Androgen Receptor Reporter Gene Research Articles

Antiandrogenic activity and bioavailability of magnolol analogs – A potential for prostate cancer therapeutics

BackgroundProstate cancer is the second most common form of cancer in men worldwide and there is a great need for novel treatment strategies, especially for castrate-resistant prostate cancers where the proliferation of the cancer cells is stimulated by androgens produced in the adrenal cortex and the cancer cells. PurposeIn this study, we have investigated the antiandrogenic properties of magnolol and ten synthetic analogs in vitro. Study design and methodsThe compounds were evaluated for cytotoxicity, antiandrogenic receptor activity, binding to the androgen receptor, effects on the production of Prostate-specific antigen (PSA), and potential to pass over a tight layer of Caco-2 cells mimicking gastrointestinal absorption. ResultsWe found that almost all investigated compounds were antiandrogenic in an androgen receptor reporter gene assay, with IC50 values ranging from 7 to 86 µM. Magnolol itself had the highest antiandrogenic potency. Five of the compounds were then evaluated for their binding to the androgen receptor and three of these compounds were found to bind to the receptor. These five compounds were also evaluated for their effect on the PSA production and four were found to decrease PSA production at non-cytotoxic concentrations. The antiandrogenic activity after passage through a layer of Caco-2 cells, mimicking gastrointestinal absorption, was also evaluated for three of the compounds. All three compounds were found to have the capacity to be transported from the apical to the basolateral side of the Caco-2 cell layer and exert antiandrogenic effects after the transport. ConclusionIn conclusion, this study shows that magnolol and analogs have antiandrogenic effects in vitro and that selected analogs can pass over a tight layer of Caco-2 cells, indicating a potential for good bioavailability after oral administration. These magnolol analogs thereby constitute an interesting group of compounds worthy of further evaluation as potential anti-prostate cancer therapeutics.

Virtual Screening and Bioactivity Evaluation of Novel Androgen Receptor Antagonists From Anti-PCa Traditional Chinese Medicine Prescriptions.

Prostate cancer (PCa), a type of malignancy that arises in the prostate gland, is the most commonly diagnosed neoplasm and the second leading cause of cancer-related deaths in men. Acquisition of resistance to conventional therapy is a major problem for PCa patient treatment. Androgen receptor (AR) signaling pathway is necessary in the pathogenesis of prostate cancer, and there is a heightened interest in finding novel AR antagonists that target AR and its regulatory pathways. In our search for novel androgen receptor antagonists, we focus on the Traditional Chinese Medicine (TCM), which has been used for thousands of years to prove effective in the treatment of cancer. In this study, we collected 653 traditional Chinese medicine prescriptions that have certain therapeutic effect to prostate cancer, including the prescriptions and even the folk prescriptions. After summarizing the frequency of herbs and gathering the natural products contained in these prescriptions, we built a natural products database to do computer-aided virtual screening and drug-like evaluation to find potential AR antagonists. Totally 25 compounds were submitted to experimental biological activity tests. Through the MTT cell proliferation experiment, 5 chemicals were found to inhibit the proliferation of LNCaP cells in a concentration-dependent manner. Especially, MoL_11 was found to have good antagonistic activity and significantly inhibit fluorescence enzyme activity by the AR reporter gene experiment. Finally, the molecular dynamics simulation method was used to study the interaction between the most active compound MoL_11 and the wild-type and F876L mutant androgen receptor (WT/F876L AR), and it was found that F876L AR could not cause resistance to MoL_11.

A 3-(4-nitronaphthen-1-yl) amino-benzoate analog as a bifunctional AKR1C3 inhibitor and AR antagonist: Head to head comparison with other advanced AKR1C3 targeted therapeutics

Drugs used for the treatment of castration resistant prostate cancer (CRPC) include Abiraterone acetate (Zytiga®) and Enzalutamide (XTANDI®). However, these drugs provide clinical benefit in metastatic disease for only a brief period before drug resistance emerges. One mechanism of drug resistance involves the overexpression of type 5 17-β-hydroxysteroid dehydrogenase (aldo-keto reductase 1C3 or AKR1C3), a major enzyme responsible for the formation of intratumoral androgens that activate the androgen receptor (AR). 3-((4-Nitronaphthalen-1-yl)amino)benzoic acid 1 is a “first-in-class” AKR1C3 competitive inhibitor and AR antagonist. Compound 1 was compared in a battery of in vitro studies with structurally related N-naphthyl-aminobenzoates, and AKR1C3 targeted therapeutics e.g. GTx-560 and ASP9521, as well as with R-bicalutamide, enzalutamide and abiraterone acetate. Compound 1 was the only naphthyl derivative that was a selective AKR1C3 inhibitor and AR antagonist in direct competitive binding assays and in AR driven reporter gene assays. GTx-560 displayed weak activity as a direct AR antagonist but had high potency in the AR reporter gene assay consistent with its ability to inhibit the co-activator function of AKR1C3. By contrast ASP9521 did not act as either an AR antagonist or block AR reporter gene activity. Compound 1 was the only compound that showed comparable potency to inhibit AKR1C3 and act as a direct AR antagonist. Compound 1 blocked the formation of testosterone in LNCaP-AKR1C3 cells, and the expression of PSA driven by the AKR1C3 substrate (4-androstene-3,17-dione) and by an AR agonist, 5α-dihydrotestosterone consistent with its bifunctional role. Compound 1 blocked the nuclear translocation of the AR at similar concentrations to enzalutamide and caused disappearance of the AR from cell lysates. R-biaclutamide and enzalutamide inhibited AKR1C3 at concentrations 200x greater than compound 1, suggesting that its bifunctionality can be explained by a shared pharmacophore that can be optimized.

Discovery of novel 5-methyl-1H-pyrazole derivatives as potential antiprostate cancer agents: Design, synthesis, molecular modeling, and biological evaluation.

Androgen receptor (AR) signaling functions as a core driving force for the progression of prostate cancer (PCa), and AR has been proved to be an effective therapeutic target even for castration-resistant prostate cancer (CRPC). Herein, structural modification via a fragments splicing strategy was performed based on two lead compounds T3 and 10e, leading to the discovery of a series of 5-methyl-1H-pyrazole derivatives. AR reporter gene assay revealed compounds A13 and A14 as potent AR antagonists. Some of the compounds in this series inhibited growth of PCa LNCaP cells more efficiently than enzalutamide. A13 and A14 also showed improved metabolic stability compared with 10e in human liver microsomes.

Downscaling procedures reduce chemical use in androgen receptor reporter gene assay

Bioactivity screening studies often face sample amount limitation with respect to the need for reliable, reproducible and quantitative results. Therefore approaches that minimize sample use are needed. Low-volume exposure and chemical dilution procedures were applied in an androgen receptor reporter gene human cell line assay to evaluate environmental contaminants and androgen receptor modulators, which were the agonist 5α-dihydrotestosterone (DHT); and the antagonists flutamide, bisphenol A, 1-hydroxypyrene and triclosan. Cells were exposed in around 1/3 of the medium volume recommended by the protocol (70μL/well). Further, chemical losses during pipetting steps were minimized by applying a low-volume method for compound dilution in medium (250μL for triplicate wells) inside microvolume glass inserts. Simultaneously, compounds were evaluated following conventional procedures (200μL/well, dilution in 24-well plates) for comparison of results. Low-volume exposure tests produced DHT EC50 (3.4–3.7×10−10M) and flutamide IC50 (2.2–3.3×10−7M) values very similar to those from regular assays (3.1–4.2×10−10 and 2.1–3.3×10−7M respectively) and previous studies. Also, results were within assay acceptance criteria, supporting the relevance of the downscaling setup for agonistic and antagonistic tests. The low-volume exposure was also successful in determining IC50 values for 1-hydroxypyrene (2.1–2.8×10−6M), bisphenol A (2.6–3.3×10−6M), and triclosan (1.2–1.9×10−6M) in agreement with values obtained through high-volume exposure (2.3–2.8, 2.5–3.4 and 1.0–1.3×10−6M respectively). Finally, experiments following both low-volume dosing and exposure produced flutamide and triclosan IC50 values similar to those from regular tests. The low-volume experimental procedures provide a simple and effective solution for studies that need to minimize bioassay sample use while maintaining method reliability. The downscaling methods can be applied for the evaluation of samples, fractions or chemicals which require minimal losses during the steps of pipetting, transference to medium and exposure in bioassays.

ODM-204, a novel dual inhibitor of CYP17A1 and androgen receptor for the treatment of castration-resistant prostate cancer.

230 Background: Castration-resistant prostate cancer (CRPC) is characterized by high androgen receptor (AR) expression and persistent activation of AR signaling axis by residual tissue androgens. Inhibiting AR and androgen biosynthesis together may be more effective than inhibiting either alone to treat CRPC. ODM-204 is a potent, orally administered investigational non-steroidal dual inhibitor of CYP17A1 and AR. In vivo, ODM-204 shows favourable pharmacokinetic/ pharmacodynamics (PK/PD) properties in intact mature male monkeys and strong antitumor activity in VCaP xenograft. Methods: The inhibition of CYP17A1 by ODM-204 was studied by using human testicular microsomes and adrenal cortex cell line. Potency to human AR was demonstrated in cells stably transfected with the full-length AR and androgen-responsive reporter gene constructs. PK/PD relationships were evaluated in intact male mature monkeys after single and multiple doses of ODM-204 (10-30 mg/kg/day) by measuring plasma PK after several time points and serum testosterone (T), dehydroepiandrosterone (DHEA), luteinizing hormone (LH), aldosterone, and cortisol 5 hours after the daily dose. Efficacy was further studied in VCaP xenograft in intact male nude mice treated with ODM-204, enzalutamide (enza), abiraterone (abi), or enza+abi-combination for 28 days. Results: ODM-204 binds to AR with a high affinity (Ki47 nM) and selectivity, and has a high potency towards CYP17A1 (IC5022 nM). In intact monkeys, ODM-204 was well tolerated and showed good oral bioavailability with linear and dose proportional pharmacokinetics. Already after a single oral dose of ODM-204, serum T and DHEA were markedly suppressed. After multiple days of dosing, LH was increased, however, changes in cortisol and aldosterone were only minimal. In VCaP xenograft model, ODM-204 showed significant antitumor activity, better than single agent enza or abi, or their combination. Conclusions: ODM-204 is a novel, dual CYP17A1 and AR inhibitor for the treatment of CRPC. A clinical phase 1/2 dose-escalation trial (NCT02344017) is ongoing in patients with mCRPC to determine PK, PD, safety and anti-tumor activity of ODM-204.

Antagonism effects of cypermethrin on interleukin-6-induced androgen receptor activation

To identify whether androgen receptor (AR) antagonism by cypermethrin involves interleukin-6 (IL-6)-induced ligand-independent AR signaling, we have developed the AR reporter gene assay. The reporter gene plasmid pMMTV-chloramphenicol transferase (CAT) was transfected into LNCaP cells. IL-6 increased expression of MMTV-CAT significantly (P<0.05). Cypermethrin decreased CAT reporter expression induced by IL-6 (50ng/ml), and the significant inhibition was detected at 10−5M (P<0.05). IL-6 induces ligand-independent activation of AR. Cypermethrin exhibits inhibitory effects on IL-6-induced ligand-independent AR signaling. We provide a novel insight into cypermethrin-mediated antagonism of the IL-6-mediated ligand-independent activation of the AR.

MP66-08 DUAL PATHWAY INHIBITION IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2015MP66-08 DUAL PATHWAY INHIBITION IN PROSTATE CANCER Eugine Lee, Susan Ha, and Susan Logan Eugine LeeEugine Lee More articles by this author , Susan HaSusan Ha More articles by this author , and Susan LoganSusan Logan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2361AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen deprivation therapy is the mainstay of aggressive prostate cancer treatment, but can result in drug resistance and disease recurrence. Our studies show that small molecule inhibition of nuclear β-catenin can inhibit transcription of the androgen receptor (AR) by decreasing β-catenin occupancy on the AR promoter. Such an approach that targets AR transcription and lowers AR levels independent of hormone ligand action is important in light of recent studies showing that AR splice variants lacking the ligand-binding domain can still promote transcription. We recently showed that a small molecule inhibitor of nuclear β-catenin, an oxazole compound called inhibitor of beta-catenin responsive transcription-3 (iCRT3), can inhibit both AR and β-catenin action, resulting in prostate cell and tumor growth inhibition. Although promising, iCRT3 was not stable enough for systemic use and its effect was dependent on direct injection into tumors. METHODS To identify new inhibitors of nuclear β-catenin with improved pharmacokinetic properties, we conducted a screen in LNCaP-abl prostate cancer cells at the Rockefeller University High-Throughput Screening Resource Center. We screened 10,353 compounds that maximize “chemical diversity space” intended to accelerate the hit-to-lead process and assayed a Wnt/β-catenin reporter activity followed by a secondary screen assaying AR reporter gene activity. RESULTS We identified 55 compounds that reproducibly inhibited the Wnt/β-catenin reporter gene and AR reporter gene, underscoring the strength of our approach to target the AR and Wnt/β-catenin pathways. We recently determined the IC50 value of these compounds by validating their ability to inhibit the Wnt/β-catenin reporter gene using concentrations ranging from 20 micromolar to 39 nanomolar. Four compounds inhibited transcription at a concentration of 1.25 micromolar and 16 showed inhibition into the nanomolar range. Among these, 11 compounds showed no toxicity in 293 cells but were toxic to LNCaP-abl cells. The characteristics of these 11 compounds also satisfied the Lipinski rules used to evaluate the potential of chemical compounds for drug development. CONCLUSIONS Our studies underscore the potential of small molecule inhibitors of nuclear β-catenin to target both the AR and Wnt/β-catenin pathways, thus killing prostate cancer cells. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e818-e819 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Eugine Lee More articles by this author Susan Ha More articles by this author Susan Logan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

ODM-204: A novel dual inhibitor of CYP17A1 and androgen receptor for the treatment of castration-resistant prostate cancer—Preclinical data.

221 Background: Castration-resistant prostate cancer (CRPC) is characterized by high androgen receptor (AR) expression and persistent activation of AR signaling axis by residual tissue/tumor androgens. Targeting AR and androgen biosynthesis together may be more effective than either alone. ODM-204 is a novel, non-steroidal dual inhibitor of CYP17A1 and AR, which has shown promising results in preclinical studies. Methods: The binding affinity of ODM-204 to wild type AR was determined in rat prostate cytosolic lysates. The potency and functional activity of ODM-204 to human AR were demonstrated in cells stably transfected with the full-length AR and androgen-responsive reporter gene constructs. In addition, assays for AR nuclear translocation and the transactivation of human AR mutants T877A, W741L, and F876L were conducted. The effects of ODM-204 on the growth of androgen-dependent VCaP and LNCaP cells in vitro and subcutaneously grafted VCaP cells in vivo with the oral dose of 50 mg/kg/day were studied. The inhibition of CYP17A1 by ODM-204 was studied in vitro by using human and rat testicular microsomes and a human adrenal cortex cell line, and in vivo in male rats coadministered with luteinizing hormone releasing hormone agonist leuprolide acetate to mimic clinical situation. Results: ODM-204 is a potent inhibitor of both AR and CYP17A1. It binds to AR with a high affinity (Ki=47 nM) and selectivity and has a high potency towards CYP17A1 (IC50=22 nM). In addition, ODM-204 inhibited testosterone-mediated nuclear translocation of AR and the mutant ARs (IC50 values for AR(T877A), AR(W741L), and AR(F876L) were 95, 277, and 6 nM, respectively), and suppressed androgen-induced cell proliferation of LNCaP (IC50=170 nM) and VCaP (IC50=280 nM) cells. In a VCaP xenograft model, ODM-204 showed significant antitumor activity (tumor growth inhibition=66%). In rats, inhibitory effects of leuprolide acetate on testosterone production and androgen-sensitive organ weights were potentiated by ODM-204. Conclusions: ODM-204 is a promising new dual CYP17A1 and AR inhibitor for the treatment of CRPC. Clinical trials in patients with mCRPC will be started in early 2015.

Crystal structures of AKR1C3 containing an N-(aryl)amino-benzoate inhibitor and a bifunctional AKR1C3 inhibitor and androgen receptor antagonist. Therapeutic leads for castrate resistant prostate cancer

Castrate resistant prostate cancer (CRPC) is associated with increased androgen receptor (AR) signaling often brought about by elevated intratumoral androgen biosynthesis and AR amplification. Inhibition of androgen biosynthesis and/or AR antagonism should be efficacious in the treatment of CRPC. AKR1C3 catalyzes the formation of potent AR ligands from inactive precursors and is one of the most upregulated genes in CRPC. AKR1C3 inhibitors should not inhibit the related isoforms, AKR1C1 and AKR1C2 that are involved in 5α-dihydrotestosterone inactivation in the prostate. We have previously developed a series of flufenamic acid analogs as potent and selective AKR1C3 inhibitors [Adeniji, A. O. et al., J. Med. Chem.2012, 55, 2311]. Here we report the X-ray crystal structure of one lead compound 3-((4-(trifluoromethyl)phenyl) amino)benzoic acid (1) in complex with AKR1C3. Compound 1 adopts a similar binding orientation as flufenamic acid, however, its phenylamino ring projects deeper into a subpocket and confers selectivity over the other AKR1C isoforms. We exploited the observation that some flufenamic acid analogs also act as AR antagonists and synthesized a second generation inhibitor, 3-((4-nitronaphthalen-1-yl)amino)benzoic acid (2). Compound 2 retained nanomolar potency and selective inhibition of AKR1C3 but also acted as an AR antagonist. It inhibited 5α-dihydrotestosterone stimulated AR reporter gene activity with an IC50=4.7μM and produced a concentration dependent reduction in androgen receptor levels in prostate cancer cells. The in vitro and cell-based effects of compound 2 make it a promising lead for development of dual acting agent for CRPC. To illuminate the structural basis of AKR1C3 inhibition, we also report the crystal structure of the AKR1C3·NADP+·2 complex, which shows that compound 2 forms a unique double-decker structure with AKR1C3.

Endocrine disrupting effects in vitro of conazole antifungals used as pesticides and pharmaceuticals

Widely used conazole antifungals were tested for endocrine disruptive effects using a panel of in vitro assays. They all showed endocrine disrupting potential and ability to act via several different mechanisms. Overall the imidazoles (econazole, ketoconazole, miconazole, prochloraz) were more potent than the triazoles (epoxiconazole, propiconazole, tebuconazole). The critical mechanism seems to be disturbance of steroid biosynthesis. In the H295R cell assay, the conazoles decreased the formation of estradiol and testosterone, and increased the concentration of progesterone, indicating inhibition of enzymes involved in the conversion of progesterone to testosterone. Prochloraz was most potent followed by econazole ∼ miconazole > ketoconazole > tebuconazole > epoxiconazole > propiconazole. In the MCF-7 cell proliferation assay, the conazoles showed anti-estrogenic effect, including aromatase inhibition, since they inhibited the response induced by both 17β-estradiol (miconazole > econazole ∼ ketoconazole > prochloraz > tebuconazole > epoxiconazole > propiconazole) and testosterone (econazole > miconazole > prochloraz > ketoconazole > tebuconazole > epoxiconazole > propiconazole). The triazoles were anti-androgenic in an androgen receptor reporter gene assay (epoxiconazole ∼ tebuconazole > propiconazole). This effect could not be evaluated for the pharmaceutical imidazoles due to cytotoxicity.

Detection of anabolic androgenic steroid abuse in doping control using mammalian reporter gene bioassays

Anabolic androgenic steroids (AAS) are a class of steroid hormones related to the male hormone testosterone. They are frequently detected as drugs in sport doping control. Being similar to or derived from natural male hormones, AAS share the activation of the androgen receptor (AR) as common mechanism of action. The mammalian androgen responsive reporter gene assay (AR CALUX ® bioassay), measuring compounds interacting with the AR can be used for the analysis of AAS without the necessity of knowing their chemical structure beforehand, whereas current chemical–analytical approaches may have difficulty in detecting compounds with unknown structures, such as designer steroids. This study demonstrated that AAS prohibited in sports and potential designer AAS can be detected with this AR reporter gene assay, but that also additional steroid activities of AAS could be found using additional mammalian bioassays for other types of steroid hormones. Mixtures of AAS were found to behave additively in the AR reporter gene assay showing that it is possible to use this method for complex mixtures as are found in doping control samples, including mixtures that are a result of multi drug use. To test if mammalian reporter gene assays could be used for the detection of AAS in urine samples, background steroidal activities were measured. AAS-spiked urine samples, mimicking doping positive samples, showed significantly higher androgenic activities than unspiked samples. GC–MS analysis of endogenous androgens and AR reporter gene assay analysis of urine samples showed how a combined chemical–analytical and bioassay approach can be used to identify samples containing AAS. The results indicate that the AR reporter gene assay, in addition to chemical–analytical methods, can be a valuable tool for the analysis of AAS for doping control purposes.

Xenohormone transactivities are inversely associated to serum POPs in Inuit

BackgroundThe persistent organic pollutants (POPs) are highly lipophilic and resistant to biodegradation and found in e.g. seafood and marine mammals. Greenlandic Inuit have high intake of marine food and thus high POP burden that varies according to local conditions and dietary preference. We do for the very first time report the serum POP related non-steroidal xenohormone activity of Inuit across Greenland.The aims were 1) to determine the integrated xenohormone bioactivities as an exposure biomarker of the actual lipophilic serum POP mixture measuring the effect on estrogen (ER) and androgen receptor (AR) transactivity in citizens from different Greenlandic districts and 2) to evaluate associations to serum POP markers (14 PCBs and 10 pesticides) and lifestyle characteristics.MethodsSerum samples from 121 men and 119 women from Nuuk, Sisimiut and Qaanaaq were extracted using SPE-HPLC fractionation to obtain the serum POP fraction free of endogenous hormones. The serum POP fraction was used for determination of xenohormone transactivity using ER and AR reporter gene assays.ResultsIn overall, the xenohormone transactivities differed between districts as well as between the genders. Associations between the transactivities and age, n-3/n-6 and smoker years were observed. The xenoestrogenic and xenoandrogenic transactivities correlated negatively to the POPs for the combined female and male data, respectively.ConclusionThe non-steroidal xenohormone transactivities can be used as an integrated biomarker of POP exposure and lifestyle characteristics. The actual serum POP mixtures antagonized the age adjusted sex hormone receptor functions. Comparison of different study populations requires in addition to age inclusion of diet and lifestyle factors.

Evaluation of androgen receptor transcriptional activities of some pesticides in vitro

Organophosphates and pyrethroids are among the most common pesticides currently in use worldwide. Several pesticides have been reported to possess hormonal activities, and thus are classified as endocrine disruptors. The present study was planned to evaluate potential androgenic and antiandrogenic activities of the pesticides. The selected chemicals are three organophosphate pesticides including dichlorvos, parathion and trichlorphon, and two pyrethroid pesticides including permethrin and cypermethrin. We evaluated the pesticides for androgen receptor (AR)-mediated mechanisms using a human AR reporter gene assay in African monkey kidney cell line CV-1 transiently transfected with the constructed reporter gene plasmid pMMTV-CAT and the hAR expression plasmid AR/pcDNA3.1. We demonstrated that parathion showed significant inhibitory effects on the transcriptional activity induced by 1 nM of DHT with IC 50 value of (2.01 ± 0.42) × 10 −7 M, though trichlorphon and dichlorvos lacked this activity. The two pyrethroid pesticides permethrin and cypermethrin exhibited lower activity than parathion with IC 50 value of (5.68 ± 2.20) × 10 −5 and (6.80 ± 2.30) × 10 −5 M, respectively. On the other hand, we failed to find AR-mediated androgenic activities of the tested chemicals. It is suggested that parathion possesses the highest activity, and permethrin and cypermethrin acted as poor antiandrogens. The present study provides insight into the mechanism of the pesticides.

Androgen receptor activities of p, p′-DDE, fenvalerate and phoxim detected by androgen receptor reporter gene assay

In this study, we have developed a transient human androgen receptor (hAR) reporter gene assay using African monkey kidney cell line CV-1. The assay displayed appropriate response to the known androgen receptor (AR) agonist 5α-dihydrotestosterone (DHT) and AR antagonist nilutamide. DHT induced AR-mediated transcriptional activity in a concentration-dependent manner with median effective concentration (EC 50) value of 3.90 × 10 −10 M. Nilutamide exhibited potent antiandrogenic activity with median inhibitory concentration (IC 50) value of 8.90 × 10 −7 M. The transcription could not be activated by glucocorticoid receptor (GR) agonist dexamethasone, which suggested that the assay system be highly specific to androgenic compounds without cross-talk to GR agonist. The assay shows acceptable repeatability to DHT with intra coefficient of variability (CV) of 9.3% and inter CV of 10.9%. We applied this assay to evaluate the androgenic and antiandrogenic activities of some pesticides including organochlorine p, p′-dichlorodiphenyldichloroethylene (DDE), pyrethroid fenvalerate and organophosphorus phoxim. The results showed that p, p′-DDE exhibited potent antiandrogenic activity. Fenvalerate acted as weak AR antagonist and phoxim did not show AR antagonistic activity. We failed to find AR agonistic effects of the three pesticides. The present study provided a promising tool to screen for AR agonists and antagonists.

Antiandrogenic effects in vitro and in vivo of the fungicide prochloraz.

The commonly used imidazole fungicide prochloraz was tested for antiandrogenic effects in vitro and in vivo. Prochloraz, but not the metabolites 2,4,6-trichlorophenoxyacetic acid or 2,4,6-trichlorophenol, inhibited the R1881-induced response in an androgen receptor reporter gene assay. In the Hershberger assay, prochloraz exposure at all dose levels (50, 100, and 200 mg/kg) given orally to castrated testosterone (T)-treated males markedly reduced weights of ventral prostate, seminal vesicles, musc. levator ani/bulbocavernosus, and bulbourethral gland. These effects were accompanied by an increase in LH and a reduction of the T(4) and TSH level. The effects on seminal vesicles, LH, T(4), and TSH were also evident in intact prochloraz-exposed young adult rats. Body weights were unaffected whereas liver weights were increased in prochloraz-treated animals. Changes in androgen-regulated gene expression were determined in ventral prostates by real-time RT-PCR. A pronounced decrease of ornithin decarboxylase and PBP C3 mRNA levels was observed for both prochloraz and flutamide. These results indicate that prochloraz antagonizes the peripheral androgen receptors resulting in decreased growth of androgen-dependent tissues and that it antagonizes central androgen receptors blocking the negative feed-back mechanism of testosterone resulting in increased LH secretion from the pituitary. The antiandrogenic effects of prochloraz were in many ways qualitatively comparable, although weaker, to the effects of flutamide. However, differential effects on levels of FSH, T(4), and TSH indicate that other modes of action apart from the pure AR antagonism might play a role in vivo.

Effects of Aroclors and individual PCB congeners on activation of the human androgen receptor in vitro

To investigate possible interactions between the human androgen receptor and PCBs in vitro, we have used a previously characterized human androgen receptor reporter gene assay in which PC-3 LUC AR+ cells respond to 5α-dihydrotestosterone (DHT, 50 pM) with enhanced luciferase activity. The effects of Aroclors, commercial mixtures of PCBs, or polychlorinated terphenyls (PCTs) (0, 0.1, 1.0, and 10.0 μM) on luciferase activity in PC-3 LUC AR+ cells were determined after exposure for 18 h in the presence and absence of DHT (50 pM). In the absence of DHT, none of the Aroclors induced luciferase activity but, in the presence of DHT (50 pM), Aroclors 1016, 1221, 1232, 1242, 1248, 1254, 1260, 5432, and 5442 acted antagonistically at concentrations that did not affect cell viability. Aroclor 5460 was without effect. Similarly, when PCBs found as human milk contaminants were assessed as individual congeners (each at 1 μM, where no cytotoxic effects were observed), none activated luciferase expression in the absence of DHT but PCBs 49, 66, 74, 105, and 118 completely antagonized the stimulation by DHT (50 pM) and PCBs 138, 153, and 156 were less effective antagonists, reducing the DHT stimulation by about 50%. Thus, 30% (by weight) of the PCBs in human milk are androgen antagonists (PCBs 66, 74, 105, and 118) and a further 25% are partial antagonists (PCBs 138, 153, and 156). A proportionally representative mixture of PCBs that contaminate human milk also caused the DHT-mediated activation of luciferase activity in PC-3 LUC AR+ cells to be reduced by more than 50%.

Interaction between tris(4-chlorophenyl)methanol and the human androgen receptor in vitro

The interaction between tris(4-chlorophenyl)methanol (TCPM), an environmental contaminant of uncertain origin, and the human androgen receptor was examined in vitro using a previously characterized human androgen receptor reporter gene assay in which PC-3 LUC AR+ cells respond to 5α-dihydrotestosterone (DHT) with enhanced luciferase activity. In two preliminary studies, TCPM (1 μM) had no effect on androgen receptor activation in the absence of DHT (50 pM), but DHT-mediated activation of the androgen receptor was decreased more than 90% by TCPM at 1 μM. This was not due to an effect of TCPM on cell viability since, with TCPM concentrations of 0.1 and 1.0 μM, cell viability was 111 and 102%, respectively, but at 10 μM TCPM, cell viability was reduced to 60% compared with controls. For kinetic studies of the effects of TCPM on androgen receptor activation, DHT was added at 0, 5, 25, 50, 75, 100, 250 and 500 pM and the TCPM concentrations used were 0, 1, 10, 100, 300 and 1000 nM. This range incorporated the levels of TCPM in human adipose tissue, where concentrations up to 55 nM (20 ng/g lipid) have been measured. TCPM increased the value of the K m(app) of the androgen receptor for DHT 4–6-fold with little or no effect on the V max(app). Slope and intercept replots revealed that TCPM inhibited the DHT-mediated activation of the androgen receptor in a competitive manner with K i(app) values of 0.26, 0.26 and 0.20 μM compared with respective K m(app) values of 9.8, 3.1 and 3.9 pM. Thus, TCPM is a competitive antagonist of human androgen receptor activation and the value of the K i(app) indicates that TCPM would be approximately 10–50 times more potent as an inhibitor than the previously identified androgen receptor antagonists p, p′-DDE and vinclozilin and also, the K i(app) values (200–260 nM) are very close to the concentration of TCPM found in the adipose tissue of some humans (55 nM).