* 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
1 To whom correspondence should be addressed at Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. Fax: +81-82-257-5329. E-mail: skitamu{at}hiroshima-u.ac.jp.
Received September 16, 2004; accepted December 7, 2004
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ABSTRACT |
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Key Words: estrogenic activity; anti-androgenic activity; thyroid hormonal activity; bisphenol A; bisphenol derivative; human breast cancer cell line MCF-7; rat pituitary cell line GH3.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 x 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, 104 109 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 x 1010 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 23 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 x 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, 104 108 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 x 1010 or 1 x 1011 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 x 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 x 107 or 1 x 108 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.
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RESULTS |
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DISCUSSION |
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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
, MtT/E-2 and a mouse fibroblast cell line, NIH3T3, transiently transfected with ER
or ß (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.
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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
1 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.
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ACKNOWLEDGMENTS |
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