Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals.

We evaluated and compared four in vitro assays to detect androgen agonists and antagonists in an international interlaboratory study. Laboratory 1 used a cell proliferation assay (assay 1) with human mammary carcinoma cells stably transfected with human androgen receptor. The other laboratories used reporter gene assays, two based on stably transfected human prostate carcinoma cells (assay 2) or human mammary carcinoma cells (assay 4), and the third based on transient transfection of Chinese hamster ovary cells (assay 3). Four laboratories received four coded compounds and two controls: two steroidal androgens, two antiandrogens, an androgenic control, 5alpha-dihydrotestosterone (DHT), and an antiandrogenic control, bicalutamide (ICI 176,334). All laboratories correctly detected the androgenic activity of 4-androsten-3,17-dione and 17alpha-methyltestosterone. For both compounds, the calculated androgenic potencies relative to the positive control (RAPs) remained within one order of magnitude. However, laboratory 3 calculated a 50-fold higher RAP for 4-androsten-3,17-dione. All assays detected and quantified the antiandrogenic effect of vinclozolin [median inhibitory concentration (IC50) values ranging from 1.1 times symbol 10(-7) M to 4.7 times symbol 10(-7) M]. In assays 2 and 3, vinclozolin showed partial androgenic activity at the highest concentrations tested. For vinclozolin, calculated antiandrogenic potencies relative to bicalutamide (RAAPs) differed no more than a factor of 10, and IC50 values matched those of bicalutamide. Similarly, we found antiandrogenic activity for tris-(4-chlorophenyl)methanol. RAAP values were between 0.086 and 0.37. Three assays showed cytotoxicity for this compound at or above 1 times symbol 10(-5) M. In summary, all assays proved sensitive screening tools to detect and quantify androgen receptor-mediated androgenic and antiandrogenic effects of these chemicals accurately, with coefficients of variation between 8 and 90%.

Effect of organochlorine pesticides on human androgen receptor activation in vitro.

Many persistent organochlorine pesticides (OCs) have been implicated in adverse effects, that is, reproductive and developmental effects, in man and in wildlife alike. It has been hypothesized that these so-called xeno-hormones could be responsible for the increased incidence in various male sexual differentiation disorders such as hypospadias, cryptorchidism, low sperm counts and quality. In this report, OCs, called endocrine disrupters, were tested for their interaction with the androgen receptor. The stable prostatic cell line PALM, which contains a human androgen receptor (hAR) expression vector and the reporter MMTV-luciferase, was used to characterize the response of hAR to OC and was compared with synthetic androgen compound R1881. We found that all the OC pesticides tested were able to shift the agonist [(3)H]-R1881 from its binding site to the AR in competitive binding assays. In addition, these compounds antagonize-in a dose-dependent manner-the AR-mediated transcription by synthetic AR ligand R1881. None of the pesticides reacted as agonists. These results demonstrate that OC endocrine activities in vivo probably result from direct and specific binding to the AR ligand-binding domain. Although the antagonistic potential of OC pesticides is lower than that of hydroxyflutamide, they are capable of disrupting the male hormone signaling pathway. Because these chemicals are extremely persistent and tend to bioaccumulate, these results support the hypothesis that the recent increase in the incidence of male sexual disorders could be due to long exposure to ubiquitous OC pesticides found in the environment.

Mutation of the androgen receptor at amino acid 708 (Gly-->Ala) abolishes partial agonist activity of steroidal antiandrogens.

Mutation of a single amino acid in the ligand-binding domain (LBD) of the human androgen receptor (hAR) can induce functional abnormalities in androgen binding, stabilization of active conformation, or interaction with coactivators. The Gly708Ala and Gly708Val substitutions are associated with partial and complete androgen insensitivity syndromes, respectively. In this work, we introduced Ala, Val, and aromatic Phe mutations at position 708 on helix H3 of the hAR-LBD and tested the functional and structural consequences on hAR activity in the presence of steroidal or nonsteroidal agonists and antagonists. The residues involved in the specific recognition of these androgen ligands were identified and analyzed in the light of in vitro biological experiments and the 3D hAR-LBD structure. Our study demonstrated that the Gly708Ala mutation influenced the agonist versus antagonist activity of the ligands and confirmed the crucial role of this residue within the ligand-binding pocket (LBP) in the modulation of androgen agonists. The Gly708Ala mutation transformed the antiandrogen cyproterone acetate (CPA), a partial agonist, into a pure antiandrogen, and the pure nonsteroidal antiandrogen hydroxyflutamide in a partial agonist. From the docking studies, we suggest that CPA acts on AR through the novel mechanism called "passive antagonism".

Pathophysiology of androgen insensitivity syndromes: molecular and structural approaches of natural and engineered androgen receptor mutations at amino acid 743.

To decipher the clues of genotype-phenotype mapping in androgen insensitivity syndromes (AIS), we integrated clinical, molecular, and structural data in an investigation into the characteristics of androgen receptor (AR) ligand binding and activation. We looked for residues substituted in AIS that are conserved among the different AR species but that diverge in the other steroid receptors, thus suggesting a role in androgen binding specificity. Of the residues fitting these characteristics, we focused on the glycine at position 743, for which natural substitutions (glutamic acid and valine) have been associated with different androgen resistance phenotypes. The consequences of both substitutions were evaluated along with the minimal glycine to alanine mutation. The gradual impairment of binding and trans-activation efficiencies in AR mutants ranging from alanine to valine and subsequently glutamic acid were highlighted by in vitro experiments. Structural analyses showed the consequences of these substitutions at the atomic level and suggested a difference in local organization among the nuclear receptor superfamily. Overall, this integrative approach provides a useful tool for further investigations into the AR structure-function relationship in AIS.

Mechanism of antiandrogen action: key role of hsp90 in conformational change and transcriptional activity of the androgen receptor.

Activation of the androgen receptor (AR) is induced by ligand binding through conformational changes leading to control of gene expression. Antiandrogens compete with androgens for AR occupancy and subsequently block at least one step in AR action. Analysis of nuclear transfer kinetics using the GFP-AR fusion protein and partial proteolysis analysis provided evidence that the ligand-bound receptor was in equilibrium between at least two distinct conformations, leading to the production of 35 and 29 kDa trypsin-resistant fragments. It also indicated that this equilibrium may regulate the rate of nuclear transfer. The slowing of nuclear transfer by antiandrogens was correlated with the amount of receptor in conformation leading to the 35 kDa trypsin-resistant fragment. To establish the role of heat shock protein (hsp) 90 activity in antiandrogenic action, the effect of geldanamycin (GA) was evaluated in both in vitro assays and live cells. We demonstrated that in vitro hsp90s are required to stabilize the receptor in the inactive conformation and that hsp90 activity is involved in the integrity and nuclear transfer of agonist- and antagonist-bound AR. Furthermore, nuclear transfer is not the only step affected by GA since this compound was also active on a constitutively nuclear AR (GFP-NLS-AR). Hsp90 inactivation impedes interaction of androgen-bound GFP-NLS-AR with nuclear components and inhibits transcriptional activity. We conclude that hsp90s are required for the acquisition of active conformation in agonist-bound AR to regulate nuclear transfer, nuclear matrix binding, and transcriptional activity. Pure antiandrogens block the transconformational change of AR in an intermediary complex unable to acquire the active conformation and to dissociate the hsp90.

Phenylphenols, biphenols, bisphenol-A and 4-tert-octylphenol exhibit alpha and beta estrogen activities and antiandrogen activity in reporter cell lines.

We previously demonstrated the interactions of different chemical compounds with estrogen receptors ERalpha and ERbeta and the androgen receptor (AR) using different reporter cell lines. In this study, we characterize the ERalpha, ERbeta and AR activity of different biphenyls using the same tools. We provide evidence that several phenyl derivatives present both estrogenic and antiandrogenic activity. The extent of hydroxylation and the position of the hydroxyl function were important in determining their estrogenicity and antiandrogenicity. Of the tested compounds, bisphenol-A and 4,4' biphenol had very high estrogenic activity, although it was lower than that of the strong estrogenic alkylphenol, 4-tert-octylphenol. Bisphenol-A and 4,4' biphenol were able to activate ERs at concentrations lower than 1 microM, whereas the other compounds only activated at concentrations above 1 microM. Interestingly, 4,4' biphenol was a better agonist for ERbeta than for ERalpha. No androgenic activity was detected for any of these compounds. Bisphenol-A, 3-OH phenylphenol, 4-OH phenylphenol and 4,4' biphenol exhibited antiandrogenic activity close to that of 4-tert-octylphenol (IC(50) approximately 5 microM). In whole cell binding assays, these compounds displaced [3H] R1881 with Ki = 10 microM. Although these Ki values seem high in comparison with that of hydroxyflutamide (0.4 microM), one must keep in mind that environmental chemicals can accumulate in adipose tissues for several years. In conclusion, these environmental chemicals may have a negative impact on androgen action during fetal and post-natal life.

A new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity in children.

The evaluation of estrogenic status is necessary for many physiological and pathological conditions in pediatric as well as adult endocrinology. Because current immunoassays exclusively measure E2--and with a sensitivity that is insufficient for prepubertal children--we developed a new recombinant cell bioassay for ultrasensitive determination of serum estrogenic bioactivity. This assay is based on human uterine cervix carcinoma cells, HeLa cells, that do not naturally express E2 receptor. These cells were transfected with plasmids encoding the human ERalpha or beta, along with an estrogen-responsive promoter fused to the luciferase gene, and called HELNalpha and HELNbeta for HeLa estrogen-responsive element luciferase neomycin alpha and beta. HELNalpha and HELNbeta are able to respond to estrogens and various compounds having estrogenic activity but, because of the importance of ERalpha in the reproductive function, we chose to work with the HELNalpha cell line. The luciferase activity we obtained was compared with an E2 standard curve specific for each serum sample and established with stripped serum. The estrogenic bioactivity was expressed in picograms of E2 equivalents, and the detection limit was < 1 pg x ml(-1) E2 equivalents. The intra and interassay error was lower than 10% and 20%, respectively. We measured estrogenic bioactivity in 18 normal prepubertal boys (age = 9.7 +/- 2.4 yr), 18 normal prepubertal girls (age = 9.2 +/- 1.7 yr) and 18 normal pubertal girls (age = 13.6 +/- 1.8 yr). The estrogenic bioactivity in the prepubertal girls was significantly higher than in the boys, i.e. 3.53 +/- 2.23 pg x ml(-1) vs. 1.44 +/- 0.87 pg x ml(-1) (P < 0.01). A significant difference was found between the pre- and pubertal girls, i.e. 3.53 +/- 2.23 pg x ml(-1) vs. 26.77 +/- 18.32 pg x ml(-1) (P < 0.01). This ultrasensitive bioassay measures total estrogenic bioactivity of serum with very high sensitivity. It has numerous potential applications in pediatric and adult endocrinology. In addition, this assay may help to evaluate excess estrogenic activity related to aromatase overexpression or contamination by environmental chemicals.

Potential action of IGF-1 and EGF on androgen receptor nuclear transfer and transactivation in normal and cancer human prostate cell lines.

This work was designed to determine whether IGF-1 and EGF modulate nuclear transfer and transactivation of the androgen receptor (AR) in human prostate cell lines (PNT1A and DU-145). We first characterized the IGF-1 and EGF receptors by ligand-binding assays with [125I] IGF-1 and [125I] EGF in a normal human prostate epithelial cell line, PNT1A. We then evaluated the effects of these growth factors on AR nuclear transfer and transcriptional activation in this cell line and in DU-145, a human prostate tumor cell line. The cell lines were cotransfected with an AR expression vector and an androgen-responsive luciferase gene driven by the mouse mammary tumor virus (MMTV-luciferase) promoter. Neither IGF-1 nor EGF could activate reporter gene in the absence of androgens. Conversely, both enhanced the magnitude of the AR response in the presence of low levels of androgen (10(-11)-10(-9) M) and this response, increased by twofold, was inhibited by hydroxyflutamide. No effect of IGF-1 and EGF was observed on the intracellular localization of the fusion protein EGFP-AR in either cell line. The fluorescence stayed cytoplasmic even after 24 h of IGF-1 or EGF treatment. Taken together, these data indicate that growth factors are unable to initiate the nuclear translocation of AR in the absence of androgens or to induce ligand-independent transcriptional activity. We observed only cross-talk in the presence of androgens and IGF-1 or EGF, leading to an over-activated AR. In conclusion, the cross-talk between AR and growth factor signaling pathways may sensitize AR to suboptimal stimulation by low levels of androgens.

Evaluation of androgenic bioactivity in human serum by recombinant cell line: preliminary results.

Besides the measurement of circulating conjugated metabolites of dihydrotestosterone (DHT), which reflects androgenic activity, only one assay to measure androgenic bioactivity in human serum has been proposed thus far. This recombinant bioassay is based on the androgen-dependent interaction between the LBD and NT domains of AR fused to the Gal 4 DNA-binding domain, but its construction is highly complex. We have developed a mammalian cell (CHO 515) bioassay that measures total androgen bioactivity in human serum. The AR-deficient Chinese hamster ovary (CHO) cells were stably transfected with pSG5-puro-hAR and pMMTV-neo-Luc. After selection with puromycin and neomycin, five highly inducible clones were isolated and one was selected. The expression of human androgen receptor (hAR) was confirmed by Western blot and steroid-binding assays on the whole cells. The transcriptional activity of the clone was measured after 24 h of incubation with increasing concentrations of various androgenic and non-androgenic steroid compounds in a 96-well plate. The EC50s for each tested androgenic steroid compound were 4 x 10(-11) M, 1.5 x 10(-11) M, 1 x 10(-9) M, 2 x 10(-10) M, 3 x 10(-10) M for testosterone, DHT, dehydroepiandrosterone (DHEA), delta5-androstenediol, and delta4-androstenedione, respectively. In the physiological concentrations of the non-androgenic steroids, estradiol, cortisol, aldosterone, and progesterone, no interference was noted with the AR transactivation level. Evaluation of androgenic bioactivity in human serum was performed by incubation of CHO 515 cells with 100 microl of patient serum, diluted at 1/100 = 1% in DMEM-F12 without phenol red. The sensitivity of the assay was < 0.3 ng/ml. The mean androgenic bioactivity expressed in testosterone equivalents was 0.6 +/- 0.2 ng/ml in normal prepubertal boys, and 12.4 +/- 2 and 1.7 +/- 0.1 ng/ml in normal pubertal boys and girls, respectively. In conclusion, this new recombinant cell bioassay is today the only assay that takes into account testosterone, DHT, DHEA, delta5-androstenediol, and delta4-androstenedione. It should be of particular use in male children with cryptorchidism, delayed puberty or hypogonadotrophic hypogonadism, i.e., in pediatric patients with low androgen levels.

Evidence that chlormadinone acetate exhibits antiandrogenic activity in androgen-dependent cell line.

Chlormadinone acetate (CMA), like other 17-hydroxyprogesterone derivatives, is thought to be a potential antiandrogen on the basis of its effect on spontaneous benign prostatic hyperplasia (BPH) in dogs. This work was undertaken to find out whether CMA presents antiandrogen activity in human androgen-dependent cell line. For this purpose, we used PALM cells, the PC-3 cell line stably transfected with human androgen receptor and a luciferase gene under transcriptional control of MMTV. Potential antiandrogenic activity was compared with that of cyproterone acetate (CPA), a standard steroidal antiandrogen. Both compounds were tested in competitive binding assays at 37 degrees C in the presence of 1 nM of [3H] R1881, a synthetic and non-metabolizable androgen. Their impact on AR transcriptional activity was evaluated by the measure of luciferase activity in the presence of R1881 with increasing concentrations of CMA or CPA (10(-8)-10(-6) M). In whole cell binding assays, competitive studies revealed that the Ki for CMA was 3.3 +/- 1.5 x 10(-8) M (versus 7.2 +/- 1.3 x 10(-8) M for CPA). Inhibition of AR transcriptional activity was 40 +/- 5% for CMA (3 x 10(-7) M) versus 59 +/- 6% for CPA at the same concentration. Moreover, CMA caused a slower import of green fluorescent protein (GFP)-AR to the nuclei of COS-7 cells than R1881. These data show that CMA exerted a competitive binding for AR and significantly decreased the AR transcriptional activity. In conclusion, this synthetic progestin presents simultaneous antiandrogenic activity that could be helpful as a new therapeutic option in women with luteal defect along with clinical signs of hyperandrogenism.