Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea
Received January 29, 2003; accepted May 5, 2003
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ABSTRACT |
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Key Words: bisphenol A (BPA); nonylphenol (NP); androgen receptor (AR); androgen; antiandrogen; endocrine disruptor.
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INTRODUCTION |
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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., 1993; Quigley et al., 1995
). 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., 1999
; Wong et al., 1993
). 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 ARandrogen binding, nuclear import, DNA binding, and/or transcriptional activation (Quigley et al., 1995
).
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., 1995) and in the saliva collected from subjects treated with dental sealants (Olea et al., 1996
). 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., 1993
) although it binds to both ER (estrogen receptor)-
and ERß with low affinity (Gaido et al., 1997
).
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., 1992; Nimrod et al., 1996
). 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., 1997
; Jager et al., 1999
; P. C. Lee, 1998
; Sharpe et al., 1995
; Yoshida et al., 2001
). 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., 1999
; Nagao et al., 2001
; Odum et al., 2000
; Tyl et al., 1999
). NP has been also reported to possess the estrogenic property in in vitro and in vivo assay systems (Jobling et al., 1996
; Legler et al., 2002
; Moffat et al., 2001
).
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.
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MATERIALS AND METHODS |
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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., 2002). 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/1834, as previously described (Ausubel et al., 1994). 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., 1994). 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., 1990). 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
-dihydroxytestosterone (DHT) in the presence and absence of increasing concentrations of unlabeled compounds. Nonspecific binding of [3H]5
-DHT was assessed by adding a 100-fold molar excess of unlabeled 5
-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 manufacturers 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., 1993), 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., 1997
). 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).
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RESULTS |
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DISCUSSION |
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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., 1994, 1995
) and subsequent expression of androgen target genes in vitro and in vivo (Kelce et al., 1995
; Wong et al., 1993
). 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., 1995; Viscardi and Max, 1993
). 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, 1998; Kousteni et al., 2001
; Yang et al., 2001
). Guo et al. (2001)
, 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., 2002
). 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, 2000), abnormal sexual development, alterations in pituitary and thyroid gland functions (Gore, 2001
), immune suppression (Straube et al., 1999
), and abnormal neurobehavioral effects (McEwen, 1987
; Takahama and Shirasaki, 2001
). Interference with androgen action during gonadal development can also cause abnormalities of the male reproductive system (Guillette et al., 1994
). 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., 1999
), 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.
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ACKNOWLEDGMENTS |
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NOTES |
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