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.     

Expression of ksdD gene encoding 3-ketosteroid-Delta1-dehydrogenase from Arthrobacter simplex in Bacillus subtilis

AIMS: To improve KSDH enzyme activity and the transformation level for androst-4-ene-3,17-dione. METHODS AND RESULTS: 3-ketosteroid-Delta(1)-dehydrogenase gene from Arthrobacter simplex was expressed in Bacillus subtilis under the control of P43 promoter. The molecular weight of expressed enzyme was about 55 kDa by SDS-PAGE analysis. The activities of intracellular and extracellular soluble enzymes examined by spectrophotometrical method were 110 +/- 0.5 mU mg(-1) and 15 +/- 0.6 mU mg(-1) of protein, respectively. The transformation rate of androst-4-ene-3,17-dione was 45.3% in the B. subtilis recombinant cells. CONCLUSIONS: The enzyme activity of KSDH expressed in B. subtilis was improved about 30-fold compared with that of Arthrobacter simplex, and the transformation level of androst-4-ene-3,17-dione by the B. subtilis recombinant cells was improved about 10-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: The recombinant B. subtilis cells used for biotransformation of steroids provide a new method for production of steroid medicine. The time required for transformation of B. subtilis is much shorter than that of other bacteria, which means it will have wider usage in biopharmaceutical industry.     

Site-directed mutagenesis under the direction of in silico protein docking modeling reveals the active site residues of 3-ketosteroid-Δ<Superscript>1</Superscript>-dehydrogenase from <Emphasis Type="Italic">Mycobacterium neoaurum</Emphasis>

3-Ketosteroid-Δ¹-dehydrogenases (KsdD) from Mycobacterium neoaurum could transform androst-4-ene-3,17-dione (AD) to androst-1,4-diene-3,17-dione. This reaction has a significant effect on the product of pharmaceutical steroid. The crystal structure and active site residues information of KsdD from Mycobacterium is not yet available, which result in the engineering of KsdD is tedious. In this study, by the way of protein modeling and site-directed mutagenesis, we find that, Y122, Y125, S138, E140 and Y541 from the FAD-binding domain and Y365 from the catalytic domain play a key role in this transformation. Compared with the wild type, the decline in AD conversion for mutants illustrated that Y125, Y365, and Y541 were essential to the function of KsdD. Y122, S138 and E140 contributed to the catalysis of KsdD. The following analysis revealed the catalysis mechanism of these mutations in KsdD of Mycobacterium. These information presented here facilitate the manipulation of the catalytic properties of the enzyme to improve its application in the pharmaceutical steroid industry.     

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.     

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。     

The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.     

Multiple sites of steroid hydroxylation by the liver microsomal cytochrome P-450 system: primary and secondary metabolism of androstenedione

To investigate the potential interaction of the various pathways of androgen hydroxylation, we have conducted studies to identify the profile of products formed during the time course of metabolism of androst-4-ene-3,17-dione (AD). Incubates containing AD, NADPH, and liver microsomes (from rats pretreated with phenobarbital) were sampled at times between 0 and 20 min and the metabolites resolved by reverse-phase (C18) high-performance liquid chromatography. By this method, the pattern of formation and of utilization of eight major primary and secondary metabolites of AD was determined. We report here the formation of two previously unidentified major metabolites of AD: 6 beta,16 alpha-dihydroxyandrost-4-ene-3,17-dione and 6 beta,16 beta-dihydroxyandrost-4-ene-3,17-dione. We propose that liver microsomal cytochromes P-450 can sequentially hydroxylate a single molecule of AD at multiple sites. These hydroxylase activities are presumably a result of multiple cytochrome P-450 isozymes acting on AD resulting in a transient time course for the appearance of some monohydroxylated metabolites. In addition, a unidirectional conversion of the metabolite 16 alpha-hydroxyandrost-4-ene-3,17-dione to 16 beta-hydroxyandrost-4-ene-3,17-dione is described. Evidence is provided to support the role of cytochrome P-450 in catalyzing this reaction.     

Stereochemistry of estrogen biosynthesis by a reconstituted aromatase cytochrome P-450 preparation from human placenta

According to the literature, the multistep reaction mechanism of estrogen biosynthesis proceeds with stereospecific loss of the equatorial 1 beta-, and axial 2 beta-protons. These results were deduced from experiments carried out, either with crude microsomes, or at best with impure enzyme extracts. However, when [1,2- 3H]4-androstene-3,17-dione of known absolute 3H-label distribution was incubated with a reconstituted enzyme system, consisting of homogeneous NADPH-cytochrome P-450 reductase and highly purified aromatase, we obtained results that can only be logically explained by a trans- and antiparallel elimination reaction of both the axially oriented C-2 beta-, and C-1-alpha protons. We further demonstrate that the reconstituted enzyme has an aromatase activity optimum at pH 7.2, and an apparent Km of 0.66 microM for NADPH and of 0.24 microM for 4-androstene-3,17-dione. Also, the enzyme requires 3 nmoles of NADPH for each nmole of estrogen that is formed.     

Molecular characterization of a first human 3(alpha-->beta)-hydroxysteroid epimerase

In this report, we describe the isolation and characterization of a cDNA encoding an enzyme that exhibits catalytic characteristics of a 3(alpha-->beta)-hydroxysteroid epimerase (3(alpha-->beta)-HSE). The enzyme overexpressed in human 293 embryonic kidney cells transforms androsterone into epi-androsterone in two steps: the oxidation of androsterone to 5 alpha-androstane-3,17-dione, followed by the reduction of the latter to epi-androsterone. The reverse reaction, 3(beta-->alpha)-hydroxysteroid epimeration, is approximately 10-fold weaker. These results are confirmed by V(max)/K(m) determination, which shows that the enzyme catalyzes the oxidation of androsterone to 5 alpha-androstane-3,17-dione and the reduction of 5 alpha-androstane-3,17-dione to epi-androsterone more efficiently than the reverse reactions. The selective catalysis of the reaction following the 3(alpha-->beta) direction is also observed in intact transfected cells in culture, which better reflect physiological conditions. In vitro assays reveal that the recombinant enzyme prefers NAD(+) and NADH as cofactors and could recognize both C-19 and C-21 3 alpha-hydroxysteroids as substrates. DNA sequence analysis predicts a protein of 317 amino acids. Tissue distribution analysis using RT-PCR reveals that the mRNA of the enzyme is expressed in various tissues, including liver, brain, prostate, adrenal, and uterus, with the most abundant expression in the liver. Because active hydroxysteroids generally exert their effect in a stereo-specific manner, 3(alpha-->beta)-HSE could thus potentially play an important role in regulating the biological activities of various steroids.     

Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase

A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring.     

Differential inhibition of androst-4-en-3,17-dione aromatization by carbon monoxide in the presence of estr-4-en-3,17-dione.

The aromatization of androst-4-en-3,17-dione or 17beta hydroxyandrost-4-en-3-one (testosterone) is not inhibited by carbon monoxide under normal incubation conditions, whereas the aromatization of corresponding 19-nor steroids (estr-4-en-3,17-dione and 17beta-hydroxyestr-4-en-3-one) is readily inhibited under the same conditions. A possible explanation was found when it was shown that androst-4-en-3,17-dione and testosterone could displace bound carbon monoxide from human placental microsomal cytochrome P-450. The 19-nor steroids did not displace carbon monoxide, even at very high concentrations. These C-18 compounds appeared to facilitate complex formation and reversed the effects of the C-19 steroids. A mutual antagonism was observed with regard to effects on the formation of the ce titrated. These observations suggested that the aromatization of androst-4-en-3,17-dione should be inhibited by carbon monoxide if sufficient concentrations of the 19-nor steroids were present in reaction flasks. This hypotheses was tested and positive results were obtained, providing strong evidence for the involvement of cytochrome P-450 in normal estrogen biosynthesis.     

Influence of the position of the double bond in steroid substrates on the efficiency of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Δ4–Δ5-isomerase

Studies of the proton-transfer reaction by Pseudomonas testosteroni 3-oxo steroid Delta(4)-Delta(5)-isomerase with Delta(5(6))- and Delta(5(10))-steroid substrates demonstrate the importance of the position of the double bond for the efficiency of the isomerization process. Thus 3-oxo-Delta(5(6))-substrates have markedly high k(cat.) values, whereas those of 3-oxo-Delta(5(10))-substrates are very low and their apparent K(m) values approach equilibrium dissociation constants. The first step in the isomerization process is: [Formula: see text] which is governed by the k(-1)/k(+1) ratio and is shown to be very similar for the two classes of substrates (3-oxo-Delta(5(6))- and -Delta(5(10))-steroids). They therefore differ in the steps distal to the initial formation of the Michaelis-Menten complex. The use of the deuterated androst-5(6)-ene-3,17-dione substrate enabled us to calculate individual rate constants k(+1) and k(-1) as well as to determine the apparent rate-limiting step in the isomerization process. With the deuterated oestr-5(10)-ene-3,17-dione substrate, no significant isotope effect was observed suggesting that a different rate-limiting step may be operative in this isomerization process. Data are presented that indicate that under optimal concentrations of the efficient androst-5(6)-ene-3,17-dione substrate, the forward reaction for ES complex formation (as defined by k(+1)) is limited only by diffusion and the apparent K(m) does not approach the equilibrium constant, suggesting that the evolution of this enzyme has proceeded close to ;catalytic perfection'.     

The time-dependent inactivation of aromatase by 17β-hydroxy-10-methylthioestra-1,4-dien-3-one

17β-Hydroxy-10-methylthioestra-1,4-dien-3-one is an active-site irreversible inhibitor of aromatase, the cytochrome P-450 dependent enzyme responsible for the conversion of androst-4-ene-3,17-dione to estrone. Two time-dependent pathways to inactivation are observed, one of which requires NADPH activation.     

Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.     

Hydroxylation of 19-norandrostenedione by adrenal cortex mitochondrial P-450(11)beta

The activity of purified bovine adrenocortical P-450(11)beta on the C18-steroid, 4-estrene-3,17-dione (19-norandrostenedione), is described. The major steroid products were separated by HPLC and identified by GC-MS, and 1H- and 13C-NMR as 11 beta-, 18- and 6 beta-hydroxylated derivatives of 19-norandrostenedione. The turnover numbers of the 11 beta-, 18- and 6 beta-hydroxylase reactions were 45, 7.5 and 1.9 (mol/min/mol of P-450(11)beta), respectively, with a common Km of 44 microM. All of these activities required the presence of the electron donating system consisting of NADPH, adrenal ferredoxin (adrenodoxin) and its reductase. These findings provide additional insights into the versatile catalytic roles of P-450(11)beta in the adrenal cortex, in which it may act on C18-19-nor-steroids in addition to its known activities on C21- and C19-steroids.     

Human glutathione transferase A3-3, a highly efficient catalyst of double-bond isomerization in the biosynthetic pathway of steroid hormones.

The cDNA of a novel human glutathione transferase (GST) of the Alpha class was cloned, and the corresponding protein, denoted GST A3-3, was heterologously expressed and characterized. GST A3-3 was found to efficiently catalyze obligatory double-bond isomerizations of Delta(5)-androstene-3,17-dione and Delta(5)-pregnene-3,20-dione, precursors to testosterone and progesterone, respectively, in steroid hormone biosynthesis. The catalytic efficiency (k(cat)/K(m)) with Delta(5)-androstene-3,17-dione was determined as 5 x 10(6) m(-1) s(-1), which is considerably higher than with any other GST substrate tested. The rate of acceleration afforded by GST A3-3 is 6 x 10(8) based on the ratio between k(cat) and the rate constant for the nonenzymatic isomerization of Delta(5)-androstene-3,17-dione. Besides being high in absolute numbers, the k(cat)/K(m) value of GST A3-3 exceeds by a factor of approximately 230 that of 3beta-hydroxysteroid dehydrogenase/isomerase, the enzyme generally considered to catalyze the Delta(5)-Delta(4) double-bond isomerization. Furthermore, GSTA3-specific polymerase chain reaction analysis of cDNA libraries from various tissues showed a message only in those characterized by active steroid hormone biosynthesis, indicating a selective expression of GST A3-3 in these tissues. Based on this finding and the high activity with steroid substrates, we propose that GST A3-3 has evolved to catalyze isomerization reactions that contribute to the biosynthesis of steroid hormones.     

Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3

Glutathione transferase (GST) A3-3 is the most efficient human steroid double-bond isomerase known. The activity with Delta(5)-androstene-3,17-dione is highly dependent on the phenolic hydroxyl group of Tyr-9 and the thiolate of glutathione. Removal of these groups caused an 1.1 x 10(5)-fold decrease in k(cat); the Y9F mutant displayed a 150-fold lower isomerase activity in the presence of glutathione and a further 740-fold lower activity in the absence of glutathione. The Y9F mutation in GST A3-3 did not markedly decrease the activity with the alternative substrate 1-chloro-2,4-dinitrobenzene. Residues Phe-10, Leu-111, and Ala-216 selectively govern the activity with the steroid substrate. Mutating residue 111 into phenylalanine caused a 25-fold decrease in k(cat)/K(m) for the steroid isomerization. The mutations A216S and F10S, separate or combined, affected the isomerase activity only marginally, but with the additional L111F mutation k(cat)/K(m) was reduced to 0.8% of that of the wild-type value. In contrast, the activities with 1-chloro-2,4-dinitrobenzene and phenethylisothiocyanate were not largely affected by the combined mutations F10S/L111F/A216S. K(i) values for Delta(5)-androstene-3,17-dione and Delta(4)-androstene-3,17-dione were increased by the triple mutation F10S/L111F/A216S. The pK(a) of the thiol group of active-site-bound glutathione, 6.1, increased to 6.5 in GST A3-3/Y9F. The pK(a) of the active-site Tyr-9 was 7.9 for the wild-type enzyme. The pH dependence of k(cat)/K(m) of wild-type GST A3-3 for the isomerase reaction displays two kinetic pK(a) values, 6.2 and 8.1. The basic limb of the pH dependence of k(cat) and k(cat)/K(m) disappears in the Y9F mutant. Therefore, the higher kinetic pK(a) reflects ionization of Tyr-9, and the lower one reflects ionization of glutathione. We propose a reaction mechanism for the double-bond isomerization involving abstraction of a proton from C4 in the steroid accompanied by protonation of C6, the thiolate of glutathione serving as a base and Tyr-9 assisting by polarizing the 3-oxo group of the substrate.     

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.     

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.     

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)