Antiandrogenic Effects of Bisphenol A and Nonylphenol on the Function of Androgen Receptor

Hyun Ju Lee, Soma Chattopadhyay, Eun-Yeung Gong, Ryun Sup Ahn and Keesook Lee1

Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea

Received January 29, 2003; accepted May 5, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
By using a yeast detection system for androgenic and antiandrogenic effects of chemicals, we identified bisphenol A (BPA) and nonylphenol (NP) as antiandrogens. In this study, we report molecular mechanisms for the antiandrogenic action of BPA and NP. In the ARhLBD-activating signal cointegrator 1 (ASC1) yeast two-hybrid system, which reflects the androgen-dependent interaction between androgen receptor (AR) and its coactivator, ASC1, BPA and NP acted as potent AR antagonists comparable to a known strong antagonist, cyproterone acetate. Ligand competition assays revealed that [3H]5{alpha}-dihydroxytestosterone (DHT) binding to AR is inhibited a maximum of 30 and 40% at approximately 5 nM of NP and 50 nM of BPA, respectively. In addition, the nuclear translocation of green fluorescent protein (GFP)-AR fusion protein in the presence of testosterone was affected by the addition of BPA and NP, which cause rather dispersed distribution of GFP-AR between the nuclear and the cytoplasmic compartments. Furthermore, in transient transfection assays, BPA and NP inhibited androgen-induced AR transcriptional activity. Taken together, the results suggest that BPA and NP affect multiple steps of the activation and function of AR, thereby inhibiting the binding of native androgens to AR, AR nuclear localization, AR interaction with its coregulator, and its subsequent transactivation. These data may help us better understand the biological alterations induced by these environmental compounds.

Key Words: bisphenol A (BPA); nonylphenol (NP); androgen receptor (AR); androgen; antiandrogen; endocrine disruptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Some environmental chemicals have been shown to affect sexual differentiation and development of laboratory animals, suggesting their implications in the increasing incidence of reproductive abnormalities in wildlife and human populations (Colborn et al., 1993Go; Danzo, 1998Go; Rajesh, 1999Go). They may have such disruptive effects by disturbing the normal function of the gonadal steroid hormones that are necessary for normal sexual development. For example, recent in vitro (Sohoni and Sumpter, 1998Go) and in vivo (Takahashi and Oishi, 2001Go) studies of endocrine disrupters suggest that environmental antiandrogens that bind to and inhibit androgen receptors (AR) may be crucial contributors to abnormal development of the male sex.

Androgens are absolutely necessary for normal male sexual development and reproduction. They also stimulate masculinization of the fetus and induce male imprinting of the developing brain (McPhaul et al., 1993Go; Quigley et al., 1995Go). Androgens act through their receptor, AR, which is a ligand-activated transcription factor and belongs to the family of steroid hormone receptors. AR is held as an inactive state, being associated with specific heat-shock proteins before exposure to androgens. Upon ligand binding, AR translocates into the nucleus and forms a complex with specific DNA sequences called androgen-responsive elements (ARE) to enhance transcription of target genes recruiting coactivators (McKenna et al., 1999Go; Wong et al., 1993Go). Molecular defects in the AR gene thus cause the syndrome of androgen insensitivity with a certain degree of abnormal sexual development, which results from the failure of AR–androgen binding, nuclear import, DNA binding, and/or transcriptional activation (Quigley et al., 1995Go).

Bisphenol A (BPA) is one of the industrial compounds that have generated concerns, due to their high production and widespread use. BPA has been found in the liquor from canned food packed in lacquer-coated cans (Brotons et al., 1995Go) and in the saliva collected from subjects treated with dental sealants (Olea et al., 1996Go). It has been previously reported to be weakly estrogenic in both in vitro and in vivo assay systems. BPA competes with [3H]-estradiol for binding to estrogen receptors (ER) from the rat uterus, induces the expression of progesterone receptors, and promotes cell proliferation in cultured human mammary cancer cells (MCF-7) (Krishnan et al., 1993Go) although it binds to both ER (estrogen receptor)-{alpha} and ERß with low affinity (Gaido et al., 1997Go).

Nonylphenol (NP) is generated from alkylphenol ethoxylates that are widely used in the production of plastics, textiles, and agricultural chemicals, and in household applications such as detergents, paints, pesticides, and cosmetics (Naylor et al., 1992Go; Nimrod et al., 1996Go). Several studies have reported adverse effects of NP on the development of the male reproductive tract when animals are perinatally exposed to NP (Boockfor et al., 1997Go; Jager et al., 1999Go; P. C. Lee, 1998Go; Sharpe et al., 1995Go; Yoshida et al., 2001Go). These effects include reduced testes size, decreased sperm production, cryptorchidism, and reduced reproductive organ weights. On the other hand, other studies, including multigenerational studies utilizing oral exposures, have found little effects of NP on male reproduction (Chapin et al., 1999Go; Nagao et al., 2001Go; Odum et al., 2000Go; Tyl et al., 1999Go). NP has been also reported to possess the estrogenic property in in vitro and in vivo assay systems (Jobling et al., 1996Go; Legler et al., 2002Go; Moffat et al., 2001Go).

In this study, we report that BPA and NP have antiandrogenic activity at multiple steps of AR activation and function. They inhibit AR-androgen binding, AR nuclear import, AR interaction with its coactivator, and its subsequent transactivation in a concentration-dependent manner. These results suggest that BPA and NP may act as antiandrogens in vivo and change the gene expression, resulting in abnormal development and function of AR target tissues.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Cyproterone acetate (minimum 97% purity) was purchased from Sigma Chemical Co. Bisphenol A (BPA) (99+% purity) and NP were obtained from Aldrich Chemical Co. High concentrations of some chemicals can have cytotoxicity, inhibiting the growth of cells or even lysing cells (Routledge et al., 1998Go). However, none of the chemicals, at the concentrations that we tested, significantly reduced the growth of cells (ex: in yeast, absorbance value at 600 nm was in the range of 0.5–1.2), which cannot account for our results as previously discussed (Sumpter, 1998Go).

Plasmids.
The LexA-ARhLBD, B42-ASC-1 and LexA-ARAF1DBDh fusion vectors, and the mammalian expression plasmid of AR (pcDNA3.mAR) were previously described (Lee et al., 2002Go). Luciferase reporter plasmids, pARE2-TATA-Luc containing two AREs of the C3 gene, and pGL3-PSA-Luc containing 5.3 kb prostate specific antigen (PSA) promoter, are kind gifts from Dr. J. J. Palvino (University of Helsinki, Finland) and Dr. C. J. Bieberich (University of Maryland, Baltimore County, Baltimore, MD), respectively. GFP-AR fusion vector was constructed by cloning the full-length AR in frame into the pEGFP vector (Clontech, Palo Alto, CA).

Yeast transformation.
Plasmids encoding LexA and B42 fusions were cotransformed into Saccaromyces cerevisiae EGY48 containing the lac-Z reporter plasmid, SH/18–34, as previously described (Ausubel et al., 1994Go). The transformants were obtained by growing them on the YPD/-His/-Trp/-Ura selection media and established as strains.

Yeast two-hybrid protein interaction assay.
Yeast cells were grown in the SD/Gal/Raf/-His/-Trp/-Ura induction media in the absence or presence of testosterone or/and the indicated chemicals. After incubation at 30°C for 3 h, an equal amount of yeast cells was harvested and resuspended in a buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, and 50 mM 2-mercaptoethanol, pH 7.0). The cells were then lysed by incubating at 30°C for 15 min with addition of the final 0.1% of SDS and 10% of chloroform. After taking the supernatants, liquid ß-galactosidase assays were carried out using o-nitrophenyl ß-D-galactopyranoside (ONPG) as a substrate, as described previously (Lee et al., 1994Go). All the experiments were repeated at least three times in duplicate.

Competitive steroid binding assays.
Whole-cell binding assays were performed as described previously (Yarbrough et al., 1990Go). Briefly, HeLa cells were transfected with pcDNA3.mAR using Superfect reagent (Qiagen, Germany). Twenty-four h prior to the binding reaction, cells were placed in serum-free and phenol red-free medium and incubated for 2 h at 37° with 5 nM [3H]5{alpha}-dihydroxytestosterone (DHT) in the presence and absence of increasing concentrations of unlabeled compounds. Nonspecific binding of [3H]5{alpha}-DHT was assessed by adding a 100-fold molar excess of unlabeled 5{alpha}-DHT. Cells were washed twice in phosphate-buffered saline (PBS), harvested in a buffer containing 2% SDS, 10% glycerol, and 10 mM Tris (pH 6.8), and radioactivity was determined by scintillation counter. Dose-response data were analyzed using the sigmoidal dose-response function of the graphical and statistical program Prism (GraphPad, San Diego, CA).

Fluorescence.
HeLa cells were plated onto gelatin-coated coverslips the day before transfection. The green fluorescent protein (GFP)-AR expression vector was transiently transfected by using Effectene reagent (Qiagen, Germany) according to the manufacturer’s instructions. After 16 h, transfected cells were fed with fresh medium containing 10% charcoal-stripped serum, and treated for 1 h with vehicle (ethanol) only, 10 nM testosterone, or 10 nM testosterone in combination with each of 10 µM other chemicals. The coverslips were then taken for picturing cells using a fluorescent microscope.

Transient transfection assays.
Both 15p-1 cells, an established Sertoli cell line from transgenic males that express the PyLT protein (Rassoulzadegan et al., 1993Go), and HepG2 cells were maintained in Dulbeccos modified Eagles medium (Life Technologies, Grand Island, MD) in the presence of 10% fetal bovine serum. Twenty-four h before transfection, cells were plated in 24-well plates, and transfected with the AR expression plasmid, a reporter plasmid, pARE2-TATA-Luc or pGL3-PSA-Luc, and the control lac-Z expression vector, pCMVß (Clontech, Palo Alto, CA) by using Superfect reagent (Qiagen, Germany). The total amount of DNA used was kept constant. Twenty-four h after transfection, the medium was replaced with fresh medium containing 10% charcoal-stripped serum and either 10 nM testosterone or vehicle (ethanol). Cells were harvested 24 h after hormone treatment, and luciferase and ß-galactosidase activities were assayed as described previously (Chen et al., 1997Go). The levels of luciferase activity were normalized to the lac Z expression. Dose-response data were analyzed using the sigmoidal dose-response function of the graphical and statistical program Prism (GraphPad, San Diego, CA).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antiandrogenic Activity of BPA and NP in ARhLBD-ASCI Yeast Cells
Since recent studies suggest that environmental antiandrogens may be crucial contributors to abnormal development of the male sex, we have tested putative endocrine disruptors, including BPA and NP (Fig. 1Go) for their antiandrogenic activity, using the ARhLBD-activating signal cointegrator 1 (ASC1) yeast system. The ARhLBD-ASC1 yeast system was previously developed for detection of androgenic and antiandrogenic activities of chemicals, using a yeast two-hybrid assay system in which the hinge-ligand binding domain (hLBD) of AR fuses to the DNA binding domain of LexA and interacts with the AR coactivators, ASC-1 fused to the activation domain of B42 in an androgen-dependent manner (Lee et al., 2002Go, 2003Go). Both BPA (IC50: 1.8 µM) and NP (IC50: 2.6 µM) significantly inhibited testosterone-induced interaction between ARhLBD and ASC-1, thereby decreasing the expression of a reporter, ß-galactosidase, in a dose-dependent manner (Fig. 2Go). The extents of their inhibition were stronger than that of cyproterone acetate (CPA) (IC50: 5.1 µM), a strong AR antagonist. The negative control, ß-estradiol, showed no such inhibitory effect. These results suggest that BPA and NP may be potent AR antagonists, and thus we further investigated their effect on the AR function.



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FIG. 1. Structural formula of bisphenol A (BPA) and nonylphenol (NP).

 


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FIG. 2. Antiandrogenic activity of BPA and NP in the ARhLBD-activating signal cointegrator 1 (ASC1) yeast system. Yeast cells were treated with various concentrations (0.1–100 µM) of BPA (A) and NP (B) in competition with 10-nM testosterone (T). Cyproterone acetate (CPA) (C), and ß-estradiol (D) were used as positive and negative controls, respectively. One-µM concentrations of each compound alone were also tested for androgen receptor (AR) agonist activity. The ß-galactosidase activities were compared with that obtained with 10 nM T only, which was set as 100. Nonspecific inhibitory effects were tested with 1–100 µM concentrations of each compound using the LexA-AR-AF1DBDh construct, which contains a constitutively active AR fragment (E). The ß-galactosidase activities were compared with that obtained with the no-compound treatment, which was set as 100. No significant nonspecific inhibitory effects of the compounds were observed. Each value represents the mean ± SE of three independent experiments.

 
Effect of BPA and NP on Androgen Binding to AR
The molecular basis for the antiandrogenic effects of BPA and NP was investigated using mouse AR expressed transiently in HeLa cells. In competitive androgen binding assays using [3H]5{alpha}-DHT (a labeled androgen), NP and BPA inhibited androgen binding to AR only partially, up to 30 and 40% at approximately 5 and 50 nM, respectively (Fig. 3Go). Furthermore, there was the lack of a dose-response relationship in their inhibition. These results suggest that BPA and NP may inhibit androgen binding to AR in a noncompetitive manner.



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FIG. 3. Effect of BPA and NP on [3H]5{alpha}-dihydroxytestosterone (DHT) binding to androgen receptor (AR). Binding inhibition was determined in HeLa cells transfected transiently with the mouse AR expression vector, pcDNA3.mAR, as described in Materials and Methods. Results are expressed as percent binding relative to [3H]5{alpha}-DHT alone and are shown for unlabeled T, DHT, CPA, BPA, and NP. The data are representative of three independent experiments.

 
Effect of BPA and NP on Dynamics of AR Nuclear Translocation
We investigated the effect of BPA and NP on dynamics of AR subcellular distribution using a GFP-AR fusion protein. When the GFP-AR was overexpressed in HeLa cells in the absence of androgen, the fusion protein was distributed in the cytoplasmic compartment as well as the nucleus, but in the presence of testosterone (T, 10 nM) GFP-AR protein was predominantly localized in the nucleus as previously described for the native AR (Jenster et al., 1991Go) (Fig. 4Go). The inhibitory effect of BPA and NP on the nuclear import of AR protein was assessed by challenging 10 nM of T with 10 µM of each compound. The distribution of GFP-AR protein in cells treated with BPA or NP together with T was similar to that in cells treated with vehicle only (no T), wherein the nuclear translocation of GFP-AR was inhibited, showing rather dispersed distribution between the nuclear and the cytoplasmic compartments. CPA, a steroidal antiandrogen, also showed a similar effect on the nuclear import of GFP-AR as previously reported (Georget et al., 2002Go; Terouanne et al., 2002Go). These results suggest that BPA and NP interfere with the nuclear translocation of AR.



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FIG. 4. Effects of BPA and NP on the dynamics of the nuclear translocation of green fluorescent protein (GFP)-AR. HeLa cells expressing the GFP-AR fusion protein were cultured in a culture dish at 37°C and the fluorescence was analyzed directly by the fluorescence microscope. The subcellular localization of the GFP-AR in living cells was observed and recorded after a 60-min treatment with vehicle (ethanol) only, 10 nM testosterone (T), or 10 µM concentration of each of BPA, NP, and cyproterone acetate in the presence of 10 nM T.

 
Effect of BPA and NP on the Transcriptional Activity of AR
The effect of BPA and NP on the androgen-induced AR transactivation was investigated by transient transfection assays with reporters pARE2-TATA-Luc (Fig. 5AGo) containing two AREs of the C3 gene and pGL3-PSA-Luc (Fig. 5BGo) containing 5.3 kb PSA promoter in 15p-1 and HepG2 cells, respectively. Antiandrogen CPA showed a dose-dependent inhibition of the reporter expressions (Fig. 5Go) although it revealed agonist activity at high concentrations (>0.1 µM) with the reporter pGL3-PSA-Luc, which contains a complex promoter (data not shown). Both BPA and NP also exhibited their inhibitory effect on the AR transactivation activity in a dose-dependent manner. With pARE2-TATA-Luc, BPA (IC50: 80 nM) inhibited the reporter expression better than CPA (IC50: 512 nM) while NP (IC50: 1.97 µM) did worse. With pGL3-PSA-Luc containing a complex promoter, BPA (IC50: 318 nM) and NP (IC50: 781 nM) were much less effective than cyproterone acetate (IC50: 4 nM). With both reporters, BPA was a stronger antagonist than NP in respect to the inhibition of androgen-induced AR transactivation.



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FIG. 5. Inhibitory effect of BPA and NP on androgen-mediated AR transcriptional activity. Transcriptional activity was determined in 15p-1 (A) and HepG2 (B) cells transiently transfected with AR expression vector and its reporter, pARE2-TATA-Luc (A) or pGL3-PSA-Luc (B), as described in Materials and Methods. Cells were treated with the indicated concentrations of BPA and NP in the presence of 10 nM T. CPA was used as a positive control. Each value represents the mean ± SE of at least three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endocrine disruptors are thought to mimic natural hormones, inhibit the action of hormones, or alter the normal regulatory function of endocrine systems. Many putative endocrine disruptors have indeed turned out to be estrogen-like and antiandrogenic chemicals. In this study, we propose that BPA and NP, previously shown to have estrogenic effects, have also antiandrogenic activity. Antiandrogenic effects of BPA and NP have been previously accessed. Sohoni and Sumpter (1998)Go reported that both BPA and NP have antiandrogenic activity in an in vitro yeast-based assay system. On the other hand, Gaido et al. (2000)Go reported that BPA has no antiandrogenic activity in HepG2 cells transfected transiently with human AR, which contradicts our results in this study. They challenged 100 nM DHT with 0.1–10 µM BPA, while we did 10 nM T with 0.01–10 µM BPA. The use of a ligand with different affinity to AR and a different ligand concentration challenged by BPA might result in the difference in the sensitivity to detect the effects of BPA.

It has been previously demonstrated that several compounds including vinclozolin and p,p'-DDE are potent environmental antiandrogens competing to inhibit the androgen binding of AR (Kelce et al., 1994Go, 1995Go) and subsequent expression of androgen target genes in vitro and in vivo (Kelce et al., 1995Go; Wong et al., 1993Go). Here, we report that BPA and NP may be also AR antagonists by affecting AR function: they inhibit AR interaction with its coactivator, androgen binding of AR, AR nuclear translocation, and androgen-induced AR transcriptional activity. However, the in vivo study is critical in the final assessment of their endocrine disrupting activity, since sufficient levels of the parent chemicals and/or metabolites should be attained in target tissues for a sufficient time to alter AR-regulated processes.

The inhibition of androgen binding to AR by BPA and NP is partial and lacks a dose-response relationship, which suggests that the manner of their inhibition may be noncompetitive. Noncompetitive inhibition of ligand binding to hormone receptors by a specific compound has been previously reported. For example, amiodarone and unsaturated fatty acid bind to the thyroid hormone receptor subtype b1 and glucocorticoid receptor, respectively (Drvota et al., 1995Go; Viscardi and Max, 1993Go). They have been proposed to bind to sites different from ligand binding sites in the receptors. It is worthwhile to investigate whether BPA and NP act in a similar manner.

Testosterone has been described for having both genomic and nongenomic actions. The ligand-activated intracellular AR is not only able to act on transcription but also exert nongenomic actions such as activation of extracellular signal-regulated kinase (ERK)1/2 and p21-activated kinase (Cato and Peterzied, 1998Go; Kousteni et al., 2001Go; Yang et al., 2001Go). Guo et al. (2001)Go, on the other hand, demonstrated that non-genomic testosterone signaling is able to exert genomic actions in context with the lipopolysaccharide (LPS) signaling pathway through p38 mitogen-activated protein kinase (MAPK). Although signaling pathways involved in the endocrine disrupters have been poorly characterized, BPA has been recently shown to induce Nur77 gene expression via MAPK activation, suggesting a link between the signaling cascade and the action of endocrine disrupters (Song et al., 2002Go). In light of such a connection, it is worth investigating the effects of BPA and NP on nongenomic testosterone signaling.

Endocrine disruptors are potentially hazardous for causing abnormalities in a variety of animal systems. Reduced fertility in males is one of the major endpoints in addition to testicular and prostate cancers (Imaida and Shirai, 2000Go), abnormal sexual development, alterations in pituitary and thyroid gland functions (Gore, 2001Go), immune suppression (Straube et al., 1999Go), and abnormal neurobehavioral effects (McEwen, 1987Go; Takahama and Shirasaki, 2001Go). Interference with androgen action during gonadal development can also cause abnormalities of the male reproductive system (Guillette et al., 1994Go). Since the AR is also required for the development of prostate cancer, and the early stages of prostate cancer cells are androgen-dependent and highly sensitive to antiandrogens (Cude et al., 1999Go), it will be interesting to investigate the effects of endocrine disruptors with antiandrogenic activity such as BPA and NP on the development and progression of prostate cancer.

In conclusion, we show in this work that BPA and NP have an antiandrogenic activity at several steps of AR function. These data may help us predict the biological alterations induced by these environmental compounds and better understand their contribution to the reproductive disorders in males.


    ACKNOWLEDGMENTS
 
This work was supported by Korea Research Foundation Grant (KRF-2002-070-C0007) and Hormone Research Center Grant (2002G0101). We thank Dr. J. J. Palvino and Dr. C. J. Bieberich for providing pARE2-TATA-Luc and pGL3-PSA-Luc constructs, respectively.


    NOTES
 
1 To whom correspondence should be addressed. Fax: +82-62-530-0500. E-mail: klee{at}chonnam.ac.kr. Back


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