Placental Microsomes Research Articles

Evidence of conversion of estradiol 17-sulfate to its 2- and 4-hydroxylated catechols by human placental microsomes.

When estradiol 17-sulfate (ES) was incubated with human placental microsomes under an NADPH-generating system, 2- and 4-hydroxyestradiol 17-sulfates (2- and 4-OH-ES) formed as the main products. By kinetic experiments, the apparent Km values of 44.0 and 360 microM and apparent Vmax values of 236 and 140 pmol/mg protein/10 min were obtained for the 2- and 4-hydroxylation, respectively. The results indicate that human placental microsomes have fairly high 2- and 4-hydroxylase activities toward ES. This paper describes the formation of 2- and 4-OH-ES and speculates on their physiological role during pregnancy.

Immunochemical specificity of placental NADPH cytochrome c ( P-450) reductase in neoplastic and non-neoplastic human tissue

NADPH cytochrome c ( P-450) reductase was purified from human placental microsomes using a combination of affinity and gel filtration chromatography. Affinity chromatography using agarose-hexane-adenosine 2′5 diphosphate resulted in two protein bands being detected by SDS-PAGE of approximate M Ws 68 and 75 kDa. Fractions containing the two proteins were pooled, and then resolved using Sephacryl S-200. Both of the purified proteins displayed enzyme activity, measured by their ability to reduce cytochrome c. The 75 kDa protein obtained was used to immunize three female New Zealand white rabbits. The IgG fraction was partly purified from rabbit sera which suppressed placental microsomal NADPH cytochrome c reductase activity by >80% using 33% ammonium sulphate. The procured antibody suppressed androstenedione aromatase activity in microsomal preparations of human placental and breast adipose tissue, and NADPH cytochrome c reductase activity in prostate (benign and malignant), MDA-MB-231 breast cancer cells, breast adipose, Hep G2 hepatoma cells and placental microsomal preparations. The extent of NADPH cytochrome c reductase inhibition varied in the order of malignant prostate < benign prostate<MDA<breast adipose<Hep G2<placenta. The results suggest that human placental NADPH cytochrome c ( P-450) reductase shares common antigenic epitopes pertinent to its capability of reducing cytochrome c in all of the above-mentioned tissues. In attempting to associate possible changes in NADPH cytochrome c reductase activity imposed by neoplasia to the obtained immunochemical cross reactivity and enzyme activity results, it was noted that microsomes obtained from MDA cells exhibited enzyme activity significantly less than that of breast adipose microsomes (1.6 and 8.1 nmol/min/mg protein, respectively) and by comparison showed 6% less homology towards the placental antibody. The results obtained for benign and malignant prostate showed no significant difference between the neoplastic states as adjudged by enzyme activity and immunochemical assays.

7-Alkoxyquinoline O-dealkylation by microsomes from human liver and placenta.

1. The O-dealkylation of seven 7-alkoxyquinoline derivatives by human hepatic and placental microsomes and the effect of maternal cigarette smoking on placental 7-alkoxyquinoline metabolism was studied. 2. None of several monoclonal antibodies to isoenzymes of cytochrome P450 had a clear effect on metabolism of the compounds by liver microsomes. 3. Maternal cigarette smoking induced the O-dealkylation of all of the 7-alkoxyquinoline derivatives, being greatest for 7-butoxy- and 7-benzyloxyquinoline. 4. Placental 7-alkoxyquinoline metabolism induced by smoking was partially inhibited by the monoclonal antibody 1-7-1 raised against 3-methylcholanthrene-induced rat liver P450. 5. None of the 7-alkoxyquinoline O-dealkylations could be assigned specifically to any known P450 isoenzyme in human liver or placenta.

Aromatase and testosterone fatty acid esters: the search for a cryptic biosynthetic pathway to estradiol esters

The estradiol fatty acid esters (lipoidal derivatives, LE 2) are extremely potent estrogens that accumulate in fat, including fat of menopausal women. These steroidal esters are protected from metabolism and are converted to the free, biologically active steroid through the action of esterases. Previous studies have shown that biosynthetic pathways in the adrenal gland exist in which steroid fatty acid esters are substrates. This led us to determine whether a cryptic aromatase pathway exists in which testosterone esters could be converted directly into LE 2. We tested a representative fatty acid ester, testosterone stearate, both as an inhibitor and as a substrate for the aromatase enzyme from human placental microsomes. This ester had neither activity. In addition, we tested [1β- 3H]testosterone acetate as a substrate for this enzyme complex, measuring the production of 3H 2O as evidence of aromatization. Although the rate of reaction was considerably slower than that of testosterone, 3H 2O was produced. However, when [2, 4, 6, 7- 3H]testosterone acetate was incubated and the steroidal products isolated, we found that hydrolysis of the substrate had occurred. Both [ 3H]-labeled testosterone and estradiol were found, and very little if any [ 3H] estradiol acetate was formed. Thus, we conclude that an aromatase pathway involving testosterone esters does not exist and that the sole source of LE 2 is through direct esterification of estradiol.

A study of metabolism of deaminated and sulfoconjugated iodothyronines by rat placental iodothyronine 5-monodeiodinase

The interaction of the rat placental type III iodothyronine 5-monodeiodinase (5-MD) with acetic acid (AA), propionic acid (PA), and sulfoconjugate (SA) derivatives of thyroid hormones has been investigated in comparison with hepatic iodothyronine type I MD. PA and AA derivatives of both T3 and T4 were potent inhibitors of 5-monodeiodination of [125I]T3 by rat placental microsomes. 3,5,3'-Triiodothyroacetic acid (T3AA) and 3,5,3'-triiodothyropropionic acid (T3PA) were comparable to T3 in their ability to inhibit 5-monodeiodination of [125I]T3, whereas T4AA and T4PA were more potent than T4. 3,5,3'-triiodothyrosulfonic acid (T3SA), T4SA, and rT3SA caused little or no inhibition of placental 5-MD activity. Among various analogs of T3 or T4, the order of relative potency of inhibition of hepatic 5'-MD was PA > AA > SA > parent iodothyronine. The metabolism of T3 and its derivatives by rat placental microsomes was studied by determining the rates of disappearance of the various substrates and the production of the metabolites generated by inner ring monodeiodination of the substrate. T3AA and T3PA were metabolized at a rate comparable to that of T3. Under the same conditions, essentially 100% of T3SA remained intact. Kinetic studies of placental inner ring monodeiodination of T3, T3AA, and T3PA demonstrated comparable values for Km (1.3, 1.8, and 2.3 nM, respectively) and maximum velocity (44, 57, and 74 fmol/micrograms.h, respectively). All derivatives of T3 studied were deiodinated by hepatic type I MD more avidly than the parent iodothyronine. Our data suggest that 1) deamination does not appreciably influence, while sulfoconjugation markedly inhibits type III 5-monodeiodination of T3; and 2) deamination may be even more conducive to degradation of thyroid hormone than sulfoconjugation.

A study of metabolism of deaminated and sulfoconjugated iodothyronines by rat placental iodothyronine 5-monodeiodinase.

The interaction of the rat placental type III iodothyronine 5-monodeiodinase (5-MD) with acetic acid (AA), propionic acid (PA), and sulfoconjugate (SA) derivatives of thyroid hormones has been investigated in comparison with hepatic iodothyronine type I MD. PA and AA derivatives of both T3 and T4 were potent inhibitors of 5-monodeiodination of [125I]T3 by rat placental microsomes. 3,5,3'-Triiodothyroacetic acid (T3AA) and 3,5,3'-triiodothyropropionic acid (T3PA) were comparable to T3 in their ability to inhibit 5-monodeiodination of [125I]T3, whereas T4AA and T4PA were more potent than T4. 3,5,3'-triiodothyrosulfonic acid (T3SA), T4SA, and rT3SA caused little or no inhibition of placental 5-MD activity. Among various analogs of T3 or T4, the order of relative potency of inhibition of hepatic 5'-MD was PA > AA > SA > parent iodothyronine. The metabolism of T3 and its derivatives by rat placental microsomes was studied by determining the rates of disappearance of the various substrates and the production of the metabolites generated by inner ring monodeiodination of the substrate. T3AA and T3PA were metabolized at a rate comparable to that of T3. Under the same conditions, essentially 100% of T3SA remained intact. Kinetic studies of placental inner ring monodeiodination of T3, T3AA, and T3PA demonstrated comparable values for Km (1.3, 1.8, and 2.3 nM, respectively) and maximum velocity (44, 57, and 74 fmol/micrograms.h, respectively). All derivatives of T3 studied were deiodinated by hepatic type I MD more avidly than the parent iodothyronine. Our data suggest that 1) deamination does not appreciably influence, while sulfoconjugation markedly inhibits type III 5-monodeiodination of T3; and 2) deamination may be even more conducive to degradation of thyroid hormone than sulfoconjugation.

Patent Evaluation: Novel Non-Steroidal Aromatase Inhibitors

SummaryNovelty: Fluoromethylphenyl derivatives bearing a heterocyclic group are disclosed as aromatase inhibitors. These compounds are potentially useful in the treatment of oestrogen-dependent illnesses such as gynaecomastia and breast cancer.Biology: No specific data are provided, however the compounds are said to inhibit aromatase activity in vitro in placental microsomes. IC50 values below 10–9 M are reported for the conversion of [14C]-androstenedione to [14C]-oestrogen. The compounds are also said to inhibit aromatase in vivo in rats with MED values between 0.001–1.0 mg/kg sc, and to promote regression of DMBA-induced mammary tumours in rats at doses of 1.0 mg/kg po.Chemistry: Synthesis of the compounds is outlined in a series of multistep preparative schemes, forming a subsidiary part of the claim and exemplified in eighteen cases. The compound 4-[α-(4-cyanophenyl)-α-fluoro-1-(1,2,3-triazolyl) methyl]benzonitrile is the only specifically claimed compound.Structure:

Aromatase activity in cultured brain cells: difference between neurons and glia

At the level of the central nervous system (CNS) of several mammalian and non-mammalian species, estrogens may be intracellularly formed from circulating androgens through the action of the aromatase complex. Estrogenic steroids play a crucial role in organizing and directing certain behavioral and neuroendocrine responses both during the fetal/neonatal life and in adulthood. Biochemical and immunocytochemical studies have shown that the aromatase is particularly concentrated in CNS areas involved in the control of reproductive functions, such as the hypothalamus, the preoptic area and the limbic system; despite this large body of evidence, the exact cellular localization of this enzymatic complex within the different cell populations of the brain is still uncertain. In the experiments described here, the presence of the aromatase has been evaluated in the two main cellular components of the brain: the neurons and the glia. In these experiments, cultures of neurons obtained from the brains of 14–15-day-old rat embryos, mixed glial cells from 1-day-old rats and type 1 astrocytes derived from cultured glial cells, have been utilized. The aromatase has been also evaluated in oligodendrocytes isolated from adult male rat brain by density gradient ultracentrifugation. The aromatase activity has been assayed by an ‘in vitro’ radiometric method which quantifies the production of tritiated water from [1β- 3H]-androstenedione as an index of estrogen formation. The validity of the method has been verified both on the placental microsomes and on rat hypothalamic tissue, in which the actual formation of estrogens has also been measured. Among the different cells tested, only neurons possess the aromatase complex to a significant degree, while mixed glial cell and astrocyte preparations have an extremely low enzymatic activity: oligodendrocytes seem to be completely inactive in the respect. The calculation of the kinectic parameters of the neuronal aromatase has shown that this enzymatic activity is linear with respect to the protein content and the incubation time. The neuronal aromatase follows a Michaelis-Menten model with a K m of 52 nM: this value is analogous to that calculated for other central or peripheral structures in which the enzyme is known to be present.

Evidence that the human placental 5-monodeiodinase is a phospholipid-requiring enzyme.

Gel filtration (GFI) of the solubilized human placental microsomes (SHPMP) performed in an Ultrogel AcA-34 column in the presence of 1 mM 3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS) plus 10 mM n-octyl-beta-D-glucopyranoside (beta-OG) demonstrated two main protein peaks. The 5-Monodeiodinase (5-MD) activity measured by the conversion of [125I]T3 to [125I]3,3'-diiodothyronine in a 2- to 18-h incubation at 37 C in the presence of 10 mM dithiothreitol was detected only in the first peak, and the specific activity was increased about 9 times over that of the starting SHPM. The fractions containing most of the 5-MD activity were filtered through a second Ultrogel AcA34 column (GFII) in the presence of 2 mM CHAPS plus 20 mM beta-OG. In these conditions, 5-MD activity was detected in low amounts only in the second peak. Cation exchange chromatography on carboxymethylcellulose-Sephadex with a starting buffer of pH 5 containing 2 mM CHAPS plus 20 mM beta-OG, followed by a pH 8 buffer, showed a very small OD peak at the void volume (P) and a second peak with about 95% of the protein (E). However, no 5-MD activity was detectable in either peak, while a nearly complete restoration of the enzyme was achieved when P and E were mixed. 5-MD was also completely restored by combination of P with the first inactive peak of GFII. When P was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no distinct protein bands were observed. After ethanol-ether extraction and digestion with H2SO4 and H2O2, inorganic phosphate was detectable only in P, suggesting the presence of phospholipids. We next studied the effect of phosphatidyl serine (PS), phosphatidyl choline (PC), or phosphatidyl ethanolamine (PE) on 5-MD activity of E (5 micrograms protein/mL). The 5-MD activity was recovered in a dose-response manner with all phospholipids studied, but PS was the most effective agent for reconstitution. At 1 microgram/mL, 5-MD activity, expressed as a percentage of the total P plus E activity, was 101% for PS, 35% for PC, and 20% for PE. The addition of rat liver or kidney microsomes (80 micrograms/mL) to E (5 micrograms/mL) provided recoveries of 79% and 48%, respectively, of the total P plus E activity. The following conclusions were reached. 1) Phospholipids are essential for the 5-MD activity of SHPMP. 2) CHAPS and beta-OG may extract phospholipids from the membranes without denaturation of the 5-MD.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity labeling of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase: evidence for bifunctional catalysis by a different conformation of the same protein for each enzyme activity.

3 beta-Hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase copurify from human placental microsomes as a single enzyme protein. The affinity-alkylating secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivates the dehydrogenase and isomerase reactions in a time-dependent manner, but which of the two activities is targeted depends on the concentration of secosteroid. At 2-5 microM secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (KI = 4.2 microM, k3 = 1.31 x 10(-2) min-1). As the secosteroid level increases from 11 to 30 microM, dehydrogenase is paradoxically inactivated at progressively slower rates, and pregnenolone no longer protects against the alkylator. The inactivation of isomerase exhibits the expected first-order kinetics (KI = 31.3 microM, k3 = 6.42 x 10(-2) min-1) at 11-30 microM secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15 microM secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5 microM). A shift from a dehydrogenase to an isomerase conformation in response to rising secosteroid levels explains these results. Analysis of the ligand-induced conformational change along with cofactor protection data suggests that the enzyme expresses both activities at a bifunctional catalytic site. According to this model, the protein begins the reaction sequence as 3 beta-hydroxysteroid dehydrogenase. The products of the first step (principally NADH) promote a change in protein conformation that triggers the isomerase reaction.

Vanadium redox cycling, lipid peroxidation and co-oxygenation of benzo(a)pyrene-7,8-dihydrodiol

Mechanism of lipid peroxidation triggered by vanadium in human term placental microsomes was reinvestigated in vitro. Production of lipid peroxyl radicals was estimated from co-oxygenation of benzo(a)pyrene and benzo(a)pyrene-7,8-dihydrodiol. Vanadyl(IV), but not vanadate(V) caused a dose-dependent co-oxygenation. Vanadate(V) required the presence of reduced nicotinamide adenine dinucleotide phosphate to trigger co-oxygenation of benzo(a)pyrene-7,8-dihydrodiol. To determine the role of pre-formed lipid hydroperoxides, the results obtained with partially peroxidized linoleic acid were compared with those of fresh linoleate. Superoxide dismutase inhibited the co-oxygenation of reaction when fresh linoleic acid was used. To further characterize the role of Superoxide anion-radical in the vanadium redox cycling, the increase of optical density of vanadate(V) dissolved in Tris buffer was measured at 328 nm during the addition of KO 2. The rate of this reaction producing peroxy-vanadyl complex was decreased by Superoxide dismutase, especially, in the presence of catalase. It is suggested that vanadium catalyzes two separate processes, both leading to enhanced lipid peroxidation: (i) initiation, dependent on Superoxide and triggered by peroxy-vanadyl; (ii) propagation, dependent on pre-formed lipid hydroperoxide and not sensitive to Superoxide dismutase. It is postulated that the vanadium-triggered initiation of lipid peroxidation may be crucial for toxicity in organs with limited endogenous lipid peroxidation.

RU54115, a tight-binding aromatase inhibitor potentially useful for the treatment of breast cancer

RU54115 is a new potent inhibitor of aromatase. In vitro, it inhibits the enzyme from human placental microsomes with a K i of 0.5 nM, which places it among the tightest reported steroidal inhibitors of aromatase. In vivo, it lowers the amount of circulating estradiol in pregnant mare serum gonadotrophin (PMSG)-primed female rats with an ED 50 of 0.4 mg/kg when given s.c. and 4 mg/kg when given orally. An oral dose of 25 mg/kg when given once daily to female rats was able to inhibit the growth of DMBA-induced mammary tumors.

Biochemical and molecular genetic analyses on placental aromatase (P-450AROM) deficiency.

Biochemical and molecular genetic studies were made on a case of placental aromatase (P-450AROM) deficiency. Of the enzymes participating in the electron transport system of placental microsomes, only aromatase activity was decreased specifically in the patient, being less than 0.3% of the normal activity. Northern and Western blotting analyses showed that the transcription of the aromatase gene and the translation of its mRNA proceeded normally in the placenta of this patient. However, aromatase cDNA isolated from a placental cDNA library of the patient was found to have an insert of 87 base pairs, encoding 29 amino acids in frame with no termination codon. The insert was located at the splicing point between exon 6 and intron 6 of the normal aromatase gene, and the extra DNA fragment was the first part of intron 6, except that its initial GT was altered to GC. These findings indicated that in this patient with aromatase deficiency, splicing between exon 6 and intron 6 did not occur at the normal position because of a point mutation in its consensus sequence and was forwarded to GT in the next cryptic consensus sequence 87 base pairs downstream according to the canonical GT/AG rule, resulting in translation of an abnormal protein molecule with 29 extra amino acids. During the transient expression in COS-7 cells, the aromatase cDNA of the patient was found to produce a protein with a trace of activity. This is the first report of a genetic defect for aromatase deficiency.

The physiological role of estradiol 17-sulfate during pregnancy

To clarify the physiological role of estradiol 17-sulfate (ES) during pregnancy, experiments were conducted and the following results were obtained: (1) serum or urinary ES levels rose as a function of gestational age; (2) placental microsomes showed fairly high 2- and 4-hydroxylase activity for ES; and (3) the catechol products, 2- and 4-hydroxy-ES, had a strong inhibitory effect upon the in vitro production of lipid peroxides. These results suggest that ES actsas a precursor to the catechol metabolites which maintain normal gestation. This is coincident with the negative correlation of serum levels in ES and lipid peroxides observed in late pregnancy.

Ridogrel: A selective inhibitor of the cytochrome P450-dependent thromboxane synthesis

Ridogrel {( E)-5-[[[(3-pyridinyl)[3-( trifluoromethyl)phenyl]methylene]amino]oxy] pentanoic acid} is a potent inhibitor of the P450-dependent human platelet thromboxane A 2 (TxA 2) synthase. Fifty percent inhibition is already achieved at 5.0 ± 0.37 nM. This ic 50 value is close to half the P450 concentration used, i.e. 10.7 nM. Ridogrel binds to human platelet microsomal P450 as proven by the type II spectral changes induced by the addition of increasing concentrations of ridogrel to solubilized microsomes. The calculated half-maximal spectral change sc 50 value) is 3.78 ± 1.79 nM. These results indicate that ridogrel binds stoichiometrically and suggest that inhibition of thromboxane synthesis may originate from liganding of its basic nitrogen to the haem-iron of P450 and from the attachment of the hydrophobic carboxylic side chain to or near the substrate binding place. Ridogrel is a selective inhibitor of the TxA 2 synthase. At a high concentration (10 μM), ridogrel has a slight, if any, effect on the P450-mediated cholesterol synthesis in human liver and hepatoma cells and androgen synthesis from 17α-hydroxy-20-dihydroprogesterone or pregnenolone in subcellular fractions from rat testes. These results indicate that ridogrel is a poor inhibitor of the P450-dependent 14α-demethylase, 17α-hydroxylase and 17,20-lyase. It has, up to 10 μM, no effect on the adrenal mitochondrial 11β-hydroxylase and cholesterol side-chain cleavage enzyme and does not inhibit aromatase activity in human placental microsomes. Ridogrel has no significant effect on the regio- and stereoselective P450-dependent oxidations of testosterone in liver microsomes from unpretreated or from 5-pregnen-3β-ol-20-one-16α-carbonitrile-, phenobarbital- or 3-methylcholanthrene-pretreated male and female Sprague-Dawley rats. It does not interfere with the reduction of testosterone into 5α-dihydrotestosterone and 5α androstane 3β,17β-diol.

Relationship between activities of cytochrome P-450 monooxygenases in human placental microsomes and binding of benzo(a)pyrene metabolites to calf thymus DNA.

Benzo(a)pyrene metabolism in human placental microsomes from smokers was studied. Benzo(a)pyrene metabolites were separated using high pressure liquid chromatographic technique. Reaction of benzo(a)pyrene with a microsomal fraction of placenta from individuals who smoke cigarettes during pregnancy yields 7,8 dihydroxy benzo(a)pyrene as a major metabolite, while 3'-hydroxy benzo(a)pyrene, 4,5 dihydroxy benzo(a)pyrene and quinones constitute minor metabolites. The activities of arylhydrocarbon hydroxylase and 7-ethoxycoumarin deethylase exhibited much higher activities in smokers than in nonsmokers. Examination of specific binding of monoclonal antibodies to cytochrome P-450 isozymes in placental microsomes revealed that cigarette smoking specifically enhanced the level of cytochrome P-450 c and d isozymes in human placental microsomes. Coincubation of 3H-benzo(a)pyrene and calf thymus DNA with placental microsomes yielded acid insoluble 3H-B(a)P from smokers, suggesting that cigarette smoking may induce placental enzymes which convert benzo(a)pyrene into ultimate metabolites to form carcinogen-DNA adducts.

Purification and Properties of Steroid Sulfatase from Human Placenta.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.

Evidence of direct conversion of testosterone sulfate to estradiol 17-sulfate by human placental microsomes.

Ring A aromatization of testosterone sulfate (TS), using human term placental microsomes as a enzyme source, was examined under a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-generating system. The aromatization to estradiol 17-sulfate (ES) without removal of the C-17 conjugate group was confirmed. Judging from the results of various experiments, including an inhibition experiment, the conversion was considered to occur by placental aromatase. Apparent Km and Vmax values for this aromatization were determined to be 37.4 microM and 330 pmol/mg protein/min respectively. The physiological role of direct conversion of TS by placenta to ES is discussed in connection with pregnancy.

Aromatase inhibition by 7-substituted steroids in human choriocarcinoma cell culture

Androstenedione analogs containing 7α-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, 7-substituted 4,6-androstadiene-3,17-diones were synthesized and demonstrated competitive inhibition of aromatase activity in human placental microsomes. 7-Substituted 1,4,6-androstatriene-3,17-diones demonstrated mechanism-based inhibition of placental aromatase acitivity. These agents were evaluated for inhibition of aromatase activity in the JAr human choriocarcinoma line. The 7-substituted 4,6-androstadiene-3,17-diones produced dose dependent inhibition of aromatase activity in the cell cultures, with IC 50 values ranging from 490 nM to 4.5 μM. However, these agents are less effective when compared to other steroidal inhibitors, such as 7α-thiosubstituted androstenediones. These results on the 7-substituted 4,6-androstadiene-3,17-diones are consistent with the data from biochemical enzyme inhibition studies using human placental aromatase. On the other hand, 7-phenethyl-1,4,6-androstatriene-3,17-dione exhibits greater inhibitory activity, with an IC 50 value of 80 nM. Other mechanism-based inhibitors, 7α-(4′-amino)phenylthio-1,4-androstadiene-3,17-dione and 4-hydroxyandrostenedione, also exhibited potent inhibition of aromatase acitivity in JAr cells. In summary, the most effective B-ring modified steroidal aromatase inhibitors are those derivatives that can project the 7-aryl substituent into the 7α-position.

A radiometric assay method for aromatase activity using [1 beta-3H]16 alpha-hydroxyandrostenedione.

[1 beta-3H]16 alpha-Hydroxyandrostenedione (16 alpha-OHA) (715 mCi/mmol) was prepared from commercially available [1 beta-3H]androstenedione (A) by the microbiological method with Streptomyces roseochromogenes and its structure and purity were determined by chromatographic and reverse isotope dilution methods. When [1 beta-3H]16 alpha-OHA was incubated with human placental microsomes and reduced nicotinamide adenine dinucleotide phosphate (NADPH), 3H2O-release into the medium was dependent upon protein concentration and incubation time. An apparent Km and Vmax of the microsomal aromatase for the [1 beta-3H]substrate were 650 nM and 34 pmol/min/mg protein, respectively. In this assay, aromatase activity could be determined as low as 0.1 nmol estrogen formation/min/mg protein. 3-Deoxyandrostenedione, a potent competitive inhibitor of the A aromatization, also blocked the 16 alpha-OHA aromatization in a competitive manner with Ki of 15 nM.