Structure-activity relationships of synthetic progestins in a yeast-based in vitro androgen bioassay

The recent identification of tetrahydrogestrinone (THG), a non-marketed designer androgen used for sports doping but previously undetectable by established mass spectrometry-based urine drug screens, and its production by a facile chemical modification of gestrinone has raised concerns about the risks of developing designer androgens from numerous marketed progestins. We therefore have used yeast-based in vitro androgen and progesterone bioassays to conduct a structure-activity study assessing the intrinsic androgenic potential of commercially available progestins and their derivatives, to identify those compounds or structures with the highest risk of forming a basis for such misapplication. Progestins had a wide range of androgenic bioactivity that was not reliably predicted for individual steroids by their progestin bioactivity. 17alpha-Hydroxyprogesterone and 19-norprogesterone derivatives with their bulky 17beta-substituents were strong progestins but generally weak androgens. 17alpha-Ethynylated derivatives of testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone such as gestrinone, ethisterone, norethisterone and norgestrel had the most significant intrinsic androgenicity of all the commercially marketed progestins. Facile chemical modification of the 17alpha-ethynyl group of each of these progestins produces 17alpha-methyl, ethyl and allyl derivatives, including THG and norbolethone, which further enhanced androgenic bioactivity. Thus by using the rapid and sensitive yeast bioassay we have screened a comprehensive set of progestins and associated structures and identified the ethynylated testosterone, 19-nortestosterone and 18-methyl-19-nortestosterone derivatives as possessing the highest risk for abuse and potential for conversion to still more potent androgens. By contrast, modern progestins such as progesterone, 17alpha-hydroxyprogesterone and 19-norprogesterone derivatives had minimal androgenic bioactivity and pose low risk.     

Virilizing effect of testosterone and its metabolites on the urovaginal septum of female fetal rats

A single injection of 50 microgram testosterone was given to fetal rats on day 17, 18, 19 or 20 of gestation. On day 21, the fetuses were removed from the mother under maternal ether anesthesia, and the length of the urovaginal septum was measured microscopically in female fetuses in order to assess the virilizing effect of testosterone. In fetuses treated with testosterone on day 17, the length of the urovaginal septum was comparable to that of oil-treated littermate controls. In fetuses treated on day 18, the length was significantly abridged compared with controls. In fetuses treated on day 19, the abridgment of the urovaginal septum was most marked. In fetuses treated on day 20, the length of the septum was again comparable to that of controls. The observations suggest that day 19 is the critical day for the virilizing effect of testosterone. Various amounts of testosterone and its metabolites including dihydrotestosterone, androstane-3 beta, 17beta-diol and androstane-3 alpha, 17beta-diol were injected into 19-day-old female fetuses, in order to test the dose relation to the virilizing effects of these steroids in terms of abridgment of the urovaginal septum. As a consequence, it was found that testosterone was the most effective for virilization.     

Contraceptive progestins. Various 11-substituents combined with four 17-substituents: 17alpha-ethynyl, five- and six-membered spiromethylene ethers or six-membered spiromethylene lactones

Norethisterone (NET) is a 19-nortestosterone derivative with progestagenic and some androgenic activity, which was used in the first generation of contraceptives. NET was succeeded by levonorgestrel (LNG) and later on by desogestrel (DSG) and gestodene (GSD). Although these latter two progestins had increased potency, there was still androgenicity with gestodene and to a lesser extent with desogestrel. New progestins were synthesized in order to further enhance progestagenic and to reduce androgenic activity. Four different chemical moieties were introduced in position 17 of 19-nortestosterone, viz. 17alpha-ethynyl, five- and six-membered spiromethylene ethers, and a six-membered-spiromethylene lactone. In combination with these structures seven different substituents were added at position 11, i.e. methylene, methyl, ethyl, ethenyl, ethynyl, 2-propenyl and 1-propynyl. All substituents except for methylene occupied the 11beta-position. All these 32 compounds were synthesized and analysed in vitro and in vivo against etonogestrel (ETG, 3-keto-desogestrel), the biologically active metabolite of desogestrel. Their relative binding potency to progesterone (PR), androgen (AR) and estrogen (ER) receptors were determined in cell lysates of human breast tumor MCF-7 cells and to glucocorticoid (GR) receptors in that of human leukemic IM-9 cells. Moreover, their relative agonistic activities were assessed in Chinese hamster ovary cell-based transactivation assays. All in vivo activities were determined in McPhail (progestagenic), ovulation inhibition (progestagenic and estrogenic), Hershberger (androgenic), hormone screening (glucocorticoid and estrogen) and Allen-Doisy (estrogenic) tests after oral and for the McPhail test also after subcutaneous administration. The progestagenic binding and transactivation potencies of all compounds in the three 17-spiro series were higher than those of the corresponding analogues in the 17alpha-ethynyl series. None of the compounds showed estrogenic or clear androgenic binding and transactivation potential except for a six-membered-spiromethylene lactone with a propynyl group. This compound showed strong androgenic binding. The glucocorticoid binding and transactivation were very low for the compounds with the 17alpha-ethynyl and the five-membered-spiromethylene ether groups, whereas both six-membered-spiro series showed, clearly with methyl and ethynyl substituents, and less pronounced with methylene and ethenyl, higher binding and transactivation values. For the 17alpha-ethynyl series, the McPhail test showed high potencies with methylene, methyl and ethenyl substituents after oral treatment or with propenyl after subcutaneous administration. The introduction of the spiro substituents in position 17 led to high potencies for other 11-substituents as well. Besides methyl, also ethyl, ethynyl and propynyl were potent substituents. With ovulation inhibition tests, the ethyl, ethenyl and ethynyl substituents were the more potent compounds in all four series. However, compounds with methyl or ethynyl additions appeared to be glucocorticoidal in the hormone screening test irrespective of the 17-substituent, while with the three spiro series even methylene and ethenyl groups became active. Androgenicity was only observed at dose levels at or above 5 mg/kg, which is 2.5-fold weaker than ETG. Moreover, estrogenicity appeared negligible with the three spiro series, while with the 17alpha-ethynyl series methyl, ethyl, ethenyl and ethynyl substituents, a very high estrogenic potential was assessed. Based on the high efficacy and low side-effects, the following compounds show a high selectivity: 17alpha-ethynyl with ethyl, ethenyl and 2-propenyl substituents, six-membered spiromethylene ether with ethyl and six-membered-spiromethylene lactone with ethyl, 2-propenyl or 1-propynyl substituents. (ABSTRACT TRUNCATED)     

Anabolic-Androgenic Steroid Effects on Sexual Receptivity in Ovariectomized Rats

Anabolic-androgenic steroid (AAS) compounds are synthetic androgens taken by athletes to increase physical strength and endurance. Recent studies in our laboratory have demonstrated that AAS administration disrupts the estrous cycle of Long–Evans rats. The present experiments examined the effects of six commonly abused AAS compounds on sexual receptivity in ovariectomized rats. Adult female Long–Evans rats received estradiol benzoate (EB; 2.0 μg/day sc) for 6 consecutive days followed by 15 days of EB concurrent with daily sc injections of 7.5 mg/kg of one of the following AAS compounds: 17α-methyltestosterone, methandrostenolone, nandrolone decanoate, stanozolol, oxymetholone, testosterone cypionate, or the oil vehicle. On Day 15, all female rats received progesterone (1.0 mg/rat) 4 h before testing. Tests for sexual receptivity were conducted on Days 3, 6, 14, and 15 of AAS treatment. Although the time course of AAS effects on sexual receptivity varied, some overall effects were clear. For example, 17α-methyltestosterone, methandrostenolone, nandrolone decanoate, and stanozolol interfered with the display of sexual receptivity on Day 14, whereas oxymetholone and testosterone cypionate had no effect. Rats in all groups displayed high levels of sexual receptivity after receiving progesterone on Day 15. Our results show that AAS compounds vary in their degree of inhibition of female sexual behavior in ovariectomized rats.     

Effects of the anabolic steroid nandrolone phenylpropionate on craniofacial growth in rats

Primary testosterone and its derivatives are anabolic steroids used in the treatment of osteoporosis and Turner syndrome. They also enhance fast-twitch muscle weight in female rats. The present study examines the effect of an anabolic steroid on craniofacial growth and development in rats. Five-week-old female Sprague-Dawley rats (125) were divided into experimental and control groups. The experimental group was injected subcutaneously with 1 mg nandrolone phenylpropionate in the interscapular region on alternate days, whereas those in the control group were injected with a vehicle, arachis oil. Rats were sacrificed at 60 and 120 days of age. Cephalometric analysis of soft X-ray cephalograms showed that chronic administration of the anabolic steroid, nandrolone phenylpropionate, resulted in: (1) about a 20% increase in body weight, (2) an increase in total skull length, (3) elongation of the maxillary and mandibular incisors, (4) an increase in the depth of the antegonial notch, and (5) downward-forward growth of the viscerocranium against the neurocranium. These results suggest that nandrolone phenylpropionate may accelerate craniofacial growth and/or induce high functional activity of the masticatory muscles in female rats. © 1994 Wiley-Liss, Inc.     

Steroids and hematopoiesis. I. The effect of steroids on in vitro erythroid colony growth: structure/activity relationships.

When substituted steroids of several classes are added to cultures of rat bone marrow cells in the presence of erythropoietin a consistent enhancement of the number of colonies of hemoglobin synthesizing cells is obtained. Maximum steroid effectiveness was found to be between 10(-6) and 10(-7) M. Representative compounds of several classes of steroids were examined for their ability to enhance colony growth, including delta 4-estrenes, delta 4-androstenes, 5alpha-H androstanes and estranes, 5beta-H estranes, pregnanes and androstanes. While testosterone and its 5alpha-H derivatives had little or no activity, many synthetic derivatives of testosterone were highly active in increasing erythroid colony growth. All 5beta-H androstanes, estranes, and all but one 5beta-H pregnane were active. Cortisol consistently inhibited colony growth and estradiol and progesterone had no significant effect.     

New aromatic esters of progesterone as antiandrogens

The in vivo and in vitro antiandrogenic activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione 1,4-bromo-17alpha-(pchlorobenzoyloxy)-4-pregnene-3,20-dione 2, 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione 3 and 4-bromo-17alpha-(p-toluoyloxy)-4-pregnene-3, 20-dione 4 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster prostate and reduced the weight of the prostate glands in gonadectomized hamsters treated with testosterone 5 (T) or dihydrotestosterone 6 (DHT) in a similar manner to that of commercially available finasteride, thus indicating a potent in vivo effect. The in vitro studies showed that steroids 1-4 have a weak inhibitory activity on 5alpha-reductase with IC50 values of: 280 (1), 2.6 (2), 1.6 (3) and 114 microM (4). The presence of Cl and Br atoms in the C-17 benzoyloxy group tends to increase the inhibitory potency of the compounds. The binding efficiency of the synthesized steroids 1-4 to the androgen receptor of the prostate gland is also evaluated. All compounds form a complex with the receptor and this explains the weight reduction of the seminal vesicles in the animals treated with DHT plus steroids 1-4.     

Steroids' transformations in Penicillium notatum culture

The application of Penicillium notatum genus for biotransformations of steroids has been investigated. The reactions observed include insertion of an oxygen atom into D-ring of steroids, 15alpha-hydroxylation of 17alpha-methyl testosterone derivatives, ester bond hydrolysis, and degradation of a testosterone derivatives side chain. Microbial production of testolactones, the biologically active compounds, was also achieved using this strain in up to 98% yield.     

Structural requirements for progesterone binding to the hepatic endoplasmic reticulum in the female rat

Microsomes isolated from the liver of the female rat specifically bind progesterone. The progesterone-microsomal complex shows highly specific characteristics. The binding is probably associated with the carbonyl groups at positions C-20 and C-3. Other steroids compete for microsomal binding sites less effectively. Competition for progesterone binding sites by other steroids in percentages: testosterone 33; testosterone propionate, 9; 17-methyltestosterone, 23.2; cortisol, 6.4; estradiol-17 beta, 1.8; 17 alpha-ethynyl estradiol, 4.7; mestranol, 1.0; norethynodrel, 4.5; ethisterone, 7.1; lynestrenol, 4.3; medroxyprogesterone, 23.3; medroxyprogesterone acetate, 15.2; 5 alpha-pregnane-3,20-dione, 47.6; 5 beta-pregnane-3,20-dione, 20.7; pregnenolone, 14.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8%; 20 beta-hydroxy-4-pregnen-3-one, 2.8; 3 beta-hydroxy-5 alpha-pregnan-20-one, 5.2; 4-pregnene-3 beta, 20 beta-diol, 2.1; 11 alpha-hydroxyprogesterone 21.0; 16 alpha-hydroxyprogesterone, 7.9; 17-hydroxyprogesterone, 26.7; 16 alpha, 17-epoxyprogesterone, 2.7; 16 alpha-methylprogesterone, 3.8; 6-methylpregnenolone, 1.2; 16 alpha-methylpregnenolone, 3.8; promegestone, 27.0. 3 beta-Hydroxy-5 beta-pregnan-20-one, 3 alpha-hydroxy-5 beta-pregnan-20-one, 5-pregnene-3 beta,20 beta-diol, 5-pregnene-3 beta, 20 alpha-diol; 5 alpha-pregnane-3 beta, 20 beta-diol, 5 alpha-pregnane-3 beta, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol, 5 beta-pregnane-3 alpha, 20 alpha-diol diacetate, 5 beta-pregnane-3 alpha, 20 beta-diol, 3 alpha, 17-dihydroxy-5 beta-pregnan-20-one, 17-hydroxypregnenolone, 6-methyl-17-hydroxypregnenolone, pregnenolone-16 alpha-carbonitrile, dihydrotestosterone and cholesterol show no competition at all. The varying degree of competition by different steroids is unrelated to their lipid solubility.     

Characterization of human fetal cord blood steroid profiles in relation to fetal sex and mode of delivery using temperature-dependent inclusion chromatography and principal component analysis (PCA)

In the present work, human male and female fetal cord blood samples were purified, selectively extracted and separated to examine a fraction of steroids ranging from polar estetrol to relatively non-polar progesterone using solid phase extraction based on C-18 tubes and beta-cyclodextrin driven temperature dependent inclusion chromatography. Resulting UV diode array chromatographic patterns revealed the presence of 27 peaks. Chromatographic patterns of UV detected steroids were analyzed using principal components analysis which revealed differences between male/female and labour/not-in-labour clusters. Quantitative analysis of nine identified steroids including: estetrol, 17beta-estradiol, estrone, estriol, cortisol, cortisone, progesterone, 20 alpha-hydroxyprogesterone and 17 alpha-hydroxyprogesterone were not significantly different between males and females. Significant differences between male and female fetuses were related to as yet unidentified compounds. Four peaks were significantly different with labour which corresponded with cortisol, cortisone and two unidentified compounds. This protocol may distinguish significant differences between clinical groups that are not readily identifiable using univariate measurements of single steroids or different low molecular mass biomarkers. Moreover, we have provided new evidence that despite the absence of testosterone there are number of steroids and low molecular mass compounds that differ between male and female fetuses.     

Production of testosterone from progesterone by rat testicular microsomes without release of the intermediates 17 alpha-hydroxyprogesterone and androstenedione

It has been shown that during the in vitro conversion of progesterone to androstenedione, 17 alpha-hydroxyprogesterone is not an obligatory intermediate which equilibrates with freely diffusible steroids in the incubation medium. Recently a cytochrome P-450 was purified that catalyzed, in addition to hydroxylase/lyase activities, reduction of androstenedione to testosterone. In order to determine whether progesterone could be transformed to testosterone without both intermediates (17 alpha-hydroxyprogesterone and androstenedione) being equilibrated with steroids in the medium, several double-label double-substrate experiments were performed. When rat microsomes were incubated with an equimolar mixture of [14C]progesterone and 17 alpha-hydroxy[3H]progesterone, androstenedione was isolated with a 11-fold higher 14C/3H ratio than 17 alpha-hydroxyprogesterone, indicating that androstenedione could not be produced from free, diffusible 17 alpha-hydroxyprogesterone. Incubation of an equimolar mixture of 17 alpha-hydroxy[3H]progesterone and [14C]androstenedione with testicular microsomes resulted in the incorporation of 3-4-fold more 17 alpha-hydroxyprogesterone into testosterone than of androstenedione, although the latter is the immediate precursor of testosterone. In an experiment in which equimolar concentrations of [3H]progesterone and [14C]androstenedione were incubated with testicular microsomes, the large pool of progesterone inhibited competitively lyase activity, but still the label of progesterone was incorporated into testosterone to the same extent as that of androstenedione. These results indicate that testosterone can be produced by immature rat testicular microsomes from added progesterone on an organized unit without the intermediates equilibrating with the incubation medium.     

In vivo MRI evaluation of anabolic steroid precursor growth effects in a guinea pig model

Anabolic steroids are widely used to increase skeletal muscle (SM) mass and improve physical performance. Some dietary supplements also include potent steroid precursors or active steroid analogs such as nandrolone. Our previous study reported the anabolic steroid effects on SM in a castrated guinea pig model with SM measured using a highly quantitative magnetic resonance imaging (MRI) protocol. The aim of the current study was to apply this animal model and in vivo MRI protocol to evaluate the growth effects of four widely used over-the-counter testosterone and nandrolone precursors: 4-androstene-3 17-dione (androstenedione), 4-androstene-3β 17β-diol (4-androsdiol), 19-nor-4-androstene-3β-17β-diol (bolandiol) and 19-nor-4-androstene-3 17-dione (19-norandrostenedione). The results showed that providing precursor to castrated male guinea pigs led to plasma steroid levels sufficient to maintain normal SM growth. The anabolic growth effects of these specific precursors on individual and total muscle volumes, sexual organs, and total adipose tissue over a 10-week treatment period, in comparison with those in the respective positive control testosterone and nandrolone groups, were documented quantitatively by MRI.     

Synthesis and biological effect of halogen substituted phenyl acetic acid derivatives of progesterone as potent progesterone receptor antagonists

In this paper, we report the relative binding affinities to the progesterone receptor (PR) of several progesterone derivatives containing an acetoxyphenyl substituent at C-17 and their structure-bioactivity relationship. The inhibitory effect to ovulation as well as their function as interrupters of endometrial maturation is also described. The biological activity of the novel steroids was determined in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with the steroidal compounds. The cytosol used for the determination of the PR, was obtained from the uteri of adult estrogen-primed rabbits and the androgen (AR), mineralocorticoid (MR) and glucocorticoid (GR) receptors were determined in the cytosolic fractions from the prostate of castrated rats and from the kidneys and livers of adenalectomized male rats. We evaluated six related steroidal compounds 8a-8f differing in the nature of the 17alpha ester side chain for the inhibition of [(3)H] R5020 binding to the PR. The IC(50) values for the displacement of [(3)H] R5020 binding to the PR and its relative binding affinities (RBAs) were determined. Progesterone and R5020 had similar IC(50) values; steroids 8a, 8f and 8c bind to the progesterone receptor with RBAs of 100%, whereas 8e, 8b and 8d have RBA values <100%. These data indicate that there is a relationship between the structure of these steroids and their binding activity to the progesterone receptors. Having demonstrated in this study that steroids 8a-8f bind to the PR, we also evaluated the receptor's selectivity, since some progesterone derivatives bind to AR, MR, GR receptors. We demonstrated that the tested steroids did not bind to the AR, MR, GR, since none of the steroids inhibited the labeled mibolerone, aldosterone or dexamethasone binding to the AR, MR or GR, respectively. These results show that the novel compounds have certain selectivity for the PR. After LHRH treatment, the mice of the control group showed the presence of ova in the oviduct, whereas the animals treated with steroids 8a, 8f, 8e and 8c with RBAs of 92-100%, did not exhibit any ovum in the oviducts. As a result of this study, it is evident that the novel steroids 8a, 8f, 8e and 8c inhibited the ovulation in these animals at dose of 0.22mg/kg. After the treatment with LHRH, the uterus of the control group showed the typical progestational activity with an enlarged endometrial thickness with secretory activity. However, the endometrium of the mice treated with steroids 8a, 8f, 8e and 8c (with RBAs of 92-100%) neither did show any enlargement of the endometrium, nor a secretory activity could be detected. The diameter of the uterus was also significantly reduced compared to those of the control group, thus indicating that compounds 8a, 8f, 8e and 8c had antagonistic activity in this tissue. The overall data showed that steroids 8a, 8f, 8e and 8c have a high and selective binding activity to the PR. Furthermore there is a relationship between the structure of these steroids and their binding activity, since the presence of fluorine atom in meta position in the acetoxyphenyl substituent at C-17, improved the binding activity as compared to that for the ortho and para positions. These data also demonstrated that 8a-8f have an anti-progestational activity in vivo, and therefore they have better characteristics than the compounds previously reported.     

Ab initio electronic structure of the progestogen norethisterone and its 5 alpha-derivatives

The steroid 17 alpha-ethynyl-19-nor-4-androsten-17 beta-ol, 3-one (Norethisterone; NET) and its 5 alpha-dihydro (5 alpha-NET), 3 alpha- and 3 beta-tetrahydro derivatives (3 alpha,5 alpha- and 3 beta,5 alpha-NET), were comparatively studied by the ab initio quantum mechanics theory. Additionally, 5 alpha-androstan-3 beta,17 beta-diol (ADIOL) was also studied. The Hartree-Fock method and the 6-31G(*) basis set were used to obtain the lowest energy conformation, geometries, electronic structure and physicochemical properties of the steroids. The results showed bond distances and valence angles similar among all steroids, but some differences in dihedral angles in the A-B-ring system were observed. The electronic structure analysis showed that NET has both frontier orbitals that is, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) located at the C4-C5 pi-bond. In A-ring reduced derivatives, the HOMO was found at the 17 beta-OH and ethynyl groups. In the case of 5 alpha-NET, the LUMO was confined to the A-ring and its C3 carbonyl group while the two NET tetrahydro-reduced derivatives showed the LUMO at the 17 beta-OH and ethynyl groups. The energy changes of the rotational barrier of the 17 beta-OH group suggest that its movement is somewhat restricted by the 17 alpha-ethynyl group. Interestingly both groups at C17 form a single electrostatic potential with high electronic density. On the other side, the 19-nor condition increases the A-ring mobility. However, the 3 beta-OH group of 3 beta,5 alpha-NET may rotate without significant energy differences as compared to the same group in ADIOL. The electronic structure of NET and its A-ring reduced derivatives explains in some extent their interaction with androgen and progesterone receptors as well as their selectivity for the estrogen alpha-receptor.     

Estrogen-2/4-hydroxylase activities in rat brain and liver microsomes exhibit different substrate preferences and sensitivities to inhibition

NADPH-dependent estrogen-2/4-hydroxylase activities in rat brain and liver microsomes were compared with respect to the utilization of different estrogens as substrates and the inhibitory effects of alpha-naphthoflavone, metyrapone and steroids. Of 6 different estrogens used as substrates, only 17 beta- and 17 alpha-estradiol were transformed relatively effectively by brain microsomes. In contrast liver microsomes utilized these two estrogens as well as ethynyl estradiol, estrone and diethylstilbestrol effectively. Estriol was a poor substrate for estrogen-2/4-hydroxylase activity in both tissues. With 40 microM 17 beta-estradiol as substrate the estrogen-2/4-hydroxylase activities in brain and liver were inhibited by alpha-naphthoflavone, metyrapone, progesterone, 17 alpha-hydroxyprogesterone and testosterone. The brain enzyme activity appeared to be more sensitive than the liver enzyme to inhibition by alpha-naphthoflavone and metyrapone. Testosterone propionate (50-100 microM) stimulated the brain enzyme activity significantly. Progesterone and 17 alpha-hydroxyprogesterone were the most effective steroidal inhibitors of brain estrogen-2/4-hydroxylase activity. In the liver the inhibitory potencies of 3 different steroids varied, depending on the estrogen used as substrate. With 17 beta-estradiol, for example, progesterone was the most potent steroidal inhibitor, while corticosterone was the most potent inhibitor when diethylstilbestrol was used as substrate. These findings indicate that rat liver microsomes can utilize a wider range of different estrogens for catecholestrogen formation than brain microsomes and suggest that the profiles of catecholestrogen-forming P-450 isozymes in the two organs differ.     

Quantitative evaluation of virilizing activity of steroids by measuring morphological changes in uro-genital region of rats.

A new technique to measure the uro-genital parameters such as the lengths of urovaginal septum, corpora cavernosa and anogenital distance on the sagittal sections of the pelvic region of female fetus of rat under microscope equipped with a micrometer was developed. In the examination of 180 normal female fetuses on the 21st day of gestation, relationships were observed between the fetal body weight and the length of urovaginal septum as well as anogenital distance, but not on the length of corpora cavernosa. Following maternal subcutaneous administration of various doses of 17alpha-methyltestosterone between the 17th and 20th day of gestation, dose-dependent abridgment in urovaginal septum length and extensions in corpora cavernosa length and anogenital distance were observed in female fetus on the 21st day's examination. When these three parametars were calculated on the relative value to the fetal body weight, however, linear relationships against log-dose were observed in all parameters. Among these three parameters the abridgment in urovaginal septum was shown to be the most sensitive. A quantitative assay of virilizing activity of steroids in female fetuses was examined on rats treated subcutaneously with 17alpha-methyltestosterone and norethandrolone. Linear regressions against the log-doses of both steroids were demonstrated in urovaginal septum length, and parallelism was noted between both regression lines. The relative potency of norethandrolone to 17alpha-methyltestosterone calculated on urovaginal septum length was 0.354 with fiducial limits of 0.293-0.44l, and it was suggested that the virilizing activities of steroids can be evaluated quantitatively.     

The effect of some sex hormones on the isovolumetric pressure curve of the rabbit's left ventricle

The effect of some sex hormones (estradiol-17α), estradiol- 17ß, estrone, progesterone and testosterone) on the mechanical activity of rabbit left ventricle was studied in concentrations within the physiological range. Investigations were carried out during the first ten minutes after the introduction of the steroid. Only progesterone and estradiol-17ß affect the isovolumetric pressure curve; the other steroids do not exhibit any effect. The effects of estradiol-17ß and progesterone counterbalance each other. Estradiol-17ß enhances the rate of pressure development and decreases the time to reach peak pressure. Progesterone decreases both the rate of pressure development and the time to reach peak pressure.     

Steroid hormone transformation in the gonads and liver of aged rats

In order to study some aspects of the steroid hormone balance in old age the following organ functions of young and senescent male and female animals were investigated: 1) The capacity of testicular (45, 68-75 and 900 day-old animals) and ovarian tissue homogenates (29, 45, 66 and 900 day-old animals) to metabolically transform the sex hormone precursor, progesterone. 2) The capacity of liver slices (60-90 and 900 day-old animals) to generate a sex-specific metabolite pattern during incubation with testosterone. 3) The activities of some enzymes of steroid metabolism, which normally show sex differences in liver cell fractions (60-90 and 900 day-old animals). The testicular capacity of senescent animals to synthesize 17 alpha-hydroxyprogesterone, androstenedione and testosterone (main pathway of androgen biosynthesis) is drastically reduced compared to that of young adult rats; the reduction also extends to the production of highly polar C19O3- and C21O3-steroids. In contrast to these deficiencies, conversion of progesterone to 20 alpha-dihydroprogesterone increases in old age, whereas the generation of 5 alpha-hydrogenated compounds from testosterone and androstenedione remains unchanged. If the group of adolescent 45 day-old animals is also taken into consideration, then the biosynthetic sequence from progesterone to testosterone exhibits a biphasic developmental course. Production rates rise from low levels only to fall back to lower rates of synthesis in old age. In no age group can the production of oestrogens in measurable quantities be detected. However, 5 alpha-hydrogenated C19O2-steroid metabolites are detected, albeit only in prepuberal animals. After puberty only progesterone, 20 alpha-dihydroprogesterone and the 5 alpha-pregnane derivatives of these two steroids can be demonstrated. The pattern of the respective metabolites undergoes an age-dependent metabolite-specific development ending (900 day-old animals) with minimal yields of products (less than 21% of progesterone is converted). The production of hydroxylated metabolites (highly polar C21O3-steroid fraction) decreases very early in life (between day 29 and 45) to values indistinguishable from those of old animals. The sexually highly differentiated metabolite pattern of hepatic testosterone metabolism typical of young adult animals (60-90 day-old) is not prominent in old age. Both sexes exhibit a retarded testosterone turnover due to a decrease in the hydroxylating activity (males being more affected than females) and a deficiency of 5 alpha-hydrogenation (females only).(ABSTRACT TRUNCATED AT 400 WORDS)     

Metabolism of testosterone and dihydrotestosterone in cultured rat hepatoma cells.

Steroid metabolism in hepatoma tissue culture (HTC) cells derived from a male rat was investigated. Steroids in ethanol were incubated with the cells for various lengths of time. Volume of ethanol never exceeded 1% of incubation volume. Thin-layer and paper chromatography were used. Incubation was with tritiated steroids. It was demonstrated that testosterone as well as dihydrotestosterone is transformed. The main enzyme activities detected were 5alpha-reduction and 3alpha-, 3beta, and 17beta-hydroxysteroid dehydrogenation. The pattern of metabolism was reproducible and varied with time, substrate concentration, and number of cells incubated. Some steroids interfered with androgen metabolism. 17beta-estradiol, 17-epitestosterone, and progesterone competed for the 17beta-hydroxyprogesterone dehydrogenase. it is concluded that 3beta and 17beta reduction in the HTC cells may be catalyzed by the same enzyme which might differ considerably from the 3beta-hydroxysteroid dehydrogenase assayed in intact liver cells. A hepatoma derived from a female rat also produced considerable amounts of 3beta-derivatives of testosterone.