Androgen and 19-norandrogen aromatization by equine and human placental microsomes

The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone occurred with both equine and human aromatases. While having no effect on mare placental microsomes, Na+ and K+ (500 mM) stimulated testosterone aromatization by human placental microsomes by 73 and 52% respectively. If indeed a single enzyme is responsible for the aromatization of testosterone and 19-nortestosterone, which seems to be the case in both equine and human placental aromatase, our results show that differences in the structure of the active sites exist between equine and human aromatases.     

Different patterns of metabolism determine the relative anabolic activity of 19-norandrogens

Testosterone, the principal androgen secreted by Leydig cells, exerts a wide range of actions including growth of the male reproductive tract (androgenic effects) and growth of non-reproductive tissues such as muscle, kidney, liver, and salivary gland (anabolic effects). As androgenic steroids were discovered some were found to have relatively more anabolic than androgenic activity. The results reviewed in this report suggest that these differences result, in part, from the differential metabolism of the steroids in individual tissues and the varied activities of the individual metabolites. In the accessory sex organs (e.g. the prostate) testosterone is 5-reduced to dihydrotestosterone (DHT) which, due to its higher affinity for androgen receptors (AR), amplifies the action of testosterone. In contrast, when 19-nortestosterone (NT) is 5-reduced, its affinity for AR decreases, resulting in a decrease in its androgenic potency. However, their anabolic potency remains unchanged since significant 5-reduction of the steroids does not occur in the muscle. 7-methyl-19-nortestosterone (MENT) does not get 5-reduced due to steric hindrance from the 7-methyl group. Therefore, the androgenic potency of MENT is not amplified as happens with testosterone. These metabolic differences are responsible for the increased anabolic activity of NT and MENT compared to testosterone. Part of the biological effects of testosterone are mediated by its aromatization to estrogens. The fact that MENT is also aromatized to 7-methyl estradiol, a potent estrogen, in vitro by human placental and rat ovarian aromatase suggests that some of the anabolic actions of MENT may be mediated by this estrogen.     

Synthesis and aromatization of 19-norandrogens in the stallion testis

The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost entirely conjugated after biosynthesis in vitro. The sequence of appearance of steroids in the peripheral blood after a single injection of 10,000 IU hCG suggests that, in the equine testis, 19-norandrogens are produced by a specific C10-19 desmolase (estrene synthetase), stimulable by hCG. 19-Nortestosterone was aromatized into estradiol-17 beta by stallion testicular microsomes. The affinity of the aromatase for 19-nortestosterone was very low compared to that for testosterone. At low and presumably physiological levels, and at a high testosterone/19-nortestosterone ratio, testosterone did not inhibit 19-nortestosterone aromatization by more than 53%. Thus, 19-nortestosterone may be aromatized in vivo in the testis in spite of the endogenous concentrations of androgens. However, the low velocity of 19-nortestosterone aromatization by testicular microsomes at roughly physiological concentrations suggests that 19-norandrogen aromatization may only participate slightly in the testicular estrogen production. These results suggest that in the equine testis, two aromatizing enzyme systems may exist: one which aromatizes both androgens and 19-norandrogens, and a minority system more specific for 19-norandrogens.     

Biotransformation XXXIX. Metabolism of testosterone, androstenedione, progesterone and testosterone derivatives in Absidia coerulea culture

The strain of Absidia coerulea was used to investigate the transformations of testosterone, androstenedione, progesterone and testosterone derivatives with additional C1–C2 double bond and/or 17-methyl group. All the examined substrates were transformed, mainly hydroxylated. 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. The first three substrates (hormones) underwent hydroxylation at C-14, and additional hydroxylation at 7 was observed in progesterone. The presence of the double bond (C1–C2) in 1-dehydrotestosterone did not influence the position of hydroxylation, but the product with additional C14–C15 double bond (at the same site as hydroxylation) was formed. 17-Methyltestosterone was hydroxylated at the 7 position, and also the dehydrogenated product (at the same site, with C6–C7 double bond) was obtained. The testosterone derivative with both C1–C2 double bond and 17-methyl group underwent hydroxylation at the 7 or 11β position, and a little amount of 14, 15 epoxide was formed.     

Use of a new radioactive ligand, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H]19-nortestosterone for the estimation of androgen receptors in rat liver cytosol

The use of the testosterone derivative, 7 alpha, 17 alpha-dimethyl[17 alpha-methyl 3H] 19-nortestosterone for the estimation of cytosolic androgen receptors in male rat liver is described. Use of this compound demonstrates binding which has a similar dissociation constant, maximum binding and steroid specificity to that seen with other synthetic testosterone derivatives. In contrast to previous data significant binding to the progesterone receptor also occurs and future studies with this ligand should employ triamcinolone acetonide to block such binding.     

7alpha,11beta-Dimethyl-19-nortestosterone: a potent and selective androgen response modulator with prostate-sparing properties

7alpha,11beta-Dimethyl-19-nortestosterone, made by 1,6-methyl addition to 17beta-acetoxy-11beta-methylestra-4,6-dien-3-one, was a highly potent and selective androgen response modulator, with enhanced androgen receptor binding, androgenic activity and anabolic:androgenic ratio over its two monomethyl homologs.     

Aromatization of testosterone and 19-nortestosterone by a single enzyme from equine testicular microsomes. Differences from human placental aromatase

A single enzyme in the stallion testis was able to aromatize both testosterone and nortestosterone. This enzyme had a much lower affinity for nortestosterone than for testosterone. In contrast to human placental estrogen synthetase, this enzyme aromatized testosterone and 19-nortestosterone with similar efficiency. The differences observed (effects of monovalent cations, inhibition of androstenedione aromatization by testosterone and 19-nortestosterone and, above all, rate of norandrogen aromatization) suggest that the aromatase in the horse testis is not the same as that in the human placenta.     

Synthesis and biological activity of brassinolide and its 22β,23β-isomer: Novel plant growth-promoting steroids

Brassinolide (2α,3α,22α, 23α-tetrahydroxy-24α-methyl-B-homo-7-oxa-5α-cholestan-6-one), a novel plant growth-promoting steroid isolated from rape pollen, and its hitherto unknown 22β, 23β-isomer were synthesized from a C-24 epimeric 60:40 mixture of 22-dehydrocampesterol (24α-methyl) and brassicasterol (24β-methyl) from oysters. The method of synthesis favored the formation of the 22β, 23β-isomer by better than 4:1. Comparative plant growth-promoting capabilities of brassinolide, both natural and synthetic, and its three side chain $\text{cis}$-glycolic isomers in the bean second internode bioassay showed that the natural and synthetic brassinolides were equally active and caused splitting of the internode at the 0.1 μg level. The least active was the 22β,23β-isomer of brassinolide. The isomers with the 22α, 23α and 24β, and the 22β, 23β and 24β configurations were highly active and were required at about 10 times the concentration of brassinolide to cause the same physiological response. In the bean first internode bioassay, an auxin-induced growth test system which employs isolated bean plant segments, the isomer with 22β, 23β and 24β configuration caused a greater response than brassinolide. Two of the four tetrahydroxy ketones obtained in the synthesis of the isomers were also active in both assays.     

Biotransformations of steroid compounds by Chaetomium sp. KCH 6651

Biotransformations of steroid compounds: androstenedione, testosterone, progesterone, pregnenolone and DHEA using Chaetomium sp. 1 KCH 6651 strain as a biocatalyst were investigated. The microorganism proved capable of selective hydroxylation of the steroid substrates. Androstenedione was converted to 14α-hydroxyandrost-4-en-3,17-dione (in over 75% yield) and 6β-hydroxyandrost-4-en-3,17-dione (in low yield), while testosterone underwent regioselective hydroxylation at 6β position. Progesterone was transformed to a single product—6β,14α-dihydroxypregnan-4-en-3,20-dione in high yield, whereas biotransformation of DHEA resulted in the formation of 7α-hydroxy derivative, which was subsequently converted to 7α-hydroxyandrost-4-en-3,17-dione.     

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.     

Tibolone is not converted by human aromatase to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE): analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.     

Identification and characterization of 19-nortestosterone in urine of meat-producing animals

To monitor the illegal use of 19-nortestosterone as an anabolizing agent in meat-production, the Belgian Institute of Veterinary Expertise applies a strategy of urine control by radioimmunoassay, positive samples being confirmed by thin-layer chromatography. We have evaluated this control strategy, using gas chromatography—mass spectrometry to confirm the presence of 19-nortestosterone, or its metabolite oestrane-diol, in positive samples from radioimmunoassay. Our results show that the effective way of proceeding remains reliable in cattle, for mature and immature males as well as non-pregnant females, and in pigs, for pregnant and non-pregnant sows. The possible presence of endogenous 19-nortestosterone in cattle, in pregnant cows urine, and in pigs, in boars and in cryptorchid pigs, impedes the control of the use of 19-nortestosterone on these samples. False-positive (not confirmed by gas chromatography—mass spectrometry) results were produced by radioimmunoassay in the urine of castrated pigs and sheep.     

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.     

Microbial transformation of 17α-cyanomethyl-17-hydroxy-4,9-estradien-3-one (STS 557) and 17α-cyanomethyl-19-nortestosterone by Mycobacterium smegmatis

Microbial transformation of the new progestagen STS 557 (17α-cyanomethyl-17-hydroxy-4,9-estradien-3-one) by $\text{Mycobacterium smegmatis}$ yielded predominantly ring A-aromatized compounds: 17α-cyanomethyl-1,3,5(10),9(11)-estratetraene-3, 17-diol, 17α-cyanomethyl-1,3,5(10)-estratriene-3, 17-diol and the corresponding 3-methyl ethers. The analogous compound without the 9(10) double bond, 17α-cyanomethyl-19-nortestosterone, was transformed mainly to 5α-hydrogenated metabolites: 17α-cyanomethyl-17-hydroxy-5α-estran-3-one, 17α-cyanomethyl-17-hydroxy-5α-1-estren-3-one, 17α-cyanomethyl-5α-estrane-3α, 17-diol, and 17α-cyanomethyl-5α-estrane-3β, 17-diol. From these results, it is concluded that 4,9-dien-3-oxo compounds are not substrates for enzymatic 5α-hydrogenation.     

Synthesis of the 7alpha-cyano-(17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones: potential radioligands for androgen and progesterone receptors

We report the preparation of the 7alpha-cyano derivative of the isomeric (17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones (IVNT) together with their binding affinity for the androgen receptor (AR) and their biodistribution in two different animal models. The cyano group was introduced at the 7alpha-position by hydrocyanation of 4,6-estradien-17beta-ol-3-one with diethylaluminum cyanide. Selective protection of the A-ring enone system as the dienol ether followed by ethynylation and deprotection under base and acid hydrolysis condition gave 7alpha-cyano-17alpha-ethynyl-19-nortestosterone. The stannyl derivatives were prepared by addition of tri-n-butylstannyl hydride and converted stereospecifically to the corresponding [125I]iodovinyl analog using [125I]NaI and H2O2. The [125I]iodovinylsteroids were intravenously administered to male rats and estrogen-primed immature female rats and tissue uptake was measured up to 6h post-injection. Co-administration of NLP-004 or ORG-2058, highly selective ligands for the progesterone receptor, to the female rats did not affect uterus uptake of the 125I-ligands. However co-injection of testosterone to DES-primed male rats induced a marked increase in prostate uptake of the 20Z-isomer of 7alpha-cyano-[125I]-IVNT. The relative binding affinity (RBA) of either 7alpha-cyano-(17alpha,20E/Z)-IVNT isomer for the AR is low (RBA=4 and 3, respectively, versus 100 for 5alpha-dihydrotestosterone (DHT)), suggesting the absence of a possible role of the AR in the localization process. These findings contrast previously reported data for the analogous 7alpha-methyl-[125I]-IVNT where co-administration of testosterone was shown to result in a 50% drop in prostate uptake. These data indicate that the addition of an electron withdrawing 7alpha-cyano group to 123I-labeled nortestosterone derivatives does not improve their potential to serve as SPECT agents for the imaging of AR densities in the prostate.     

Radioimmunoassay of 7 alpha-methyl-19-nortestosterone and investigation of its pharmacokinetics in animals

A method for the measurement of 7 alpha-methyl-19-nortestosterone (7MENT) in serum/plasma by radioimmunoassay (RIA) is described. The antiserum, raised against 7 alpha-methyl-19-nortestosterone-3-O-oxime-bovine serum albumin, had a low titer (final dilution = 1:4500) and low affinity (Ka = 1.17 x 10(9) l/mol) but showed little or no cross-reactivity with several of the steroids tested. The sensitivity of the RIA was 28.2 pg/ml and the mean recovery of added cold steroid was 86 to 100%. Intra- and inter-assay coefficients of variation ranged from 4.3 to 7.3% and 7.3 to 8.4%, respectively. This RIA was used to follow plasma 7MENT levels after a single i.v. injection of the steroid in rats and rabbits. The metabolic clearance rates (MCR) of 7MENT as determined from the plasma disappearance curve for rats and rabbits were 50 l/day and 336 l/day, respectively. The MCR of 7MENT in rats and rabbits lies in the same range as for testosterone. When compared to other nortestosterone derivatives such as norethisterone, 7MENT is metabolized relatively faster.     

Medroxyprogesterone acetate has opposite effects on the androgen binding protein concentrations in serum and epididymis

In the rat, the effects of progestin and androgen administration on serum, testicular and epididymal androgen binding protein (rABP) concentrations were determined and related to the organ weight and morphology. Adult rats were treated with medroxyprogesterone acetate (MPA; 17 alpha-acetoxy-6 alpha-methylprogesterone), testosterone propionate (TP) and mibolerone (MB; 7 alpha, 17 alpha-dimethyl-19-nortestosterone). MPA reduced testicular and epididymal weights and the concentrations of serum follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone. During MPA treatment testicular and epididymal ABP content declined in parallel with organ weights and hormone concentrations, whereas serum ABP concentrations increased. Combinations of MPA and TP reduced testicular and epididymal ABP, but the reductions were less than with MPA alone; this combined treatment also elevated serum AMP. Both MB and TP reduced ABP in the male reproductive tract, but unlike MPA did not increase the concentration of this protein in serum. The results suggest that MPA acts directly on Sertoli cells resulting in increased ABP release into the blood. The comparison was made of steady state polyacrylamide gel electrophoresis (SS-PAGE) and radioimmunoassay (RIA) methods of estimating rABP. The potency ratio of testicular ABP estimated by the two methods (RIA:SS-PAGE) was three times higher than this ratio in the epididymis in normal and hormonally treated animals. Due to differences in end points, these observations imply that these assays do not quantify the molecules in the same way in one or both of these tissues. The results indicate, however, that both assays are suitable for following rABP concentration in animals with altered hormonal states.     

Oxidation of Dihydrotestosterone by Human Cytochromes P450 19A1 and 3A4

Dihydrotestosterone is a more potent androgen than testosterone and plays an important role in endocrine function. We demonstrated that, like testosterone, dihydrotestosterone can be oxidized by human cytochrome P450 (P450) 19A1, the steroid aromatase. The products identified include the 19-hydroxy- and 19-oxo derivatives and the resulting Δ(1,10)-, Δ(5,10)-, and Δ(9,10)-dehydro 19-norsteroid products (loss of 19-methyl group). The overall catalytic efficiency of oxidation was ～10-fold higher than reported for 3α-reduction by 3α-hydroxysteroid dehydrogenase, the major enzyme known to deactivate dihydrotestosterone. These and other studies demonstrate the flexibility of P450 19A1 in removing the 1- and 2-hydrogens from 19-norsteroids, the 2-hydrogen from estrone, and (in this case) the 1-, 5β-, and 9β-hydrogens of dihydrotestosterone. Incubation of dihydrotestosterone with human liver microsomes and NADPH yielded the 18- and 19-hydroxy products plus the Δ(1,10)-dehydro 19-nor product identified in the P450 19A1 reaction. The 18- and 19-hydroxylation reactions were attributed to P450 3A4, and 18- and 19-hydroxydihydrotestosterone were identified in human plasma and urine samples. The change in the pucker of the A ring caused by reduction of the Δ(4,5) bond is remarkable in shifting the course of hydroxylation from the 6β-, 2β-, 1β-, and 15β-methylene carbons (testosterone) to the axial methyl groups (18, 19) in dihydrotestosterone and demonstrates the sensitivity of P450 3A4, even with its large active site, to small changes in substrate structure.