©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Androgen Receptor Antagonist versus Agonist Activities of the Fungicide Vinclozolin Relative to Hydroxyflutamide (*)

(Received for publication, February 13, 1995; and in revised form, May 22, 1995)

Choi-iok Wong (1) William R. Kelce (5) Madhabananda Sar (2) Elizabeth M. Wilson (3) (4)(§)

From the  (1)Laboratories for Reproductive Biology and the Departments of Biology, (2)Cell Biology and Anatomy, (3)Pediatrics, and (4)Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599 and the (5)Developmental Toxicology Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Research Triangle Park, North Carolina 27711

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanism of antiandrogenic activity of vinclozolin (3-(3,5-dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione), a dicarboximide fungicide under investigation for its potential adverse effects on human male reproduction, was investigated using recombinant human androgen receptor (AR). The two primary metabolites of vinclozolin in plants and mammals are M1 (2-[[3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid) and M2 (3`,5`-dichloro-2-hydroxy-2-methylbut-3-enanilide). Both metabolites, in a dose-dependent manner, target AR to the nucleus and inhibit androgen-induced transactivation mediated by the mouse mammary tumor virus promoter. M2 is a 50-fold more potent inhibitor than M1 and only 2-fold less than hydroxyflutamide. In the presence of dihydrotestosterone (50 nM), M2 (0.2-10 µM) inhibits androgen-induced AR binding to androgen response element DNA. In the absence of dihydrotestosterone, concentrations of 10 µM M2 or hydroxyflutamide promote AR binding to androgen response element DNA and activation of transcription. Agonist activities of M2 and hydroxyflutamide occur at 10-fold lower concentrations with the mutant AR (Thr to Ala) endogenous to LNCaP human prostate cancer cells. The results indicate that androgen antagonists can act as agonists, depending on ligand binding affinity, concentration, and the presence of competing natural ligands.


INTRODUCTION

The human androgen receptor (AR) (^1)is a member of the steroid hormone receptor family of ligand-activated transcriptional regulatory proteins required for normal male sex development. Androgens through their receptor stimulate masculinization of the fetus and induce male imprinting of the developing brain. Molecular defects in the AR gene cause the syndrome of androgen insensitivity, which results from failure of AR androgen binding, nuclear import, DNA binding, and/or transcriptional activation(1) . Certain antiandrogens, such as hydroxyflutamide, bind AR with moderate affinity, promote nuclear import(2) , but inhibit androgen-mediated transcriptional activity by failing to promote DNA binding, whereas others, such as cyproterone acetate, promote DNA binding at moderate concentrations and induce partial agonist activity(3) .

Vinclozolin is a dicarboximide fungicide registered in the United States and Europe for use on fruits, vegetables, ornamental plants, and turf grasses. Administration of vinclozolin to adult male rats causes Leydig cell hyperplasia and atrophy of the prostate and seminal vesicles(4) , whereas administration to pregnant rats causes incomplete development of the male reproductive tract (i.e. cleft phallus and hypospadias) in male pups(5, 6) , indicating antiandrogenic activity. Two major ring-opened metabolites of vinclozolin (i.e. the butenoic acid M1 and the enanilide M2, Fig. 1) predominate in plants and soil (7, 8, 9) as well as in rodent fluid and tissue extracts following in vivo exposure (10, 11) . It was shown previously that vinclozolin, M1, and M2 have little effect on the androgen-metabolizing enzyme 5alpha-reductase. In addition, vinclozolin was a poor inhibitor of androgen binding to rat AR in cell-free extracts, whereas M1 and M2 were effective competitors (10) , suggesting that the antiandrogenic effects of vinclozolin are mediated by M1 and/or M2. It has not been established, however, the degree to which environmental levels of vinclozolin, M1, or M2 induce adverse developmental effects.


Figure 1: Structural formulas of vinclozolin, metabolites M1 and M2, and hydroxyflutamide.



In this report, the mechanism of transcriptional inhibition by vinclozolin and its metabolites is shown to be inhibition of androgen-induced DNA binding and subsequent transactivation. A surprising and important result of the study is that at high concentrations in the absence of DHT, vinclozolin metabolite M2 and the classical androgen antagonist metabolite, hydroxyflutamide, are agonists, since they increase AR DNA binding and transcriptional activity. The results reveal that the androgen antagonist activities of these nonsteroidal aromatic compounds are concentration-dependent and may be influenced by the binding of native androgens to the AR dimer.


EXPERIMENTAL PROCEDURES

Materials

Monkey kidney COS-1 and CV1 cells were from the American Type Culture Collection; Dulbecco's modified essential medium with high glucose with or without phenol red, Grace medium supplemented with yeastolate and lactalbumin hydrolysate, and Ex-cell 400 or 401 with L-glutamime were from JRH Biosciences; fetal calf serum for mammalian and insect cell cultures were from Life Technologies, Inc.; Spodoptera frugiperda Sf9 cells were from Invitrogen Corp., San Diego CA; antibiotics and gentamicin were from Life Technologies, Inc.; D-luciferin was from Analytical Luminescence; CV1 cell lysis buffer was from Ligand Pharmaceuticals; [^3H]methyltrienolone ([17alpha-methyl-^3H]R1881, 85.5 Ci/mmol) was from DuPont NEN; Texas red-conjugated goat anti-rabbit IgG was from Molecular Probes, Inc., Eugene OR; vinclozolin was from Crescent Chemical Co., Hauppauge, NY; vinclozolin metabolite M2 was from BASF AG; vinclozolin metabolite M1 was synthesized using alkaline hydrolysis of vinclozolin and purified as described previously(10) ; hydroxyflutamide was provided by R. O. Neri, Schering Corp., Bloomfield, NJ; unlabeled steroids were from Sigma, and buffers and chemicals were from Fisher, EM Science, and Sigma.

Vinclozolin Metabolism

Serum and cell extract concentrations of vinclozolin, M1, and M2 were determined by high-performance liquid chromatography/diode array detection as described previously (7, 8, 9, 10) using linuron (N`-(3,4-dichlorophenyl)-N-methoxy-N-methylurea) as internal standard to correct for procedural losses. In animal studies, adult male rats were dosed orally with 30 or 100 mg of vinclozolin in corn oil/kg of body weight/day or with vehicle alone for 30 days, after which time the animals were killed. Care of the animals was in accordance with institutional guidelines.

Competitive Steroid Binding Assays

Whole cell binding assays were performed as described previously(12) . COS-1 cells (10^5/well of 12-well plates) were transfected with 1 µg of pCMVhAR DNA/well using diethylaminoethyl dextran. Twenty-four h prior to the binding reaction, cells were placed in serum-free, phenol red-free medium and incubated for 2 h at 37 °C with 5 nM [^3H]R1881 in the presence and absence of increasing concentrations of unlabeled compounds. Nonspecific binding of [^3H]R1881 was assessed by adding a 100-fold molar excess of unlabeled R1881. Cells were washed twice in phosphate-buffered saline (PBS), harvested in 200 µl of 2% SDS, 10% glycerol, and 10 mM Tris, pH 6.8, and radioactivity determined by scintillation counting.

Immunocytochemistry

COS cells (10^5 cells/well of two-chamber slide) were transfected with 1 µg/well pCMVhAR using diethylaminoethyl dextran as described previously(13) . Hormones were added 24 and 1 h prior to washing with PBS. Cells were air-dried, fixed in 95% ethanol at -20 °C for 10 min, washed in PBS, treated in 5% bovine serum albumin in PBS, pH 7.4, for 30 min, and incubated with AR52 IgG (2 µg/ml) overnight at 4 °C. Cells were washed in PBS and incubated with Texas red-conjugated goat anti-rabbit IgG (1:500) for 60 min at room temperature. The stained cells were examined using a Nikon Optiplot 2 microscope with an EP1 fluorescence attachment.

Androgen-dependent Transcription Assays

Transcriptional activity was assessed by transient cotransfection of monkey kidney CV1 cells (0.4 10^6/6-cm dish) with 50-100 ng of pCMVhAR expression vector and 5 µg of mouse mammary tumor virus luciferase reporter vector (provided by Ronald M. Evans, Salk Institute, La Jolla, CA) using the calcium phosphate precipitation method(13, 14) . Twenty-four and 48 h after transfection, the indicated concentrations of DHT and antiandrogens were added with fresh medium and 5 h after the last addition, cells were harvested in 0.6 ml of lysis buffer. Relative light units of a 0.1-ml aliquot were determined using a Monolight 2010 Analytical Luminescence Laboratory luminometer.

DNA Mobility Shift Assays

Sf9 insect cells expressing recombinant full-length wild-type and mutant LNCaP prostate cancer cell line human AR baculoviruses were incubated for 42 h at 26 °C with the indicated concentrations of ligands; cells were harvested and extracted in a high salt containing buffer as described previously(3) . The P-labeled 27-base pair oligonucleotide contained the androgen response element of the 0.5 kb first intron region of the rat prostatein C3 subunit gene (15) and was analyzed in the DNA mobility shift assay as described previously(3) .


RESULTS

Competitive Binding and Metabolism

The molecular basis for the antiandrogenic effects of vinclozolin was investigated using recombinant human AR transiently expressed in monkey kidney COS cells. In a competitive androgen binding assay using [^3H]R1881 (a radiolabeled synthetic androgen), M2 was a slightly weaker competitor than the antiandrogen, hydroxyflutamide, but considerably more effective than M1 or vinclozolin (Fig. 2). Half-maximal inhibition of 5 nM [^3H]R1881 binding occurred at approximately 500 nM M2 and 10-20 µM M1 or vinclozolin. Strongest competitive binding was observed with the synthetic androgen R1881 and the natural androgen, DHT. Competitive inhibition by cyproterone acetate was greater than that of M2 or hydroxyflutamide. The similar effectiveness of M2 and hydroxyflutamide in competing for [^3H]R1881 binding to AR is supported by structural similarity between these two nonsteroidal aromatic compounds (see Fig. 1).


Figure 2: Competitive inhibition of [^3H]R1881 binding to AR by unlabeled vinclozolin, its metabolites M1 and M2, and hydroxyflutamide and cyproterone acetate. Binding inhibition was determined in COS cells transiently transfected with the human AR expression vector, pCMVhAR, as described under ``Experimental Procedures.'' Results expressed as percent binding relative to [^3H]R1881 alone are shown for unlabeled R1881 (up triangle, filled), dihydrotestosterone (DHT, bullet), cyproterone acetate (black square), hydroxyflutamide (*), M2 (-bullet-), vinclozolin (), and M1 () and are representative of three independent experiments.



Metabolism of vinclozolin, M1, and M2 was assessed by high-performance liquid chromatography with on-line uv absorbance diode array detection as described under ``Experimental Procedures.'' During a 24-h 37 °C incubation at 50 µM, vinclozolin was metabolized to 87% M1 and 12% M2 in monkey kidney COS cells and 94% M1 and 6% M2 in monkey kidney CV1 cells, the cell lines used to assess AR ligand binding and transcriptional activity, respectively. M1 and M2 were stable during the incubations, with more than 98% of the original compounds retained during the cell cultures. A similar pattern of vinclozolin metabolism was observed in Sf9 insect cells used for the expression of recombinant baculovirus. Thus, vinclozolin metabolism in primate and insect cells parallels that observed in rats, where M1 and M2 are the predominant metabolites of the fungicide.

Subcellular Distribution

AR transiently expressed in COS cells is perinuclear in the cytoplasm in the absence of androgen and nuclear in the presence of 50 nM DHT as reported previously (13, 14) and shown in Fig. 3, A and B. Significant AR nuclear staining (40-50%) was observed with exposure to 0.5 µM M2 (Fig. 3C), 10 µM M1 (Fig. 3G), and 10 µM vinclozolin (Fig. 3I), closely paralleling the ability of these ligands to compete for AR androgen binding (see Fig. 2). M1 and vinclozolin at 1 µM resulted in absence of detectable nuclear staining (Fig. 3, F and H), whereas 1 µM M2 caused nearly complete AR nuclear localization (Fig. 3D). At 10 µM, M2 AR nuclear transport was indistinguishable from that observed with 50 nM DHT (Fig. 3, E and B).


Figure 3: Immunocytochemical staining of AR in transfected COS cells in the presence of dihydrotestosterone, vinclozolin, or its metabolites, M1 and M2. Immunocytochemical staining was performed as described under ``Experimental Procedures.'' Cells expressing AR were untreated (A) or exposed for 24 h at 37 °C to 50 nM dihydrotestosterone (DHT, B), 0.5 µM M2 (C), 1 µM M2 (D), 10 µM M2 (E), 1 µM M1 (F), 10 µM M1 (G), 1 µM vinclozolin (Vin, H), and 10 µM vinclozolin (Vin, I). The regions shown are representative of the overall staining pattern determined in four experiments. Magnification: 720.



Mechanism of AR Inhibition by Vinclozolin

The effect of vinclozolin and its metabolites on DHT-induced transcriptional activity was investigated by transient cotransfection of monkey kidney CV1 cells with the human AR expression vector and a mouse mammary tumor virus promoter-luciferase reporter vector used previously to measure androgen-induced transcriptional responses to AR(13) . Transcriptional activity induced with 0.1 nM DHT was inhibited about 80% by 10 µM M1, 0.2 µM M2, 1 µM vinclozolin, or 0.2 µM hydroxyflutamide (Fig. 4). The greater transcriptional inhibition by M2 and hydroxyflutamide compared with vinclozolin and M1 parallel their more effective competitive inhibition of [^3H]R1881 binding to AR (see Fig. 2) and their ability to promote AR nuclear transport (see Fig. 3and (2) ). The parent compound vinclozolin and the major metabolite M1 were about 10- and 100-fold less potent, respectively, than M2 in inhibiting AR transcriptional activity (Fig. 4).


Figure 4: Transcriptional inhibitory effects of increasing concentrations of vinclozolin, metabolites M1 and M2, and hydroxyflutamide on DHT-induced transcriptional activity. Transcriptional activity was determined in transiently transfected CV1 cells as described under ``Experimental Procedures'' at 0.1 nM DHT and the indicated ligand concentrations. Optical readings are shown with standard error, and -fold induction is indicated at the bottom relative to the activity determined in the absence of DHT. p5 represents results obtained when the parent expression vector pCMV5 lacking AR sequence which was cotransfected with the luciferase reporter vector. The data shown are representative of at least four independent determinations.



The intermediate effectiveness of vinclozolin relative to M1 and M2 in inhibiting DHT-induced transcription in the cotransfection assay (Fig. 4) is consistent with its metabolism to M1 and M2 in CV1 cells (94% M1 and 6% M2). In adult male rats treated orally with 30 and 100 mg of vinclozolin/kg of body weight/day for 30 days, the major serum metabolite was M1; serum levels averaged 107 nM and 1.6 µM vinclozolin, 1.7 µM and 10 µM M1, and 22 nM and 270 nM M2, respectively. The higher concentration of the less potent metabolite M1 suggests that it contributes, with M2, to the antiandrogenic effects of vinclozolin.

The mechanism of transcriptional inhibition was investigated by determining the effect of each ligand on androgen-induced AR binding to androgen response element DNA. It was shown previously that human AR expressed from baculovirus in Sf9 cells requires intracellular exposure to androgen to induce high affinity, sequence-specific DNA binding activity (3) as shown in Fig. 5(lanes 1 and 2). Inhibition of DNA binding induced by 50 nM DHT required 10 µM vinclozolin (Fig. 5, lane 6), 10 µM M1 (Fig. 5, lane 10), or 0.2-0.5 µM M2, with essentially complete inhibition at 1-10 µM M2 (Fig. 5, lanes 11-14). M2 was 2-3-fold less effective than hydroxyflutamide in blocking androgen-induced AR DNA binding (Fig. 5, lanes 15-18). The results suggest that the mechanism of antagonism by these ligands is a concentration-dependent inhibition of androgen-induced AR DNA binding, with M2 being the most effective antiandrogen of the vinclozolin metabolites.


Figure 5: Inhibition of DNA binding by baculovirus-expressed recombinant AR. Sf9 cells expressing wild-type AR were incubated in the absence (lane 1) or presence of 50 nM dihydrotestosterone (DHT) either alone (lanes 2, 19, and 20) or with increasing concentrations of vinclozolin (Vin, lanes 3-6), M1 (lanes 7-10), M2 (lanes 11-14), or hydroxyflutamide (OH-FL, lanes 15-18). DNA mobility shift assays were performed with an androgen response element as described under ``Experimental Procedures.'' The upper band represents specific AR binding to P-labeled androgen response element DNA and is shifted to a slower migration (lane 20) with the addition of AR52 antipeptide antibody (AB) described previously(29) . The middle bands represent nonspecific DNA binding and are detected with extracts from Sf9 cells not exposed to recombinant baculovirus (not shown). At the bottom is the upper portion of the free labeled oligonucleotide band. The concentration of competing unlabeled ligand ranged from 0.2 to 10 µM as indicated. The data shown are representative of three independent experiments.



Antagonist versus Agonist Activity

When M2 and hydroxyflutamide were added at high concentrations (10 µM) in the absence of androgen to Sf9 cells expressing AR, AR DNA binding was observed (Fig. 6, lanes 12 and 16) raising the possibility that these antagonists are agonists at high concentrations in the absence of androgen. (Note the absence of AR DNA binding at these concentrations in Fig. 5in the presence of DHT.) The luciferase transcription assay was therefore repeated using high concentrations of these compounds in the absence of DHT. As shown in Fig. 7, concentrations of 10 and 50 µM M2 or hydroxyflutamide, and to a lesser extent, vinclozolin, induced AR-mediated transcriptional activation from 20- to 48-fold. In the absence of AR expression, no agonist effects were observed, ruling out inductions through non-AR-mediated pathways. The results reveal the striking ability of the androgen antagonists, M2 and hydroxyflutamide, to act as AR agonists at high concentrations in the absence of DHT.


Figure 6: Enhancement of AR DNA binding by high concentrations of M2 and hydroxyflutamide in the absence of DHT. Incubations of Sf9 cells expressing AR were performed in the absence (lanes 19 and 20) or presence of 0.05 µM dihydrotestosterone (DHT, lanes 17 and 18) or at increasing concentrations of vinclozolin (Vin, lanes 1-4), metabolites M1 (lanes 5-8), M2 (lanes 9-12), or hydroxyflutamide (OH-FL, lanes 13-16) between 0.2 and 10 µM. The DNA mobility shift assay was performed as described under ``Experimental Procedures.'' The upper band is specific for ARbulletDNA complex formation, since it is shifted to a slower migration with the addition of antibody AR52 (AB, lane 18) but is undetectable in the absence of DHT (lane 20). The data shown are representative of four independent experiments.




Figure 7: Agonist activity of high concentrations of vinclozolin, its metabolites, and hydroxyflutamide with wild-type AR. Agonist activity was determined by transient cotransfection using the parent expression vector lacking AR coding sequence (p5) or pCMVhAR coding for wild-type AR and the luciferase reporter vector as described under ``Experimental Procedures'' and in the legend of Fig. 4. Shown are the optical units obtained following incubations with 0.1 nM DHT and increasing concentrations of metabolites M1 and M2, vinclozolin (Vin), and hydroxyflutamide (OH-FL) in a range from 1 to 50 µM. -Fold induction was determined relative to the activity observed in the absence of added ligand and is shown numerically at the bottom.



Relative ligand binding affinity, androgen response element DNA binding, and agonist activity of M2 and hydroxyflutamide were investigated further using an AR mutant that codes for the same amino acid sequence as the AR mutation in the human LNCaP human prostate cancer cell line. In these cells, a single base mutation within the AR gene region coding for the steroid binding domain changes threonine 877 to alanine and increases AR binding affinity for and agonist activity of hydroxyflutamide(16, 17, 18) . M2 was two to three times more effective as a competitive inhibitor of [^3H]R1881 binding to LNCaP AR compared with wild-type AR, a binding difference similar to that observed with hydroxyflutamide (Fig. 8). Similarly, DNA binding of baculovirus expressed LNCaP mutant AR was induced by concentrations of M2 10-fold lower than required for DNA binding of wild-type AR (Fig. 9, lane 3). Hydroxyflutamide induced DNA binding of the LNCaP mutant AR at a 50-fold lower concentration than required for wild-type AR (Fig. 9, lanes 5-7). The extent of expression of wild-type and LNCaP recombinant AR was similar based on immunoblot analysis (data not shown). Similarly, the transcriptional response to M2 with LNCaP AR was 9-fold greater than with wild-type AR (Fig. 10). Transcriptional activity induced by 1 nM DHT was 44- and 68-fold with the wild-type and mutant receptors, respectively (Fig. 10). The results suggest that M2 and hydroxyflutamide can act as agonists at high concentrations (10 µM) in the absence of androgen both in cells expressing wild-type AR and at lower concentrations (0.2-1 µM) in cells expressing mutant ARs with ligand binding specificity of the threonine 877 to alanine mutation.


Figure 8: Competitive inhibition of [^3H]R1881 binding to wild-type and LNCaP AR by M2 and hydroxyflutamide. Whole cell binding assays were performed in transiently transfected COS cells as described under ``Experimental Procedures'' and the legend of Fig. 2using wild-type (WT) and LNCaP prostate cancer pCMVhAR expression vector DNA and incubating with 5 nM [^3H]R1881 and increasing concentrations of unlabeled R1881, M2, and hydroxyflutamide (OH-FL) as competitor. The data are expressed as percent of total binding observed in the presence of 5 nM [^3H]R1881 alone and are representative of three independent experiments.




Figure 9: Increased DNA binding of baculovirus expressed LNCaP prostate cancer cell line mutant AR after incubation with M2 and hydroxyflutamide. DNA mobility shift assays were performed as described under ``Experimental Procedures.'' Sf9 cells expressing the mutant LNCaP AR from recombinant baculovirus were incubated with increasing concentrations of M2 (lanes 1-4) or hydroxyflutamide (OH-FL, lanes 5-8) between 0.2 and 10 µM or with (lanes 9-10) or without (lanes 11-12) 50 nM DHT. The upper band represents specific AR-DNA binding, since it is shifted to a slower migration by the addition of AR52 IgG antibody (AB, lane 10) and is undetected in the absence of DHT but presence of AR52 antibody (lane 12). The middle band represents nonspecific DNA binding by Sf9 cell extracts, and the band at the bottom of the gel is the upper portion of the free labeled oligonucleotide. Approximately 15,000 cpm were applied to each lane. The data shown are representative of three independent experiments.




Figure 10: Increased agonist activity of M2 and hydroxyflutamide with the LNCaP prostate cancer cell line mutant AR. Wild-type (WT) and the LNCaP mutant AR (Thr to Ala) expression vector DNAs were transiently expressed into CV1 cells with the luciferase reporter vector as described under ``Experimental Procedures'' and incubated with 0.1 and 1 µM M2 and hydroxyflutamide (OH-FL). Optical units are compared with activity determined in the presence of 1 nM DHT. -Fold induction was determined relative to the activity determined in the absence of added ligand and is indicated numerically at the bottom.




DISCUSSION

Metabolites M1 and M2 of the fungicide vinclozolin are potential antiandrogens that inhibit AR-mediated transcriptional activity in a concentration-dependent manner by blocking androgen-induced AR binding to androgen response element DNA. M2 has a potency similar to a structurally related antiandrogen, hydroxyflutamide, whereas the major metabolite M1 is less active but could contribute to antiandrogenic potency because of its higher concentration; M1 reaches 40-75 times higher concentrations than M2 in rats administered vinclozolin. An unexpected result was the agonist activity of M2 and hydroxyflutamide in the absence of androgen. These ligands at high concentrations apparently induce a receptor conformation compatible with AR DNA binding and transcriptional activation. With the LNCaP cell mutant AR (Thr to Ala), hydroxyflutamide and M2 had agonist activity at lower concentrations due to increased binding affinity of the mutant AR for these compounds.

The results raise the possibility that mixed ligand dimers, i.e. agonist (natural androgens) and antagonist bound in the same dimer, are required for antagonism, whereas same ligand dimers of sufficiently high affinity promote receptor activation. A similar hypothesis was suggested for type II antagonists of PR(19) . Based on trypsin digestion patterns, antihormones are believed to induce inappropriate receptor conformations of PR (20) but not AR(21) . Mixed ligand dimers may be transcriptionally inactive because the two ligands induce incompatible conformational states, each of which could be active in same ligand dimers. In support of distinct ligand-induced receptor conformations, steroid binding domain mutations altered the antagonist/agonist relationship in PR (22) and ER(23) . Antagonist and agonist activities appear to be determined by receptor binding affinity, ligand concentration, and the presence or absence of competing high affinity natural ligands. Tissue-specific differences in ligand metabolism would then contribute to the antagonist or agonist activity of a particular compound. Differences may also exist among natural enhancers and/or promoters of androgen regulated genes.

It remains to be established the extent to which the general public and occupational workers are exposed to the widely used fungicide, vinclozolin, or whether active metabolites reach concentrations sufficient for antiandrogenic or androgenic activity. Daily oral dosing of rats results in serum M2 levels sufficient for antagonist but not agonist activity. On the other hand, flutamide administered in high doses to prostate cancer patients may be detrimental due to the agonist potential of the active metabolite, hydroxyflutamide. Plasma levels of hydroxyflutamide can reach 78 ng/ml (8 µM) (24) which is within the agonist range. Prostate cancer patients can experience improvement upon discontinuing flutamide treatment, a phenomenon known as flutamide withdrawal syndrome(25, 26) . Similar improvement is reported in some breast cancer patients removed from tamoxifen(27) . High level, long term exposure to flutamide, particularly in men undergoing androgen withdrawal therapy for prostate cancer, could cause proliferation of androgen-responsive cells. AR mutations like the prostate cancer cell line LNCaP mutant AR, reported in 6 of 24 advanced prostate cancer specimens(28) , can enhance the agonist effects of certain antiandrogens like M2 and hydroxyflutamide because of increased binding affinity.

The results raise concern about the potential biological effects of excess exposure to the fungicide, vinclozolin, that could potentially influence normal male sexual differentiation and/or fertility. Furthermore, the use of high dose flutamide treatment in men with prostate cancer may be detrimental in some patients undergoing androgen withdrawal therapy. Finally, a relationship is suggested between antagonist and agonist activities based on the possible formation of mixed ligand or same ligand dimers, respectively.


FOOTNOTES

*
This work was supported by Grants HD16910 and P30-HD18968 from the NICHD Center for Population Research and by Grant NS17479 from the NINDS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratories for Reproductive Biology, CB#7500 MacNider Bldg., 374 Medical Science Research Bldg., University of North Carolina, Chapel Hill, NC 27599. Tel.: 919-966-5159; Fax: 919-966-2203.

(^1)
The abbreviations and trivial names used are: AR, androgen receptor; vinclozolin, (3-(3, 5-dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione; M1, (2-[[3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3butenoic acid; M2, 3`,5`-dichloro-2-hydroxy-2-methylbut-3-enanilide; R1881, methyltrienolone; PBS, phosphate-buffered saline; DHT, dihydrotestosterone; LNCaP, lymph node-derived human prostate carcinoma cell line; hydroxyflutamide, alpha,alpha,alpha-trifluoro-2-methyl-4`-nitro-m-lactotoluidide (SCH16423).


ACKNOWLEDGEMENTS

We thank Dr. Frank S. French for critical reading of the manuscript and Malcolm V. Lane, Jon A. Kemppainen, and Michelle Cobb for technical assistance.


REFERENCES

  1. French, F. S., Lubahn, D. B., Brown, T. R., Simental, J. A., Quigley, C. A., Yarbrough, W. G., Tan, J. S., Sar, M., Joseph, D. R., Evans, B. A. J., Hughes, I. A., Migeon, C. J., and Wilson, E. M. (1990) Recent Prog. Horm. Res. 46,1-42 [Medline] [Order article via Infotrieve]
  2. Kemppainen, J. A., Lane, M. V., Sar, M., and Wilson, E. M. (1992) J. Biol. Chem. 267,968-974 [Abstract/Free Full Text]
  3. Wong, C., Zhou, Z., Sar, M., and Wilson, E. M. (1993) J. Biol. Chem. 268,19004-19012 [Abstract/Free Full Text]
  4. van Ravenzwaay, B. (1992) Data submission to United States Environmental Protection Agency from BASF Corp., MRID 425813-02
  5. Gray, L. E., Jr., Ostby, J. M., and Marshall, R. (1993) Biol. Reprod. 48, Suppl. 1, 97 (Abstr. 154)
  6. Gray, L. E., Ostby, J. S., and Kelce, W. R. (1994) Toxicol. Appl. Pharmacol. 129,46-52 [CrossRef][Medline] [Order article via Infotrieve]
  7. Szeto, S. Y., Burlinson, N. E., Rahe, J. E., and Oloffs, P. C. (1989) J. Agric. Food Chem. 37,523-529
  8. Szeto, S. Y., Burlinson, N. E., Rahe, J. E., and Oloffs, P. C. (1989) J. Agric. Food Chem. 37,529-534
  9. Szeto, S. Y., Burlinson, N. E., Rettig, S. J., and Trotter, J. (1989) J. Agric. Food Chem. 37,1103-1108
  10. Kelce, W. R., Monosson, E., Gamcsik, M. P., Laws, S. C., and Gray, L. E. (1994) Toxicol. Appl. Pharmacol. 126,276-285 [CrossRef][Medline] [Order article via Infotrieve]
  11. Hawkins, D. R., Kirkpatrick, D., Dean, G. M., Cheng, K., and Riseborough, J. (1990) United States Environmental Protection Agency Guideline Number 85-1 submission from BASF Corp., MRID 418243-07
  12. Yarbrough, W. G., Quarmby, V. E., Simental, J. A., Joseph, D. R., Sar, M., Lubahn, D. B., Olsen, K. L., French, F. S., and Wilson, E. M. (1990) J. Biol. Chem. 265,8893-8900 [Abstract/Free Full Text]
  13. Zhou, Z., Sar, M., Simental, J. A., Lane, M. V., and Wilson, E. M. (1994) J. Biol. Chem. 269,13115-13123 [Abstract/Free Full Text]
  14. Simental, J. A., Sar, M., Lane, M. V., French, F. S., and Wilson, E. M. (1991) J. Biol. Chem. 266,510-518 [Abstract/Free Full Text]
  15. Tan, J. A., Marschke, K. B., Ho, K. C., Perry, S. T., Wilson, E. M., and French, F. S. (1992) J. Biol. Chem. 267,4456-4466 [Abstract/Free Full Text]
  16. Harris, S. E., Sarris, M. A., Rong, Z., Hall, J., Judge, S., French, F. S., Joseph, D. R., Lubahn, D. B., Simental, J. A., and Wilson, E. M. (1991) Molecular and Cellular Biology of Prostate Cancer (Karr, J. P., Coffey, D. S., Smith, R. G., and Tindall, D. J., eds) pp. 315-330, Plenum Press, New York
  17. Veldscholte, J., Ris-Stalpers, C., Kuiper, G. G. J. M, Jenster, G., Berrevoets, C., Claassen, E., van Rooij, H. C. J., Trapman, J., Brinkmann, A. O., and Mulder, E. (1990) Biochem. Biophys. Res. Commun. 173,534-540 [Medline] [Order article via Infotrieve]
  18. Veldscholte, J., Voorhorst-Ogink, M. M., Bolt-de Vries, J., van Rooij, H. C. J., Trapman, J., and Mulder, E. (1990) Biochim. Biophys. Acta 1052,187-194 [Medline] [Order article via Infotrieve]
  19. Edwards, D. P., Prendergast, P., Weigel, N. L., Beck, C. A. (1994) Ninth International Congress on Hormonal Steroids, Dallas, TX , September 24-29 , Abstract S 192, p. 65
  20. Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M. J., and O'Malley, B. W. (1992) J. Biol. Chem. 267,19513-19520 [Abstract/Free Full Text]
  21. Kallio, P. J., Janne, O. A., and Palvimo, J. J. (1994) Endocrinology 134,998-1001 [Abstract]
  22. Garcia, T., Benhamou, B., Gofflo, D., Verbezac, A., Philibert, D., Chambon, P., and Gronemeyer, H. (1992) Mol. Endocrinol. 6,2071-2078 [Abstract]
  23. Jiang, S. Y., Langan-Fahey, S. M., Stella, A. L., McCague, R., and Jordan, V. C. (1992) Mol. Endocrinol. 6,2167-2174 [Abstract]
  24. Physicians' Desk Reference (1993) Eulexin, Medical Economics Data , pp. 2185-2186, Montvale, NJ
  25. Kelly, W. K., and Scher, H. I. (1993) J. Urol. 149,607-609 [Medline] [Order article via Infotrieve]
  26. Scher, H. I., and Kelly, W. K. (1993) J. Clin. Oncol. 11,1566-1572 [Abstract]
  27. Belani, C. P., Pearl, P., Whitley, N. O., and Aisner, J. (1989) Arch. Intern. Med. 149,449-450 [Abstract]
  28. Gaddipati, J. P., McLeod, D. G., Heidenberg, H. B., Sesterhenn, I. A., Finger, M. J., Moul, J. W., and Srivastava, S. (1994) Cancer Res. 54,2861-2864 [Abstract]
  29. Quarmby, V. E., Kemppainen, J. A., Sar, M., Lubahn, D. B., French, F. S., and Wilson, E. M. (1990) Mol. Endocrinol. 4,1399-1407 [Abstract]

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