(Received for publication, February 13, 1995; and in revised form, May 22, 1995)
From the
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.
The human androgen receptor (AR) ()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
5-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.
Figure 2:
Competitive inhibition of
[H]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 [
H]R1881 alone are
shown for unlabeled R1881 (
), dihydrotestosterone (DHT,
),
cyproterone acetate (
), hydroxyflutamide (*), M2
(-
-), 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.
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.
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.
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 ARDNA
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
[H]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
[H]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 [
H]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 [
H]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.
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.