Dissociation of 19-hydroxy- 19-oxo-, and aromatizing-activities in human placental microsomes through the use of suicide substrates to aromatase

Suicide substrates of aromatase were used as chemical probes to determine if free 19-hydroxyandrost-4-ene-3,17-dione (19-OHA) and 19-oxoandrost-4-ene-3,17-dione (19-oxoA) are obligatory intermediates in the aromatization of androst-4-ene-3,17-dione (androstenedione) to oestrone by human placental aromatase. A radiometric-HPLC assay was used to monitor 19-hydroxy, 19-oxo-, and aromatized products formed in incubations of [14C]androstenedione and human placental microsomes. When microsomes were preincubated with the suicide substrates 10 beta-mercapto-estr-4-ene-3,17-dione (10 beta-SHnorA), or 17 beta-hydroxy-10 beta-mercaptoestr-4-ene-3-one (10 beta-SHnorT), it was found that 19-hydroxy-, 19-oxo- and aromatase activities were inhibited in parallel. However, when the suicide substrates 4-hydroxyandrost-4-ene-3,17-dione (4-OHA) and 19-mercaptoandrost-4-ene-3,17-dione (19-SHA) were preincubated with placental microsomes, significantly greater inhibition of formation of oestrogens was observed in comparison to the inhibition of formation of 19-hydroxy- and 19-oxo-metabolites. Furthermore, significantly more time-dependent inhibition of 19-oxoA formation was observed in comparison to inhibition of 19-OHA formation with these same inhibitors. These results suggest that 19-hydroxy- and 19-oxo-androstenediones are not free, obligatory intermediates in the aromatization of androstenedione by human placental aromatase, but rather are products of their own autonomous cytochrome P-450-dependent, microsomal enzymatic activities.     

Selective 19-hydroxylase inhibition by an aromatase inhibitor, 4-hydroxyandrostenedione

19-Nor-deoxycorticosterone is a newly recognized mineralocorticoid which has been associated with some forms of genetic, experimental, and human hypertension. To further examine this relationship, specific inhibitors of 19-nor-deoxycorticosterone biosynthesis must be developed. Since 19-hydroxylation is the pivotal step in both 19-nor-deoxycorticosterone biosynthesis and aromatization of androgens to estrogens, we evaluated an aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione on the inhibition of 19-hydroxylation in both rat and human adrenal mitochondria in vitro and 19-nor-deoxycorticosterone production and blood pressure in spontaneously hypertensive rats in vivo. Adrenal mitochondria from 48 male Sprague-Dawley rats and 1 patient with an aldosterone-producing adenoma were incubated in the presence of deoxycorticosterone substrate both with and without 4-hydroxyandrost-4-ene-3,17-dione. 4-Hydroxyandrost-4-ene-3,17-dione produced significant inhibition of 19-hydroxy-deoxycorticosterone production in both rat and human adrenal mitochondria, with a smaller and not significant inhibition of corticosterone and 18-hydroxy-corticosterone. 4-Hydroxyandrost-4-ene-3,17-dione given subcutaneously to spontaneously hypertensive rats lowered 19-nor-deoxycorticosterone by 69% and completely abolished hypertension compared to Wistar-Kyoto controls. These data demonstrate that 4-hydroxyandrost-4-ene-3,17-dione is a specific inhibitor of 19-hydroxylase, that it lowers 19-nor-deoxycorticosterone production and prevents hypertension in the spontaneously hypertensive rat. These studies reinforce the possible pathogenic significance of 19-nor-deoxycorticosterone in hypertension in spontaneously hypertensive rats.     

Tritium release from [19-3H]-19,19-difluoroandrost-4-ene-3,17-dione during inactivation of aromatase

Aromatase is a cytochrome P-450 enzyme involved in the conversion of androst-4-ene-3,17-dione to estrogen via sequential oxidations at the 19-methyl group. Previous studies from this laboratory showed that 19,19-difluoroandrost-4-ene-3,17-dione (5) is a mechanism-based inactivator of aromatase. The mechanism of inactivation was postulated to involve enzymic oxidation at, and hydrogen loss from, the 19-carbon. The deuteriated analogue 5b has now been synthesized and shown to inactivate aromatase at the same rate as the nondeuteriated parent (5). We conclude that C19-H bond cleavage is not the rate-limiting step in the overall inactivation process caused by 5. [19-3H]-19,19-Difluoroandrost-4-ene-3,17-dione (5b) with specific activity of 31 mCi/mmol was also synthesized to study the release of tritium into solution during the enzyme inactivation process. Incubation of [19-3H]19,19-difluoroandrost-4-ene-3,17-dione with human placental microsomal aromatase at differing protein concentrations resulted in time-dependent NADPH-dependent, and protein-dependent release of tritium. This tritium release is not observed in the presence of (19R)-10 beta-oxiranylestr-4-ene-3,17-dione, a powerful competitive inhibitor of aromatase. We conclude that aromatase attacks the 19-carbon of 19,19-difluoroandrost-4-ene-3,17-dione, as originally postulated.     

Metabolism of 19-methyl substituted steroids and a proposal for the third aromatase monooxygenation

The article summarizes the results of recent studies on the metabolism of 10-ethylestr-4-ene-3,17-dione, 10-[(1R)-1-hydroxyethyl]-,and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione, in placenta. These compounds are the 19-methyl analogs of androstenedione, 19-hydroxyandrostenedione, and 19-oxoandrostenedione, respectively. No conversion of 10-ethylestr-4-ene-3,17-dione to either estrogens or oxygenated metabolites was detected. Both 10-[(1R)-1-hydroxyethyl]- and 10-[(1S)-1-hydroxyethyl]estr-4-ene-3, 17-dione were oxygenated to 10-(1,1-dihydroxyethyl)estr-4-ene-3,17-dione and isolated following in situ dehydration as 10-acetylestr-4-ene-3,17-dione. Evidence for the involvement of aromatase in these conversions is discussed. No conversion of 10-acetylestr-4-ene-3,17-dione to either estrogens or other oxygenated products was detected. These results lead us to propose a new mechanism for the third aromatase monooxygenation. We propose that the third oxygenation is initiated by 1β-hydrogen abstraction at C₁ of 19,19-dihydroxyandrostenedione, followed by homolytic cleavage of the C₁₀−C₁₉ bond with concurrent formation of a Δ^1(10),4−3-ketosteroid and a C₁₉ carbon radical, and terminated by oxygen rebound at C₁₉.     

The constitutive 7-ethoxycoumarin 0-deethylase of human placental microsomes: relationship to the intermediary steps in steroid aromatization

All oxidative functions of aromatase, i.e., estrogen production, 19-oxygenated androgen production and 7-ethoxycoumarin deethylation, were inhibited in parallel in placental microsomes from non-smokers by the mechanism-based, time-dependent inactivators (suicide substrates) 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione. In contrast, the aromatase suicide substrate androst-4-ene-3,6,17-trione had little or no effect on the conversion of androst-4-ene-3,17-dione to 19-hydroxyandrost-4-ene-3,17-dione or on the conversion of the latter to 3,17-dioxoandrost-4-en-19-al while severely limiting the capacity for estrogen production from androst-4-ene-3,17-dione and 19-hydroxyandrost-4-ene-3,17-dione in such microsomal preparations. Androst-4-ene-3,6,17-trione, therefore, appears to uncouple the 19-hydroxylation of androgens from estrogen synthesis. This agent also produced only a minimal inhibition of 7-ethoxycoumarin deethylation, indicating that this major constitutive transformation of a xenobiotic chemical is associated with the steroid 19-hydroxylating function of the aromatase system.     

Metabolism of 19-methyl-substituted steroids by human placental aromatase

The 19-methyl analogues of androstenedione and its aromatization intermediates (19-hydroxyandrostenedione and 19-oxoandrostenedione) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substituent on the enzyme's regiospecificity. Neither the androstenedione analogue [10-ethylestr-4-ene-3,17-dione (1c)] nor the 19-oxoandrostenedione analogue [10-acetylestr-4-ene-3,17-dione (3c)] was converted to estrogens or oxygenated metabolites by placental microsomes. In contrast, both analogues of 19-hydroxyandrostenedione [10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione (2c) and 10-[(1R)-1-hydroxyethyl]estr-4-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring O2 and either NADH or NADPH. No change in enzyme regiospecificity was detected. The absolute configuration of 2e was determined by X-ray crystallography. Experiments with 18O2 established that 3c generated from 2c retained little 18O (less than 3%), while 3c arising from 2e retained a significant amount of 18O (approximately equal to 70%). All four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (KI = 81 nM, 11 microM, 9.9 microM, and 150 nM for 1c, 2c, 2e, and 3c, respectively). Pretreatment of microsomes with 4-hydroxyandrostenedione (a suicide inactivator of aromatase) abolished the metabolism of 2c and 2e to 3c, as well as the type I difference spectrum elicited by 2c and 2e.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.

The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.     

Biotransformation of progesterone to 14 alpha-hydroxypregna-1,4-diene-3,20-dione, a novel fungal metabolite, by Colletotrichum antirrhini

The fermentation of progesterone by Colletotrichum antirrhini SC 2144 was examined. Instead of 15 alpha-hydroxyprogesterone, the reported product, this fungus converted progesterone to androst-4-ene-3,17-dione, androsta-1,4-diene-3,17-dione, 14 alpha-hydroxyandrosta-1,4-diene-3,17-dione, 11 alpha-hydroxypregn-4-ene-3,20-dione, 14 alpha-hydroxypregn-4-ene-3,20-dione, and a hitherto undescribed compound, 14 alpha-hydroxypregna-1,4-diene-3,20-dione.     

Metabolism of androst-4-ene-3,17-dione by subcellular fractions of rat adrenal tissue with particular reference to microsomal C19-steroid 5α-reductase

The location and some characteristics of rat adrenal C(19)-steroid 5alpha-reductase were investigated by using [7alpha-(3)H]androst-4-ene-3,17-dione and [7alpha-(3)H]testosterone as substrates. The enzymes system was shown to be NADPH-dependent and associated with the microsomal fraction. In addition, some evidence was also obtained for the existence of a separate NADH-dependent system in the soluble fraction. Further investigation of androst-4-ene-3,17-dione metabolism by subcellular fractions indicated the presence of NADH-dependent 3alpha- and 3beta-hydroxy steroid dehydrogenase systems in the microsomal pellet. This pellet also appeared to contain an NADH-dependent 17beta-hydroxy steroid dehydrogenase system, and a similar though separate system was detected in the cytosol. Malate (20mm) effectively inhibited the microsomal C(19)-steroid 5alpha-reductase, which showed similar values for K(m) and V(max.) when either androst-4-ene-3,17-dione or testosterone was used as substrate. Cytochrome c was added to all incubation mixtures used for the determination of these values to inhibit the formation of metabolites other than 5alpha-androstane-3,17-dione and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) respectively. It was also found that corticosterone did not inhibit the 5alpha-reduction of androst-4-ene-3,17-dione under these conditions, indicating that separate enzymes exist for the 5alpha-reduction of C(19)- and C(21)-steroids in the rat adrenal.     

Biotransformation of 3b-Hydroxyandrost-5-en-17-one by Cell Suspension Cultures of Catharanthus roseus

Catharanthus roseus (L.) G. Don cell suspension cultures were used to transform 3b-hydroxyandrost-5-en-17-one, the products were isolated by chromatographic methods. Their structures were established by means of NMR and MS spectral analyses. Nine metabolites were respectively elucidated as: androst-4-ene-3,17-dione (Ⅰ), 6a-hydroxyandrost-4-ene-3,17-dione (Ⅱ), 6a,17b-dihydroxyandrost-4-en-3-one (Ⅲ), 6b-hydroxyandrost-4-ene-3,17-dione (Ⅳ), 17b-hydroxyandrost-4-en-3-one (Ⅴ), 15a,17b-dihydroxyandrost-4-en-3-one (Ⅵ), 15b,17b-dihydroxyandrost-4-en-3-one (Ⅶ), 14a-hydroxyandrost-4-ene-3,17-dione (Ⅷ), 17b-hydroxyandrost-4-ene-3,16-dione (Ⅸ). It is the first time to obtain the above compounds by biotransformation with Catharanthus roseus cell cultures.     

The constitutive 7-ethoxycoumarin O-deethylase activity of human placental microsomes: relationship to aromatase

The constitutive 7-ethoxycoumarin deethylase activity of human placental microsomes from non-smokers was acutely inhibited by a number of androgens which serve as substrates for and/or competitive inhibitors of estrogen synthesis by the aromatase activity of these preparations. 10 beta-(2-Propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione, androgen derivatives which produce a mechanism-based, time-dependent inactivation of placental aromatase caused a cofactor-dependent decay in deethylase activity which paralleled the loss of aromatase activity caused by these agents and which was antagonized by aromatase substrates. Conversely, 7-ethoxycoumarin antagonized the time-dependent action of 10 beta-(2-propynyl)estr-4-ene-3,17-dione and 4-hydroxyandrost-4-ene-3,17-dione on aromatase and inhibited competitively the aromatization of 4-androstene-3,17-dione. The Ki for 7-ethoxycoumarin was equivalent to its Km as substrate for deethylation. It is concluded that a common oxidase species is responsible for both the aromatase and constitutive 7-ethoxycoumarin deethylase activities of human placental microsomes.     

Studies on the Microbiological Transformation of Steroids

An attempt was made to clarify how Pellicularia filamentosa f. sp. microsclerotia IFO 6298 capable of hydroxylating C₂₁-steroids at the C-19 position converts C₁₉-steroids, especially monohydroxyderivatives of androst-4-ene-3, 17-dione. Such substrates as 11β-hydroxyandrost-4-ene-3,17-dione (I), androst-4-ene-3, 11, 17-trione (II), androsta-1,4-diene-3, 17-dione (III), 11β-hydroxyandrosta-1,4-diene-3,17-dione (IV), 14α-hydroxyandrost-4-ene-3, 17-dione (V), 15α-hydroxyandrost-4-ene-3, 17-dione (VI) and 9α-hydroxyandrost-4-ene-3, 17-dione (VII) were converted by the organism. All the main and several minor products were then isolated and identified. As a result it is concluded that this organism converts I and II into 14α-hydroxyandrost-4-ene-3,11,17-trione, III and IV into 14α-hydroxyandrosta-1,4-diene-3,1l,17-trione, V into 11α 14α dihydroxyandrost-4-ene-3, 17-dione (main) and 11β, 14α-dihydroxyandrost-4-ene-3, 17-dione (minor, a tentative structure), VI into 11β, 15α-dihydroxyandrost-4-ene-3,17-dione (main) and 15α-hydroxyandrost-4-ene-3,11,17-trione (minor, a tentative structure) and VII into 9α, 14α-dihydroxyandrost-4-ene-3, 17-dione (main) and 6β, 9α-dihydroxyandrost-4-ene-3,17-dione (minor).

In addition, the structural requirement of substrate for the 19-hydroxylation catalyzed by the organism and the influence of a hydroxyl group on steroid nucleus upon the 11β- and 14α-hydroxylations and the 11β-OH-dehydrogenation was discussed.     

Synthesis and analytics of 2,2,3,4,4-d5-19-nor-5alpha-androsterone--an internal standard in doping analysis

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.     

A new radioimmunoassay for serum 16 alpha-hydroxyandrost-4-ene-3,17-dione with specific antiserum

A radioimmunoassay system for serum 16 alpha-hydroxyandrost-4-ene-3,17-dione was developed with the use of rabbit antiserum against 16 alpha-hydroxyandrost-4-ene-3,17-dione-3-(O-carboxymethyl)oxime which was conjugated with bovine serum albumin. The antiserum was highly specific for 16 alpha-hydroxyandrost-4-ene-3,17-dione, with cross reactions to other steroids being less than 0.8% except for androst-4-ene-3,17-dione(3.4% cross reaction). Use of LH-20 column chromatography, however, clearly separated these two steroids. Pregnancy sera were measured with this assay system after an addition of labelled internal standard, extraction and separation by column chromatography. The lower limit of detection for 16 alpha-hydroxyandrost-4-ene-3,17-dione was 2 pg/tube. The mean recovery rate of the added standard was 98.3 +/- 8.8% (mean +/- SE). Intra- and inter-assay coefficients of variation were 8.6% (n = 6) and 12.1% (n = 7), respectively.     

Synthesis of 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol and of 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol, human metabolites of norethindrone

A convenient synthesis of both 5 beta,17 alpha-19-norpregn-20-yne-3 beta,17-diol (1) and 5 beta,17 alpha-19-norpregn-20-yne-3 alpha,17-diol (2) in multigram quantities from estr-4-ene-3,17-dione is reported. Full characterization of these often-cited human metabolites of norethindrone is presented for the first time.     

A synthesis of 7α-hydroxyandrost-4-ene-3, 17-dione

The first convenient chemical synthesis of 7α-hydroxyandrost-4-ene-3,17-dione is reported. Androsta-4,6-diene-3,17-dione was converted into its 6α,7α-epoxy-derivative; reduction of the epoxide with aluminium amalgam gave 7α-hydroxyandrost-4-ene-3,17-dione. This reducing agent is more efficient than chromous acetate for the purpose.     

Hormonal steroids in the tissue of induced rat mammary tumours

The possible presence of steroids in the tissue of induced hormone-dependent rat mammary tumours was investigated. The method used involves a preliminary extraction of tumours followed by chemical separation and thin-layer chromatography. The identified compounds were cholesterol, androst-4-ene-3,17-dione, 5β-androst-1-ene-3,17-dione, androsta-1,4-diene-3,17-dione and oestrone. This is the first report of the presence of these steroids in the tissue of an experimental tumour of a non-endocrine organ. In particular 5β-androst-1-ene-3,17-dione has not previously been identified from natural sources.     

Microbial transformation of 3-hydroxy-5,6-cyclopropanocholestanes--an alternative route to 6-methylsteroids

Incubation of 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholestane (4), 3beta-hydroxy-5,6beta-cyclopropano-5beta-cholestane (5), and 3beta-hydroxy-5,6alpha-cyclopropano-5alpha-cholest-7-e ne (6) with Mycobacterium sp. (NRRL B-3805) gave a mixture of side chain cleaved 17-keto steroids as the major products in 52, 57, and 69% yields, respectively. Among these 17-keto steroids, the cyclopropyl ring eliminated product, androst-4-ene-3,17-dione (9), was isolated in 6, 4, and 8% yields, respectively. A cyclopropyl ring migration product, 6alpha,7alpha-cyclopropanoandrost-4-ene-3,17-dione (16), was isolated from the incubation mixture of 6 in 4% yield, also 10% yield of 16 was obtained when 5, 6alpha-cyclopropano-5alpha-androst-7-ene-3,17-dione (12) was incubated. The cyclopropyl ring opening and subsequent reduction followed by oxidation of the two major biotransformation products, 5, 6beta-cyclopropano-5beta-androsta-3,17-dione (10) and 5, 6alpha-cyclopropano-5alpha-androsta-3,17-dione (7), gave 6beta- and 6alpha-methylandrost-4-ene-3,17-dione in 60, and 45% yields, respectively.     

Specific antisera suitable for solid-phase radioimmunoassay of 11beta-hydroxyandrost-4-ene-3,17-dione

Specific antiserum has been developed for use in measuring 11β-hydroxyandrost-4-ene-3, 17-dione by radioimmunoassay (RIA). Rabbit antiserum was generated by employing the conjugate prepared by coupling 6β,11β-dihydroxyandrost-4-ene-3,17-dione 6-hemisuccinate with bovine serum albumin. The antiserum bound 68% of 50 picograms of 11β-hydroxyandrost-4-ene-3,17-dione-[1,2,6,7-³H] during characterization at a dilution of 1:12,500. Among the numerous steroids tested for cross-reactivity, 5α-androstane-3,17-dione, androst-4-ene-3,17-dione, and 11β-hydroxy-5α-androstane-3, 17-dione showed 2%, 5%, and 30% cross-reactivity respectively. The Rivanol-treated antiserum was coupled to Enzacryl AA, in order to study the feasibility of a solid-phase RIA, and this complex showed 50% binding with the labeled antigen at a dilution of 1:3000. The complex retained high specificity and should prove useful in a simple solid-phase RIA.