22 research outputs found
Androgen Receptor Characteristics in Skin Fibroblasts from Hirsute Women
Hormonal measurements in some women with hirsutism often reveal little or no elevation in androgen levels to explain the disorder. Thus, it has been postulated that increased sensitivity of the hair follicle to androgen may contribute to the development of hirsutism in such patients. We, therefore, sought androgen receptor abnormalities in skin fibroblasts cultured from 10 hirsute women (ages 17ā43) and normal or mildly elevated plasma testosterone levels (28ā82 ng/dl). Androgen receptor content (Ro) and binding affinity (Kd) in cultured pubic skin fibroblasts were measured using a dispersed, whole cell assay. Ten such cell lines from these women were compared with 19 pubic skin cell lines from 9 normal volunteers (6 males and 3 females) and from 10 other subjects (males with gynecomastia or hypospadias). There was no statistically significant difference in the mean androgen receptor content (11,600 Ā± 2700 (SE) sites/cell fibroblasts vs 7900 Ā± 700 sites/cell or binding affinity (2.0 Ā± 0.3 (SE) Ć 10[sup-9] M vs 1.5 Ā± 0.2 Ć 10-9 M, respectively) between the patients' fibroblasts and those of the controls.We conclude that hirsutism cannot be explained by abnormalities in fibroblast androgen receptor number or affinity. These observations do not exclude the possibility that other mechanisms might lead to increased peripheral androgen sensitivity in such patients
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%
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A dispersed-whole cell method for the determination of androgen receptors in human skin fibroblasts
A method is described for the determination of binding capacity (R
o) and dissociation constant (K
d) of androgen receptors in dispersed, whole, cultured human skin fibroblasts. The cells, obtained from punch biopsies or operative specimens of skin, are grown to confluence in monolayers, harvested, washed, and dispersed in medium. Binding is assessed by incubating the cells with [
3H]dihydrotestosterone
3
3
The following trivial names were used: dihydrotestosterone (17Ī²-hydroxy-5Ī±-androstan-3-one), methyltrienolone (R1881, 17Ī²-hydroxy-17-methyl-estra-4,9,11-trien-3-one), R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R2956 (17Ī²-hydroxy-2,2,17-trimethylestra-4,9, 11-trien-3-one), androstanediol (5Ī±-androstane-3Ī±,17Ī²-diol), cyproterone acetate (6-chloro-17 acetoxy-1, 2Ī±-methylene pregna-4,6-diene-3,20-diene), and dexamethasone (9-fluoro-11Ī², 17,21-trihydroxy-16Ī±-methylpregna-1,4-diene-3, 20-dione).
(DHT) at 22Ā°C for one hour followed by washing, centrifugation and counting. Non-specific binding is determined by adding radioinert methyltrienolone (R1881). Scatchard plots are constructed using 6 concentration points; binding is expressed as sites/cell. The method is precise (R
o = 12,105 Ā± 4305 (S.D.) sites/cell; K
d = 1.0 Ā± 0.7 (S.D.) Ć 10
ā9M, n = 14 for one prepuce cell line) and independent of cell passage number. Binding is linear with respect to cell number and shows those characteristics commonly attributed to androgen receptors: high affinity, low binding capacity, saturable nuclear binding, androgen specificity, and an 8S peak in fibroblast cytosol using sucrose density gradients. Cell lines shown by other published methods to lack androgen receptors had no detectable DHT binding with this method. This assay technique has the advantages of simplicity, rapidity, and precision