Targeting the stromal androgen receptor in primary prostate tumors at earlier stages.

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates.

A test of rats' tolerance for 3beta-acetoxyandrosta-1,5-dien-17-one ethylene ketal (ADEK), a new anti-androgen.

Following the demonstration that the androgen activity of androsta-5-ene-3beta,17beta-diol (Adiol) is not inhibited by the anti-androgens currently used to treat prostate cancer, we sought agents that would inhibit the androgenic function of Adiol as well as of dihydrotestosterone. The steroid 3beta-acetoxyandrosta-1,5-dien-17-one ethylene ketal (ADEK) met this criterion. Its tolerance was assessed in rats by oral and by subcutaneous administration for four weeks.

Oligozoospermia with normal fertility in male mice lacking the androgen receptor in testis peritubular myoid cells.

Androgens and the androgen receptor (AR) play important roles in the testes. Previously we have shown that male total AR knockout (T-AR-/y) mice revealed incomplete germ cell development and lowered serum testosterone levels, which resulted in azoospermia and infertility. However, the consequences of AR loss in particular types of testicular cells remain unclear.

Redox reactions of dehydroepiandrosterone and its metabolites in differentiating 3T3-L1 adipocytes: A liquid chromatographic-mass spectrometric study.

Dehydroepiandrosterone is known to depress fatty acid formation in differentiating 3T3-L1 adipocytes. The metabolism of dehydroepiandrosterone and four of its natural metabolites in differentiating adipocytes was studied by liquid chromatography-mass spectrometry. Adipocytes rapidly converted dehydroepiandrosterone to androst-5-ene-3beta,17beta-diol.

C19-steroids as androgen receptor modulators: design, discovery, and structure-activity relationship of new steroidal androgen receptor antagonists.

Dehydroepiandrosterone (DHEA), the most abundant steroid in human circulating blood, is metabolized to sex hormones and other C19-steroids. Our previous collaborative study demonstrated that androst-5-ene-3beta,17beta-diol (Adiol) and androst-4-ene-3,17-dione (Adione), metabolites of DHEA, can activate androgen receptor (AR) target genes. Adiol is maintained at a high concentration in prostate cancer tissue; even after androgen deprivation therapy and its androgen activity is not inhibited by the antiandrogens currently used to treat prostate cancer patients.

A postulated role for 16 alpha-hydroxydehydroepiandrosterone in the prevention of respiratory distress syndrome.

The concentrations of 16-hydroxylated steroids, especially 16 alpha-hydroxydehydroepiandrosterone (16 alpha-hydroxyDHEA) in amniotic fluid and infants' blood are elevated many fold at normal birth time as compared with mid-term concentrations and those found in prematurely born infants. It is logical to postulate that 16 alpha-hydroxyDHEA may be the natural inducer of lung maturation and preventor of respiratory distress syndrome. Because the infant born at normal gestational terminus has a very high concentration of blood 16 alpha-hydroxyDHEA, treating premature infants with amounts of this steroid to provide blood concentrations that are normal in full-term infants should be a well-tolerated procedure and should avoid the developmental problems associated with glucocorticoid treatments.

C(19)-5-ene steroids in nature.

Dehydroepiandrosterone (DHEA), produced from cholesterol in the adrenals, is the most abundant steroid in our circulation. It is present almost entirely as the sulfate ester, but the free steroid is the form that serves as a precursor of estrogens and androgens, as well as 7- and 16-oxygenated derivatives. Mammalian tissues reduce the 17-keto Group of DHEA to produce androstenediol-a weak estrogen and full-fledged androgen.

Development and validation of a high performance liquid chromatography assay for 17alpha-methyltestosterone in fish feed.

17alpha-Methyltestosterone (MT) is used to manipulate the gender of a variety of fish species. A high performance liquid chromatography (HPLC) internal standard method for the determination of 17alpha-methyltestosterone in fish feed using 3beta-methoxy-17beta-hydroxyandrost-5-en-7-one as internal standard (IS) has been developed. The method has been validated for the quantitation of MT in fish feed using 245 nm UV absorbance as the parent wavelength and 255 nm as a qualifier wavelength.

Identification of steroid derivatives that function as potent antiandrogens.

We have hypothesized that some steroid derivatives bind to the androgen receptor (AR) with very low androgenic activity and therefore potentially function as better AR antagonists than clinically used antiandrogens, such as flutamide. Indeed, we previously found such a compound, 3beta-acetoxyandrosta-1,5-diene-17-one ethylene ketal (ADEK), with some estrogenic activity. Here we report the identification of 2 additional steroid derivatives, 3beta-hydroxyandrosta-5,16-diene (HAD) and androsta-1,4-diene-3,17-dione-17-ethylene ketal (OAK), as new potent antiandrogens.

Infertility with defective spermatogenesis and hypotestosteronemia in male mice lacking the androgen receptor in Sertoli cells.

Androgens and the androgen receptor (AR) play important roles in male fertility, although the detailed mechanisms, particularly how androgen/AR influences spermatogenesis in particular cell types, remain unclear. Using a Cre-Lox conditional knockout strategy, we generated a tissue-specific knockout mouse with the AR gene deleted only in Sertoli cells (S-AR(-/y)). Phenotype analyses show the S-AR(-/y) mice were indistinguishable from WT AR mice (B6 AR(+/y)) with the exception of testes, which were significantly atrophied.

Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex.

Dehydroepiandrosterone (DHEA) is a neurosteroid with potential effects on neurogenesis and neuronal survival in humans. However, most studies on DHEA have been performed in rodents, and there is little direct evidence for biological effects on the human nervous system. Furthermore, the mechanism of its action is unknown.

Inactivation of androgen receptor coregulator ARA55 inhibits androgen receptor activity and agonist effect of antiandrogens in prostate cancer cells.

Antiandrogens given to antagonize androgen receptor (AR) activity gradually lose their efficacy as antagonists and eventually function as agonists to promote (instead of block) AR-mediated growth of prostate cancer cells. The mechanisms of how antiandrogens acquire this agonist activity during hormonal therapy are largely unknown. Here, we report that expression of a dominant-negative AR-associated protein 55 (dARA55) coregulator, inhibits AR transcriptional activity and reduces the agonist activity of antiandrogens.

3 beta-acetoxyandrost-1,5-diene-17-ethylene ketal functions as a potent antiandrogen with marginal agonist activity.

The majority of available antiandrogens have been reported to possess agonist activity to induce prostate-specific antigen, which might result in antiandrogen withdrawal syndrome. Here we report the identification of 3 beta-acetoxyandrost-1,5-diene-17-ethylene ketal (ADEK) from dehydroepiandrosterone metabolites and derivatives as a potent antiandrogen. We found ADEK could interrupt androgen binding to the androgen receptor (AR) and suppress androgen-induced transactivations of WT AR and a mutant AR in prostate cancer cells.

Transformations of DHEA and its metabolites by rat liver.

Because dehydroepiandrosterone (DHEA) has a wide variety of weak beneficial effects in experimental animals and humans, we searched for metabolites of this steroid in the hope of finding more active compounds that might qualify for the title "steroid hormone." Incubation of DHEA with rat liver homogenate fortified with energy-yielding substrates resulted in rapid hydroxylation at the 7alpha-position of the molecule and subsequent conversion to other 7-oxygenated steroids in the sequence DHEA --> 7alpha-hydroxyDHEA --> 7-oxoDHEA --> 7beta-hydroxyDHEA, with branching to diols, triols, and sulfate esters. The ability of these metabolites to induce the formation of liver thermogenic enzyme activity increased from left to right in that sequence.

Ergosteroids VII: perchloric acid-induced transformations of 7-oxygenated steroids and their bio-analytical applications--a liquid chromatographic-mass spectrometric study.

Sulfate esters of 7-oxo-delta(5)-steroids can be selectively and quantitatively hydrolyzed to the corresponding free steroids in the presence of carboxylic acid esters by solvolysis with perchloric acid in ethyl acetate at room temperature. Sulfates as well as carboxylic acid esters, methyl ethers, and ketals can be quantitatively converted to the corresponding 3,5-diene-7-one derivatives by heating with perchloric acid in methanol at 65 degrees C. The dienes have a strong UV absorption with maximum centered around 284 nm.

Generation and characterization of androgen receptor knockout (ARKO) mice: an in vivo model for the study of androgen functions in selective tissues.

By using a cre-lox conditional knockout strategy, we report here the generation of androgen receptor knockout (ARKO) mice. Phenotype analysis shows that ARKO male mice have a female-like appearance and body weight. Their testes are 80% smaller and serum testosterone concentrations are lower than in wild-type (wt) mice.

Analysis of ergosteroids. VIII: Enhancement of signal response of neutral steroidal compounds in liquid chromatographic-electrospray ionization mass spectrometric analysis by mobile phase additives.

The signal response of moderately polar to nonpolar neutral steroidal compounds in positive ion mode was significantly improved in electrospray ionization mode by addition of volatile organic acids (trifluoroacetic acid, acetic and formic) at concentrations much lower than those normally employed for high-performance liquid chromatographic separations of ionic compounds. Each of the three acids enhanced the sensitivity, the order being: formic acid (approximately 50-200 ppm, v/v) > acetic acid (100-500 ppm) > trifluoroacetic acid (5-20 ppm). Higher concentrations caused decrease in the sensitivity.

Molecular differences caused by differentiation of 3T3-L1 preadipocytes in the presence of either dehydroepiandrosterone (DHEA) or 7-oxo-DHEA.

The effects of dehydroepiandrosterone (DHEA) and 7-oxo-DHEA on the cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 preadipocyte cells are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell sorting shows that preadipocyte cells treated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus DHEA comprise a population distribution of predominantly large cells with reduced adiposity. In contrast, cells treated with MDI plus 7-oxo-DHEA comprise a population distribution of almost equal proportions of small and large cells that have an adiposity equivalent to cells differentiated with MDI alone.

Ergosteroids. VI. Metabolism of dehydroepiandrosterone by rat liver in vitro: a liquid chromatographic-mass spectrometric study.

Because relatively large amounts of dehydroepiandrosterone (DHEA) are required to demonstrate its diverse metabolic effects, it is postulated that this steroid may be converted to more active molecules. To search for the possible receptor-recognized hormones. DHEA was incubated with whole rat liver homogenate and metabolite appearances were studied by LC-MS as a function of time to predict the sequence of their formation.

Ergosteroids V: preparation and biological activity of various D-ring derivatives in the 7-oxo-dehydroepiandrosterone series.

Our previous finding that D-ring seco derivatives of dehydroepiandrosterone retained biologic activity (Reich et al., Steroids 1998;63:542-53) motivated us to synthesize and test a number of steroids in which the D-ring is retained but altered in various ways. Several new steroids were synthesized and characterized by (1)H and (13)C NMR spectroscopy.