Transformations of testosterone and related steroids by Botrytis cinerea

Botrytis cinerea (strain AM235) was used to investigate the transformations of testosterone and related steroids. It was found that the position and stereochemistry of the introduced hydroxyl group, as well as the yield of products, depended on the structure of the substrate. Botrytis cinerea converts the examined substrates mainly to 7 alpha-hydroxy derivatives. 1-Dehydrotestosterone was also significantly hydroxylated at a 14 alpha-position.     

Transformations of 4- and 17alpha-substituted testosterone analogues by Fusarium culmorum

A series of 4- and/or 17alpha-substituted testosterone analogues has been incubated with the hydroxylating fungus Fusarium culmorum AM282. It was found that 19-norandrostenedione, 19-nortestosterone, 4-methoxytestosterone, 4-methyltestosterone, and 4-chloro-17alpha-methyltestosterone were hydroxylated exclusively or mainly at the 6beta-position. The mixtures of 6beta-, 15alpha-, and 12beta- or 11alpha-monohydroxy derivatives were obtained from 17alpha-methyltestosterone and 17alpha-ethyl-19-nortestosterone--the substrates with alkyl group at C-17alpha. 4-Chlorotestosterone was predominantly hydroxylated at 15alpha-position, but the reaction was accompanied by the reduction of 4-en-3-one system, which proceeded in the sequence: reduction of ketone to 3beta-alcohol and then reduction of the double 4,5 bond. The results obtained indicate an influence of stereoelectronic and steric effects of substitutes on regioselectivity of the hydroxylation of 4-en-3-one steroids by F. culmorum.     

Structural characteristics of anabolic androgenic steroids contributing to binding to the androgen receptor and to their anabolic and androgenic activities: Applied modifications in the steroidal structure

Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone introduced for therapeutic purposes providing enhanced anabolic potency with reduced androgenic effects. Androgens mediate their action through their binding to the androgen receptor (AR) which is mainly expressed in androgen target tissues, such as the prostate, skeletal muscle, liver and central nervous system. This paper reviews some of the wide spectrum of testosterone and synthetic AAS structure modifications related to the intended enhancement in anabolic activity. The structural features of steroids necessary for effective binding to the AR and those which contribute to the stipulation of the androgenic and anabolic activities are also presented.     

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.     

Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study

Anabolic steroids are synthetic derivatives of testosterone and are characterized by their ability to cause nitrogen retention and positive protein metabolism, thereby leading to increased protein synthesis and muscle mass. There are disagreements in the literature in regards to the interaction of anabolic steroids with the androgen receptor (AR) as revealed by competitive ligand binding assays in vitro using cytosolic preparations from prostate and skeletal muscle. By use of tissue extracts, it has been shown that some anabolic steroids have binding affinities for the AR that are higher than that of the natural androgen testosterone, while others such as stanozolol and methanedienone have significantly lower affinities as compared with testosterone. In this study we show that stanozolol and methanedienone are low affinity ligands of the rat recombinant AR as revealed by a ligand binding assay in vitro, however, based on a cell-based AR-dependent transactivation assay, they are potent activators of the AR. We also show that a single injection of stanozolol and methanedienone causes a rapid cytosolic depletion of AR in rat skeletal muscle. Based on these results, we conclude that anabolic steroids with low affinity to AR in vitro, can in fact in vivo act on the AR to cause biological responses.     

5alpha-reduced C21 steroids are substrates for human cytochrome P450c17

The 5alpha-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5alpha-reduced C(19) steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5alpha-reduced C(21) steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C(21) steroids to C(19) steroids via its 17alpha-hydroxylase and 17,20-lyase activities. hCYP17 17alpha-hydroxylates 5alpha-pregnan-3,20-dione, but little androstanedione is formed by 17,20-lyase activity. hCYP17 also 17alpha-hydroxylates 5alpha-pregnan-3alpha-ol-20-one and the 5alpha-pregnan-3alpha,17alpha-diol-20-one intermediate is rapidly converted to androsterone by 17,20-lyase activity. Furthermore, 5alpha-pregnan-3alpha,17alpha-diol-20-one is a better substrate for the 17,20-lyase reaction than the preferred substrate 17alpha-hydroxypregnenolone and cytochrome b(5) stimulates androsterone formation only 3-fold. Both 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3alpha,17alpha-diol-20-one bind to hCYP17 with higher affinity than does progesterone. We conclude that 5alpha-reduced, 3alpha-hydroxy-C(21) steroids are excellent, high-affinity substrates for hCYP17. The brisk metabolism of 5alpha-pregnan-3alpha,17alpha-diol-20-one to androsterone by CYP17 explains how, when 5alpha-reductases are present, the testis can produce C(19) steroids androsterone and androstanediol from 17alpha-hydroxyprogesterone without the intermediacy of androstenedione and testosterone.     

Synthesis of steroid-containing oligonucleotides and their alkylating derivatives

New alkylating derivatives of oligonucleotides carrying a steroid (cholesterol, testosterone or ergosterol) residue have been synthesized, the residue being introduced via its hydroxyl group into the triester oligonucleotide block in the presence of triisopropylbenzenesulphonyl chloride and N-methylimidazole. Covalent attachment of steroids to oligonucleotides increases their hydrophobicity and does not influence the melting temperature of their complementary complexes. The data obtained showed that the oligonucleotide derivatives, bearing both an alkylating group of nitrogen mustard and a steroid residue, can be used as reagents for specific modification of nucleic acids.     

Virilizing activities of various steroids in female rat fetuses

Virilizing activities of nineteen steroids were examined by a method measuring the abridgment of urovaginal septum length of female rat fetuses directly under microscope, following oral administration of the steroids to the mothers for 4 days during the late period of gestation. The characteristics of the virilizing activities of the steroids seemed to depend on their chemical structures. No virilizing activities were observed in progesterone, retroprogesterone, chlormadinone acetate and nandrolone phenpropionate. The introduction of 6-methyl and 17-acetoxy groups into progesterone exhibited a marked virilizing activity. With testosterone and its derivatives, the introduction of 17alpha-ethynyl group into testosterone slightly decreased the virilizing activity compared with the parent compound. On the contrary, the introduction of [3, 2-C] pyrazole ring in addition to the saturation of A-ring strikingly increased the virilizing activity. The most active virilizing steroid in this group was stanozolol followed by oxymetholone, methyl-testosterone and dimethysterone. Testosterone propionate was assumed to be less active than methyltestosterone by this route. With 19-nortestosterone derivatives, the virilizing activities of compounds with 17alpha-ethynyl group were moderate, but the presence of 17alpha-ethyl group caused a marked increase of virilizing activity. While, nandrolone phenpropionate with long side chain at C-17 exhibited no significant virilizing activity. The present results show the possibility of the evaluation on a potential virilizing activity of compounds in the female fetus, and also suggest that a dossociation between the virilizing activity and the androgenic activity may exist among some compounds.     

Individual and seasonal variation in fecal testosterone and cortisol levels of wild male tufted capuchin monkeys, Cebus apella nigritus

This study tested the "challenge hypothesis" and rank-based predictions for temporal steroid production in male tufted capuchin monkeys, Cebus apella. Fecal samples (n = 209) collected from six wild males were analyzed for testosterone and cortisol concentration by enzyme immunoassay. The temporal pattern in male steroid production was compared to female sexual activity and rates of male aggression. The top-ranking adult male did not differ from other adult males in testosterone or cortisol concentration. Mean adult testosterone was significantly higher than mean subadult testosterone throughout the year. There was a clear elevation of testosterone and cortisol in both adult and subadult males during the peak of adult female sexual activity after the birth season. In fact, the magnitude of increase in testosterone was higher than predicted for a species with low male-male aggression. However, there was no difference between nonbreeding baseline testosterone levels during the birth season, and the "breeding" baseline of testosterone in males found during asynchronous female sexual activity. Of all behavioral indices examined, the distribution of female-maintained consortships was the best predictor of mean adult male testosterone concentrations. Although in many species, elevated testosterone coincides with increased male-male aggression, in the present study, the sustained high-magnitude increase in steroids during the peak of adult female sexual activity was associated with a relatively low rate of male-male intragroup aggression.     

Aromatic esters of progesterone as 5alpha-reductase and prostate growth inhibitors

The aim of this study was to determine the biological activity of 4 steroidal derivatives (9a, 9b and 10a, 10b) prepared from the commercially available 17alpha acetoxyprogesterone, where 9a, 9b, have the Delta(4)-3-oxo structure and 10a and 10b an epoxy group at C-4 and C-5. These steroids were tested as inhibitors of 5alpha-reductase enzyme, which is present in androgen-dependent tissues and converts testosterone to its more active reduced metabolite dihydrotestosterone. The pharmacological effect of these steroids was demonstrated by the significant decrease of the weight of the prostate gland of gonadectomized hamsters treated with testosterone plus finasteride or with steroids 10a and 10b. For the studies in vitro the IC(50) values were determined by measuring the steroid concentration that inhibits 50% of the activity of-5alpha-reductase. In this study we also determined the capacity of these steroids to bind to the androgen receptor present in the rat prostate cytosol. The results from this work indicated that compounds 9a, 9b, 10a, and 10b inhibited the 5alpha reductase activity with IC(50) values of 360, 370, 13 and 4.9 nM respectively. However these steroids did not bind to the androgen receptors since none competed with labeled mibolerone. Steroid 10b, an epoxy steroidal derivative containing bromine atom in the ester moiety, was the most active inhibitor of 5alpha-reductase enzyme, present in human prostate homogenates with an IC(50) value of 4.9 nM and also showed in vivo pharmacological activity since it decreased the weight of the prostate from hamsters treated with testosterone in a similar way as finasteride.     

Mass spectrometry and stability of t-butyldimethylsilylethers of some estrogens and androstanes.

In addition to using radioimmunoassays for the determination of estrogens and other steroids, the possibility of using mass fragmentography for analysis was investigated. t-Butyldimethylsilyl chloride was selected as a reagent for derivatisation because it forms rather stable silylethers. In all the mass-spectra obrained from the steroid derivatives, one pronounced peak suitable for mass fragmentography was always present. Some of the spectra of the investigated estrogens, as well as testosterone, 3 alpha-hydroxy-5 alpha-androstan-17-one and 17 alpha-methyl-17 beta-hydroxy-5 alpha-androstan-3-one are discussed. The stability of various t-butyldimethylsilylethers and the rate of enolization of testosterone and progesterone in the presence of the silylation-agent under different conditions were established.     

Partial filling affinity capillary electrophoresis with cationic poly(vinylpyrrolidone)-based copolymer coatings for studies on human lipoprotein-steroid interactions

Human plasma lipoproteins have strong hydrophobic interactions with steroids and their fatty acyl derivatives such as estradiol fatty acyl esters. In this work, affinity capillary electrophoresis with the partial filling technique was applied to study the hydrophobic interactions between lipoproteins, which are nanometer-sized particles, and nonconjugated steroids. The capillaries were first rinsed with one of two novel poly(vinylpyrrolidone) (PVP)-based cationic copolymers that were strongly adsorbed onto the fused-silica surface via electrostatic interactions. This surface treatment greatly suppressed the adsorption of lipoproteins. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) particles were then employed in the coated capillaries as pseudostationary phase in the partial filling mode. The changes in corrected migration times of steroids increased linearly with the filling time of the lipoproteins. The affinity constants between the steroids and lipoproteins were calculated, and the most hydrophobic steroid studied, progesterone, had stronger affinity than testosterone or androstenedione toward both LDL and HDL. Affinity between steroids and LDL was stronger than that between steroids and HDL. Interactions between the steroids and lipoproteins were mainly nonspecific with particle lipid components, whereas some were site specific with the apolipoproteins. The developed technique has great potential for determination of the affinity of various compounds toward lipoproteins.     

Transformations of steroid esters by Fusarium culmorum

The course of transformations of the pharmacological steroids: testosterone propionate, 4-chlorotestosterone acetate, 17beta-estradiol diacetate and their parent alcohols in Fusarium culmorum AM282 culture was compared. The results show that this microorganism is capable of regioselective hydrolysis of ester bonds. Only 4-ene-3-oxo steroid esters were hydrolyzed at C-17. 17beta-Estradiol diacetate underwent regioselective hydrolysis at C-3 and as a result, estrone--the main metabolite of estradiol--was absent in the reaction mixture. The alcohols resulting from the hydrolysis underwent oxidation at C-17 and hydroxylation. The same products (6beta- and 15alpha-hydroxy derivatives) as from testosterone were formed by transformation of testosterone propionate, but the quantitative composition of the mixtures obtained after transformations of both substrates showed differences. The 15alpha-hydroxy derivatives were obtained from the ester in considerably higher yield than from the parent alcohol. The presence of the chlorine atom at C-4 markedly reduced 17beta-saponification in 4-chlorotestosterone acetate. Only 3beta,15alpha-dihydroxy-4alpha-chloro-5alpha-androstan-17-one (the main product of transformation of 4-chlorotestosterone) was identified in the reaction mixture. 6beta-Hydroxy-4-chloroandrostenedione, which was formed from 4-chlorotestosterone, was not detected in the extract obtained after conversion of its ester.     

Highly hydroxylated steroids of the starfish Archaster typicus from the Vietnamese waters

Five new steroidal compounds, including an unusual glucoside, along with several known steroids were isolated from the starfish Archaster typicus collected in shallow waters of Quang Ninh province (Vietnam). Three new compounds are 27-nor-cholestane derivatives and the other two are 24,26-dihydroxycholestane derivatives. A biogenesis pathway for the unusual side chain of 27-nor-cholestane derivatives is proposed. Isolated compounds presented moderate toxic effects in the sperm- and 8-blastomere tests on embryonal development of the sea urchin Strongylocentrotusintermedius.     

Biosynthesis of lipoidal derivatives of pregnenolone and dehydroisoandrosterone by the adrenal

The incubation of pregnenolone or dehydroisoandrosterone with bovine or rat adrenal homogenates leads to the formation of nonpolar metabolites of these steroids. The enzymatically prepared compounds have properties that are similar to the endogenous lipoidal derivatives of pregnenolone found in bovine adrenals (Hochberg, R. B., Bandy, L., Ponticorvo, L., and Lieberman, S. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 941-945) in that they are much less polar than the parent steroid and yield the parent steroid as a product of treatment with alkali. The lipoidal derivatives of both dehydroisoandrosterone and pregnenolone proved to be chromatographically heterogeneous. 17 alpha-Hydroxypregnenolone, 17 alpha-hydroxyprogesterone, progesterone, and testosterone were not converted into lipoidal derivatives when incubated with the bovine adrenal homogenate.     

Increased sibling competition does not increase testosterone or corticosterone levels in nestlings of the spotless starling (Sturnus unicolor)

Nestling begging in passerine birds is a complex behaviour that is shaped by a multitude of ecological factors and could be physiologically mediated by varying levels of steroid hormones. Previous research has shown links between sibling competition and testosterone and corticosterone in several bird species. The spotless starling (Sturnus unicolor) is a medium sized passerine in which nestlings compete intensively for resources, often resulting in marked size hierarchies that can have profound effects on their fitness. We tested the hypothesis that an increase in sibling competition levels would result in increases in testosterone and corticosterone in this species. To this end we conducted a brood size manipulation, creating small, medium and large broods. This manipulation had the expected effect on morphology: nestling size and mass decreased with increasing brood size. Androgen levels varied in response to brood size manipulation but, contrary to expectations, the largest concentrations were found in reduced brood sizes. Corticosterone levels increased with increasing brood size, but this effect disappeared when we corrected for the time taken to process nestlings. Cell-mediated immune response was found to decrease with increasing brood size and testosterone levels. The results suggest that the proposed link between testosterone and corticosterone and sibling competition does not hold in this species, and underlines the diversity of species-specific responsiveness to steroids.     

Biocatalyst mediated production of 6β,11α-dihydroxy derivatives of 4-ene-3-one steroids

Biotransformation of steroids with 4-ene-3-one functionality such as progesterone (I), testosterone (II), 17α-methyltestosterone (III), 4-androstene-3,17-dione (IV) and 19-nortestosterone (V) were studied by using a fungal system belonging to the genera of Mucor (M881). The fungal system efficiently and quantitatively converted these steroids in regio- and stereo-selective manner into corresponding 6β,11α-dihydroxy compounds. Time course experiments suggested that the transformation was initiated by hydroxylation at 6β- or 11α-(10β-hydroxy in case of V) to form monohydroxy derivatives which upon prolonged incubation were converted into corresponding 6β,11α-dihydroxy derivatives. The fermentation studies carried out using 5 L table-top fermentor with substrates (I and II) clearly indicates that 6β,11α-dihydroxy derivatives of steroids with 4-ene-3-one functionality can be produced in large scale by using M881.     

Synthesis and release of steroids in intestines from cynomolgus monkeys (Macaca fascicularis)

To examine the synthesis and release of steroids in intestinal tissues from cynomolgus monkeys (Macaca fascicularis), we performed the following experiments: 1) incubated prepared intestinal tissues with [(3)H]testosterone to study the conversion to other steroids; 2) used a radioimmunoassay to determine steroid levels in six segments of intestinal tissues and contents (duodenum, jejunum, ileum, cecum, colon, and rectum); 3) localized testosterone in the six intestinal segments by immunofluorescence histochemistry; and 4) determined steroid levels in feces from males and females of various ages by radioimmunoassay to examine a correlation between steroid levels and age or sex. In prepared intestinal tissues, testosterone was converted into androstenedione, 5 alpha-dihydrotestosterone, and an unidentified substance; all of these steroids were detected in all segments of the intestinal tissues and contents by radioimmunoassay. Immunofluorescence showed that testosterone was located in all segments of intestinal epithelia. Androstenedione, testosterone, 5 alpha-dihydrotestosterone, and the unidentified substance were also detected in feces, and their levels were not affected by the age or sex of the animal. The present findings in cynomolgus monkeys led us to conclude that 1) steroids were synthesized in the intestines; 2) intestinal steroids were released from the six intestinal tissues to the intestinal cavities and excreted outside the body with feces; and 3) intestinal steroids were released irrespective of age or sex of the animal. Intestinal steroids seem to be paracrine or exocrine agents and to have different characteristics from classical serum steroids.     

Age-dependent changes in the concentration of active sex steroids,precursors, metabolites,and regulatory agents in blood serum of male subjects

We measured blood concentration of active and non-active sex steroids, metabolites, and precursors and compared to changes in protein and peptide hormones controlling the reproductive axis (total 14 hormones and hormone-like substances) in male subjects aged 18 to 72 y.o. We found a significant decrease in serum concentration of precursors for active sex steroids (pregnenolone, progesterone, dehydroepiandrosterone, and DHEA-sulfate), free testosterone, androstenedione (non-active metabolite of testosterone) as well as 5α-dihydrotestone after the age of 35. However, the level of total testosterone and estradiol (another active testosterone metabolite) remained steady. The systems regulated production of active sex steroids resisted a higher load associated with age and caused the increase in luteinizing and follicle-stimulating hormones in hypophysis and activin in steroidogenic glands directly correlating with age; negative correlation for these hormones was confirmed with certain sex steroids explaining the negative feedback. Decrease in level of hypopheseal adrenocorticotropic hormone with age demonstrated a more substantial role for adrenal glands compared to that of testicles in reduction of blood concentration of active sex steroids. In general, despite the reduced activity of steroidogenic glands in 60-to 70-year old male subjects the level of testosterone and estradiol remained unchanged due to associated growth of level of luteinizing and follicle-stimulating hypopheseal hormones as well as activin in steroidogenic glands that stimulated biosynthesis of sex steroids. Also androgen effects were inhibited due to the reduced level of free (unbound) testosterone and 5α-dihydrotestone.     

Behavior and physiological effects of supplemental episodes of testosterone, its precursors and metabolite in rats

Androgen-sensitive behavior and physiology were examined in gonadally intact male rats receiving daily injections of steroids (0.4 mg/kg) in two pharmaceutical forms for one month. When steroids were injected as aqueous solutions of hydroxypropyl-beta-cyclodextrin complexes, a form which insures a rapid distribution through the organism, testerosterone strongly increased behavioral parameters, while testosterone precursors (dehydroepiandrosterone and 4-androstene-3, 17-dione) and metabolite (5-alpha-dihydrotestosterone) decreased them. These results suggest that it is not possible to produce an effective testosterone pulse by metabolic conversion through supplemental pulses of precursors. The treatments did not affect sperm counts in epididymis. The size of ventral prostate was increased only after the administration of 4-androstene-3,17-dione or 5-alpha-dihydrotestosterone, not after testosterone or dehydroepiandrosterone. When steroids were injected in oil, a pharmaceutical form which distributes steroids slowly and in a protracted manner, testosterone led to an enlargement of the prostate in addition to the increase in behavioral parameters seen with the complexed form. The suppression in behavior and prostate enlargement by other steroids were more pronounced than when these were administered in complexed forms. Obviously, some of the adverse effects of the presently used depot steroid preparations are of pharmacokinetic origin.