Substitution of synthetic chimpanzee androgen receptor for human androgen receptor in competitive binding and transcriptional activation assays for EDC screening.

The potential effect of receptor-mediated endocrine modulators across species is of increasing concern. In attempts to address these concerns, we are developing androgen and estrogen receptor binding assays using recombinant hormone receptors from a number of species across different vertebrate classes. The United States Environmental Protection Agency (USEPA) Office of Science Coordination and Policy (OSCP) requested that we develop a nonhuman mammalian receptor-binding assay for possible use in their Endocrine Disruptor Screening Program (EDSP). Since the chimpanzee androgen receptor is very similar to that of humans and thus possesses properties which could be exploited in future endocrine studies, we synthesized and expressed this gene in eukaryotic expression plasmids, baculovirus expression vectors and replication deficient adenovirus. In all ligand-binding and transcriptional activation assays tested, the chimpanzee receptor performed essentially identically to the human receptor. This suggests that the chimpanzee gene could substitute for the human gene in endocrine screening assays.

Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening.

The discovery of xenobiotics that interfere with androgen activity has highlighted the need to assess chemicals for their ability to modulate dihydrotestosterone (DHT)-receptor binding. Previous test systems have used cells transfected with plasmid containing a reporter gene. Here we report the use of transduction for gene delivery and assessment of the modulation of DHT-induced gene activation. Transduction, the ability of replication-defective viruses to deliver biologically competent genes, is a well understood biological process, which has been utilized to repair defective genes in humans as well as to express exogenous genes in rodent models. Human breast carcinoma cells (MDA-MB-453) containing endogenous copies of the androgen (hAR) and glucocorticoid (GR) receptors were transduced with replication-defective human adenovirus type 5 containing the luciferase (Luc) reporter gene driven by the AR- and GR-responsive glucocorticoid-inducible hormone response element found with the mammary tumor virus LTR (Ad/MLUC7). In a second set of experiments, CV-1 cells were transduced as above with MMTV-luc and also hAR. Cells were subcultured in 96-well plates, transduced with virus, exposed to chemicals, incubated for 48 h, lysed, and assayed for luciferase. Luc gene expression was induced in a dose-dependent manner by DHT, estradiol, and dexamethasone (MDA only) and inhibited by AR antagonist hydroxyflutamide (OHF), hydroxy-DDE, HPTE (2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane), a methoxychlor metabolite, and M1 and M2 (vinclozolin metabolites). The transduced cells responded to AR agonists and antagonists as predicted from our other studies, with a very robust and reproducible response. Over all replicates, 0.1 nM DHT induced luc expression by about 45-fold in CV-1 cells (intra-assay CV = 20%) and 1micromolar OHF inhibited DHT by about 80%. In the transduced MDA cells, 0.1 nM DHT induced luc by about 24-fold (intra-assay CV = 33%), which was inhibited by OHF by about 85%. DHT-induced luciferase activity peaked in both cell lines between 1 and 100 nM, displaying about 64- and 115-fold maximal induction in the CV-1 and MDA 453 cells, respectively. For agonists, a two-fold induction of luc over media control was statistically significant. For AR antagonists, a 25-30% inhibition of DHT-induced luc expression was typically statistically significant. Comparing the two assays, the transduced CV-1 cells were slightly more sensitive to AR-mediated responses, but the transduced MDA 453 cells were more responsive to GR agonists. In summary, these assays correctly identified the endocrine activity of all chemicals examined and displayed sensitivity with a relatively low variability and a high-fold induction over background. Adenovirus transduction for EDC screening has the potential to be employed in a high-throughput mode, and could easily be applied to other cell lines and utilized to deliver other receptors and reporter genes.

The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat.

Phthalate esters (PE) such as DEHP are high production volume plasticizers used in vinyl floors, food wraps, cosmetics, medical products, and toys. In spite of their widespread and long-term use, most PE have not been adequately tested for transgenerational reproductive toxicity. This is cause for concern, because several recent investigations have shown that DEHP, BBP, DBP, and DINP disrupt reproductive tract development of the male rat in an antiandrogenic manner. The present study explored whether the antiandrogenic action of DEHP occurs by (1) inhibiting testosterone (T) production, or by (2) inhibiting androgen action by binding to the androgen receptor (AR). Maternal DEHP treatment at 750 mg/kg/day from gestational day (GD) 14 to postnatal day (PND) 3 caused a reduction in T production, and reduced testicular and whole-body T levels in fetal and neonatal male rats from GD 17 to PND 2. As a consequence, anogenital distance (AGD) on PND 2 was reduced by 36% in exposed male, but not female, offspring. By GD 20, DEHP treatment also reduced testis weight. Histopathological evaluations revealed that testes in the DEHP treatment group displayed enhanced 3ss-HSD staining and increased numbers of multifocal areas of Leydig cell hyperplasia as well as multinucleated gonocytes as compared to controls at GD 20 and PND 3. In contrast to the effects of DEHP on T levels in vivo, neither DEHP nor its metabolite MEHP displayed affinity for the human androgen receptor at concentrations up to 10 microM in vitro. These data indicate that DEHP disrupts male rat sexual differentiation by reducing T to female levels in the fetal male rat during a critical stage of reproductive tract differentiation.

Vinclozolin and p,p'-DDE alter androgen-dependent gene expression: in vivo confirmation of an androgen receptor-mediated mechanism.

Vinclozolin and p,p'-DDE induce antiandrogenic developmental effects in vivo and are potent inhibitors of androgen receptor (AR) binding and AR-dependent gene expression in vitro. To determine whether this molecular mechanism is operative in vivo, the effects of these compounds on two androgen-regulated prostatic mRNAs were studied. Rats were sham operated or castrated and immediately implanted with one or two empty 2.5-cm silastic capsules or with one (1x) or two (2x) 2.5-cm capsules containing testosterone (T). T-implanted rats were treated by gavage for 4 days with vehicle (corn oil), vinclozolin (200 mg/kg/day), p,p'-DDE (200 mg/kg/day), or the antiandrogen flutamide (100 mg/kg/day) as a positive control. Vinclozolin, p,p'-DDE, and flutamide all induced a reciprocal decline in seminal vesicle (p < 0.01) and prostate (p < 0.01) weight as well as a reduction in immunohistochemical staining of AR in epididymal nuclei compared to vehicle-treated T-implanted controls. Specific AR antagonism was assessed by determining the ability of these chemicals to induce a testosterone-repressed prostatic message (i.e., TRPM-2) and/or repress a testosterone-induced prostatic message (i.e., prostatein subunit C3). Densitometry scans of Northern blots indicated that vinclozolin, p,p'-DDE, and flutamide each induced TRPM-2 mRNA and repressed C3 mRNA compared to vehicle-treated T-implanted controls. These antiandrogenic effects were competitively reduced in castrate rats implanted with two 2.5-cm T capsules (2x), where serum T levels were elevated more than twofold above physiological levels. Taken together, these data indicate that vinclozolin and p,p'-DDE act as antiandrogens in vivo by altering the expression of androgen-dependent genes.