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.     

In equine testis microsomes, are testosterone and 19-nortestosterone aromatized by the same enzyme? [Poster 05]

Testosterone and 19-nortestosterone were aromatized with about the same velocity by equine testis microsomes. Coincubations of the two substrates suggest that a single enzyme system would be responsible for their aromatization. This enzyme system had a lower affinity for 19-nortestosterone than for testosterone.     

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.     

Equine testicular aromatase: substrates specificity and kinetic characteristics.

1. In the stallion, estrogens were synthesized and sulfated in vivo by the testis. 2. The equine testicular enzyme aromatized androgens and 19-norandrogens with similar velocity, but not 16 alpha-hydroxytestosterone or epitestosterone in contrast to the human placental aromatase. 3. One single enzyme was implicated in the aromatization of androstenedione, testosterone, 19-norandrostenedione and 19-nortestosterone by ETMES. 4. During the process of androstenedione aromatization by ETMES, 19-hydroxyandrostenedione and 19-oxoandrostenedione were released and 4-hydroxyandrostenedione was a competitive inhibitor causing an additional irreversible enzyme inactivation which is what occurs with HPMES. 5. Dihydrotestosterone was a potent competitive inhibitor of aromatase activity.     

Catalytic differences between porcine blastocyst and placental aromatase isozymes.

Two isozymes of porcine aromatase, the placental and the blastocyst forms, were expressed in CHO cells using the mammalian cell transfection method. Using an 'in-cell' assay (a 3H-water release method), catalytic parameters of the porcine placental aromatase were found to be very similar to those of the human enzyme; however, the activity of the blastocyst isozyme was found to be one-thirtieth that of the placental isozyme. Product isolation assay (using testosterone as the substrate) revealed that the major steroid products were 17beta-estradiol and 19-nortestosterone. The product ratio of estradiol/19-nortestosterone was found to be 94 : 6 for the porcine placental form, 6 : 94 for the porcine blastocyst form, and 92 : 8 for the human wild-type aromatase. Therefore, the porcine blastocyst aromatase isozyme catalyzes mainly androgen 19-desmethylation rather than aromatization. In addition, inhibition profile analyses on the placental and blastocyst isozymes were performed using three steroidal inhibitors [4-hydroxyandro-stenedione (4-OHA), 7alpha-(4'-amino)phenylthio-1, 4-androstandiene-3,17-dione (7alpha-APTADD), and bridge (2, 19-methyleneoxy) androstene-3,17-dione (MDL 101,003)], and four nonsteroidal inhibitors [aminoglutethimide (AG), CGS 20267, ICI D1033, and vorozole (R83842)]. While the two isozymes of porcine aromatase share 93% amino-acid sequence identity, our results indicate that the two porcine aromatase isozymes have distinct responses to various aromatase inhibitors.     

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.     

Aromatization of 19-norandrogens by porcine Leydig cells

The aromatization of norandrogens was investigated with highly purified preparations of Leydig cells from mature male pigs. Cell incubations with norandrostenedione and nortestosterone gave rise to large amounts of estrone sulfate in the medium as determined directly by a specific radioimmunoassay (RIA). Estrogen production was at least equal to that seen with androstenedione and testosterone as substrates. Similar findings were made with cells in culture for 5 days before addition of the androgen substrates in a 4h-test of aromatase activity. Stimulation of estrogen formation was noted when cells were exposed for 48 h to either hCG (0.5 i.u.) or FGF-beta (10 ng) daily, as a pretreatment, before adding androstenedione for the test of aromatase activity. Little or no increase was seen with norandrostenedione or nortestosterone as substrate. Further evidence for estrogen production was obtained from HPLC separations of metabolites of cell incubations with norandrostenedione and [14C]nortestosterone monitored by RIA and radioactivity, respectively. It is suggested that norandrogens could serve as important substrates for aromatization in the boar testes.     

Studies on cytochrome P-450-dependent microsomal enzymes of testicular androgen and estrogen biosynthesis in a urodele amphibian, Necturus

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.     

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.     

Novel properties of human placental aromatase as cytochrome P-450: purification and characterization of a unique form of aromatase

Aromatase has been purified to homogeneity from human placental microsomes based on detection of its catalytic activities in the eluates from columns of octylamino-Sepharose 4B, hydroxylapatite, Mono S, hydroxylapatite HCA, and Mono Q. The purified preparation shows only one band corresponding to the apparent subunit molecular weight of 51,000 daltons on sodium dodecyl sulfate-polyacrylamide gel. The aromatase in the presence of NADPH and NADPH-cytochrome P-450 reductase converts testosterone to 17 beta-estradiol with the high specific activity of 103 nmol/min/mg of protein. However, whether the preparation is reduced by sodium dithionate chemically or by NADPH and the reductase enzymatically, its reduced, CO-difference spectrum has no peak at about 450 nm and has only a small peak at about 420 nm, probably due to its inactivation in spite of the catalytically full activity in the same preparation. The absolute spectrum of the aromatase exhibits a Soret peak at 423 nm in the absence of testosterone and addition of testosterone to the aromatase sample makes its absorption peak shift gradually from 423 to 393 nm (high spin type peak), which is a usual characteristic in the spectrum of cytochrome P-450. The reconstituted aromatase system efficiently catalyzes aromatization of 4-androstenedione, 19-hydroxy-4-androstenedione as well as testosterone. 16 alpha-Hydroxy-4-androstenedione and 16 alpha-hydroxytestosterone are also aromatized less efficiently and 19-nortestosterone is aromatized least efficiently. The reconstituted aromatase could scarcely oxidize various xenobiotics examined, suggesting a strict and narrow substrate specificity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the aromatase system associated with the mitochondrial fraction of human placenta

The aromatase system associated with the mitochondrial fraction of human term placenta, present at 35–50% the specific activity of the microsomal enzyme, is substantially the same as the microsomal enzyme as determined by the following: 1) The rate of aromatization of androstenedione, 19-nortestosterone, and 16α-hydroxytestosterone in mitochondria was a nearly constant proportion of the microsomal rate; 2) Sensitivity to carbon monoxide was the same; 3) The magnitude of cytochrome P-450 Type I spectral interactions with androgen substrates was a constant proportion in mitochondria and microsomes; 4) Sensitivity to an antibody raised against hepatic microsomal NADPH-cytochrome c reductase was the same. When inner and outer mitochondrial membrane subfractions were prepared, the predominant aromatase activity was associated with the outer membrane preparation. This aromatase activity could not be accounted for by microsomal contamination as determined by inosine diphosphatase activity, a microsomal marker. After correction, the rate of aromatization in the outer membrane preparation was almost six times that in the inner membranes and three times that of the whole mitochondrial fraction     

3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.     

The Impact of Exogenic Testosterone and Nortestosterone-Decanoate Toxicological Evaluation Using a Rat Model

The impact of exogenic testosterone (T): 1.5 and 3.0 mg/kg.bw) and 19-nortestosterone 17-decanoate (ND): 1.5 and 7.5 mg/kg.bw) in castrated male rats was evaluated based on: (a) weight increase of the androgen target tissues, respecting the Hershberger methodology; (b) the 17α and β-testosterone, 17 α and β-estradiol and 17 α and β-nortestosterone levels using the GC-MS/MS technique; and (c) observation of the serum free thyroxine levels (T₄). Results revealed that T and ND significantly increased the weight of androgen target tissues as follows: ND was more influential on seminal vesicles, levator ani-bulbocavernosus muscle (LABC) and Cowper''s glands and T (at a dose of 3.0 mg/kg.bw) influenced the weight of the ventral prostate and glans penis. Serum samples analyzed for steroid hormone levels showed the presence of 17β-testosterone, 17β-estradiol and 17β-nor-testosterone, in castrated male rats injected with testosterone and nortestosterone, but no significant differences were found between thyroid responses and thyroid hormone levels. The results of this research proved the disrupting activity of T and ND when administered in high doses and the useful application of the Hershberger bioassay in the case of ND.     

14-Dehydro-19-nortestosterone and its 7 -methyl derivative

14-Dehydro-19-nortestosterone and its 7α-methyl derivative were synthesized. The former was found to be approximately 100 and the latter 1000 times as active as testosterone in chick comb (local application) assays. In rat assays (subcutaneous), 14-dehydro-19-nortestosterone was approximately one-half as active as, or equal to testosterone in the ventral prostate or levator ani assays respectively, whereas its 7α-methyl derivative still retained its high potency (100 times as active as testosterone) in either type of assay.     

Synthesis of nitrile derivatives of estrogens

The first time synthesis of 7alpha- and 11beta-nitrile estradiol is described. Reaction of 7alpha-cyano-19-nortestosterone with copper(II)bromide in acetonitrile at room temperature results in aromatization of the A-ring. Treatment of 11beta-cyano-19-nortestosterone-17-one under similar condition does not induce A-ring aromatization but rather results in bromination at the 2beta-position. However A-ring aromatized products are obtained when the latter compound is treated with Ac2O-Py-AcOCl, NBS and HCl.     

Androgen and 19-norsteroid profiles in human preovulatory follicles from stimulated cycles: an isotope dilution-mass spectrometric study

Follicular fluid was aspirated from preovulatory follicles of women under ovarian stimulation for in vitro fertilization and analyzed by a highly specific technique based on gas chromatography-mass spectrometry associated with stable isotope dilution. 19-Nortestosterone and 19-norandrostenedione were identified and quantified for the first time in human follicular fluid. There was a strong positive correlation between 19-nortestosterone and estradiol-17 beta and between 19-norandrostenedione and estrone concentrations, thus indicating a common cellular origin. The accumulation of 19-norsteroids in follicular fluid confirms that they are weakly active intermediates in the multistep enzymatic conversion of androgen to estrogen. Testosterone concentrations were significantly lower than those obtained by radioimmunoassay; cross-reaction with substantially higher levels of 19-nortestosterone seems to be at the origin of this discrepancy. Androstenedione concentrations were similar to those reported in the literature and it was therefore confirmed that an estradiol/androstenedione concentration ratio above 20 is favourable for oocyte cleavage. Other and some newly estimated androgens are: testosterone sulfate, 5-androstene-3 beta, 17 beta-diol 3-sulfate and disulfate, dihydrotestosterone sulfate, epitestosterone, 19-hydroxyandrostenedione, 5 alpha-androstane-3 alpha, 17 beta-diol, 5 alpha-androstane-3 beta, 17 beta-diol, 5 alpha-androstane-3,17-dione and androsterone. Dehydroepiandrosterone sulfate was by far the most abundant androgen in this type of follicles.     

Steroid metabolism in the gonads of a patient with testicular feminization. II. Metabolism of c19- and c18-steroids in vitro.

The metabolism of C19- and C18-steroids, in particular, the aromatization of androstenedione and testosterone, the interconversion of androgens to estrogens and the 5alpha-reductase activity of a right abdominal (r) and a left inguinal (l) testis of a patient with testicular feminization, are reported. Aromatization and 5alpha-reductase activity were also evaluated in tissue from the left ductus diferens (ld). The following results were obtained: 1. aromatization of androstenedione to estrone 2.52% (r), 0.02% (l), 0.94% (ld); 2. aromatization of testosterone to estradiol 0.58% (r), 2.88% (l); 3. conversion of androstenedione to testosterone 95.65% (r), 98.07% (l); 4. conversion of testosterone to androstenedione 33.14% (r), 53.65% (l); 5. conversion of estrone to estradiol85.29% (r), 100% (l), 6. conversion of estradiol to estrone 33.12% (r), 32.33% (l); 7.5alpha-reduction of testosterone to 5alpha-dihydrotestosterone 12.01% (r), 13.64% (l) and 4.10% (ld). A lack of 5alpha-reductase activity was not found in the tissues examined as stated in the literature. Estrogen production in these testes was demonstrated by the aromatization of androstenedione and testosterone to estrone and estradiol and is reflected in the difference of the estradiol concentration measured in spermatic and peripheral blood of the same patient (168 versus 33 pg/ml).     

Substrate specificity of the placental microsomal aromatase

Using an accurate and sensitive assay for the human placental aromatase we have found apparent Km values for androstenedione (4-androstene-3,17-dione) and testosterone to be 14 ± 4.0 nM and 41 ± 12 nM respectively. These values were significantly different (p < 0.001). Analyses at substrate concentrations 5–10 fold above and below the Km values did not indicate any anomalous kinetic behavior. Mixed substrate experiments were consistent with a single enzyme metabolizing both steroids: each competitively inhibited the aromatization of the other, and the “Ki” values were the same as their apparent Km values. Sodium chloride (1.2M) significantly increased the rate of testosterone aromatization by decreasing its Km value and had no significant effect on the aromatization of androstenedione. However, in the presence of this salt testosterone still inhibited the aromatization of androstenedione competitively with a “Ki” equal to its apparent Km. Our data is therefore consistent with the proposal that human placental microsomes contain a single “high affinity” site for the aromatization of androstenedione and testosterone.     

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.