Determination of rat 5alpha-reductase type 1 isozyme activity and its inhibition by novel steroidal oxazolines.

The 5alpha-reductase type 1 isozyme is a key enzyme in the metabolism of the androgen steroid hormones and inhibitors of this enzyme represent a new pharmacological treatment for several androgen dependent diseases. We developed a radiosubstrate in vitro incubation method for the determination of 5alpha-reductase type 1 activity using rat liver microsomes as an enzyme source. With this method we have studied the inhibiting activity of novel (5' S)-17beta-(4,5-dihydrooxazol-5-yl)androst-5-en-3-one compounds containing various derivatized phenyl substituents coupled to the exo -heterocyclic moiety. Tests revealed moderate inhibitory actions compared to finasteride, nevertheless, results provide interesting structure-activity relationship data.

Flash labeling of a nuclear receptor domain (D domain of ultraspiracle) fused to tetracysteine tag.

Biarsenical fluorescein compounds feature unique fluorescence characteristics and special binding mechanism to tetracysteine tags with certain structures and these dyes offer a feasible method for site specific labeling of heterologously expressed proteins. We aimed FlAsH fluorescent labeling of tetracysteine fused hinge region of the ultraspiracle from Drosophila melanogaster (DmUSP-D domain) to facilitate functional studies of this receptor domain. A CCPGCC tetracysteine motif was integrated between His6, Gateway attB1, and Flag tags and attached to the N-terminus of the DmUSP-D. The fusion protein was expressed in Esherichia coli and the FlAsH labeling was performed in bacterial extracts, under conditions which are compatible with receptor function. The dye was bound to the tetracysteine tag with high affinity and complex stability and the labeling proved to be specific for the target fusion protein. Results indicate that FlAsH labeling of the internal CCPGCC motif can be a valuable tool for the functional characterisation of any nuclear receptor domains.

HPLC-RIA analysis of the ectopic cortisol production in a cancerous pancreas tumor.

Steroidal pathophysiology of a malignant, ACTH-producing pancreas tumor was investigated via HPLC-RIA determinations of intratissular concentrations of eleven main steroid hormones. The tumor specimen underwent extraction procedure with ethyl acetate and the extract was purified on a C18 minicolumn. Steroids were isolated by HPLC (C18-silica reversed phase stationary phase and methanol-water eluent system) and quantified by specific RIAs. Cortisol content of the tumor specimen was 15,700 pmol/g, the further steroid hormones were found in much lower concentrations (< 1.5-28 pmol/g). The extremely high cortisol concentration in the tissue witnesses the synthesis of the main glucocorticoid steroid in the ACTH-producing pancreas tumor and suggests a stimulating paracrine effect of ACTH on cortisol production. The present data verify that the determination of intratissular steroid concentrations by HPLC-RIA methods may identify even the most peculiar hormone sources and the hormone profiles facilitate studying pathophysiology of ectopic endocrine tumors.

HPLC-RIA analysis of steroid hormone profile in a virilizing stromal tumor of the ovary.

The pathological steroid biosynthesis of a virilizing ovarian tumor was examined via high performance liquid chromatography-radioimmunoassay (HPLC-RIA) determination of the intratissular steroid concentrations. Sex cord-stromal tumor of the ovary was obtained surgically from an 18-year-old female patient with extremely high androst-4-ene-3,17-dione (4-en-dione) and testosterone (Test) blood serum levels. The tissue specimen was extracted with ethyl acetate and the extract was then purified on a C18 mini-column with methanol-water eluents. Steroids were isolated by reversed-phase HPLC on a C18 silica gel column with 51%, 55% and 64% v/v methanol-water eluents. Steroids in the collected eluent fractions were detected by the radioactivity of tritiated internal standards and then quantified by specific RIAs. In the tumor specimen, very high 17alpha-hydroxyprogesterone (17-OH-Prog; 6300 fmol/g), dehydro-epiandrosterone (2870 fmol/g), androst-4-ene-3,17-dione (3000 fmol/g), testosterone (5700 fmol/g) concentrations, and less progesterone (PROG; 320 fmol/g) and androst-5-ene-3beta,17beta-diol (5-en-diol; 320 fmol/g), were determined. Tissue levels of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), 5alpha-androstane-3beta,17beta-diol (3beta-diol), and 17beta-estradiol were found to be 71, 20, 28, and 12 fmol/g, respectively. Steroid profile analysis verified a pathological steroid biosynthesis in the ovarian tumor and suggested that the 17alpha-hydroxylase (17alpha-H), 17,20-lyase (17,20-L), and 3beta-hydroxysteroid dehydrogenase/Delta5-4-isomerase (Delta5-3beta-HSD) activities were particularly elevated in this tumorous tissue. Present data demonstrate that the analysis of intratissular steroid profile by a HPLC-RIA method may valuably contribute to the steroidal pathophysiology of endocrine tumors.

Testosterone-secreting gonadotropin-responsive adrenal adenoma and its treatment with the antiandrogen flutamide.

A 55-year-old woman with virilization had an appreciably elevated testosterone level, which was not suppressed by dexamethasone, but was increased by stimulation with human chorionic gonadotropin (hCG). Ultrasonography and computed tomography revealed an adenoma 2.5-3.0 cm in diameter in the right adrenal gland. The patient was treated with the antiandrogen flutamide in a daily dose of 500 mg for 4 months. A substantial regression of her hirsutism was observed during flutamide administration, but the serum testosterone level remained high. Right adrenalectomy was performed. Histologically, the tumor proved to be an adrenocortical adenoma of zona reticularis type. The adenoma tissue contained specific hCG receptors (187 fmol/g). The steroid concentration in the tumor tissue was examined by means of high pressure liquid chromatography-radioimmunoassay (HPLC-RIA). A significantly increased testosterone content was detected, and the levels of its precursors, androstenedione and dehydroepiandrosterone, were also elevated. Following adrenalectomy, serum testosterone concentration decreased to the normal level. The mechanism of the inappropriate regulation in the testosterone production of the adrenal tumor has not been fully elucidated.

Configurational analysis and relative binding affinities of 16-methyl-5alpha-androstane derivatives.

The four possible isomers 16beta-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 1, 16alpha-hydroxymethyl-5alpha-androstane-3beta,17beta-diol 2, 16beta-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 3 and 16alpha-hydroxymethyl-5alpha-androstane-3beta,17alpha-diol 4 with proven configuration were converted into the corresponding 16beta-methyl-5alpha-androstane-3beta,17beta-diol 5, 16alpha-methyl-5alpha-androstane-3beta,17beta-diol 6, 16beta-methyl-5alpha-androstane-3beta,17alpha-diol 7, 16alpha-methyl-5alpha-androstane-3beta,17alpha-diol 8, furthermore into the 16beta-methyl-17beta-hydroxy-5alpha-androstane-3-one 13, 16alpha-methyl-17beta-hydroxy-5alpha-androstan-3-one 14, 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3-one 15 and 16alpha-methyl-17alpha-hydroxy-5alpha-androstan-3-one 16. The steric structures of the resulting epimers were determined by means of 1H-, and 13C-NMR spectroscopy. In this way, comparison was possible with the C-16 epimers 5, 6 and 13, 14 prepared earlier by a different route, and the series of isomers could be completed with the steric structures of 16beta-methyl-17alpha-hydroxy-5alpha-androstan-3beta-ol 7 and 16alpha-methyl-17alpha-hydroxy-5alpha 8 and with their 3-keto derivatives 15 and 16. The relative binding affinities of the 16-methyl-5alpha-androstane-3beta,17-diols 5, 6, 7, 8 and 17-hydroxy-16-methyl-5alpha-androstan-3-ones 13, 14, 15, 16 were studied. The introduction of a 16-methyl substituent into 5alpha-androstane molecules substantially decreases the binding affinity to the androgen receptor and 16alpha-methyl derivatives were always bound more weakly than the 16beta-methyl isomers.

Molecular forms of alpha-melanocyte-stimulating hormone in pheochromocytoma tissue.

The molecular forms of alpha-melanocyte-stimulating hormone (MSH) in pheochromocytoma tissues have been characterized using reversed-phase HPLC and radioimmunoassay. Six alpha-MSH-related peptides were detected. Three of the six peaks had elution times identical to those of synthetic desacetyl-alpha-MSH, alpha-MSH and diacetyl-alpha-MSH. The remaining three forms of the alpha-MSH-like immunoreactivity suggest a different processing of pro-opiomelanocortin in this tissue than in the intermediate lobe of the pituitary gland. Current results confirm that alpha-MSH are present in human pheochromocytoma and suggest that alpha-MSH has role in the pathophysiology of this tissue.

Progesterone in Periplaneta americana and Neobellieria bullata adults from the procuticle phase until first progeny production.

A significant amount of progesterone-like immunoreactive material (150 ng/g) was measured by EIA in the procuticle phase of adult of both sexes of Periplaneta americana. This peak markedly decreased to 1-10 ng/g during sclerotization and was unlikely to be of dietary origin. In the case of 0-hr-old P. americana adults 96-98% of progesterone-like material was localized in the digestive tract and Malpighian tubules. In contrast, a relatively low level of progesterone-like immunoreactive material was measured in 0-hr-old Neobellieria bullata adults. Activity of 3beta-HSD/isomerase converting pregnenolone to progesterone was high (22-43 fmol/mg protein/20 min) in 0-hr-old P. americana adults and significantly fell during sclerotization. High progesterone levels (13-16 ng/g), measured by HPLC-RIA, coexist with high levels of 3beta-HSD/isomerase activity. Orally active human contraceptives (ethisterone, ethynodiol, ethynodiol diacetate, lynestrenol, mestranol, norgestrel, norethynodrel, tamoxifen citrate, and mifepristone) which act on mammalian steroid receptors had no significant effects on progeny production in either polytrophic or meroistic insect ovaries even at concentration of 5000 mg/kg.

Activity and inhibition of 3-beta-hydroxysteroid dehydrogenase/delta-5-4-isomerase in human skin.

Activity and inhibition of 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase, a key example of biosynthesis of androgenic steroids, in human skin were studied. Whole-width dermal tissue specimens excised from various regions of the male and female body were investigated with an in vitro radioenzyme assay method using dehydroepiandrosterone as substrate. The Michaelis-Menten constant of the enzyme was found to be Km = 10nM and the maximal velocity was Vmax = 0.625 pmol produced 4-androstene-3,17-dione/mg protein/20 min. Activity of 3 beta-hydroxysteroid dehydrogenase/delta 5-4-isomerase in male inguinal skin (n = 8) was 0.132-0.412, in female abdominal skin (n = 4) 0.140-0.255, in perineal skin (n = 4) 0.138-0.962 pmol/mg protein/20 min. The synthetic steroids cyproterone acetate, 4-MA and epostane proved to be potent inhibitors, IC50 values were 150, 6.2 and 1.45 nM, respectively.

In vitro inhibition of testicular delta 5-3 beta-hydroxysteroid dehydrogenase and prostatic 5 alpha-reductase activities in rats and humans by strogen forte extract.

Clinical findings indicate that Strogen forte (a standardized extract of the plant Sabalis serrulata) can be use successfully in the medical treatment of benign prostatic hyperplasia. The aim of the present study was to investigate the possible inhibitory effects of the Strogen forte extract on rat and human testicular delta 5-3 beta-hydroxysteroid dehydrogenase (delta 5-3 beta-HSD) and prostatic 5 alpha-reductase (5 alpha-R). Strogen forte proved to be a direct inhibitor of medium effectivity, with similar IC50 values for rat testicular delta 5-3 beta-HSD (400 +/- 23 micrograms/ml) and human testicular delta 5-3 beta-HSD (212 +/- 8.6 micrograms/ml). 5 alpha-R activities were analysed by in vitro incubation of rat and human prostatic tissue homogenates with 14C-labelled testosterone as substrate in KRPG-DTT medium (pH = 7.4) with NADPH coenzyme, in air, at 37 degrees C, in the presence of Strogen forte extract. The results clearly demonstrate that Strogen forte is a potent inhibitor of prostatic 5 alpha-R, with IC50 values of 385 +/- 35.6 micrograms/ml for rat and 245 +/- 64.6 micrograms/ml for human prostatic 5 alpha-R. The present study has revealed that this plant extract inhibits not only prostatic 5 alpha-R, but also testicular delta 5-3 beta-HSD.

Effects of testosterone on the rat renal medullary vasopressin receptor concentration and the antidiuretic response.

The renal concentrating ability declines with age in humans and animals. Studies suggest that the concentrating defect is due to a decrease in renal vasopressin sensitivity. With ageing, expression of the renal vasopressin V2 receptor in rat is impaired; the normal receptor expression is restored by testosterone treatment. The effect of testosterone on the renal sensitivity to vasopressin was investigated in young rats. Male rats after orchidectomy and chronic antiandrogen cyproterone acetate treatment, and female rats after chronic testosterone phenylpropionate treatment, were used. The plasma arginine-vasopressin (AVP) and testosterone concentrations, and the antidiuretic responses to AVP and the V2 agonist deamino-[8-D-arginine]-vasopressin (dDAVP) after volume loading were measured, and the renal [3H]AVP binding density was determined. The plasma AVP level decreased slightly, but not significantly, in male rats after orchidectomy and cyproterone acetate treatment, but did not alter in female rats after testosterone treatment. The AVP and dDAVP sensitivities decreased in male rats after orchidectomy and cyproterone acetate administration, and increased in female rats treated with testosterone, as compared with the animals with a normal gonadal function. [3H]AVP binding to the renal inner medullary membranes was decreased following orchidectomy or antiandrogen treatment in male rats, and increased in testosterone-treated female rats. The results suggest that testosterone may play a physiological role in maintenance of the V2 vasopressin receptor expression and hence in the normal urinary concentrating ability in rat.

[Mercury concentrations in blood and urine in persons with and without amalgam fillings]. / Uber die Quecksilberkonzentrationen in Blut und Urin von Personen mit und ohne Amalgamfüllungen.

Using flameless atom-absorption spectroscopy, the mercury concentration in the urine and in the blood of 102 persons was determined in a double-blind experiment. The subjects were divided into four groups: persons with and without amalgam fillings as well as persons with and without occupational contact with mercury and amalgam. Mercury concentrations in the blood and the urine were the same in relation to the presence or absence of amalgam fillings. The individual values varied, depending on the nutrition, and did not correlate with the number of amalgam fillings present in the individual case. Persons with occupational contact with mercury and amalgam excreted slightly more mercury in the urine. Even these values however were far below the upper limits of the range of normalcy.