Saccharomyces cerevisiae BLYAS, a New Bioluminescent Bioreporter for Detection of Androgenic Compounds

A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 ± 4.6) × 10⁻⁹ M, respectively. The lower limit of detection in response to DHT was 2.5 × 10⁻⁹ M, and in response to testosterone it was 2.5 × 10⁻¹⁰ M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment.     

Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17beta-estradiol over the concentration range of 1.2 x 10(-8) through 5.6 x 10(-12) M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 +/- 1.1) x 10(-10) and (2.4 +/- 1.0) x 10(-10) M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10(-11) to 2.8 x 10(-9) M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.     

Identification of the major oxidative 3alpha-hydroxysteroid dehydrogenase in human prostate that converts 5alpha-androstane-3alpha,17beta-diol to 5alpha-dihydrotestosterone: a potential therapeutic target for androgen-dependent disease

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.     

Use of Saccharomyces cerevisiae BLYES Expressing Bacterial Bioluminescence for Rapid, Sensitive Detection of Estrogenic Compounds

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17β-estradiol over the concentration range of 1.2 × 10⁻⁸ through 5.6 × 10⁻¹² M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) × 10⁻¹⁰ and (2.4 ± 1.0) × 10⁻¹⁰ M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10⁻¹¹ to 2.8 × 10⁻⁹ M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.     

The non-aromatizable androgen, dihydrotestosterone, induces antiestrogenic responses in the rainbow trout

In order to satisfy government mandates, numerous studies have been performed categorizing potential endocrine disrupting chemicals as (anti)estrogens or (anti)androgens. We report here that dihydrotestosterone (DHT), a potent, non-aromatizable androgen receptor agonist, induces antiestrogenic responses through direct and/or indirect modulation of vitellogenin (Vg), steroid hormone and total cytochrome P450 levels. DHT and two weak, aromatizable androgens, DHEA and androstenedione (0.05-50 mg/kg per day), were fed to juvenile trout for 2 weeks. DHEA and androstenedione significantly increased blood plasma Vg by up to 30- and 45-fold, respectively (P<0.05, t-test). 17beta-Estradiol (E2) increases were also observed with both androgens, albeit with lower sensitivity. DHT markedly decreased Vg and E2 levels, suggesting that DHEA and androstenedione increased Vg and E2 via conversion to E2 and not by estrogen receptor agonism. DHEA and androstenedione had no effect on total cytochrome P450 content, while DHT significantly decreased P450 content in a dose dependent fashion. These results indicate that alterations in metabolism mediated by androgen receptor binding may be responsible for the Vg and E2 decreases by DHT. In an attempt to decipher between receptor and non-receptor androgenic mechanisms of the observed DHT effects, DHT (0, 50 or 100 mg/kg per day) and flutamide (0-1250 mg/kg per day), an androgen receptor antagonist, were fed to juvenile rainbow trout for 2 weeks. Flutamide alone was as effective as DHT in decreasing E2 and Vg levels in males but did not significantly reverse DHT induced Vg decreases in either sex (P>0.05, F-test). DHT decreases in total P450 content were partially attenuated in males by flutamide co-treatment, but not females, suggesting a partial androgenic mechanism to the P450 decreases as well as a fundamental sex difference responding to androgen receptor binding. Moreover, flutamide alone decreased P450 content by up to 30% in males and 40% in females. These effects may be mediated through direct androgen receptor binding irrespective of whether the binding is agonistic or antagonistic. This study indicates that androgen receptor agonists/antagonists can elicit significant antiestrogenic effects that may not necessarily be mediated through classic receptor binding mechanisms and signal transduction pathways.     

7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens

CNNT. There was a good correlation between bioactivity and binding affinity to AR for the 7alpha-substituted androgens compared to T. In contrast, relative to their binding affinity to AR, the androgenic potency of DHT and 19-NT was lower compared to T. The reason for the lower in vivo androgenic activity of 19-NT is attributable to its enzymatic conversion to 5alpha-reduced-19-NT in the prostate. In the case of DHT, the lower bioactivity could be attributed to its faster metabolic clearance rate relative to T. The correlation was further investigated in vitro by co-transfection of rat ARcDNA expression plasmid and a reporter plasmid encoding the chloramphenicol acetyl transferase (CAT) gene driven by an androgen inducible promoter into CV-1 cells. All the androgens led to a dose-dependent increase in the CAT activity. MENT was found to be the most potent followed by DHT, 19-NT, T, and CNNT. The specificity of the androgenic response was confirmed by its inhibition with hydroxyflutamide, an antiandrogen. Thus, there was a good correlation between binding affinity and in vitro bioactivity in the transient transfection assay for the androgens. This suggests that the in vivo bioactivity of androgens could be influenced not only by binding affinity to receptors but also by factors such as absorption, binding to serum proteins and metabolism. However, the high potency of MENT is primarily related to its higher affinity to AR.     

Stimulation of aromatase activity by dihydrotestosterone in human skin fibroblasts

In order to study the regulation of aromatase activity by androgens in cultured fibroblasts derived from genital skin of normal prepubertal boys, aromatase activity was evaluated in the presence of various concentrations of non-aromatizable androgen DHT(5 alpha-dihydrotestosterone). The estrogen formation was assayed by an enzymatic method, after 24 h incubation of the cells with 10(-6) M androstenedione. Aromatase activity was stimulated 3- to 20-fold by DHT at concentrations 10(-10) and 10(-9) M. It was necessary to preincubate the cells with DHT for 48 h in order to bring about this stimulation. The stimulatory effect was not significant after preincubation for only 24 h. The basal value of aromatase activity was in the range of 8 +/- 1.2 pmol/mg protein/day (mean +/- SEM), while the maximal stimulation 1043 +/- 46 pmol/mg protein/day was obtained at the concentration of 10(-8) M DHT. This stimulation was partially blocked with cyproterone acetate at level of 20 +/- 4 pmol/mg protein/day; stimulation of aromatase activity by DHT could thus be mediated by the androgen receptor. This stimulatory effect was prevented by incubation of the cells with cycloheximide or actinomycin D, suggesting that DHT acts to increase aromatase activity in cultured fibroblasts by inducing the synthesis of new proteinaceous material. In vitro regulation of aromatase activity by androgens could contribute to a new approach to the extraglandular formation of estrogen.     

Self-administration of estrogen and dihydrotestosterone in male hamsters

Anabolic-androgenic steroids (AAS) are drugs of abuse. Previous studies have shown that male and female hamsters self-administer testosterone (T) and other AAS, suggesting that androgens are reinforcing in a context where athletic performance is irrelevant. AAS are synthetic derivatives of T, which may be aromatizable to estrogen and/or reducible to dihydrotestosterone (DHT). However, we do not know which metabolites of T are reinforcing. To determine if DHT, estradiol (E(2)), or DHT + E(2) are reinforcing, we tested intracerebroventricular (icv) self-administration in male hamsters. The hypothesis was that androgen reinforcement is sensitive to both androgenic and estrogenic T metabolites. If so, hamsters would self-administer DHT, E(2), and DHT + E(2). Twenty four castrated male hamsters (n = 8/group) received icv cannulas and sc T implants for physiologic androgen replacement. One week later, hamsters self-administered DHT (0.1, 1.0, 2.0 microg/microl), E(2) (0.001, 0.01, 0.02 microg/microl), or DHT + E(2), each for 8 days in increasing concentration (4 h/day). Operant chambers were equipped with an active and inactive nose-poke. At the medium concentration, hamsters self-administered DHT (active nose-poke: 47.9 +/- 13.9 responses/4 h vs. inactive: 18.7 +/- 4.8), E(2) (active: 44.8 +/- 14.9 vs. inactive: 16.6 +/- 2.6), and DHT + E(2) (active: 19.1 +/- 2.4 vs. inactive: 10.4 +/- 2.4, P < 0.05). At the highest concentration, males self-administered DHT (active: 28.3 +/- 7.7 vs. inactive: 15.0 +/- 3.5, P < 0.05) and DHT + E(2) (active: 22.6 +/- 3.8 vs. inactive: 11.6 +/- 2.5, P < 0.05), but not E(2). Hamsters did not self-administer the lowest concentrations of DHT, E(2), or DHT + E(2). These results support our hypothesis that both androgenic and estrogenic T metabolites are reinforcing. Together, they do not exert synergistic effects.     

Antiproliferative actions of the synthetic androgen, mibolerone, in breast cancer cells are mediated by both androgen and progesterone receptors

Androgen signaling, mediated by the androgen receptor (AR), is a critical factor influencing growth of normal and malignant breast cells. Given the increasing use of exogenous androgens in women, a better understanding of androgen action in the breast is essential. This study compared the effects of 5alpha-dihydrotestosterone (DHT) and a synthetic androgen, mibolerone, on estradiol (E(2))-induced proliferation of breast cancer cells. DHT modestly inhibited E(2)-induced proliferation and mibolerone significantly inhibited proliferation in T-47D cells. The effects of both androgens could be reversed by an AR antagonist, suggesting that their actions were mediated, in part, by AR. Whereas high physiological doses (10-100nM) of DHT reduced E(2)-mediated induction of the estrogen-regulated gene progesterone receptor (PR) to basal levels, mibolerone at lower doses (1nM) eliminated PR expression, suggesting that mibolerone may also act via the PR. In the AR positive, PR-negative MCF-7 cells, mibolerone had modest effects on E(2)-induced proliferation, but was a potent inhibitor of proliferation in the AR positive, PR positive MCF-7M11 PRA cells. The effects of mibolerone in breast cancer cells were similar to those of the progestin, medroxyprogesterone acetate. Our results demonstrate that mibolerone can have both androgenic and progestagenic actions in breast cancer cells.     

Androgen concentration and partial characterization of 5alpha reductase in the epididymis of the rhesus monkey.

The 5alpha reductase activity ofthe monkey epididymis was studied. The enzyme was found in particulate subcellular fractions, its distribution closely resembling that of the microsomal marker enzyme NADPH: cytochrome c reductase, suggesting an association of 5alpha reductase with membranes of the endoplasmic reticulum. Maximal enzyme activity was found at pH 5.4 and at 32--37 C. The crude nuclear preparation had a Km: 0.315 x 10(-6)M and Vmax: 168 pmoles/mg protein/h. The microsomal enzyme had a Km: 0.243 x 10(-6)M and Vmax: 828 pmoles/mg protein/h. Neither enzyme preparation was affected by addition to the incubation media of dihydrotestosterone (DHT) or 5alpha-androstane-3alpha,17beta-diol. The endogenous androgen concentration in the epididymides of 2 different monkeys, in ng/g wet weight was: DHT 20.81 +/- 1.98; T: 9.0L +/- 2.83; diol: 3.03 +/- 0.41.     

Androgen binding as evidenced by a whole cell assay system using cultured canine prostatic epithelial cells

The androgen receptor content in the prostate has been usually evaluated using subcellular fractions without taking into account cellular and functional heterogeneity of the gland. Using enriched populations of immature canine prostatic epithelial cells cultured in primary monolayers, a whole cell assay system was developed to measure androgen receptors. Tritiated dihydrotestosterone (DHT) and/or methyltrienolone (R1881) in serum-free medium were used as ligands and Triamcinolone acetonide (0.5 microM) was added to prevent the binding of R1881 to other types of receptors. The amount of radiolabelled ligand specifically bound to the cells was determined at equilibrium. Specific binding was proportional to the number of cells seeded. Scatchard analysis revealed the presence of at least two types of binding sites. The Kd for the high affinity binding site was 2 x 10(-9) M. Competition studies indicated that this component was specific for androgens; Methyltrienolone, Mibolerone and the antiandrogen RU 23908 were the most efficient competitors. They were followed by DHT, 5 alpha-androstane-3 alpha, 17 beta-diol, testosterone, estradiol and estrone. Progesterone, 5 alpha-androstane-3 beta, 17 beta-diol and epitestosterone were not inhibitors. The level of specific binding was 11.0 +/- 7.6 fmol of bound R1881 per 10(6) cells (n = 34) or 2075 +/- 1434 fmol per mg of DNA; these values correspond to an average of 6624 +/- 4577 sites per cell. Thus, using this whole cell assay system, specific and androgen receptors were detected in immature prostatic epithelial cells in culture. This assay will therefore be useful to study the interrelationship between androgen binding activity and specific cell functions.     

Studies on steroid hormone refeptors (5 α=dihidrotestosterone,estradiol, and dexamethasone) in cultured human fibroblasts and amniotic fluid cells

Summary Cultured human fibroblasts and amniotic fluid cells (AF cells) were examined for the presence of steroid hormone receptors. In both cell types, the androgen (DHT) or glucocorticoid (dexamethasone) receptor was detected, but not the estrogen receptor. Binding parameters in fibroblasts were: for androgen: K_D=3.7×10^-9M, B_max=13 fmol/mg; for dexamethasone: K_D=4.5×10^-9M, B_max=120 fmol/mg. Binding parameters in AF cells were: for androgen: K_D=4×10^-9M, B_max=8 fmol/mg; for dexamethasone: K_D=1.9×10^-8M, B_max=375 fmol/mg. Cultured cells derived from the gonads (of a patient with 17-ketosteroid reductase deficiency) seem to have more receptors than cells from extragenital body parts (B_max=21 fmol). With the aid of gel chromatography, the molecular weight of the androgen receptor was estimated to be 30–40 000D.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105, 000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1, 2-³h]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M ? 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1, 2-α-methylene-6-chloro-pregn-4, 6-diene-17α-o1–3, 20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

MCF-7; a human breast cancer cell line with estrogen, androgen, progesterone, and glucocorticoid receptors.

We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.     

Androgen receptors in rat testis

Testosterone (T) and 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; DHT) are bound to specific cytoplasmic receptors (CR) in 105,000 × g supernatant fractions of seminiferous tubules from hypophysectomized rats following the intravenous injection of [1,2-³H]testosterone. CR is clearly different from the testicular androgen binding protein (ABP) by electrophoretic mobility, temperature stability and rate of dissociation of steroid-CR complex, but very similar to the cytoplasmic receptors of epididymis and ventral prostate. Under these labeling conditions, the nuclei of seminiferous tubules also contain radioactive T and DHT bound to protein. These androgen-protein complexes, which can be extracted with 0.4 M — 1 M KC1, have a sedimentation coefficient of 3–4 S. Binding of radioactive T and DHT to both cytoplasmic and nuclear receptors $\text{in vivo}$ is specific for androgen target tissues and abolished by simultaneous injection of unlabeled T, DHT and cyproterone acetate (1,2-α-methylene-6-chloro-pregn-4, 6-diene-17α-ol-3,20-diene-17-acetate), but not by cortisol. It is suggested that receptors for testosterone and DHT in the seminiferous tubules are involved in the mediation of the androgenic stimulus to the germ cells.     

Characterisation of gene expression patterns in 22RV1 cells for determination of environmental androgenic/antiandrogenic compounds

Alteration of androgen receptor function due to hormonally active compounds in the environment, may be responsible for impaired reproductive function in aquatic wildlife. Based on human prostate carcinoma 22RV1 cells, a cell culture expression system was established to test effects of putative androgenic/antiandrogenic compounds on endogenous gene expression. 22RV1 cells were shown to express human androgen receptor, but not human progestin (hPR) or human oestrogen receptor (hER) alpha and beta. Six androgen-regulated genes (ARGs) were chosen to determine androgenic/antiandrogenic action using highly sensitive real-time RT-PCR. Results showed that gene expression is altered in a time-dependent manner. After stimulation of cells by DHT (10nM), synthetic androgen R1881 (1 nM), or organic pesticides (difenoconazole, fentinacetate, tetramethrin) TMPRSS2 mRNA expression was down-regulated by the factor 0.6 after 24h of DHT treatment. Similar results were obtained when cells were assayed for mRNA expression of PSA after fentinacetate and R1881 stimulation. In contrast, TMPRSS2 expression was up-regulated by the factor 0.9 when cells were stimulated by tetramethrin. Final goal of the work is a sensitive determination of differential gene expression by different compounds under study, achievement of substance-specific expression patterns and function related analysis of potential androgens/antiandrogens.