Effects of 2,4-D and DCP on the DHT-Induced Androgenic Action in Human Prostate Cancer Cells
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《毒物学科学杂志》
School of Life Sciences & Biotechnology, Korea University, 1, 5-Ka, Anam-dong, Sungbuk-ku, Seoul 136-701, Korea
ABSTRACT
2,4-Dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (DCP) are used extensively in agriculture as herbicides, and are suspected of potential endocrine disruptor activity. In a previous study, we showed that these compounds exhibited synergistic androgenic effects by cotreatment with testosterone in the Hershberger assay. To elucidate the mechanisms of the synergistic effects of these compounds on the androgenicity of testosterone, the androgenic action of 2,4-D and DCP was characterized using a mammalian detection system in prostate cancer cell lines. In in vitro assay systems, while 2,4-D or DCP alone did not show androgenic activity, 2,4-D or DCP with 5-dihydroxytestosterone (DHT) exhibited synergistic androgenic activities. Cotreatment of 10 nM 2,4-D or DCP with 10 nM DHT was shown to stimulate the cell proliferation by 1.6-fold, compared to 10 nM DHT alone. In addition, in transient transfection assays, androgen-induced transactivation was also increased to a maximum of 32-fold or 1.28-fold by cotreatment of 2,4-D or DCP with DHT, respectively. However, 2,4-D and DCP exerted no effects on either mRNA or protein levels of AR. In a competitive AR binding assay, 2,4-D and DCP inhibited androgen binding to AR, up to 50% at concentrations of approximately 0.5 μM for both compounds. The nuclear translocation of green fluorescent protein-AR fusion protein in the presence of DHT was promoted as the result of the addition of 2,4-D and DCP. Collectively, these results that 2,4-D and DCP enhanced DHT-induced AR transcriptional activity might be attributable, at least in part, to the promotion of AR nuclear translocation.
Key Words: 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4-dichlorophenol (DCP); androgen receptor (AR); 22Rv1; endocrine disruptor.
INTRODUCTION
The phenoxy compounds 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (DCP) were widely used as a hormonal herbicide to control the growth of broadleaf plants and as an intermediate for pesticide manufacturing, respectively. 2,4-D has been shown to exert toxic effects on both animals and humans, ranging from embryotoxicity and teratogenicity to neuro-, immuno-, and hepatotoxicity (Barnekow et al., 2000; Charles et al., 2001; Rosso et al., 2000). 2,4-D has also been reported to cause positive for chromosomal aberrations and sperm head abnormalities in two in vivo assays in mice (Amer and Aly, 2001; Venkov et al., 2000). Amer and Aly (2001) reported the induction of sperm head abnormalities by treatment with these substances, but only at very high in vivo concentrations (1/2 LD50). Tordon 75 D, which contains the 2,4-D and 4-amino-3,5,6-trichloropicolinic acid (picloram) caused severe reduction in testicular weight in some animals given high doses (Oakes et al., 2002). Although several studies have reported 2,4-D-induced testicular changes in rats, the precise mechanisms underlying these phenomena have yet to be fully understood, and it remains unclear whether phenoxy compounds affect endocrine systems in other species.
In a previous study, we have demonstrated that the administration of 2,4-D (50 mg/kg/day, po) or DCP (100 mg/kg/day, po) to rats caused an increase in the weights of ventral prostate, Cowpers gland, and glands penis. In addition, these increases in the weight of androgen-dependent tissues were synergistically potentiated when rats were simultaneously treated with 2,4-D or DCP and low dose of testosterone (1 mg/kg, sc) (Kim et al., 2002).
Some chemicals, including bisphenol A, nonylphenol, and fenthion, have been reported to be able to interact directly with AR and to activate AR-dependent transcription in mammalian cells (Lee et al., 2003; Shigeyuki et al., 2003). These chemicals that mimic the biologic activity of androgen are called environmental endocrine disruptors. Only a few chemicals have exhibited androgenic activity, and no report has been available on the effects of phenoxy compounds on AR-mediated actions, even though testicular changes caused by these phenoxy compounds have been reported in some animals.
Androgens act via an interaction with the androgen receptor (AR), a ligand dependent transcription factor. The homodimer of the androgen-bound receptors is translocated into the nucleus, where it binds to a specific DNA motif, the androgen response element (ARE), located in the regulatory region of target genes, and activates or represses the transcription of the androgen-regulatory genes (Chen and Evans, 1995; Gnanaragasam et al., 2000; Onate et al., 1995). Some environmental chemicals cause the androgenic endocrine disruption as a result of failure of AR-androgen binding, nuclear import, DNA binding, and/or transcriptional activation (Lee et al., 2003; Wong et al., 1995).
The purposes of this study were to more precisely characterize the synergistic androgenic action of 2,4-D or DCP with DHT in vitro, and to understand the molecular mechanisms of phenoxy compound action mediated AR activation. In this report, although 2,4-D and DCP (Fig. 1) inhibited the AR-DHT binding, these compounds stimulated DHT-mediated cell proliferation, DHT-induced AR nuclear importing, and its subsequent transactivation. This is the first report that provides clear evidence for the androgenic actions of phenoxy compounds using a mammalian system.
MATERIALS AND METHODS
Chemicals.
5-Dihydroxytestosterone (minimum purity >99%) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2,4-Dichlorophenoxyacetic acid and dichlorophenol were purchased from Aldrich Chemical Co. (St. Louis, MO). The purity of 2,4-D and DCP was more than 98%. Restriction and modifying enzymes were obtained from Takara (Shiga-ken, Japan). AR (C19) antibody was obtained from Santa Cruz Biotechnolgy (Santa Cruz, CA). Horseradish peroxidase conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Zymed Laboratories (San Francisco, CA).
Construction of plasmids.
The MMTV fragment was excised from pMSG (Amersham Pharmacia Biotech) by HindIII-SmaI sites and inserted in the HindIII-SmaI sites of the pBluescript II SK (+) vector (pMMTV-SK). To generate promoter-reporter construct pMMTV-Luc, then, the MMTV fragment was then excised from pMMTV-SK by SmaI-Kpn I and inserted in the SmaI-Kpn I of the pGL3 basic vector (Promega). The expression vector for the human full-length AR (pCMV-hAR) was constructed by inserting the human androgen receptor cDNA into the CMV vector. Chimeric GFP-AR was generated by inserting the human full-length AR (excised by BglII-XbaI) into the BglII-XbaI site of the 3'-end of the coding region of the modified aequorea GFP (pEGFP-C2, CLONTECH Laboratories, Inc. Palo Alto, CA).
Cell culture.
Two human prostate cancer cell lines were used: AR expressed 22Rv1 (cat # CRL-2505) and androgen-independent PC3 (cat # CRL-1435), obtained from the American Type Culture Collection (Rockville, MD). The 22Rv1 cells are derived from a relapsed tumor and have been characterized as a valuable tool for the study of prostate cancer progression (Clifford et al., 2002). A representative androgen-insensitive cell line, PC3 was used for validation. The cells were routinely maintained in RPMI 1640 medium (Gibco BRL) containing 10% FBS, penicillin (100 units/ml), 1% L-glutamine and streptomycin (100 μg/ml) in an atmosphere containing 5% CO2 at 37°C. For in vitro AR-binding assay, COS-1 (African green monkey kidney cells; ATCC, Rockville, MD) were grown in DMEM medium (Gibco BRL) containing 10% FBS, penicillin (100 units/ml), 1% L-glutamine and streptomycin (100 μg/ml) in an atmosphere containing 5% CO2 at 37°C.
Cell proliferation assay.
The 22Rv1 cells were seeded in 96-well plates at an initial concentration of 3 x 103 cells per well. After 24 h the 5-dihydrotestosterone (DHT) was added to the experimental medium (phenol red-free RPMI 1640 containing 10% dextran charcoal-stripped FBS: dcs-FBS). The bioassay was terminated on the fourth day and the cells were fixed and stained with sulforhodamine-B (SRB) as described (Villalobos et al., 1995). Briefly, the cells were treated with 10% trichloroacetic acid and incubated at 4°C for 1 h and then washed with PBS. To stain the proteins 50 μl of 0.4% SRB was added to each well for 1 h at room temperature. The wells were washed several times with 1% acetic acid and air dried. Bound dye was solubilized with the 100 μl of 10 mM Tris base (pH 10.5) in a shaker for 5 min. The amount of SRB product in each well was determined by reading the plates on a plate-reading spectrophotometer (Dynatech MRX, Chantilly, VA) at 570 nm. The assays were repeated at least three times for each cell line.
Transactivation assays.
The 22Rv1 and PC3 cells (1.2 x 105) were seeded in 24-well plates and transfected with 1 μg of mouse mammary tumor virus luciferase vector (MMTV-Luc) and pCMV-hAR expression vector or with MMTV-Luc alone using the ExGene 500 (MBI Fermentas, St. Leon-Rot, Germany). Twenty-four hours after transfection, the indicated concentrations of DHT and other chemicals were added with experimental medium. After additional 24 h-incubation, the cells were lysed using a passive lysis buffer (Promega, Madison, WI). The extracts were assayed for luciferase activity using dual-luciferase reporter system (Promega, Madison, WI), and subsequently measured on a Liquid Scintillation Counter Luminometer (Bioscan, Inc., U.S.A). Luciferase activity was normalized for transfection efficiency using pRL-TK as an internal control. Cell cytotoxicity was determined by the colorimetric MTS cell cytotoxicity assay according to the manufacturer's protocol (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI).
RNA isolation and RT-PCR.
Total RNA was isolated from 22Rv1 using TRIzol reagent (Life Technologies, Inc., Grand Island, NY) and cDNA was synthesized according to the manufacturer's protocol. Briefly, total RNA was reverse-transcribed using MMLV-reverse transcriptase (Promega, USA) and oligo dT primer using the following conditions: 70°C for 10 min and 42°C for 1 h. Expression of genes was investigated by PCR using the following primers: hAR 5'-AAGCTCAAGGATGGAAGTGCAGT-3' (sense) and 5'-CATGGACACCGACACTGCCT-3' (antisense) generating a 751 bp product; -actin 5'-CCATGGAGAGCCTCTGTGGATAT-3' (sense) and 5'-TGAGGATCTGATCTGTCCACAAG-3' (antisense) generating a 415 bp product. Subsequently, the PCR products were subjected to gel electrophoresis on 1% agarose gels containing 0.5 μg/ml of ethidium bromide and the intensities of the bands were evaluated with imaging software TINA (Raytest Isotopenmessgerte, Straubenhardt, Germany).
Immunoblotting.
22Rv1 cells (6 x 105 cells/60 mm dish) were plated and grown in phenol red-free RPMI 1640 containing 5% dcs-FBS media for three days. After treatment with DHT and/or 2,4-D and/or DCP as indicated, the cells were harvested in 100 μl lysis buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA] that contained a cocktail of protease inhibitors. The protein samples were loaded onto 10% acrylamide gel and subjected to gel electrophoresis. The protein samples were transferred to nitrocellulose membranes. The membranes were blocked for 1 h in 5% nonfat dry milk in PBST. The membranes were incubated overnight at 4°C in 5% milk/PBST containing primary antibodies to AR (Santa Cruz, CA) or -actin (sigma). The membranes were washed and then incubated with anti-mouse peroxidase secondary antibody (Zymed Laboratories, San Francisco) for 1 h at room temperature. The signal was detected using a chemiluminescense detection system (Pierce, Rockford, lL).
Competitive steroid binding assays.
Whole-cell binding assays were performed as described previously (Kim et al., 2002; Lambright et al., 2000). Briefly, COS-1 cells were transfected with pCMV-hAR using Lipofectamine reagent (Invitrogen, San Diego, CA). Twenty-four h prior to the binding reaction, the cells were placed in serum-free and phenol red-free medium and then incubated for 2 h at 37°C with 5 nM [3H]5-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 the radioactivity was determined using a scintillation counter.
Fluorescence imaging.
The PC3 cells were cultured in 2 x 2 cm2 Lab-Tek chamber slides (Nunc Inc., Naperville, IL) the day before transfection. The GFP-AR expression vector was transiently transfected using ExGene500 reagent (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. After 30 h, the transfected cells were fed with fresh medium containing 10% dcs-FBS, and treated for 30 min with the vehicle (ethanol), 10 nM DHT, or 10 nM DHT in combination with each of other chemicals also at 10 nM. The cells were then fixed with 10% formaldehyde at room temperature for 10 min. The fixed cells were rinsed twice with PBS (5 min each wash) and air dried. Fluorescence analysis was conducted using a Bio-Rad MRC1024 confocal laser system connected to a Zeiss Axioplan2 fluorescence microscope. For the evaluation of GFP-AR localization, the relative nuclear and cytoplasmic fluorescence in >100 cells per condition from three experiments was determined and scored according to a 5-grade nuclear localization score: exclusive nuclear fluorescence (4); nuclear fluorescence exceeding cytoplasmic fluorescence (3); equivalent nuclear and cytoplasmic fluorescence (2); cytopasmic fluorescence exceeding nuclear fluorescence (1); and exclusive cytoplasmic fluorescence (0). The translocation scores represent the mean ± SEM from three experiments in which >100 cells per condition per experiment were scored (Mario et al., 1998).
RESULTS
Effects of the Phenoxy Compounds on the Proliferation of Prostate Cancer Cells
22Rv1 cells were cultured in the RPMI1640 medium containing various concentrations of 2,4-D, DCP, or DHT for four days. The rate of cell growth was then determined by SRB assay. As shown in Figure 2, DHT increased cell growth in a dose-dependent manner. The maximum cell yields by treatment with DHT (10 nM) were about 1.5-fold. However, 2,4-D and DCP (Fig. 2A) treatment did not increase the proliferation rate of 22Rv1 cells. To investigate the combined effects of these phenoxy compounds with DHT on prostate cell proliferation, the 22Rv1 cells were treated with various concentrations of 2,4-D and DCP in the presence of 10 nM DHT, and the rate of cell growth was determined. Whereas stimulation with DHT alone showed a 1.5-fold increase in cell growth rate, cotreatment with 2,4-D or DCP and DHT exhibited a 2,5-fold increase in cell growth rate (Fig. 2B). This suggests that the phenoxy compounds acted either additively or synergistically with DHT on the proliferation of prostate cancer cells.
Effects of 2,4-D and DCP on DHT-Activated Transcription
We next examined the effects of 2,4-D and DCP on AR-mediated transcription with a luciferase reporter gene assay in which 22Rv1 cells had been transiently transfected with pMMTV-Luc. An approximately 8-fold induction of luciferase activity was observed in the cells incubated in the presence of 10 nM DHT (Fig. 3A). Treatment with various doses of 2,4-D or DCP did not increased luciferase activity (Fig. 3A).
To investigate the combined effects of these phenoxy compounds with DHT on AR-mediated transcription, two prostate cancer cell-lines, 22Rv1 and PC3/AR+ were treated with 2,4-D and DCP in the presence of DHT, respectively, and luciferase activity was measured (Figs. 3B and 3C), respectively. Surprisingly, although stimulation with DHT (10 nM) alone showed an 8-fold increase in luciferase activity in the 22Rv1 cells, cotreatment with 2,4-D (100 nM) or DCP (0.1 nM) and DHT exhibited a 32-fold or a 12.8-fold increase in the luciferase activity, respectively (Fig. 3B). In order to ensure that the synergistic effect of 2,4-D and DCP on DHT-activated transactivation was not specific to the 22Rv1 cells, PC3/AR+ transiently transfected with a human AR-expressing vector, pCMV-hAR, were treated with 2,4-D or DCP in the presence of DHT, and luciferase activity was measured. As shown in Figure 3C, similar synergistic effects of 2,4-D and DCP on DHT-activated transactivation were also observed in PC3/AR+ cells. In the presence of 10 nM 2,4-D or DCP, luciferase acitivity showed about a 3.2-fold or a 1.2-fold increase, respectively, compared to 10 nM DHT alone (Fig. 3C). These results indicate that the phenoxy compounds acted additively or synergistically with DHT to activate the AR-dependent transcription.
Effects of 2,4-D and DCP on AR Expression
As AR is the most important regulatory transcription factor for DHT-activated transcription, the effects of phenoxy compounds on AR expression were also investigated. In order to determine whether 2,4-D and DCP are capable of modulating the expression of AR, RT-PCR analysis and Western blotting were performed. The 22Rv1 cells were treated with 10 nM DHT combined with either 100 nM 2,4-D, or 0.1 nM DCP and the total RNA was subjected to RT-PCR analysis. As shown in Figure 4A, the combination of 2,4-D or DCP with DHT did not significantly modulate the expression of AR mRNA as measured by the expression of -actin mRNA.
Cells treated with 10 nM DHT exhibited a 50% increase in the levels of AR protein compared to the control (Fig. 4B). Western blot analysis indicated that the levels of AR protein in 22Rv1 cells did not change with cotreatment of 10 nM DHT and 100 nM 2,4-D or 0.1 nM DCP as compared to 10 nM DHT alone (Fig. 4B). These results suggested that the combination of phenoxy compounds and DHT exerted no synergistic effects on the levels of AR gene expression.
Effects of 2,4-D and DCP on Androgen Binding to AR
The AR-binding affinities of the phenoxy compounds were investigated using recombinant human AR transiently expressed in monkey kidney COS cells. The COS-1+hAR assays are recommended as the in vitro AR binding assays with the greatest priority for validation in a Tier 1 endocrine disruptor screening battery. In competitive androgen binding assays using [3H]5-DHT (a labeled androgen), 2,4-D and DCP inhibited androgen binding to AR, both up to 50% at approximately 0.5 μM (Fig. 5A). Competitive inhibition by DCP was greater than that of 2,4-D. Strongest competitive binding was observed with the natural androgen, DHT. Western blot analysis indicated that the levels of AR protein in COS cells did not change with treatment of chemicals (Fig. 5B). These results indicate that the 2,4-D and DCP had some affinity to AR although lower than DHT. Although 2,4-D and DCP inhibited DHT binding to the AR, the 2,4-D or DCP also exhibited a synergistic effect on the androgenic action, suggesting that a variety of signaling pathways for AR mediated androgenic action may be involved. The possibility that the androgenic effects of the 2,4-D and DCP may be independent of AR binding cannot be excluded.
Effect of 2,4-D and DCP on the Dynamics of AR Nuclear Translocation
In order to know how 2,4-D and DCP have synergistic effects with DHT to activate the AR-dependent transcription, we examined the effects of 2,4-D and DCP on AR translocation using a GFP-AR fusion protein. We first tested the ability of this chimeric protein for androgen-dependent transactivation of the pMMTV-Luc system in AR-negative PC3 cells. In the presence of 10 nM DHT, cells transfected with the GFP-AR expression vector showed about one-third transactivaition function as compared with the pCMV-hAR (data not shown). However, transactivation function of both of these AR proteins was almost totally dependent on the presence of the androgen. To visualize whether 2,4-D and DCP could modulate the DHT-dependent nuclear translocation of hAR, PC3 cells were transiently transfected with GFP-hAR and treated with either 0 (vehicle only, control), 10 nM DHT only as a positive control, 10 nM of 2,4-D or 10 nM of DCP with or without DHT for 20 min. In the absence of DHT, the GFP-AR fusion protein was distributed to the cytoplasmic compartment, but in the presence of DHT (10 nM) GFP-AR protein was became localized in the nucleus. In the presence of 2,4-D or DCP alone, GFP-AR was distributed to the cytoplasmic compartment (Fig. 6). Surprisingly, cotreatment with 2,4-D or DCP (10 nM) and DHT (10 nM) promoted the nuclear translocation of AR (Fig. 6). The bar graphs in Figure 6 show the nuclear translocation scores from 0 to 4 for GFP-AR in cells treated with DHT alone (lane 2), 2,4-D alone (lane 3), DCP alone (lane 4) and the promotion of DHT-mediated translocation by 2,4-D (lane 5) and DCP (lane 6), as determined for several hundred cells per condition. These data suggest that 2,4-D and DCP exerted a synergistic effect with DHT resulting in a markedly augmented activation of AR-mediated transcription via facilitation of AR nuclear translocation.
DISCUSSION
Recently, many investigations have been focused on chemicals that disrupt the normal functions of the endocrine system, the glands and hormones that regulate the growth and development of animals. Although much work remains to be done, experimental evidence does suggest that 2,4-D and DCP disrupts the animal endocrine systems (Florsheim and Velcoff, 1962; Liu et al., 1996; Rawlings et al., 1998). Studies have shown that farmers exposed to 2,4-D have low-quality sperm. In addition, farmer-applicators in areas of high use of 2,4-D have more children with circulatory/respiratory, urogenital, and musculoskeletal/integumental birth defects than unexposed men (Cox, 1999). We previously reported that 2,4-D and DCP cause an increase of weight of androgen-dependent tissues in Hershberger assay and inhibit the cytochrome P450 3A4-mediated metabolism of testosterone (Kim et al., 2002). To elucidate the molecular mechanism of androgenic action of phenoxy compound, the roles of 2,4-D and DCP in the androgenic actions were examined in this study.
To date, several in vitro screening systems for the detection of androgenic actions of chemicals were established using Chinese hamster ovary cells, human breast cancer cells, and androgen-insensitive or androgen-sensitive human prostate cancer cells (Terouanne et al., 2000; Vickie et al., 2002; Vinggaard et al., 1999). Among these systems, the 22Rv1 cells have recently become useful for the determination of the mechanism of action and for the screening of chemicals for androgen agonist and antagonist activity (Hartel et al., 2003). In a previous study, we compared the prostate cancer cell lines for androgen receptor-mediated reporter gene assays using LNCaP, 22Rv1, and PC3/AR+. Among three cell lines, 22Rv1 cell line was responded with the most sensitivity to androgenic chemicals and generated highly reproducible results (Kim et al., submitted manuscript). Using these 22Rv1 cells, we found that simultaneous treatment with DHT and 2,4-D or DCP caused highly enhanced both cell proliferation (Fig. 2B) and AR-mediated transcription (Figs. 3B and 3C). These results are consistent with our previous in vivo research (Kim et al., 2002).
In addition to the parent compound, DCP, not only a metabolite but also a manufacture intermediate of 2,4-D, may also be found as an environmental contaminant. DCP was the most abundant phenolic compound found in the Humber river water, in eastern England (House et al., 1997). Here, we showed that DCP induced AR-mediated transcriptional activity at lower doses than 2,4-D. These results raise concern about the endocrine toxicity of 2,4-D as an important potential hazard, even if it were metabolized.
Interestingly, 2,4-D and DCP combined with DHT can enhance AR transactivation via the facilitation and promotion of AR nuclear translocation (Fig. 6). Androgen receptors are ligand-regulated transcription factors that must move through the cytoplasm, traverse the nuclear pores, and subsequently move within the nucleus to reach their sites of action. Their nuclear localization of steroid receptors is determined by the shuttling of receptors into and out of the nucleus occurs constantly (Dauvois et al., 1993; DeFranco et al., 1995). Most of the unliganded AR under the steady-state condition resides in the cytoplasm and, upon hormone exposure, rapidly migrates into the nucleus. Nuclear migration of AR starts within 15 min following hormone treatment, and the receptor is completely translocated into the nucleus within 60 min. Furthermore, androgen withdrawal releases the receptor from its chromatin association and exports it back into the cytoplasmic compartment for recycling (almost four cycles) when the hormone is reintroduced (Rakesh et al., 2000). Therefore, the same receptor may be capable of mediating multiple rounds of hormonal signaling, suggesting that the more rapid nuclear translocation of AR means the more transactivation of targent gene. 2,4-D and DCP may be capable of promoting the translocation of AR from the cytosol to the nucleus, which, in turn, might result in the binding of the target genes promoter, and an increased transcription. Several factors have been reported to explain the mechanism of nuclear translocation of androgen receptor. Davies et al. (2002) reported that the switch between immunophilins FKBP51 and FKBP52 in the heterocomplexes controls the steroid receptor subcellular localization and nuclear transport. Hsp90 or associated proteins (Georget et al., 2002; Thomas et al., 2004) seem to play an important role in nuclear transfer of AR. In addition, ATP and actin polymerization are necessary for androgen-induced AR translocation (Hayley et al., 2004; Tang and DeFranco, 1996). Further investigation is required to ascertain which factors are involved in the promotion of AR nuclear translocation by the phenoxy compound.
When chemicals exert androgenic activity, various signal pathways may be involved. In competitive AR binding assay, 2,4-D and DCP inhibited DHT binding to AR (Fig. 5). This result implies another possibility that alternative signaling mechanism of the synergistic effects of phenoxy compound on DHT could be involved. Transcriptional upregulation by the AR occurs through homodimer binding of the receptor to DNA sequences (Ham et al., 1988). This takes place alongside interaction of the receptor with basal transcription factors such as TFIIF, the TATA-box binding protein (McEwan and Gustafsson, 1997) and with coactivators that potentiate its activity (Voegel et al., 1996; Yeh and Chang, 1996), or with mitogen-activated protein kinases MAPKs (Heike et al., 1999). Heike et al. (1999) reported that treatment of androgen responsive prostate cancer cells with DHT leads to a rapid activation of mitogen-activated protein kinases MAPKs (also called extracellular signal-regulated kinases or Erks) and the AR-mediated activation of MAP kinase results in enhanced activity of the transcription factor Elk-1. Since several studies have reported that treatment of 2,4-D, either alone or in the presence of steroid hormone, induced MAPK phosphorylation through MAPKK (Mizoguchi et al., 1994; Stebbins et al., 2004), the phenoxy compound in combination with DHT may activate MAPK phosphorylation in mammalian cells, leading to the enhancement of the AR-mediated transcription.
In summary, we showed that 2,4-D and DCP in combination with DHT have androgenic activity in cell proliferation and androgen-induced transactivation, possibly through promoted AR translocation to the nucleus. Now we are studying to explore which cofactor and signal pathway could lead to the promotion of AR nuclear translocation by the phenoxy compound. This is the first report that provided clear evidence for androgenic actions of phenoxy compounds using a mammalian system.
ACKNOWLEDGMENTS
This research was supported by an Endocrine disruptors research, Korea Food and Drug Administration (ED2001-10) and in part by a grant from the Korea Science and Engineering Foundation (KOSEF R03-2001-000-00024-0).
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ABSTRACT
2,4-Dichlorophenoxyacetic acid (2,4-D) and its metabolite 2,4-dichlorophenol (DCP) are used extensively in agriculture as herbicides, and are suspected of potential endocrine disruptor activity. In a previous study, we showed that these compounds exhibited synergistic androgenic effects by cotreatment with testosterone in the Hershberger assay. To elucidate the mechanisms of the synergistic effects of these compounds on the androgenicity of testosterone, the androgenic action of 2,4-D and DCP was characterized using a mammalian detection system in prostate cancer cell lines. In in vitro assay systems, while 2,4-D or DCP alone did not show androgenic activity, 2,4-D or DCP with 5-dihydroxytestosterone (DHT) exhibited synergistic androgenic activities. Cotreatment of 10 nM 2,4-D or DCP with 10 nM DHT was shown to stimulate the cell proliferation by 1.6-fold, compared to 10 nM DHT alone. In addition, in transient transfection assays, androgen-induced transactivation was also increased to a maximum of 32-fold or 1.28-fold by cotreatment of 2,4-D or DCP with DHT, respectively. However, 2,4-D and DCP exerted no effects on either mRNA or protein levels of AR. In a competitive AR binding assay, 2,4-D and DCP inhibited androgen binding to AR, up to 50% at concentrations of approximately 0.5 μM for both compounds. The nuclear translocation of green fluorescent protein-AR fusion protein in the presence of DHT was promoted as the result of the addition of 2,4-D and DCP. Collectively, these results that 2,4-D and DCP enhanced DHT-induced AR transcriptional activity might be attributable, at least in part, to the promotion of AR nuclear translocation.
Key Words: 2,4-dichlorophenoxyacetic acid (2,4-D); 2,4-dichlorophenol (DCP); androgen receptor (AR); 22Rv1; endocrine disruptor.
INTRODUCTION
The phenoxy compounds 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenol (DCP) were widely used as a hormonal herbicide to control the growth of broadleaf plants and as an intermediate for pesticide manufacturing, respectively. 2,4-D has been shown to exert toxic effects on both animals and humans, ranging from embryotoxicity and teratogenicity to neuro-, immuno-, and hepatotoxicity (Barnekow et al., 2000; Charles et al., 2001; Rosso et al., 2000). 2,4-D has also been reported to cause positive for chromosomal aberrations and sperm head abnormalities in two in vivo assays in mice (Amer and Aly, 2001; Venkov et al., 2000). Amer and Aly (2001) reported the induction of sperm head abnormalities by treatment with these substances, but only at very high in vivo concentrations (1/2 LD50). Tordon 75 D, which contains the 2,4-D and 4-amino-3,5,6-trichloropicolinic acid (picloram) caused severe reduction in testicular weight in some animals given high doses (Oakes et al., 2002). Although several studies have reported 2,4-D-induced testicular changes in rats, the precise mechanisms underlying these phenomena have yet to be fully understood, and it remains unclear whether phenoxy compounds affect endocrine systems in other species.
In a previous study, we have demonstrated that the administration of 2,4-D (50 mg/kg/day, po) or DCP (100 mg/kg/day, po) to rats caused an increase in the weights of ventral prostate, Cowpers gland, and glands penis. In addition, these increases in the weight of androgen-dependent tissues were synergistically potentiated when rats were simultaneously treated with 2,4-D or DCP and low dose of testosterone (1 mg/kg, sc) (Kim et al., 2002).
Some chemicals, including bisphenol A, nonylphenol, and fenthion, have been reported to be able to interact directly with AR and to activate AR-dependent transcription in mammalian cells (Lee et al., 2003; Shigeyuki et al., 2003). These chemicals that mimic the biologic activity of androgen are called environmental endocrine disruptors. Only a few chemicals have exhibited androgenic activity, and no report has been available on the effects of phenoxy compounds on AR-mediated actions, even though testicular changes caused by these phenoxy compounds have been reported in some animals.
Androgens act via an interaction with the androgen receptor (AR), a ligand dependent transcription factor. The homodimer of the androgen-bound receptors is translocated into the nucleus, where it binds to a specific DNA motif, the androgen response element (ARE), located in the regulatory region of target genes, and activates or represses the transcription of the androgen-regulatory genes (Chen and Evans, 1995; Gnanaragasam et al., 2000; Onate et al., 1995). Some environmental chemicals cause the androgenic endocrine disruption as a result of failure of AR-androgen binding, nuclear import, DNA binding, and/or transcriptional activation (Lee et al., 2003; Wong et al., 1995).
The purposes of this study were to more precisely characterize the synergistic androgenic action of 2,4-D or DCP with DHT in vitro, and to understand the molecular mechanisms of phenoxy compound action mediated AR activation. In this report, although 2,4-D and DCP (Fig. 1) inhibited the AR-DHT binding, these compounds stimulated DHT-mediated cell proliferation, DHT-induced AR nuclear importing, and its subsequent transactivation. This is the first report that provides clear evidence for the androgenic actions of phenoxy compounds using a mammalian system.
MATERIALS AND METHODS
Chemicals.
5-Dihydroxytestosterone (minimum purity >99%) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 2,4-Dichlorophenoxyacetic acid and dichlorophenol were purchased from Aldrich Chemical Co. (St. Louis, MO). The purity of 2,4-D and DCP was more than 98%. Restriction and modifying enzymes were obtained from Takara (Shiga-ken, Japan). AR (C19) antibody was obtained from Santa Cruz Biotechnolgy (Santa Cruz, CA). Horseradish peroxidase conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Zymed Laboratories (San Francisco, CA).
Construction of plasmids.
The MMTV fragment was excised from pMSG (Amersham Pharmacia Biotech) by HindIII-SmaI sites and inserted in the HindIII-SmaI sites of the pBluescript II SK (+) vector (pMMTV-SK). To generate promoter-reporter construct pMMTV-Luc, then, the MMTV fragment was then excised from pMMTV-SK by SmaI-Kpn I and inserted in the SmaI-Kpn I of the pGL3 basic vector (Promega). The expression vector for the human full-length AR (pCMV-hAR) was constructed by inserting the human androgen receptor cDNA into the CMV vector. Chimeric GFP-AR was generated by inserting the human full-length AR (excised by BglII-XbaI) into the BglII-XbaI site of the 3'-end of the coding region of the modified aequorea GFP (pEGFP-C2, CLONTECH Laboratories, Inc. Palo Alto, CA).
Cell culture.
Two human prostate cancer cell lines were used: AR expressed 22Rv1 (cat # CRL-2505) and androgen-independent PC3 (cat # CRL-1435), obtained from the American Type Culture Collection (Rockville, MD). The 22Rv1 cells are derived from a relapsed tumor and have been characterized as a valuable tool for the study of prostate cancer progression (Clifford et al., 2002). A representative androgen-insensitive cell line, PC3 was used for validation. The cells were routinely maintained in RPMI 1640 medium (Gibco BRL) containing 10% FBS, penicillin (100 units/ml), 1% L-glutamine and streptomycin (100 μg/ml) in an atmosphere containing 5% CO2 at 37°C. For in vitro AR-binding assay, COS-1 (African green monkey kidney cells; ATCC, Rockville, MD) were grown in DMEM medium (Gibco BRL) containing 10% FBS, penicillin (100 units/ml), 1% L-glutamine and streptomycin (100 μg/ml) in an atmosphere containing 5% CO2 at 37°C.
Cell proliferation assay.
The 22Rv1 cells were seeded in 96-well plates at an initial concentration of 3 x 103 cells per well. After 24 h the 5-dihydrotestosterone (DHT) was added to the experimental medium (phenol red-free RPMI 1640 containing 10% dextran charcoal-stripped FBS: dcs-FBS). The bioassay was terminated on the fourth day and the cells were fixed and stained with sulforhodamine-B (SRB) as described (Villalobos et al., 1995). Briefly, the cells were treated with 10% trichloroacetic acid and incubated at 4°C for 1 h and then washed with PBS. To stain the proteins 50 μl of 0.4% SRB was added to each well for 1 h at room temperature. The wells were washed several times with 1% acetic acid and air dried. Bound dye was solubilized with the 100 μl of 10 mM Tris base (pH 10.5) in a shaker for 5 min. The amount of SRB product in each well was determined by reading the plates on a plate-reading spectrophotometer (Dynatech MRX, Chantilly, VA) at 570 nm. The assays were repeated at least three times for each cell line.
Transactivation assays.
The 22Rv1 and PC3 cells (1.2 x 105) were seeded in 24-well plates and transfected with 1 μg of mouse mammary tumor virus luciferase vector (MMTV-Luc) and pCMV-hAR expression vector or with MMTV-Luc alone using the ExGene 500 (MBI Fermentas, St. Leon-Rot, Germany). Twenty-four hours after transfection, the indicated concentrations of DHT and other chemicals were added with experimental medium. After additional 24 h-incubation, the cells were lysed using a passive lysis buffer (Promega, Madison, WI). The extracts were assayed for luciferase activity using dual-luciferase reporter system (Promega, Madison, WI), and subsequently measured on a Liquid Scintillation Counter Luminometer (Bioscan, Inc., U.S.A). Luciferase activity was normalized for transfection efficiency using pRL-TK as an internal control. Cell cytotoxicity was determined by the colorimetric MTS cell cytotoxicity assay according to the manufacturer's protocol (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI).
RNA isolation and RT-PCR.
Total RNA was isolated from 22Rv1 using TRIzol reagent (Life Technologies, Inc., Grand Island, NY) and cDNA was synthesized according to the manufacturer's protocol. Briefly, total RNA was reverse-transcribed using MMLV-reverse transcriptase (Promega, USA) and oligo dT primer using the following conditions: 70°C for 10 min and 42°C for 1 h. Expression of genes was investigated by PCR using the following primers: hAR 5'-AAGCTCAAGGATGGAAGTGCAGT-3' (sense) and 5'-CATGGACACCGACACTGCCT-3' (antisense) generating a 751 bp product; -actin 5'-CCATGGAGAGCCTCTGTGGATAT-3' (sense) and 5'-TGAGGATCTGATCTGTCCACAAG-3' (antisense) generating a 415 bp product. Subsequently, the PCR products were subjected to gel electrophoresis on 1% agarose gels containing 0.5 μg/ml of ethidium bromide and the intensities of the bands were evaluated with imaging software TINA (Raytest Isotopenmessgerte, Straubenhardt, Germany).
Immunoblotting.
22Rv1 cells (6 x 105 cells/60 mm dish) were plated and grown in phenol red-free RPMI 1640 containing 5% dcs-FBS media for three days. After treatment with DHT and/or 2,4-D and/or DCP as indicated, the cells were harvested in 100 μl lysis buffer [50 mM Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA] that contained a cocktail of protease inhibitors. The protein samples were loaded onto 10% acrylamide gel and subjected to gel electrophoresis. The protein samples were transferred to nitrocellulose membranes. The membranes were blocked for 1 h in 5% nonfat dry milk in PBST. The membranes were incubated overnight at 4°C in 5% milk/PBST containing primary antibodies to AR (Santa Cruz, CA) or -actin (sigma). The membranes were washed and then incubated with anti-mouse peroxidase secondary antibody (Zymed Laboratories, San Francisco) for 1 h at room temperature. The signal was detected using a chemiluminescense detection system (Pierce, Rockford, lL).
Competitive steroid binding assays.
Whole-cell binding assays were performed as described previously (Kim et al., 2002; Lambright et al., 2000). Briefly, COS-1 cells were transfected with pCMV-hAR using Lipofectamine reagent (Invitrogen, San Diego, CA). Twenty-four h prior to the binding reaction, the cells were placed in serum-free and phenol red-free medium and then incubated for 2 h at 37°C with 5 nM [3H]5-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 the radioactivity was determined using a scintillation counter.
Fluorescence imaging.
The PC3 cells were cultured in 2 x 2 cm2 Lab-Tek chamber slides (Nunc Inc., Naperville, IL) the day before transfection. The GFP-AR expression vector was transiently transfected using ExGene500 reagent (MBI Fermentas, St. Leon-Rot, Germany) according to the manufacturer's instructions. After 30 h, the transfected cells were fed with fresh medium containing 10% dcs-FBS, and treated for 30 min with the vehicle (ethanol), 10 nM DHT, or 10 nM DHT in combination with each of other chemicals also at 10 nM. The cells were then fixed with 10% formaldehyde at room temperature for 10 min. The fixed cells were rinsed twice with PBS (5 min each wash) and air dried. Fluorescence analysis was conducted using a Bio-Rad MRC1024 confocal laser system connected to a Zeiss Axioplan2 fluorescence microscope. For the evaluation of GFP-AR localization, the relative nuclear and cytoplasmic fluorescence in >100 cells per condition from three experiments was determined and scored according to a 5-grade nuclear localization score: exclusive nuclear fluorescence (4); nuclear fluorescence exceeding cytoplasmic fluorescence (3); equivalent nuclear and cytoplasmic fluorescence (2); cytopasmic fluorescence exceeding nuclear fluorescence (1); and exclusive cytoplasmic fluorescence (0). The translocation scores represent the mean ± SEM from three experiments in which >100 cells per condition per experiment were scored (Mario et al., 1998).
RESULTS
Effects of the Phenoxy Compounds on the Proliferation of Prostate Cancer Cells
22Rv1 cells were cultured in the RPMI1640 medium containing various concentrations of 2,4-D, DCP, or DHT for four days. The rate of cell growth was then determined by SRB assay. As shown in Figure 2, DHT increased cell growth in a dose-dependent manner. The maximum cell yields by treatment with DHT (10 nM) were about 1.5-fold. However, 2,4-D and DCP (Fig. 2A) treatment did not increase the proliferation rate of 22Rv1 cells. To investigate the combined effects of these phenoxy compounds with DHT on prostate cell proliferation, the 22Rv1 cells were treated with various concentrations of 2,4-D and DCP in the presence of 10 nM DHT, and the rate of cell growth was determined. Whereas stimulation with DHT alone showed a 1.5-fold increase in cell growth rate, cotreatment with 2,4-D or DCP and DHT exhibited a 2,5-fold increase in cell growth rate (Fig. 2B). This suggests that the phenoxy compounds acted either additively or synergistically with DHT on the proliferation of prostate cancer cells.
Effects of 2,4-D and DCP on DHT-Activated Transcription
We next examined the effects of 2,4-D and DCP on AR-mediated transcription with a luciferase reporter gene assay in which 22Rv1 cells had been transiently transfected with pMMTV-Luc. An approximately 8-fold induction of luciferase activity was observed in the cells incubated in the presence of 10 nM DHT (Fig. 3A). Treatment with various doses of 2,4-D or DCP did not increased luciferase activity (Fig. 3A).
To investigate the combined effects of these phenoxy compounds with DHT on AR-mediated transcription, two prostate cancer cell-lines, 22Rv1 and PC3/AR+ were treated with 2,4-D and DCP in the presence of DHT, respectively, and luciferase activity was measured (Figs. 3B and 3C), respectively. Surprisingly, although stimulation with DHT (10 nM) alone showed an 8-fold increase in luciferase activity in the 22Rv1 cells, cotreatment with 2,4-D (100 nM) or DCP (0.1 nM) and DHT exhibited a 32-fold or a 12.8-fold increase in the luciferase activity, respectively (Fig. 3B). In order to ensure that the synergistic effect of 2,4-D and DCP on DHT-activated transactivation was not specific to the 22Rv1 cells, PC3/AR+ transiently transfected with a human AR-expressing vector, pCMV-hAR, were treated with 2,4-D or DCP in the presence of DHT, and luciferase activity was measured. As shown in Figure 3C, similar synergistic effects of 2,4-D and DCP on DHT-activated transactivation were also observed in PC3/AR+ cells. In the presence of 10 nM 2,4-D or DCP, luciferase acitivity showed about a 3.2-fold or a 1.2-fold increase, respectively, compared to 10 nM DHT alone (Fig. 3C). These results indicate that the phenoxy compounds acted additively or synergistically with DHT to activate the AR-dependent transcription.
Effects of 2,4-D and DCP on AR Expression
As AR is the most important regulatory transcription factor for DHT-activated transcription, the effects of phenoxy compounds on AR expression were also investigated. In order to determine whether 2,4-D and DCP are capable of modulating the expression of AR, RT-PCR analysis and Western blotting were performed. The 22Rv1 cells were treated with 10 nM DHT combined with either 100 nM 2,4-D, or 0.1 nM DCP and the total RNA was subjected to RT-PCR analysis. As shown in Figure 4A, the combination of 2,4-D or DCP with DHT did not significantly modulate the expression of AR mRNA as measured by the expression of -actin mRNA.
Cells treated with 10 nM DHT exhibited a 50% increase in the levels of AR protein compared to the control (Fig. 4B). Western blot analysis indicated that the levels of AR protein in 22Rv1 cells did not change with cotreatment of 10 nM DHT and 100 nM 2,4-D or 0.1 nM DCP as compared to 10 nM DHT alone (Fig. 4B). These results suggested that the combination of phenoxy compounds and DHT exerted no synergistic effects on the levels of AR gene expression.
Effects of 2,4-D and DCP on Androgen Binding to AR
The AR-binding affinities of the phenoxy compounds were investigated using recombinant human AR transiently expressed in monkey kidney COS cells. The COS-1+hAR assays are recommended as the in vitro AR binding assays with the greatest priority for validation in a Tier 1 endocrine disruptor screening battery. In competitive androgen binding assays using [3H]5-DHT (a labeled androgen), 2,4-D and DCP inhibited androgen binding to AR, both up to 50% at approximately 0.5 μM (Fig. 5A). Competitive inhibition by DCP was greater than that of 2,4-D. Strongest competitive binding was observed with the natural androgen, DHT. Western blot analysis indicated that the levels of AR protein in COS cells did not change with treatment of chemicals (Fig. 5B). These results indicate that the 2,4-D and DCP had some affinity to AR although lower than DHT. Although 2,4-D and DCP inhibited DHT binding to the AR, the 2,4-D or DCP also exhibited a synergistic effect on the androgenic action, suggesting that a variety of signaling pathways for AR mediated androgenic action may be involved. The possibility that the androgenic effects of the 2,4-D and DCP may be independent of AR binding cannot be excluded.
Effect of 2,4-D and DCP on the Dynamics of AR Nuclear Translocation
In order to know how 2,4-D and DCP have synergistic effects with DHT to activate the AR-dependent transcription, we examined the effects of 2,4-D and DCP on AR translocation using a GFP-AR fusion protein. We first tested the ability of this chimeric protein for androgen-dependent transactivation of the pMMTV-Luc system in AR-negative PC3 cells. In the presence of 10 nM DHT, cells transfected with the GFP-AR expression vector showed about one-third transactivaition function as compared with the pCMV-hAR (data not shown). However, transactivation function of both of these AR proteins was almost totally dependent on the presence of the androgen. To visualize whether 2,4-D and DCP could modulate the DHT-dependent nuclear translocation of hAR, PC3 cells were transiently transfected with GFP-hAR and treated with either 0 (vehicle only, control), 10 nM DHT only as a positive control, 10 nM of 2,4-D or 10 nM of DCP with or without DHT for 20 min. In the absence of DHT, the GFP-AR fusion protein was distributed to the cytoplasmic compartment, but in the presence of DHT (10 nM) GFP-AR protein was became localized in the nucleus. In the presence of 2,4-D or DCP alone, GFP-AR was distributed to the cytoplasmic compartment (Fig. 6). Surprisingly, cotreatment with 2,4-D or DCP (10 nM) and DHT (10 nM) promoted the nuclear translocation of AR (Fig. 6). The bar graphs in Figure 6 show the nuclear translocation scores from 0 to 4 for GFP-AR in cells treated with DHT alone (lane 2), 2,4-D alone (lane 3), DCP alone (lane 4) and the promotion of DHT-mediated translocation by 2,4-D (lane 5) and DCP (lane 6), as determined for several hundred cells per condition. These data suggest that 2,4-D and DCP exerted a synergistic effect with DHT resulting in a markedly augmented activation of AR-mediated transcription via facilitation of AR nuclear translocation.
DISCUSSION
Recently, many investigations have been focused on chemicals that disrupt the normal functions of the endocrine system, the glands and hormones that regulate the growth and development of animals. Although much work remains to be done, experimental evidence does suggest that 2,4-D and DCP disrupts the animal endocrine systems (Florsheim and Velcoff, 1962; Liu et al., 1996; Rawlings et al., 1998). Studies have shown that farmers exposed to 2,4-D have low-quality sperm. In addition, farmer-applicators in areas of high use of 2,4-D have more children with circulatory/respiratory, urogenital, and musculoskeletal/integumental birth defects than unexposed men (Cox, 1999). We previously reported that 2,4-D and DCP cause an increase of weight of androgen-dependent tissues in Hershberger assay and inhibit the cytochrome P450 3A4-mediated metabolism of testosterone (Kim et al., 2002). To elucidate the molecular mechanism of androgenic action of phenoxy compound, the roles of 2,4-D and DCP in the androgenic actions were examined in this study.
To date, several in vitro screening systems for the detection of androgenic actions of chemicals were established using Chinese hamster ovary cells, human breast cancer cells, and androgen-insensitive or androgen-sensitive human prostate cancer cells (Terouanne et al., 2000; Vickie et al., 2002; Vinggaard et al., 1999). Among these systems, the 22Rv1 cells have recently become useful for the determination of the mechanism of action and for the screening of chemicals for androgen agonist and antagonist activity (Hartel et al., 2003). In a previous study, we compared the prostate cancer cell lines for androgen receptor-mediated reporter gene assays using LNCaP, 22Rv1, and PC3/AR+. Among three cell lines, 22Rv1 cell line was responded with the most sensitivity to androgenic chemicals and generated highly reproducible results (Kim et al., submitted manuscript). Using these 22Rv1 cells, we found that simultaneous treatment with DHT and 2,4-D or DCP caused highly enhanced both cell proliferation (Fig. 2B) and AR-mediated transcription (Figs. 3B and 3C). These results are consistent with our previous in vivo research (Kim et al., 2002).
In addition to the parent compound, DCP, not only a metabolite but also a manufacture intermediate of 2,4-D, may also be found as an environmental contaminant. DCP was the most abundant phenolic compound found in the Humber river water, in eastern England (House et al., 1997). Here, we showed that DCP induced AR-mediated transcriptional activity at lower doses than 2,4-D. These results raise concern about the endocrine toxicity of 2,4-D as an important potential hazard, even if it were metabolized.
Interestingly, 2,4-D and DCP combined with DHT can enhance AR transactivation via the facilitation and promotion of AR nuclear translocation (Fig. 6). Androgen receptors are ligand-regulated transcription factors that must move through the cytoplasm, traverse the nuclear pores, and subsequently move within the nucleus to reach their sites of action. Their nuclear localization of steroid receptors is determined by the shuttling of receptors into and out of the nucleus occurs constantly (Dauvois et al., 1993; DeFranco et al., 1995). Most of the unliganded AR under the steady-state condition resides in the cytoplasm and, upon hormone exposure, rapidly migrates into the nucleus. Nuclear migration of AR starts within 15 min following hormone treatment, and the receptor is completely translocated into the nucleus within 60 min. Furthermore, androgen withdrawal releases the receptor from its chromatin association and exports it back into the cytoplasmic compartment for recycling (almost four cycles) when the hormone is reintroduced (Rakesh et al., 2000). Therefore, the same receptor may be capable of mediating multiple rounds of hormonal signaling, suggesting that the more rapid nuclear translocation of AR means the more transactivation of targent gene. 2,4-D and DCP may be capable of promoting the translocation of AR from the cytosol to the nucleus, which, in turn, might result in the binding of the target genes promoter, and an increased transcription. Several factors have been reported to explain the mechanism of nuclear translocation of androgen receptor. Davies et al. (2002) reported that the switch between immunophilins FKBP51 and FKBP52 in the heterocomplexes controls the steroid receptor subcellular localization and nuclear transport. Hsp90 or associated proteins (Georget et al., 2002; Thomas et al., 2004) seem to play an important role in nuclear transfer of AR. In addition, ATP and actin polymerization are necessary for androgen-induced AR translocation (Hayley et al., 2004; Tang and DeFranco, 1996). Further investigation is required to ascertain which factors are involved in the promotion of AR nuclear translocation by the phenoxy compound.
When chemicals exert androgenic activity, various signal pathways may be involved. In competitive AR binding assay, 2,4-D and DCP inhibited DHT binding to AR (Fig. 5). This result implies another possibility that alternative signaling mechanism of the synergistic effects of phenoxy compound on DHT could be involved. Transcriptional upregulation by the AR occurs through homodimer binding of the receptor to DNA sequences (Ham et al., 1988). This takes place alongside interaction of the receptor with basal transcription factors such as TFIIF, the TATA-box binding protein (McEwan and Gustafsson, 1997) and with coactivators that potentiate its activity (Voegel et al., 1996; Yeh and Chang, 1996), or with mitogen-activated protein kinases MAPKs (Heike et al., 1999). Heike et al. (1999) reported that treatment of androgen responsive prostate cancer cells with DHT leads to a rapid activation of mitogen-activated protein kinases MAPKs (also called extracellular signal-regulated kinases or Erks) and the AR-mediated activation of MAP kinase results in enhanced activity of the transcription factor Elk-1. Since several studies have reported that treatment of 2,4-D, either alone or in the presence of steroid hormone, induced MAPK phosphorylation through MAPKK (Mizoguchi et al., 1994; Stebbins et al., 2004), the phenoxy compound in combination with DHT may activate MAPK phosphorylation in mammalian cells, leading to the enhancement of the AR-mediated transcription.
In summary, we showed that 2,4-D and DCP in combination with DHT have androgenic activity in cell proliferation and androgen-induced transactivation, possibly through promoted AR translocation to the nucleus. Now we are studying to explore which cofactor and signal pathway could lead to the promotion of AR nuclear translocation by the phenoxy compound. This is the first report that provided clear evidence for androgenic actions of phenoxy compounds using a mammalian system.
ACKNOWLEDGMENTS
This research was supported by an Endocrine disruptors research, Korea Food and Drug Administration (ED2001-10) and in part by a grant from the Korea Science and Engineering Foundation (KOSEF R03-2001-000-00024-0).
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