3-甾酮-1-脱氢酶基因的表达及甾体转化分析

本文利用带有P43启动子的表达分泌载体pWB980,实现了简单节杆菌3-甾酮-1-脱氢酶在枯草芽孢杆菌中的表达,表达出的目的蛋白的分子量为55KDa。利用分光光度法检测得到胞内和胞外可溶性部分的酶活分别为110±0.5mU和15±0.6mU每毫克蛋白, 比出发菌株简单节杆菌提高了将近30倍。重组芽孢杆菌对甾体底物4-AD的转化率为45.3％,比出发菌株简单节杆菌提高了近10倍。利用枯草芽孢杆菌对甾体底物进行脱氢为甾体药物的生产开辟了一个新的途径。     

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.     

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.     

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.     

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.     

Studies of the catalytic mechanism of an active-site mutant (Y14F) of delta 5-3-ketosteroid isomerase by kinetic deuterium isotope effects

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the conversion of androst-5-ene-3,17-dione to androst-4-ene-3,17-dione by a stereoselective transfer of the 4 beta-proton to the 6 beta-position. The rate-limiting step has been shown to be the concerted enolization of the enzyme-bound substrate comprising protonation of the 3-carbonyl oxygen by Tyr-14 and abstraction of the 4 beta-proton by Asp-38 [Xue, L., Talalay, P., & Mildvan, A. S. (1990) Biochemistry 29, 7491-7500]. Primary, secondary, solvent, and combined kinetic deuterium isotope effects have been used to investigate the mechanism of the Y14F mutant, which lacks the proton donor and is 10(4.7)-fold less active catalytically than the wild-type enzyme. With [4 beta-D]androst-5-ene-3,17-dione as a substrate in H2O, a lag in product formation is observed which approaches, by a first-order process, the rate observed with protonated substrate. With the protonated substrate in D2O, a burst in product formation is detected by derivative analysis of the kinetic data which approaches the rate observed with the 4 beta-deuterated substrate in D2O. The absence of such lags or bursts with the protonated substrate in H2O or with the 4 beta-deuterated substrate in D2O, as well as the detection of buffer catalysis by phosphate at pH 6.8, indicates that one or more intermediates dissociate from the enzyme and partition to substrate 31.6 times faster than to product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats

Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats.     

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.     

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.     

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.     

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.     

Steroidal Metabolites Transformed by <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> Cultures Block Breast Cancer Estrogen Biosynthesis

Suspension of cultured cells of Marchantia polymorpha have the potential to hydrogenate the olefinic bonds present in androst-1,4-dien-3,17-dione (boldione, 1) to afford dihydroandrost-3,17-dione derivatives including: androst-4-ene-3,17-dione (androstenedione, 4-AD, 2), 5α-androstane-3,17-dione (androstenedione, AD, 4), and the less abundant metabolite 5α-androst-1-ene-3,17-dione (1-androstenedione, 1-AD, 3). After isolation and purification, these metabolites were characterized on the basis of spectroscopic analyses using 1D and 2D NMR as well as mass spectrometry. Cytotoxicity of the biotransformation products against breast adenocarcinoma cells (MCF-7) was assessed by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and cell death (apoptosis or necrosis) was assayed by acridine orange/ethidium bromide staining. Aromatase (cytochrome P450 19 enzyme, CYP19) inhibitory activity was measured by a tritiated water release assay and by direct measurement of bio-transformed steroids using the tritium labeled substrate ³H-androst-4-ene-3,17-dione. CYP19 mRNA expression in MCF-7 cells was analyzed by real-time PCR. Steroidal products 3 and 4 revealed a highly significant inhibition of MCF-7 cell growth that was predominantly due to apoptosis not necrosis. Steroidal products 3 and 4 are both potent inhibitors of aromatase activity and CYP19 mRNA expression, while 2 is a known substrate for aromatase. These data establish that metabolites 3 and 4 are potent chemical agents against breast cancer via aromatase inhibitory mechanism. Results were interpreted via virtual docking of the biotransformation products to the human placental aromatase active site.     

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.     

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.     

Microbial transformation of androst-4-ene-3,17-dione by Beauveria bassiana

The microbial transformation of androst-4-ene-3,17-dione (I) by the fungus Beauveria bassiana CCTCC AF206001 has been investigated using pH 6.0 and 7.0 media. Two hydroxylated metabolites were obtained with the pH 6.0 medium. The major product was 11alpha-hydroxyandrost-4-ene-3,17-dione (II) whereas the minor product was 6beta,11alpha-dihydroxyandrost-4-ene-3,17-dione (III). On the other hand, four hydroxylated and/or reduced metabolites were obtained with the pH 7.0 medium. The major product was 11alpha,17beta-dihydroxyandrost-ene-3-one (V) and the minor products were 17beta-hydroxyandrost-ene-3-one (IV), 6beta,11alpha,17beta-trihydroxyandrost-ene-3-one (VI) and 3alpha,11alpha,17beta-trihydroxy-5alpha-androstane (VII). The products were purified by chromatographic methods, and were identified on the basis of spectroscopic methods. This fungus strain is clearly an efficient biocatalyst for 11alpha-hydroxylation and reduction of the 17-carbonyl group.     

Conversion of soybean sterols into 3,17-diketosteroids using actinobacteria Mycobacterium neoaurum, Pimelobacter simplex, and Rhodococcus erythropolis

Abstract-Soybean sterols were converted into androst-4-ene-3,17-dione (AD) and 9alpha-hydroxyandrost-4-ene-3,17-dione (9-OH-AD) using three actinobacterium strains. The transformation of a microcrystallic substrate (particle size 5-15 nm) or the transformation in the presence of randomly methylated beta-cyclodextrin (MCD) were carried out by Mycobacterium neoaurum with a phytosterol load of 30 g/l over 144 h with an AD content of 14.5 and 15.2 g/l, respectively. AD obtained in the presence of MCD was transformed into ADD (13.5 g/l) by Pimelobacter simplex cells over 3 h and into 9-OH-AD by Rhodococcus erythropolis cells after 22 h without the isolation of AD from the cultural liquid. The technical product ADD was obtained in 75% yield, based on phytosterol. It contained as impurity 1.25% of AD and 1.5% of 1,2-dehydrotestosterone. In a control experiment-the process of 1,2-dehydrogenation of 20 g/l AD in the water solution of MCD-no by products were isolated. Thus, it is more expedient to introduce the 1,2-double bond into pure AD, whereas R. erythropolis strain with low destructive activity towards steroid nucleus can be used in the mixed culture with M. neoaurum. The crystal product contained, according to HPLC, 80% of 9-OH-AD, and 1.5 AD was combined. The yield of 9-OH-AD (m.p. 218-220 degrees C) based on transformed phytosterol was 56%.     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

Hepatic microsomal metabolism of androst-4-ene-3,17-dione: Relative importance of ring hydroxylation and aromatization in control and induced rat liver

The purpose of these studies was to determine whether oestrogen production is a quantitatively important pathway in the hepatic microsomal metabolism of androst-4-ene-3,17-dione. The effects of the enzyme inducing agents phenobarbitone and β-naphthoflavone on microsomal cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation and aromatization was investigated in the rat in vitro. In microsomal fractions from untreated rats the ratio of hydroxylated products to aromatized (oestrogenic) metabolites was 33:1. Phenobarbitone pretreatment of rats increased total hydroxylation by about 20% but did not change the ratio of hydroxylated to aromatized products (27:1). In contrast, β-naphthoflavone induction decreased total hydroxylation to about 35% of control but did not affect total aromatization. Thus the ratio of hydroxylation to aromatization was significantly lower than in control microsomes (17:1).The principal aromatized products were oestriol and 2-hydroxyoestradiol-17β, with oestradiol-17β and its 4-hydroxy metabolite as minor products; no oestrone was observed. In further studies of the microsomal metabolism of oestrone, the major product was oestradiol-17β whereas hydroxylated metabolites were only minor products. Oestradiol-17β, in contrast, was hydroxylated to a considerable extent. These findings suggest that oestrone is a better substrate for the microsomal 17β-oxidoreductase than it is for cytochrome P-450. It therefore appears likely that any oestrone formed from the aromatization of androst-4-ene-3,17-dione would be readily converted to oestradiol-17β which, in turn, is subject to cytochrome P-450-mediated hydroxylation. Although the liver is a site of C₁₉-steroid aromatization, it appears unlikely that this organ could contribute significantly to serum oestrogen levels since microsomal hydroxylases are readily able to convert aromatized products to biologically inactive metabolites.     

Steroid-1-dehydrogenase of <Emphasis Type="Italic">Mycobacterium</Emphasis> sp. VKM Ac-1817D strain producing 9α-hydroxy-androst-4-ene-3,17-dione from sitosterol

The strain of Mycobacterium sp. VKM Ac-1817D forms 9α-hydroxy-androst-4-ene-3,17-dione (9-OH-AD) as a major product from sitosterol. The formation of 9-OH-AD was accompanied with its partial destruction due to residual steroid-1-dehydrogenase (St1DH) activity. The activity was found to be induced by androst-4-ene-3,17-dione (AD), while other intermediates of sitosterol oxidation did not influence 1(2)-dehydrogenation. The enzyme is located mainly in the cytosolic fraction. The cytosolic St1DH (dimer, M _r∼58 kDa) was partially purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose, and gel filtration on Bio-Gel A-0.5M. It expressed the St1DH activity toward both AD and 9-OH-AD.     

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.