* Endocrinology-Reproductive Physiology Program, School of Pharmacy, and
Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53705
Received December 5, 2003; accepted February 9, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); urogenital sinus (UGS); prostatic epithelial bud formation; primary UGS mesenchymal cell culture; development; androgen receptor signaling pathway; C57BL/6J mouse fetus; prostate; hydroxyflutamide.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interruption of androgen signaling, such as by in utero exposure to the 5-reductase inhibitor finasteride or to the competitive antiandrogen flutamide, can inhibit prostatic bud formation in rats (Hib and Ponzio, 1995
). Similarly, in utero exposure to the 5
-reductase inhibitor 6-methylene-4-pregnene-3,20-dione inhibits prostatic epithelial bud formation in mice (Iguchi et al., 1991
). Androgen signaling can also be interrupted by mutations that render the AR nonfunctional (Brown et al., 1990
; Quigley et al., 1992
; Thiele et al., 1999
), or by mutations in 5
-reductase (Mahendroo and Russell, 1999
). Such mutations cause prostate abnormalities or agenesis, as well as multiple other alterations in human sexual development (Griffin et al., 1995
). AR coactivators and coregulators are also important for androgen signaling. For example, steroid receptor coactivator (SRC-1) deficiency inhibits prostate growth and development (Xu et al., 1998
), and a dominant negative mutant of the AR coregulator ARA54 inhibits androgen-dependent prostate cancer cell proliferation (Miyamoto et al., 2002
).
Ventral, dorsolateral, and anterior prostate development in C57BL/6 mice are inhibited by in utero and lactational TCDD exposure (Ko et al., 2002; Lin et al., 2002a
). These effects are mediated by the receptor for TCDD, the aryl hydrocarbon receptor (AHR; Carlson and Perdew, 2002
; Gasiewicz and Park, 2003
), as shown by the lack of inhibited prostate growth in TCDD-exposed Ahr-knockout mice (Lin et al., 2002a
). The nature and severity of the effects of TCDD on wild-type mice varies from lobe to lobe, but the ventral prostate is the most severely affected (Ko et al., 2002
; Lin et al., 2002a
). The critical period for TCDD exposure includes or occurs primarily during in utero development (Lin et al., 2002b
). TCDD inhibits prostate development in the absence of any consistent decrease in postnatal plasma androgen concentrations or postnatal testicular androgen content (Ko et al., 2002
; Lin et al., 2002a
).
In C57BL/6J mice, in utero TCDD exposure can cause complete absence of prostatic epithelial buds from the ventral surface of the UGS (Lin et al., 2003). Failure of these buds to form accounts for the complete agenesis of ventral prostate main ducts in TCDD-exposed mice (Ko et al., 2002
). The number of dorsolateral prostatic epithelial buds is also reduced by in utero TCDD exposure (Lin et al., 2003
), which accounts for the reduced numbers of dorsal and lateral prostate main ducts (Ko et al., 2002
). The inhibition of prostatic epithelial bud formation appears to be caused primarily by a direct effect of TCDD on the UGS (Lin et al., in press
), and is mediated by AHR in UGS mesenchyme, not in UGS epithelium (Ko et al., in press
).
The underlying mechanisms by which TCDD exposure inhibits prostatic epithelial bud formation during gestation are unknown. An intriguing possibility is that inhibition of prostatic epithelial bud formation results from an inhibition of androgen signaling in UGS mesenchymal cells. It is not known if TCDD binds to the AR, but mechanisms which have been suggested to be involved in the ability of TCDD to inhibit androgen signaling in other experimental systems include (1) decreased AR gene expression (Ohsako et al., 2001; Theobald et al., 2000
), (2) competition between AR and AHR for coactivators such as RIP140 and SRC-1 (Kumar and Perdew, 1999
; Kumar et al., 1999
), and (3) direct interaction of activated AHR with androgen responsive elements (Jana et al., 1999
).
The objective of the present study was to determine if the TCDD-induced inhibition of prostatic epithelial bud formation by the UGS results from an inhibition of androgen signaling or from effects of TCDD that may be independent of androgens. Androgen levels and metabolism were examined prenatally. Primary UGS mesenchymal cells transiently transfected with an androgen-responsive reporter gene were used to assess effects of TCDD on androgen signaling. Effects of TCDD on the androgen-regulated expression of genes for AR and 5-reductase type II were also examined in the UGS. The findings suggest that TCDD inhibits prostatic epithelial bud formation by a mechanism that does not involve reduced androgenic stimulation of the UGS or inhibition of AR signaling pathways in the UGS.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To produce timed pregnant females, groups of three 90120-day-old dams were housed overnight with one male per cage. Pregnant females were given a single dose of either vehicle (95% corn oil/5% acetone, v/v) or 5 µg TCDD/kg by gavage on gestation day (GD) 13. Dams were euthanized by CO2 overdose so UGS tissue could be obtained from male fetuses. All animal procedures were conducted under protocols approved by the University of Wisconsin-Madison Animal Care and Use Committee.
Testicular testosterone content.
Steroids were extracted from testes with ethyl acetate. Organic phases were evaporated to dryness under a gentle nitrogen stream and reconstituted in 100% ethanol. Testicular testosterone content was measured by enzyme immunoassay according to the protocol of the kit supplier (Assay Designs, Inc., Ann Arbor, MI). The assay range was 7.82000 pg testosterone/ml.
In vitro organ culture of the UGS.
GD 14 fetuses were excised from euthanized dams, immediately placed on ice, and sexed by inspection of the gonads. The UGS was dissected from male fetuses after removal of the urinary bladder, Wolffian ducts, and remnant Müllerian ducts. Isolated UGSs were embedded in 500 µl of 0.6% SeaPlaque agarose in Dulbecco's Modified Eagle's medium (DMEM) supplemented with 5% charcoal-dextran-stripped fetal bovine serum (CD-FBS; Hyclone, Logan, UT) and 1% antibiotics-antimycotics (Gibco BRL, Grand Island, NY). After addition of these components the agarose gels were solidified at 4°C for 10 min, then 500 µl of DMEM/5% CD-FBS with 1% antibiotics-antimycotics were added to the agarose gel. Cultured UGSs were exposed to 0.1% (v/v) ethanol, 108 M DHT, or 108 M DHT + 105 M hydroxyflutamide (OH-flutamide), which was a gift of Dr. C. Tendler (Procter and Gamble Co., Cincinnati, OH), or 108 M DHT + 109 M TCDD. UGS organ cultures were maintained for either three days (GD 1416) or five days (GD 1418) in a humidified 37°C, 5% CO2/95% air incubator, and culture medium was replaced every 48 h.
Bud counting and scanning electron microscopy.
After in vitro culture, each UGS was harvested and saved individually in calcium- and magnesium-free Hanks' balanced salt solution (HBSS; Sigma Chemical Co., St. Louis, MO) so that mesenchyme could be separated from epithelium. Mesenchyme was enzymatically dissociated from epithelium in a solution of 1% trypsin (Difco, Sparks, MD) in HBSS for 90 min at 4°C followed by gentle mechanical manipulation with fine forceps. Isolated UGS epithelium was fixed in 2.5% glutaraldehyde, then buds were counted under a light microscope at approximately 40-fold magnification by focusing up and down through the sample at each of several orientations. UGSs with more than about 10 buds were counted at least twice. UGSs were then mounted and evaluated by scanning electron microscopy as previously described (Lin et al., 2003).
Radiolabeled testosterone metabolites in UGS organ culture medium.
UGSs obtained from vehicle- and TCDD-exposed male fetuses on GD 16 were immediately placed in DMEM (Gibco BRL) with 5% CD-FBS. UGSs were cultured at 37°C in an atmosphere of 95% air and 5% carbon dioxide in the presence of 108 M [1,2,6,73H]-testosterone (Amersham, Arlington Heights, IL). After 24 and 48 h the medium was removed and stored at 20°C prior to solvent extraction and thin layer chromatographic analysis of radiolabeled testosterone metabolites, as previously described (Theobald et al., 2000).
Preparation of UGS mesenchymal primary cells.
After being cultured in vitro for three days UGSs were partially digested with trypsin as described above. Epithelium was then removed from the mesenchyme by teasing the tissues apart with fine forceps. Mesenchymes were incubated in 1% collagenase (Sigma) in HBSS at 37°C for 30 min followed by incubation in 0.1% DNAse (Sigma) in HBSS at 37°C for 10 min. Single cell suspensions were prepared by gentle pipetting of the treated tissue. Cell suspensions were centrifuged at 2500 x g for 2 min at room temperature and rinsed twice with DMEM supplemented with 5% CD-FBS. Isolated primary UGS mesenchymal cells were cultured in 5% CD-FBS-supplemented DMEM.
Primary UGS mesenchymal cell culture and transient transfection.
Primary UGS mesenchymal cells were seeded in 96-well plates at 5 x 104 cells/well and transfected by a calcium phosphate coprecipitation method (Moehlenkamp and Johnson, 1999). Cells were transfected with 30 ng of an androgen-responsive reporter plasmid, mouse mammary tumor virus luciferase reporter (MMTV-luciferase) plasmid, or TCDD-responsive reporter plasmids. The MMTV-luciferase plasmid was a gift from Dr. A. O. Brinkmann (Erasmus University Medical Center, Rotterdam, The Netherlands). TCDD-responsive reporter plasmids, pGud1.1-Luc (containing the mouse cytochrome P4501A1 promoter) and pCYP1B1-Luc (containing the mouse cytochrome P4501B1 promoter) were provided by Drs. M. S. Denison (University of California, Davis, CA) and C. R. Jefcoate (University of Wisconsin, Madison, WI), respectively. CMVß-gal plasmid (2 ng/well) was used to correct for transfection efficiency. For the androgen-responsive reporter assay, cells were treated for 12 h with 0.1% ethanol (vehicle for DHT and OH-flutamide) and 0.1% DMSO (vehicle for TCDD), 108 M DHT, 108 M DHT + 105 M OH-flutamide, or 108 M DHT + 109 M TCDD. For the TCDD-responsive reporter assay, cells were treated for 12 h with DMSO (final concentration, 0.1%) or 109 M TCDD + 108 M DHT. Cells were lysed in 100 µl of protein extraction buffer (100 mM KPO4, pH 7.4, 1.5 mM MgSO4, 1.0 mM dithiothreitol, and 0.1% Triton X-100) + 4.0 mM ATP. Luciferase activity was determined by adding 210 µl of luciferase assay buffer (100 mM KPO4, pH 7.4, 4.0 mM ATP, and 1.5 mM MgSO4) to 30 µl of cell lysate per well (Ahlgren-Beckendorf et al., 1999). Luminescence was determined using an ML2250 microtiter plate luminometer (Dynatech Laboratories, Chantilly, VA). ß-Galactosidase activity was measured by the Galacto-Light (Applied Biosystems, Foster City, CA) chemiluminescent reporter assay. Cell lysates (30 µl) were added to 200 µl of ß-galactosidase reaction buffer (100 mM sodium phosphate, pH 8.0, 1 mM MgCl2, 0.1 M ß-mercaptoethanol, and 0.6 µg of o-nitrophenyl-ß-galactoside per ml). Data are expressed as a ratio of luciferase to ß-galactosidase activities measured in separate aliquots of cell lysate from each well.
Real-time RT-PCR mRNA quantification.
Total RNA was prepared from in vitro cultured UGSs using RNeasy Mini Kits (Qiagen, Valencia, CA) according to the manufacturer's instructions. Synthesis of cDNA was conducted using Omniscript reverse transcriptase (Qiagen) in 20 µl reaction tubes that contained 250 ng total RNA, following the manufacturer's protocol. Samples were then analyzed by real-time RT-PCR using a GeneAmp 5700 system (PE Biosystems, Foster City, CA). The reaction was carried out in a final volume of 20 µl: 10 µl SYBR Green, 1 µl of 5 µM forward primer, 1 µl of 5 µM reverse primer, 1 µl template (cDNA), and 7 µl of deionized water. Primer sequences, product sizes, and annealing temperatures are shown in Table 1. The analysis was performed on five samples in each treatment group, in triplicate. PCR was performed for 40 cycles with each cycle consisting of 1 min of denaturation at 95°C, 1 min of annealing at the appropriate temperature for each gene, and 2 min of extension at 72°C. At the end of the PCR run, a melting curve was performed to verify the presence of a single amplicon. Amplified PCR product was analyzed using GeneAmp 5700 software (PE Biosystems).
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Effects of OH-Flutamide and TCDD on Prostatic Epithelial Bud Formation in Vitro
The ability of the pure antiandrogen OH-flutamide to block DHT-induced prostatic epithelial bud formation in vitro was tested and compared with the effects of TCDD. As expected, no prostatic buds were found when male UGSs obtained on GD 14 were cultured without DHT for five days (vehicle group, Figs. 3 and 4). UGSs cultured with 108 M DHT averaged about 20 prostatic buds after five days of culture (Fig. 4), distributed over their ventral, dorsolateral, and anterior surfaces (Fig. 3). When UGSs were cultured with DHT and 105 M OH-flutamide, an average of only about four prostatic buds (Fig. 4) were found on the combined ventral, dorsolateral, and anterior surfaces (Fig. 3). UGSs cultured with DHT and 109 M TCDD averaged about six prostatic buds (Fig. 4). The inhibition of budding by TCDD appeared to be more severe on the ventral surface than on the remaining surfaces (Fig. 3), but conclusive evidence of a significant difference in budding patterns between OH-flutamide and TCDD was not obtained.
|
|
When mesenchymal cells were isolated from UGSs that had been cultured with DHT but not TCDD, incubation without DHT resulted in low luciferase activity (Fig. 5A). DHT increased luciferase activity about six-fold; OH-flutamide reduced DHT-induced luciferase activity to the background level; and TCDD had no effect on DHT-induced luciferase activity (Fig. 5A).
|
These experiments demonstrate that TCDD, unlike OH-flutamide, is not antiandrogenic in this experimental system following either acute exposure (12 h of primary UGS mesenchymal cell culture) or chronic exposure (three days of UGS organ culture plus 12 h of UGS mesenchymal cell culture).
AhR-Dependent Luciferase Activity in UGS Mesenchymal Cells
To determine if the lack of effect of TCDD on androgen signaling by primary UGS mesenchymal cells was due to inability of these cells to respond to TCDD, primary UGS mesenchymal cells were transfected with TCDD-responsive luciferase reporter plasmids containing either a CYP1A1 or a CYP1B1 promoter (Garrison, 1996; Zhang et al., 1998
). These cells were prepared from GD 14 UGSs that had been cultured in vitro with 108 M DHT for three days. The primary mesenchymal cells were then transfected with reporter plasmids and incubated for 12 h with 108 M DHT and either 0.1% DMSO or 109 M TCDD. TCDD increased luciferase activity about 1.7-fold in cells transfected with the CYP1A1 promoter and about 1.8-fold in cells transfected with the CYP1B1 promoter (Fig. 6), demonstrating that the AhR signaling pathway was intact and functional.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TCDD could also inhibit androgenic stimulation of the UGS, and thereby inhibit prostatic budding, by inhibiting the conversion of testosterone to DHT within the UGS. However, in utero TCDD exposure did not inhibit formation of DHT from testosterone by UGSs cultured in vitro. And mRNA levels for the enzyme that catalyzes this reaction, 5-reductase type II, were not reduced either. These results, in combination with the testicular testosterone content results, suggest that TCDD does not decrease DHT concentrations in the UGS.
We had previously shown that exogenous DHT cannot prevent TCDD from inhibiting prostatic epithelial bud formation in vivo (Lin et al., 2003). That experiment demonstrated that inhibited budding is not due solely to insufficient DHT, but it did not rule out the possibility that inadequate DHT concentrations might be one of several factors causing the inhibition. Results presented in this report suggest that effects of in utero TCDD exposure on prostatic epithelial bud formation are not due to insufficient DHT within the UGS.
OH-Flutamide as a Positive Control to Test the Hypothesis that TCDD Inhibits Prostatic Budding by Inhibiting Androgen Signaling
OH-Flutamide was incorporated into these experiments because it is a competitive androgen receptor antagonist (Kemppainen et al., 1992) that also reduces androgen receptor concentrations (List et al., 2000
), and because its parent molecule flutamide can completely inhibit prostatic epithelial bud formation in the rat (Hib and Ponzio, 1995
). For OH-flutamide to be a valid positive control in our experiments it would also have to inhibit prostatic epithelial bud formation by mouse UGS in vitro, as TCDD does (Lin et al., in press
). When control UGSs were incubated in vitro with DHT and either OH-flutamide or TCDD, the inhibitory effect of OH-flutamide on budding was at least as severe as that of TCDD if not more so. OH-Flutamide was therefore used as a positive control for interference with androgen signaling in all succeeding experiments in which androgen-dependent effects of TCDD were examined.
Differences between Effects of OH-Flutamide and TCDD on the UGS and on UGS-Derived Cells
One method used to test the hypothesis that TCDD inhibits prostatic epithelial bud formation by interfering with androgen signaling was to determine if TCDD and OH-flutamide have comparable effects on UGS mesenchymal cells transfected with the androgen-responsive MMTV-luciferase reporter plasmid. AR-antagonistic effects of OH-flutamide have been demonstrated in several cell lines transfected with MMTV-reporter plasmids (Hartig et al., 2002; List et al., 2000
; Warriar et al., 1993
, 1994
), but it was unclear if AR would remain functional in isolated UGS primary mesenchymal cells because Gupta (1999)
had to transfect these cells with AR in order to obtain an androgen response. In our lab, luciferase activity was induced by DHT, and OH-flutamide inhibited this induction, without introduction of exogenous AR. To our knowledge, this is the first report that there is a functional endogenous AR signaling pathway in primary cultures of UGS mesenchymal cells.
Unlike OH-flutamide, TCDD did not inhibit AR signaling in the MMTV-luciferase reporter gene assay. This suggests that TCDD does not act on UGS mesenchymal cells to inhibit androgen signaling, and therefore that TCDD inhibits prostatic epithelial bud formation by mechanisms other than interference with androgen signaling. This result is consistent with a lack of correlation between AHR signaling pathway activation and antiandrogenic effects when Chinese hamster ovary cells were transiently cotransfected with AR and MMTV-luciferase plasmids and treated with AHR agonists (Vinggaard et al., 2000). In contrast, 107 M TCDD was slightly antiandrogenic in PC-3 prostate carcinoma cells cotransfected with AR and MMTV-luciferase plasmids (Schrader and Cooke, 2000
), and 108 but not 109 M TCDD inhibited androgen-regulated transcriptional activity in LNCaP prostate cancer cells transfected with MMTV-luciferase plasmid (Jana et al., 1999
). Our experiments were all done at a substantially lower TCDD concentration (109 M), however, and in pilot experiments, even 1010 M TCDD inhibited prostatic epithelial bud formation.
The lack of effect of TCDD on androgen-dependent gene expression in primary cultures of UGS mesenchymal cells was not due to an inherent inability of these cells to respond to TCDD, because the same concentration of TCDD significantly induced gene expression when these cells were transfected with CYP1A1 and CYP1B1 reporter constructs.
The other method we used to test the hypothesis that TCDD inhibits prostatic epithelial bud formation by interfering with androgen signaling was to determine if TCDD and OH-flutamide have comparable or different effects on the expression of endogenous androgen-responsive genes. DHT is known to decrease AR mRNA abundance (Prins and Woodham, 1995) and to increase 5
-reductase type II mRNA abundance (Berman et al., 1995
). OH-flutamide has been shown to antagonize the androgen-induced decrease in AR mRNA abundance in a human prostate cancer cell line (Ravenna et al., 1995
), while in utero flutamide exposure reduced 5
-reductase type II mRNA abundance in neonatal male rat brain (Poletti et al., 1998
). And in utero and lactational TCDD exposure decreased AR mRNA abundance and increased 5
-reductase type II mRNA abundance in the ventral prostate of 49-day-old rats (Ohsako et al., 2001
). In the present study, OH-flutamide inhibited the ability of DHT to decrease AR mRNA abundance, and OH-flutamide inhibited the ability of DHT to increase 5
-reductase type II mRNA abundance in mouse UGS organ cultures, as expected. In contrast, TCDD had no effect on the DHT-regulated transcription of either AR or 5
-reductase type II mRNAs in cultured UGSs, even though it severely inhibited the in vitro budding response to DHT under the same conditions. These results suggest that the inhibitory effect of TCDD on prostatic bud formation in the UGS is not associated with a generalized inhibition of AR-mediated gene transcription in the UGS.
Possible Mechanisms by Which TCDD Inhibits Prostatic Epithelial Bud Formation
Our observations that in utero TCDD exposure has no apparent effect on testicular testosterone content prenatally or on the conversion of testosterone to DHT by the UGS, and that TCDD does not inhibit DHT-stimulated luciferase activity in UGS mesenchymal cells or affect the transcription of two DHT-regulated endogenous genes in the UGS, strongly suggest that TCDD does not inhibit prostatic epithelial bud formation by inhibiting the initial events in androgen signaling. The MMTV-luciferase reporter construct we used contains the consensus androgen response element (Ham et al., 1988; Roche et al., 1992
) and has often been used to examine androgenicity or antiandrogenicity (Schrader and Cooke, 2000
; Tamura et al., 2003
; Térouanne et al., 2002
; Vinggaard et al., 1999
). While the lack of effect of TCDD on DHT-stimulated MMTV-luciferase activity or on AR and 5
-reductase type II mRNA levels do not entirely rule out the possibility that TCDD might inhibit some aspect of the initial events in androgen signaling, they strongly suggest that TCDD inhibits prostatic epithelial bud formation by affecting downstream AR signaling events and/or by inhibiting androgen-independent signaling pathways.
One example of a mechanism by which TCDD may inhibit downstream effects of AR signaling is via effects on the Sonic hedgehog (Shh) signaling pathway. Shh is an androgen-dependent gene whose expression is required for prostate development (Podlasek et al., 1999a). Further research is needed to determine if TCDD inhibits budding by altering the activity of the Shh signaling pathway, or of other androgen-dependent signaling pathways, in the UGS.
Nonandrogen regulated growth factors such as epidermal growth factor, transforming growth factor-, bone morphogenetic protein 4, and fibroblast growth factor 10 play a role in prostatic epithelial bud formation (Lamm et al., 2001
; Mizuno and Saito, 1996
; Saito and Mizuno, 1995
; Thomson and Cunha, 1999
). In addition, the homeobox genes Hoxa-10, Hoxa-13 and Hoxd-13 are not androgen-dependent but act as regulators of budding (Podlasek et al., 1997
, 1999b
,c
). Accordingly, TCDD may affect these or other androgen-independent mechanisms involved in prostatic bud formation. Gene expression analysis is currently being used to examine these possibilities.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 Present address: NCI/FCRDC, Building 538 Room 206, Ft. Detrick, Frederick, MD 21702
2 To whom correspondence should be addressed at the School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705-2222. Fax: (608) 265-3316. E-mail: repeterson{at}pharmacy.wisc.edu
Portions of this research were presented at the 42nd Annual Meeting of the Society of Toxicology, March 2003, Salt Lake City, UT. This article is Contribution 351 from the Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53726-4087.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bard, D. R., Lasnitzki, I., and Mizuno, T. (1979). Metabolism of testosterone by the epithelium and mesenchyme of the rat urogenital sinus. J. Endocrinol. 83, 211218.[Abstract]
Berman, D. M., Tian, H., and Russell, D. W. (1995). Expression and regulation of steroid 5-reductase in the urogenital tract of the fetal rat. Mol. Endocrinol. 9, 15611570.[Abstract]
Brown, T. R., Lubahn, D. B., Wilson, E. M., French, F. S., Migeon, C. J., and Corden, J. L. (1990). Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity. Mol. Endocrinol. 4, 17591772.[Abstract]
Carlson, D. B., and Perdew, G. H. (2002). A dynamic role for the Ah receptor in cell signaling? Insights from a diverse group of Ah receptor interacting proteins. J. Biochem. Mol. Toxicol. 16, 317325.[CrossRef][ISI][Medline]
Cooke, P. S., Young, P., and Cunha, G. R. (1991). Androgen receptor expression in developing male reproductive organs. Endocrinology 128, 28672873.[Abstract]
Cunha, G. R., and Lung, B. (1978). The possible influence of temporal factors in androgenic responsiveness of urogenital tissue recombinants from wild-type and androgen-insensitive (Tfm) mice. J. Exp. Zool. 205, 181193.[ISI][Medline]
Garrison, P. M., Tullis, K., Aarts, J. M. M. J. G., Brouwer, A., Giesy, J. P., and Denison, M. S. (1996). Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam. Appl. Toxicol. 30, 194203.[CrossRef][ISI][Medline]
Gasiewicz, T. A., and Park, S.-K. (2003). Ah receptor: Involvement in toxic responses. In Dioxins and Health, 2nd ed. (A. Schecter and T. A. Gasiewicz, Eds.), pp. 491532. John Wiley and Sons, Hoboken, NJ.
Griffin, J. E., McPhaul, M. J., Russell, D. W., and Wilson, J. D. (1995). The androgen resistance syndromes: Steroid 5-reductase 2 deficiency, testicular feminization, and related disorders. In The Metabolic and Molecular Bases of Inherited Disease, 7th ed. (C. R. Scrivner, A. L. Beaudet, W. S. Sly, and D. Valle, Eds.), pp. 29672988. McGraw-Hill, St. Louis.
Gupta, C. (1999). Modulation of androgen receptor (AR)-mediated transcriptional activity by EGF in the developing mouse reproductive tract primary cells. Mol. Cell. Endocrinol. 152, 169178.[CrossRef][ISI][Medline]
Ham, J., Thomson, A., Needham, M., Webb, P., and Parker, M. (1988). Characterization of response elements for androgens, glucocorticoids and progestins in mouse mammary tumour virus. Nucleic Acids Res. 16, 52635276.[Abstract]
Hartig, P. C., Bobseine, K. L., Britt, B. H., Cardon, M. C., Lambright, C. R., Wilson, V. S., and Gray, L. E., Jr. (2002). Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening. Toxicol. Sci. 66, 8290.
Hib, J., and Ponzio, R. (1995). The abnormal development of male sex organs in the rat using a pure antiandrogen and a 5-reductase inhibitor during gestation. Acta Physiol. Pharmacol. Ther. Latinoam. 45, 2733.[Medline]
Iguchi, T., Uesugi, Y., Takasugi, N., and Petrow, V. (1991). Quantitative analysis of the development of genital organs from the urogenital sinus of the fetal male mouse treated prenatally with a 5-reductase inhibitor. J. Endocrinol. 128, 395401.[Abstract]
Jana, N. R., Sarkar, S., Ishizuka, M., Yonemoto, J., Tohyama, C., and Sone, H. (1999). Cross-talk between 2,3,7,8-tetrachlorodibenzo-p-dioxin and testosterone signal transduction pathways in LNCaP prostate cancer cells. Biochem. Biophys. Res. Commun. 256, 462468.[CrossRef][ISI][Medline]
Kemppainen, J. A., Lane, M. V., Sar, M., and Wilson, E. M. (1992). Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones. J. Biol. Chem. 267, 968974.
Ko, K., Moore, R. W., and Peterson, R. E. (in press). Aryl hydrocarbon receptors in urogenital sinus mesenchyme mediate the inhibition of prostatic epithelial bud formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol.
Ko, K., Theobald, H. M., and Peterson, R. E. (2002). In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in the C57BL/6J mouse prostate: Lobe-specific effects on branching morphogenesis. Toxicol. Sci. 70, 227237.
Kumar, M. B., and Perdew, G. H. (1999). Nuclear receptor coactivator SRC-1 interacts with the Q-rich subdomain of the AhR and modulates its transactivation potential. Gene Expr. 8, 273286.[ISI][Medline]
Kumar, M. B., Tarpey, R. W., and Perdew, G. H. (1999). Differential recruitment of coactivator RIP140 by Ah and estrogen receptors. Absence of a role for LXXLL motifs. J. Biol. Chem. 274, 2215522164.
Lamm, M. L. G., Podlasek, C. A., Barnett, D. H., Lee, J., Clemens, J. Q., Hebner, C. M., and Bushman, W. (2001). Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate. Dev. Biol. 232, 301314.[CrossRef][ISI][Medline]
Lasnitzki, I., and Mizuno, T. (1977). Induction of the rat prostate gland by androgens in organ culture. J. Endocrinol. 74, 4755.[Abstract]
Lasnitzki, I., and Mizuno, T. (1980). Prostatic induction: Interaction of epithelium and mesenchyme from normal wild-type mice and androgen-insensitive mice with testicular feminization. J. Endocrinol. 85, 423428.[Abstract]
Lin, T.-M., Ko, K., Moore, R. W., Simanainen, U., Oberley, T. D., and Peterson, R. E. (2002a). Effects of aryl hydrocarbon receptor null mutation and in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on prostate and seminal vesicle development in C57BL/6 mice. Toxicol. Sci. 68, 479487.
Lin, T.-M., Rasmussen, N. T., Moore, R. W., Albrecht, R. M., and Peterson, R. E. (2003). Region-specific inhibition of prostatic epithelial bud formation in the urogenital sinus of C57BL/6 mice exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Sci. 76, 171181.
Lin, T.-M., Rasmussen, N. T., Moore, R. W., Albrecht, R. M., and Peterson, R. E. (in press). 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits prostatic epithelial bud formation by acting directly on the urogenital sinus. J. Urol.
Lin, T.-M., Simanainen, U., Moore, R. W., and Peterson, R. E. (2002b). Critical windows of vulnerability for effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on prostate and seminal vesicle development in C57BL/6 mice. Toxicol. Sci. 69, 202209.
List, H.-J., Smith, C. L., Martinez, E., Harris, V. K., Danielsen, M., and Riegel, A. T. (2000). Effects of antiandrogens on chromatin remodeling and transcription of the integrated mouse mammary tumor virus promoter. Exp. Cell Res. 260, 160165.[CrossRef][ISI][Medline]
Mahendroo, M. S., and Russell, D. W. (1999). Male and female isoenzymes of steroid 5-reductase. Rev. Reprod. 4, 179183.
Miyamoto, H., Rahman, M., Takatera, H., Kang, H.-Y., Yeh, S., Chang, H.-C., Nishimura, K., Fujimoto, N., and Chang, C. (2002). A dominant-negative mutant of androgen receptor coregulator ARA54 inhibits androgen receptor-mediated prostate cancer growth. J. Biol. Chem. 277, 46094617.
Mizuno, T., and Saito, M. (1996). Induction of prostatic buds in the urogenital sinus of androgen receptor-deficient Tfm mouse embryos. C. R. Seances Soc. Biol. 190, 497501.
Moehlenkamp J. D., and Johnson J. A. (1999). Activation of antioxidant/electrophile-responsive elements in IMR-32 human neuroblastoma cells. Arch. Biochem. Biophys. 363, 98106.[CrossRef][ISI][Medline]
Ohsako, S., Miyabara, Y., Nishimura, N., Kurosawa, S., Sakaue, M., Ishimura, R., Sato, M., Takeda, K., Aoki, Y., Sone, H., Tohyama, C., and Yonemoto, J. (2001). Maternal exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the development of reproductive organs of male rats: Dose-dependent increase of mRNA levels of 5-reductase type 2 in contrast to decrease of androgen receptor in the pubertal ventral prostate. Toxicol. Sci. 60, 132143.
Podlasek, C. A., Barnett, D. H., Clemens, J. Q., Bak, P. M., and Bushman, W. (1999a). Prostate development requires Sonic hedgehog expressed by the urogenital sinus epithelium. Dev. Biol. 209, 2839.[CrossRef][ISI][Medline]
Podlasek, C. A., Clemens, J. Q., and Bushman, W. (1999b). Hoxa-13 gene mutation results in abnormal seminal vesicle and prostate development. J. Urol. 161, 16551661.[ISI][Medline]
Podlasek, C. A., Duboule, D., and Bushman, W. (1997). Male accessory sex organ morphogenesis is altered by loss of function of Hoxd-13. Dev. Dyn. 208, 454465.[CrossRef][ISI][Medline]
Podlasek, C. A., Seo, R. M., Clemens, J. Q., Ma, L., Maas, R. L., and Bushman, W. (1999c). Hoxa-10 deficient male mice exhibit abnormal development of the accessory sex organs. Dev. Dyn. 214, 112.[CrossRef][ISI][Medline]
Poletti, A., Negri-Cesi, P., Rabuffetti, M., Colciago, A., Celotti, F., and Martini, L. (1998). Transient expression of the 5-reductase type 2 isozyme in the rat brain in late fetal and early postnatal life. Endocrinology 139, 21712178.
Prins, G. S., and Woodham, C. (1995). Autologous regulation of androgen receptor messenger ribonucleic acid in the separate lobes of the rat prostate gland. Biol. Reprod. 53, 609619.[Abstract]
Quigley, C. A., Evans, B. A. J., Simental, J. A., Marschke, K. B., Sar, M., Lubahn, D. B., Davies, P., Hughes, I. A., Wilson, E. M., and French, F. S. (1992). Complete androgen insensitivity due to deletion of exon C of the androgen receptor gene highlights the functional importance of the second zinc finger of the androgen receptor in vivo. Mol. Endocrinol. 6, 11031112.[Abstract]
Ravenna, L., Lubrano, C., Di Silverio, F., Vacca, A., Felli, M. P., Maroder, M., D'Eramo, G., Sciarra, F., Frati, L., Gulino, A., and Petrangeli, E. (1995). Androgenic and antiandrogenic control on epidermal growth factor, epidermal growth factor receptor, and androgen receptor expression in human prostate cancer cell line LNCaP. Prostate 26, 290298.[ISI][Medline]
Roche, P. J., Hoare, S. A., and Parker, M. G. (1992). A consensus DNA-binding site for the androgen receptor. Mol. Endocrinol. 6, 22292235.[Abstract]
Saito, M., and Mizuno, T. (1995). Epidermal growth factor (EGF) can induce prostatic buds in the absence of androgens. C. R. Seances Soc. Biol. 189, 637641.
Schrader, T. J., and Cooke, G. M. (2000). Examination of selected food additives and organochlorine food contaminants for androgenic activity in vitro. Toxicol. Sci. 53, 278288.
Tamura, H., Yoshikawa, H., Gaido, K. W., Ross, S. M., DeLisle, R. K., Welsh, W. J., and Richard, A. M. (2003). Interaction of organophosphate pesticides and related compounds with the androgen receptor. Environ. Health Perspect. 111, 545552.[ISI][Medline]
Térouanne, B., Paris, F., Servant, N., Georget, V., and Sultan, C. (2002). Evidence that chlormadinone acetate exhibits antiandrogenic activity in androgen-dependent cell line. Mol. Cell. Endocrinol. 198, 143147.[CrossRef][ISI][Medline]
Theobald, H. M., Roman, B. L., Lin, T.-M., Ohtani, S., Chen, S.-W., and Peterson, R. E. (2000). 2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits luminal cell differentiation and androgen responsiveness of the ventral prostate without inhibiting prostatic 5-dihydrotestosterone formation or testicular androgen production in rat offspring. Toxicol. Sci. 58, 324338.
Thiele, B., Weidemann, W., Schnabel, D., Romalo, G., Schweikert, H.-U., and Spindler, K.-D. (1999). Complete androgen insensitivity caused by a new frameshift deletion of two base pairs in exon 1 of the human androgen receptor gene. J. Clin. Endocrinol. Metab. 84, 17511753.
Thomson, A. A., and Cunha, G. R. (1999). Prostatic growth and development are regulated by FGF10. Development 126, 36933701.
Timms, B. G., Mohs, T. J., and Didio, L. J. A. (1994). Ductal budding and branching patterns in the developing prostate. J. Urol. 151, 14271432.[ISI][Medline]
Vinggaard, A. M., Hnida, C., and Larsen, J. C. (2000). Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro. Toxicology 145, 173183.[CrossRef][ISI][Medline]
Vinggaard, A. M., Joergensen, E. C. B., and Larsen, J. C. (1999). Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals. Toxicol. Appl. Pharmacol. 155, 150160.[CrossRef][ISI][Medline]
Warriar, N., Pagé, N., Koutsilieris, M., and Govindan, M. V. (1993). Interaction of antiandrogen-androgen receptor complexes with DNA and transcription activation. J. Steroid Biochem. Mol. Biol. 46, 699711.[CrossRef][ISI][Medline]
Warriar, N., Pagé, N., Koutsilieris, M., and Govindan, M. V. (1994). Antiandrogens inhibit human androgen receptor-dependent gene transcription activation in the human prostate cancer cells LNCaP. Prostate 24, 176186.[ISI][Medline]
Xu, J., Qiu, Y., DeMayo, F. J., Tsai, S. Y., Tsai, M.-J., and O'Malley, B. W. (1998). Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 279, 19221925.
Zhang, L., Savas, Ü., Alexander, D. L., and Jefcoate, C. R. (1998). Characterization of the mouse Cyp1B1 gene. Identification of an enhancer region that directs aryl hydrocarbon receptor-mediated constitutive and induced expression. J. Biol. Chem. 273, 51745183.