA in the concentration range of 1 × 10−6 to 1 × 10−4 M. TMBPA also weakly induced growth hormone release, but DMBPA was ineffective. The effects of DMBPA and TMBPA at 1 × 10−4 M could not be judged due to the cytotoxicity of these compounds (Fig. 6). BPA, BPF, BPS, BPAF, BPAD, and BPB also showed no activity (data not shown). TBBPA had the highest activity, followed by TCBPA and TMBPA. These results indicate that some BPA derivatives show thyroid hormone-like activity, and that a 4-hydroxyl group is essential for this activity, as well as for estrogenic and anti-androgenic activities. Bulky 3- and 5-substituents play an important role in the activity. However, other BPA derivatives were negative in the assay. In contrast, when the inhibitory effects of TBBPA, TCBPA, TMBPA, DMBPA, BPA, and BPB on the hormonal activity of T3 towards GH3 cells were examined, these compounds at 1 × 10−5 and 1 × 10−4 M showed no antagonistic action towards growth hormone production induced by the thyroid hormone (data not shown). These results suggest that TBBPA, TCBPA, TMBPA, and DMBPA act as thyroid hormone agonists, but not antagonists. FIG. 6. View largeDownload slide Thyroid hormonal activity of bisphenol A (BPA), tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA), and 3,3′-dimethylbisphenol A (DMBPA) in terms of GH production in GH3 cells. Each bar represents the mean ± SD of four experiments. Thyroid hormonal activity was expressed as a relative activity with respect to the control using GH3 cells. T3; L-3,5,3′-triiodothyronine. *p < 0.05, **p < 0.01 compared with control. FIG. 6. View largeDownload slide Thyroid hormonal activity of bisphenol A (BPA), tetrabromobisphenol A (TBBPA), tetrachlorobisphenol A (TCBPA), tetramethylbisphenol A (TMBPA), and 3,3′-dimethylbisphenol A (DMBPA) in terms of GH production in GH3 cells. Each bar represents the mean ± SD of four experiments. Thyroid hormonal activity was expressed as a relative activity with respect to the control using GH3 cells. T3; L-3,5,3′-triiodothyronine. *p < 0.05, **p < 0.01 compared with control. DISCUSSION In this study, we examined the relationship between the structure of BPA derivatives and endocrine-disrupting activity, i.e., estrogenic, anti-estrogenic, anti-androgenic, and thyroid hormonal activities. Minimum structural requirements for estrogenic activity of BPA derivatives seems to be a 4-hydroxyl group on the A-phenyl ring and a hydrophobic moiety at the 2-position of the propane moiety, judging from the activity estimated in this study. However, a 2-hydroxyl or 3-hydroxyl group attached to a phenyl ring of biphenyl or benzophenone has been reported to be effective for estrogenic activity, and the order of the activity is 4-hydroxyl>3-hydroxyl>2-hydroxyl in both cases (Blair et al., 2000; Kawamura et al., 2003; Kitamura et al., 2003b; Paris et al., 2002; Soto et al., 1997). In our preliminary study using MCF-7 reporter assay, 2-hydroxydiphenylmethane and 3-hydroxydiphenylamine showed activity, with EC50 values of 32.4 μM (0.32 μM for 4-hydroxydiphenylmethane) and 4.2 μM (2.0 μM for 4-hydroxydiphenylamine), respectively. It is possible that a 2- or 3-hydroxyl group also contributes to the estrogen receptor affinity of bisphenol derivatives. Moreover, substituents at the 3,5-positions of the A-phenyl ring and at the methylene bridge markedly influence the estrogenic activity. TCBPA, TMBPA, and DMBPA showed high activity, but TBBPA had lower activity. Estrogenic activity of TCBPA has already been reported (Fukazawa et al., 2002; Kuruto-Niwa et al., 2002; Olsen et al., 2003). Hydrophobic substituents in place of the 1-methyl group of the propane moiety increased the hormonal activity, as seen in BPAF and BPB, but a hydrophilic group, such as a hydroxymethyl or carboxylic acid group, decreased the activity. Hydrophobicity at the methylene bridge of BPA derivatives is an important factor for the estrogenic activity (Fig. 7). FIG. 7. View largeDownload slide Structural requirement of bisphenol A and related compounds for endocrine-disrupting activity. FIG. 7. View largeDownload slide Structural requirement of bisphenol A and related compounds for endocrine-disrupting activity. For estrogen receptor ligand activity, xenobiotics require an unhindered hydroxyl group on an aryl ring and a hydrophobic group attached para to the hydroxyl group (Blair et al., 2000; Elsby et al., 2000; Fang et al., 2000; Hong et al., 2002; Nishihara et al., 2000). The key structural requirement for estrogenic activity of bisphenol derivatives is the phenolic hydroxyl group. It is reported that 3-hydroxyl group of E2 interacts with Glu353 and Arg394 at the binding pocket of human estrogen receptor α via hydrogen bonding, and the 17β-hydroxyl group interacts with His524, based on a crystallographic analysis of estrogen receptor α bound with E2 (Brzozowski et al., 1997; Shiau et al., 1998). Perhaps the 4-hydroxyl group of BPA also interacts with these amino acids. TCBPA and TMBPA exhibited higher estrogenic activity than did BPA. 3,5-Chloro and methyl substituents of BPA may assist tight fitting of the ligand into the ligand-binding pocket. However, a lower activity was observed in the case of TBBPA. This may be due to steric hindrance by the bulky bromo substituent. Regarding the estrogenic activity of TBBPA, Christiansen et al. (2000) reported that the vitellogenin level of male rainbow trout did not increase after ip injection of TBBPA. Olsen et al. (2003) reported that the estrogenic activity of TBBPA is lower than that of BPA. The activity of 3-hydroxy-BPA (BPA catechol) was lower than that of BPA. The reason may be steric hindrance, because the 3-hydroxyl group should be effective for estrogenic activity. The hydrophobic moiety of the protein, consisting of Met323, Ala350, Leu346, Phe404, Leu428, etc., is also important for the interaction with the hydrophobic B, C, and D rings of E2 (Brzozowski et al., 1997; Shiau et al., 1998). The hydrophobic propane and B-ring moieties of BPA may bind tightly with the hydrophobic binding site of the estrogen receptor. Attachment of hydrophilic substituents at the methylene bridge markedly decreased the estrogenic activity. This may be due to a decrease in the stability of the interaction with the hydrophobic protein site. In contrast, we observed antagonistic activity of TMBPA and TBBPA against the estrogenic activity of E2, but other BPA derivatives lacked this activity. TMBPA showed very high estrogenic activity among the test compounds. TMBPA and TBBPA acted as both agonist and antagonist at the estrogen receptor. The structural requirements for antagonistic action need further study. In the present study, MCF-7 was primarily used for examining estrogenic activity. Although this cell line has been widely used to screen estrogenic activity in environmental chemicals (Soto et al., 1995), assay data from a single cell line may contain both false negative and false positive results which are related to certain cross-talk pathways in the cell. Therefore, we also utilized other cell lines, including a rat pituitary cell line expressing a high level of ER α, MtT/E-2 and a mouse fibroblast cell line, NIH3T3, transiently transfected with ER α or β (Fujimoto et al., 2004; Maruyama et al., 1999). We generally confirmed the estrogenic activity of compounds with one of these cell lines after screening in MCF-7. It is noteworthy that ERE-dependent transcriptional activation of BPB and BPA reached more than 200% of the maximal E2 effect. Such “supramaximal” effects have been reported with genistein and other phytoestrogens, although the mechanism involved is not clear (Kuiper et al., 1998). It is reasonable that estrogenic activity was decreased by transformation to further oxidized metabolites, or conjugates with glucuronic acid or sulfuric acid at the hydroxyl group (Elsby et al., 2001; Matthews et al., 2001; Nakagawa and Suzuki, 2001; Pottenger et al., 2000; Snyder et al., 2000). In this study, BPA catechol, BPA ol, and BPA carboxylic acid, candidates for BPA metabolites formed by liver microsomal enzymes, showed decreased activity. However, Yoshihara et al. (2001, 2004) reported that when BPA was incubated with liver microsomes and cytosol together, the native estrogenic activity was enhanced. The enhanced activity might be due to a dimerized type of metabolite, 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene, which is a potent estrogen (Fig. 8). Further study is necessary to establish the effect of metabolic modification on the activity of BPA. The in vivo estrogenic activity of BPA has been reported (Ashby et al., 2000; Kim et al., 2001; Matthews et al., 2001; Tinwell et al., 2000): the weight of the ovary in ovariectomized rats dosed with BPA was increased compared to that in rats dosed with vehicle alone. The estrogenic activity of BPA in rats in vivo seems to be due to both BPA itself and its metabolites. However, there are some reports indicating that BPA does not show estrogenic activity in vivo (Coldham et al., 1997; Gould et al., 1998). In the present study, we examined the estrogenic activity of BPA, TCBPA, and TBBPA in vivo by means of uterotrophic assay in ovariectomized mice. These compounds were positive in this study. However, the activity of TCBPA, which showed the highest activity among the bisphenol derivatives tested in the reporter assay conducted in this study, was lower than that of BPA. Perhaps, this lower activity of TCBPA in vivo is due to greater metabolic inactivation as compared with BPA. Alternatively, metabolic activation as in the case of BPA may not occur with TCBPA. In contrast, TBBPA showed a significant estrogenic activity in vivo in ovariectomized mice, in spite of having little activity in in vitro assay. TBBPA might be resistant to metabolic inactivation by glucuronidation or sulfation at the 4,4′-dihydroxyl groups due to steric hindrance. It is clearly necessary to consider the activity of metabolites produced from the parent compounds in assessing the hormonal toxicity of environmental contaminants, including bisphenol derivatives. FIG. 8. View largeDownload slide Postulated metabolic pathways and the effect of metabolic modification on the estrogenic and anti-androgenic activities of bisphenol A. FIG. 8. View largeDownload slide Postulated metabolic pathways and the effect of metabolic modification on the estrogenic and anti-androgenic activities of bisphenol A. In this study, we found that BPA, BPB, and BPS are potent anti-androgens. However, BPA catechol, BPA ol, BPA carboxylic acid, DPM, and TBBPA were inactive. BPA is reported to have an inhibitory effect on the androgenic activity of DHT in a yeast-based assay (Sohoni and Sumpter, 1998). BPA also has binding affinity for androgen receptor in PALM cells (Paris et al., 2002). The hydroxyl group on the A-phenyl ring of BPA is essential for the anti-androgenic activity as well as estrogenic activity. In this study, anti-androgenic activity of bisphenol derivatives was restricted to 4-hydroxyl derivatives. However, a 2-hydroxyl or 3-hydroxyl group has been reported to be effective in phenylphenols and hydroxybenzophenones (Kitamura et al., 2004; Paris et al., 2002). The activity of phenylphenols was in the order of 3-hydroxyl>4-hydroxyl>2-hydroxyl, while that of hydroxybenzophenones was in the order of 3-hydroxyl>2-hydroxyl>4-hydroxyl. Substitution at the 3,5-positions markedly altered anti-androgenic activity. TMBPA showed the highest activity among the test compounds, but TBBPA and TCBPA showed little activity (Fig. 7). The crystal structures of DHT bound to rat androgen receptor (Sack et al., 2001) and of R1881 bound to human androgen receptor (Matias et al., 2000) showed that the 3-carbonyl group of DHT interacts with Arg752, and the 17β-hydroxyl group interacts with Asn705 and Thr877 at the ligand binding site consisting of 18 amino acids of the receptor, and the hydrophobic moiety of the protein fixes the B, C, and D rings of DHT. The 3-carbonyl and 17β-hydroxyl groups of DHT are essential for agonistic action. BPA derivatives may act as antagonistic agents by interfering with the interaction of DHT with Arg752 of the receptor owing to the presence of the 4-hydroxyl group and hydrophobic moiety of BPA. The 4′-hydroxyl group may also interfere with the binding of the 17β-hydroxyl group of DHT to the androgen receptor. Hydrophilic substituents at the methylene bridge of BPA may disturb the interaction with a hydrophobic moiety of the protein in the same way as with the estrogenic receptor. Unexpectedly, p-isopropylphenol, which lacks the B-phenyl ring of BPA, exhibited anti-androgenic activity. Clearly the B-phenyl ring is not essential for this activity, unlike estrogenic activity. Substitution by a bulky group at the 3,5-positions of the A-ring seems not to hinder the interaction with androgen receptor. We have already reported the agonistic activity of TBBPA and TCBPA on the thyroid hormonal activity of T3 (Kitamura et al., 2002). In the present study, we found that DMBPA and TMBPA, in addition to TBBPA and TCBPA, show thyroid hormonal activity, though other BPA derivatives do not. Moriyama et al. (2002) reported that BPA acts as an antagonist of the thyroid hormone action of T3. We could not observe agonistic or antagonistic action of BPA. Ishihara et al. (2003) also did not observe affinity of BPA for thyroid hormone receptors. Perhaps the thyroid hormonal potency of TBBPA, TCBPA, DMBPA, and TMBPA found in this study is due to their structural resemblance to thyroid hormones. The rat pituitary cell line GH3, isolated from a rat pituitary tumor, has been widely used as a standard pituitary cell model, in which the growth hormone secretion depends markedly on thyroid hormones, but little on estrogen (Kitagawa et al., 1987). In the present study using GH3 cells, we observed agonistic activities of TBBPA, TCBPA, DMBPA, and TMBPA toward thyroid hormone, but we could not detect anti-thyroid hormonal action of these compounds. A 4-hydroxyl group and double substitution by halogen or methyl group at 3,5-positions of the A-phenyl group were essential for thyroid hormonal activity in this study. Relatively large substituents at the 3,5-positions of the phenyl ring, besides the 4-hydroxyl group, seem to be necessary for thyroid hormonal activity (Fig. 7). Thyroid hormone receptor shows a rigid substrate specificity compared with estrogenic and androgenic receptors, because of the relatively smaller size of the active site. Wagner et al. (1995, 2001) conducted crystallographic analyses of rat thyroid hormone receptor α1 and its ligands, and reported that the 4′-hydroxyl group of thyroid hormones interacts with His381 of thyroid hormone receptor via hydrogen-bounds, and the 3′-iodo group interacts with the hydrophobic pocket formed by Gly290, Gly291, Phe215, and Met388. The thyroid hormonal activity decreased in the order of bromine, chlorine, and methyl substituents at the 3,5-positions of BPA. Weaker binding ability, due to these smaller-size substituents compared with iodine, with the hydrophobic pocket in the active site may be a reason for the decreased activity. Various polybrominated biphenyls, polybrominated diphenyl ethers or their metabolites with such structural features may compete with thyroid hormones for binding to the thyroid hormone receptor. Thyroid hormone disruption could occur through interaction with serum transport proteins, such as transthyretin and thyroid-binding globulin. Some PCBs, for instance, have been reported to bind to transthyretin with very high affinity. Although we previously found that both TCBPA and TBBPA are able to bind thyroid hormone receptor, the interactions of these compounds with serum transport proteins and other metabolic components should be examined in the future. The 3,5-substituents of BPA markedly influenced the endocrine-disrupting activity. TCBPA exhibited the highest estrogenic activity among the test compounds. In contrast, TBBPA exhibited the highest thyroid hormonal activity. Some 3,5-substituted BPAs showed combined endocrine-disrupting activity. TCBPA, TMBPA, and TBBPA showed both estrogenic and thyroid hormonal activities. TMBPA and TBBPA showed both estrogenic and anti-estrogenic activities. TMBPA and DMBPA showed higher anti-androgenic activity. However, anti-androgenic activity was not observed with TBBPA or TCBPA. As 3,5-substituted BPA derivatives are widely used as industrial materials or found as environmental contaminants, it is necessary to consider other possible toxicities. In contrast, BPA, BPB, BPAF, HPP, and BPF exhibited both high estrogenic and anti-androgenic activities. The endocrine-disrupting activity of BPA derivatives in vivo may be based on combinations of endocrine-disrupting actions, as observed in vitro. Hydroxy-PCBs were also reported to show both estrogenic and anti-thyroid hormonal activities (Cheek et al., 1999; Connor et al., 1997; Korach et al., 1988). The possibility of multiple endocrine-disrupting activities in animals in vivo should be taken into consideration in assessing the toxicity of environmental contaminants, including BPA derivatives. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (13027256) from the Japanese Ministry of Education, Science, Sports and Culture, and a Grant-in-Aid for Scientific Research (C16590092) from the Japan Society for the Promotion of Science. References Andersen, H. R., Andersson, A.-M., Arnold, S. F., Autrup, H., Barfoed, M., Beresford, N. A., Bjerregaard, P., Christiansen, L. B., Gissel, B., Hummel, R., Jørgensen, E. B., Korsgaard, B., Le Guevel, R., Leffers, H., McLachlan, J., Møller, A., Nielsen, J. B., Olea, N., Oles-Karasko, A., Pakdel, F., Pedersen, K., Perez, P., Skakkeboek, N. E., Sonnenschein, C., Soto, A. M., Sumpter, J. P., Thorpe, S. M., and Grandjean, P. ( 1999). 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Google Scholar Author notes *Graduate School of Biomedical Sciences and †Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan Toxicological Sciences vol. 84 no. 2 © The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

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Published: Jan 5, 2005

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