Glucosylation of phosphorylpolyisoprenol and sterol at the plasma membrane of soya-bean (Glycine max) protoplasts.

The mechanism of isomerization of delta 5-3-ox steroids to delta 4-3-oxo steroids was examined by using the membrane-bound 3-oxo steroid delta 4-delta 5-isomerase (EC 5.3.3.1) and the 3 beta-hydroxy steroid dehydrogenase present in the microsomal fraction obtained from full-term human placenta. (1) Methods for the preparation of androst-5-ene-3 beta, 17 beta-diol specifically labelled at the 4 alpha-, 4 beta- or 6-positions are described. (2) Incubations with androst-5-ene-3 beta, 17 beta-diol stereospecifically 3H-labelled either in the 4 alpha- or 4 beta-position showed that the isomerization reaction occurs via a stereospecific elimination of the 4 beta hydrogen atom. In addition, the complete retention of 3H in the delta 4-3-oxo steroids obtained from [4 alpha-3H]androst-5-ene-3 beta, 17 beta-diol indicates that the non-enzymic contribution to these experiments was negligible. (3) To study the stereochemistry of the insertion of the incoming proton at C-6, the [6-3H]androst-4-ene-3, 17-dione obtained from the oxidation isomerization of [6-3H]androst-5-ene-3 beta, 17 beta-diol was enzymically hydroxylated in the 6 beta-position by the fungus Rhizopls stolonifer. Retention of 3H in the 6 alpha-position of the isolated 6 beta-hydroxyandrost-4-ene-3, 17-dione indicates that in the isomerase-catalysed migration of the C(5) = C(6) double bond, the incoming proton from the acidic group on the enzyme must enter C-6 from the beta-face, forcing the existing 3H into the 6 alpha-position.     

Constituents of human meconium--I. Identification of 3-hydroxy-etianic acids

The monohydroxylated fraction of bile acids of human meconium was analyzed by capillary GC-MS. In the sulfate-glucuronide fraction three saturated, and one unsaturated C20 steroidal acids were found. These acids were identified as 3 alpha-hydroxy-5 alpha-, 3 alpha-hydroxy-5 beta-,3 beta-hydroxy-5 alpha-androstane-17 beta-carboxylic, and 3 beta-hydroxyandrost-5-ene-17 beta-carboxylic based on the unequivocal GC-MS comparison with standards of all possible epimers at C-3, 5 and 17. The amount of the major C20 acid, 3 alpha-hydroxy-5 alpha-androstane-17 beta-carboxylic, in meconium was 0.2 nmol/g, i.e. 5 to 10 times the amount of lithocholic acid. To prevent the oxidation of 21-hydroxy-20-oxopregnanes to C20 acids meconium was extracted in the presence of sodium borohydride. In the absence of this reducing agent the amount of 3 beta-hydroxyandrost-5-ene-17 beta-carboxylic acid was increased and its 17 alpha-epimer could be detected. This indicates partial artifactual formation of this C20 acid from 21-hydroxypregnenolone, which is known to be present in human meconium. The amount of the saturated C20 acids was unaffected by the presence of sodium borohydride in the extraction medium, and their native occurence in human meconium was further confirmed by the absence of their 17 alpha-epimers in extracts obtained both with and without borohydride. The probable metabolic origin of C20 acids in the fetal-placental-maternal unit is discussed.     

Synthesis of 17 beta-[(1S)-1-hydroxy-2-propynyl]- and 17 beta-[(1R)-1-hydroxy-2-propynyl]androst-4-en-3-one. Potential suicide substrates of 20 alpha- and 20 beta-hydroxysteroid dehydrogenases.

The title compounds have been synthesized for evaluation as potential suicide substrates of 20 alpha- and 20 beta-hydroxysteroid dehydrogenases. Synthesis was achieved by the following route. Acetylenedimagnesium bromide was reacted with 3 beta-hydroxyandrost-4-ene-17 beta-carboxaldehyde to give 17 beta-[(1R,S)-1-hydroxy-2-propynyl] androst-4-en-3 beta-ol. Separation of the R and S diols was achieved by HPLC (high pressure liquid chromatography). Selective oxidation of the 3 beta-hydroxyl group with Jones reagent at 0 degrees gave the title compounds. Further oxidation with Jones reagent converted each acetylenic alcohol to the conjugated acetylenic ketone, 17 beta-(1-oxo-2-propynyl)androst-4-en-3-one.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β-triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

17Beta-hydroxysteroid dehydrogenase activity in Streptomyces hydrogenans.

Streptomyces hydrogenans converts 17beta-hydroxyandrost-4-ene-3-one (testosterone) to androst-4-ene-3,17-dione (androstenedione) in good yields. Time-dependence of the conversion, steroid uptake and release have been studied in vivo. Steroid analysis was done by thin-layer chromatography and recrystallization to constant specific radioactivity. After sonification of the cells the postulated 17beta-hydroxysteroid dehydrogenase activity was recovered in the 105 000 g supernatant. The enzyme was enriched by gel filtration on Sephadex G-200. It required NAD+ as cofactor. Its activity could be studied photometrically, because there are no further testosterone-netabolites. If S. hydrogenans was cultured in the presence of testosterone, estradiol or 5alphaH-dihydrotestosterone, the activity of 17beta-hydroxysteroid dehydrogenase increased.     

Neighboring group participation. Part 15. Stereoselective synthesis of some steroidal tetrahydrooxazin-2-ones, as novel presumed inhibitors of human 5alpha-reductase

During the alkaline methanolysis of 3beta-acetoxy-21-chloromethyl-pregn-5-ene-20beta-N-phenylurethane, and its p-substituted phenyl derivatives, cyclization occurs, in the course of which 17beta-[3-(N-phenyl)tetrahydrooxazin-2-on-6-yl]androst-5-en-3beta-ol and its p-substituted phenyl derivatives are formed. The cyclization takes place with (N(-)-6) neighboring group participation. Oppenauer oxidation of the 3beta-hydroxy-exo-heterocyclic steroids yielded the corresponding delta4-3-ketosteroids. The structures of the new compounds were proved by IR, 1H and 13C NMR spectroscopy, using up-to-date measuring techniques such as 2D-COSY, HMQC, and HMBC. The inhibitory effects (CI50) of the delta4-3-ketosteroids on 5alpha-reductase were studied.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β?triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

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.     

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.     

Two novel C29-5beta-sterols from the stems of Opuntia dillenii

Two novel C29-5beta-sterols, opuntisterol [(24R)-24-ethyl-5beta-cholest-9-ene-6beta,12alpha-diol] (1) and opuntisteroside [(24R)-24-ethyl-6beta-[(beta-d-glucopyranosyl)oxy]-5beta-cholest-9-ene-12alpha-ol] (2), together with nine known compounds, beta-sitosterol (3), taraxerol (4), friedelin (5), methyl linoleate (6), 7-oxositosterol (7), 6beta-hydroxystigmast-4-ene-3-one (8), daucosterol (9), methyl eucomate (10) and eucomic acid (11), were isolated from the stems of Opuntia dillenii collected in Guizhou Province, China. Their structures were elucidated mainly by spectroscopic analysis. The absolute configuration of 1 were deduced from comparative 1H NMR data of the (S)- and (R)-methoxyphenyl acetate derivatives. Compounds 6-8, 10 and 11 were isolated from O. dillenii for the first time.     

Triterpenoids from Sanguisorba officinalis

Seven triterpenoids, i.e., 3beta-[(alpha-L-arabinopyranosyl)oxy]-19beta-hydroxyurs-12,20(30)-dien-28-oic acid (1), 3beta-[(alpha-L-arabinopyranosyl)oxy]-urs-11,13(18)-dien-28-oic acid beta-D-glucopyranosyl ester (2), 2alpha,3alpha,23-trihydroxyurs-12-en-24,28-dioic acid 28-beta-D-glucopyranosyl ester (3), 3beta-[(alpha-L-arabinopyranosyl)oxy]-urs-12,19(20)-dien-28-oic acid (4), 3beta-[(alpha-L-arabinopyranosyl)oxy]-urs-12,19(29)-dien-28-oic acid (5), 3beta-[(alpha-L-arabinopyranosyl)oxy]-19alpha-hydroxyolean-12-en-28-oic acid (6), 2alpha,3beta-dihydroxy-28-norurs-12,17,19(20),21-tetraen-23-oic cid (7), together with three known ones (8-10), were isolated from the roots of Sanguisorba officinalis. Their structures were determined by spectroscopic and chemical methods. Compounds 7 and 10 showed marginal inhibition activity against the growth of tumor cell lines.     

Concerning the pathway from 19-oxoandrost-4-ene-3,17-dione to estrone

The conversion of a molecule of 19-oxoandrost-4-ene-3,17-dione [1a] to estrone [2a] by human placental aromatase requires a molecule of oxygen and of NADPH. An atom of this molecule of oxygen is incorporated into the extruded formic acid derived from C-19 of [1a]. It was proposed that the 0₂ is utilized for the enzymatic 2β-hydroxylation of [1a] and the released intermediate 2β-hydroxy-19-oxoandrost-4-ene-3, 17-dione [5a]aromatizes nonenzymatically. Should [5a] be an obligatory intermediate of estrogen biosynthesis, then all the oxygen of its 2β-hydroxyl must be incorporated into the extruded formic acid. We have previously synthesized [2β-¹⁸0;19-³H][5c] and proved that none of its 2β-¹⁸0 was incorporated in the formic acid extruded in the aromatization. On this basis we concluded that [5a] can not be an obligatory precursor of estrogen biosynthesis.

The trapping of radioactive androst-4-ene-2β,3β,17β,19-tetrol in a reductively terminated incubation of a mixture of radioactive androst-4-ene-3, 17-dione and [5a] with crude placental aromatase was interpreted as evidence in support of the intermediacy of [5a]. We confirmed that the tetrol can indeed be trapped in the reductively terminated incubations. However, considering that the crude placental enzyme preparation very likely contains numerous activated oxygen species capable of a variety of oxidation reactions, most of which may not be related to estrogen elaboration, and in view of our results quoted above, the origin and the eventual biosynthetic role of the parent compound of the tetrol remains to be determined.     

The stereospecific removal of a C-19 hydrogen atom in oestrogen biosynthesis

1. The synthesis of a number of 19-substituted androgens is described. 2. A method for the partially stereospecific introduction of a tritium label at C-19 in 19-hydroxyandrost-5-ene-3beta,17beta-diol was developed. The 19-(3)H-labelled triol produced by reduction of 19-oxoandrost-5-ene-3beta,17beta-diol with tritiated sodium borohydride is tentatively formulated as 19-hydroxy[(19-R)-19-(3)H]androst-5-ene-3beta,17beta-diol and the 19-(3)H-labelled triol produced by reduction of 19-oxo[19-(3)H]-androst-5-ene-3beta,17beta-diol with sodium borohydride as 19-hydroxy[(19-S)-19-(3)H]-androst-5-ene-3beta,17beta-diol. 3. In the conversion of the (19-R)-19-(3)H-labelled compound into oestrogen by a microsomal preparation from human term placenta more radioactivity was liberated in formic acid (61.6%) than in water (38.4%). In a parallel experiment with the (19-S)-19-(3)H-labelled compound the order of radioactivity was reversed: formic acid (23.4%), water (76.2%). 4. These observations are interpreted in terms of the removal of the 19-S-hydrogen atom in the conversion of a 19-hydroxy androgen into a 19-oxo androgen during oestrogen biosynthesis. 5. It is suggested that the removal of C-19 in oestrogen biosynthesis occurs compulsorily at the oxidation state of a 19-aldehyde with the liberation of formic acid.     

Microbiological degradation of bile acids. The preparation of hexahydroindane derivatives as substrates for studying cholic acid degradation.

Relatively large amounts of 3-(3aalpha-hexahydro-7abeta-methyl-1,5-dioxoindan-4alpha-yl)propionic acid (IIb), which is believed to be one of the intermediates involved in the degradation of cholic acid (I), were needed to identify is further degradation products. A simple method for the preparation of this compound was then investigated. Arthrobacter simplex could degrade-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIa) to 3-(1beta-carboxy-3aalpha-hexahydro-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVa) in good yield, the structure of which was established by partial synthesis. It was therefore expected that, if a similar degradation by this organism occurred with 17alpha-hydroxy-3-oxoandrost-4-ene-17beta-carboxylic acid (IIIb), which is easily obtained by chemical oxidation of commercially availabe 17alpha-hydroxydeoxycorticosterone, the resulting product, 3-(1beta-carboxy-3aalpha-hexahydro-1alpha-hydroxy-7abeta-methyl-5-oxoindan-4alpha-yl)propionic acid (IVb), could be readily converted chemically into the required dioxocarboxylic acid, (IIb). Exposure of compound (IIIb) to A. simplex produced, as expected, compound (IVb) which was then oxidized with NaBiO3 to give a reasonable yield of compound (IIb).     

HPLC analysis of PAMAM dendrimer based multifunctional devices

6-OXO, a new nutritional supplement commercially available on the internet, is sold as an aromatase-inhibitor and contains androst-4-ene-3,6,17-trione as active ingredient. This anabolic steroid is a prohibited substance in sports. Androst-4-ene-3,6,17-trione is metabolised to androst-4-ene-6alpha-ol-3,17-dione and androst-4-ene-6alpha,17beta-diol-3-one. A fast, sensitive and accurate LC/MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. The method is capable of determining the stereochemical position of the hydroxy-group at C-6 of the metabolites and consists of a liquid-liquid extraction step with diethylether after enzymatic hydrolysis, followed by separation on a reversed phase column. Ionisation of the analytes is carried out using atmospheric pressure chemical ionisation. The limit of quantification of the method was 5 ng/mL for all compounds. The accuracy ranged from 14.8 to 1.3% for androst-4-ene-3,6,17-trione, 9.4 to 1.6% for androst-4-ene-6alpha-ol-3,17-dione and 4.1 to 3.2% for androst-4-ene-6alpha,17beta-diol-3-one in the range of 5-1000 ng/mL. Using this method androst-4-ene-6alpha-ol-3,17-dione was identified as a major urinary metabolite, whereas androst-4-ene-6alpha,17beta-diol-3-one as a minor metabolite. While the parent compound is predominantly excreted in conjugated form, both metabolites are solely excreted as conjugates.     

Hepatic metabolism of 3-oxoandrost-4-ene-17 beta-carboxylic acid in the adult rat: formation of carboxyl-linked glucuronides both in vivo and in vitro.

The hepatic metabolism of 3-oxoandrost-4-ene-17 beta-carboxylic acid (etienic acid), a probable acidic catabolite of deoxycorticosterone, was investigated using rats prepared with an external biliary fistula. Metabolic products were identified by GC-MS after hydrolysis with beta-glucuronidase and by proton nuclear magnetic resonance after chromatographic purification of protected glucuronides. About 80% of the injected dose was secreted into bile in 20 hours. Three fully reduced etianic acids (3 alpha-hydroxy-5 alpha-, 3 beta-hydroxy-5 alpha-, 3 alpha-hydroxy-5 beta-androstan-17 beta-carboxylic acids) were identified as were several of their di- and trihydroxylated congeners. Glucuronides of these reduced and/or hydroxylated metabolites constituted over half of the recovered dose, with carboxyl-linked glucuronides predominating over 3-hydroxyl-linked glucuronides. The mode of glucuronidation correlated well with the ability of liver microsomes to form the corresponding compounds in vitro from the set of four 3,5-diastereomeric etianic acids.     

Neighboring group participation Part 17 Stereoselective synthesis of some steroidal 2-oxazolidones, as novel potential inhibitors of 17alpha-hydroxylase-C(17,20)-lyase

During the alkaline methanolysis of 3beta-acetoxy-21-chloropregn-5-ene-20beta-N-phenylurethane (4a), and its 4-monosubstituted (4b-e) and 3,5-disubstituted (4f) phenyl derivatives, cyclization occurs, in the course of which 17beta-[3-(N-phenyl)-2-oxazolidon-5-yl]androst-5-en-3beta-ol (5a) and its substituted phenyl derivatives (5b-f) are formed. The cyclization takes place with (N(-)-5) neighboring group participation. The reaction of 3beta-acetoxy-21-azidopregn-5-en-20beta-ol (3d) with triphenylphosphine gave 3beta-acetoxy-21-phosphiniminopregn-5-en-20beta-ol, which reacted in situ with carbon dioxide with the participation of the sterically favored 20beta-OH to give the unsubstituted steroidal cyclic carbamate (8). Oppenauer oxidation of the 3beta-hydroxy-exo-heterocyclic steroids (5a-f, 9) yielded the corresponding Delta(4)-3-ketosteroids (7a-f, 10). The inhibitory effects (IC(50)) of these compounds on rat testicular C(17,20)-lyase were investigated with an in vitro radioligand incubation technique. The N-unsubstituted 17beta-(2-oxazolidon-5-yl)-androst-4-en-3-one derivative (10) was found to be a potent inhibitor (IC(50)=3.0 microM).     

The biosynthesis of some androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue

1. The formation of androst-16-enes from [4-(14)C]progesterone has been investigated with long-term incubations and short-term kinetic studies. After 4hr., 1.7 and 10.3% respectively of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes were formed in boar testis minces, but much smaller yields were obtained in boar adrenal. Both tissues formed small quantities of androsta-4,16-dien-3-one. 2. The amounts of androst-4-ene-3,17-dione and testosterone isolated were small, suggesting that androst-16-ene formation may occur preferentially in the boar testis. 3. In the absence of tissue no radioactive androst-16-enes were formed. 4. Incubation of both [4-(14)C]pregnenolone and [7alpha-(3)H]progesterone resulted in 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes containing (3)H/(14)C ratios of near unity and confirmed that both C(21) steroids were precursors. A similar incubation with 17alpha-hydroxy[4-(14)C]-progesterone and [7alpha-(3)H]progesterone gave the same Delta(16)-alcohols, but they contained only (3)H, indicating that side-chain cleavage of pregnenolone and progesterone occurred before 17alpha-hydroxylation. 5. Dehydroepiandrosterone, testosterone, testosterone acetate and 16-dehydroprogesterone were not found to be precursors of Delta(16)-steroids. 6. A pathway is proposed for the biosynthesis of 3alpha- and 3beta-hydroxy-5alpha-androst-16-enes from pregnenolone and progesterone; this may involve androsta-4,16-dien-3-one as an intermediate, but excludes 17alpha-hydroxyprogesterone, testosterone and dehydroepiandrosterone.     

The biotransformation of hyodeoxycholic acid by Pseudomonas sp. NCIB 10590 under anaerobic conditions

The bacterial degradation of hyodeoxycholic acid under anaerobic conditions was studied. The major acidic product has been identified as 6 alpha-hydroxy-3-oxochol-4-ene-24-oic acid whilst the major neutral product has been identified as 6 alpha-hydroxyandrosta-1,4-diene-3,17-dione. The minor acidic products were 3,6-dioxochola-1,4-diene-24-oic acid, 3-oxochol-5-ene-24-oic acid, 3-oxochol-4-ene-24-oic acid, 3-oxochola-1,4-diene-24-oic acid and 6 alpha-hydroxy-3-oxochola-1,4-diene-24-oic acid and the minor neutral products were androst-4-ene-3,17-dione, androst-4-ene-3,6,17-trione, androsta-1,4-diene-3,6,17-trione, androsta-1,4-diene-3,17-dione, 17 beta-hydroxyandrosta-1,4-diene-3-one and 6 alpha-hydroxyandrost-4-ene-3,17-dione. Evidence is presented which suggests that under aerobic conditions, one pathway of hyodeoxycholic acid metabolism exists whilst under anaerobic conditions an extra biotransformation pathway becomes operative involving the induction of a 6 alpha-dehydroxylase enzyme. A biochemical pathway of hyodeoxycholic acid metabolism by bacteria under anaerobic conditions is discussed incorporating a scheme involving such an enzyme.     

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.