A comparison of three methods of hydrolysis for estrogen conjugates

The efficiencies for estrogen conjugate hydrolysis were compared between enzyme hydrolysis, acid solvolysis and a new method, ammonolysis. Samples included: 1) crystalline 1,3,5(10)-estratriene-3, 17 beta-diol disulfate (estradiol 3,17-disulfate), 2) squirrel monkey urine collected following an intravenous injection of [2,4,6,7-H] 1,3,5(10)-estratriene-3,17 beta-diol (estradiol) and 3) a pool of human pregnancy urine. Ammonolysis demonstrated a significant increase over the other techniques in "free" estrogen yields, specifically, from estradiol 3,17-disulfate.     

Deuterium-labeled steroids for study in humans. II. Preliminary studies on estrogen production rates in pre- and post-menopausal women.

6,7-Dideuterio-3-hydroxy-1,3,5(10)-estratrien-17-one (dideuterio-estrone) and 4-deuterio-1,3,5(10)-estratriene-3,17 beta-diol (monodeuterio-17 beta-estradiol) were used for the estimation of estrogen production rates in pre- and post-menopausal women. The results are concordant with those obtained by radioisotope administration as reported in the literature. This preliminary study suggests that one or more steroids labeled with one or multiple deuterium and/or other stable isotopes may be employed for the measurement of production rates of steroid hormones which are derived from multiple precursors.     

Hydroxysteroid dehydrogenases of Pseudomonas testosteroni. Separation of a 17 beta-hydroxysteroid dehydrogenase from the 3(17) beta-hydroxysteroid dehydrogenase and comparison of the two enzymes.

When a crude extract of Pseudomonas testosteroni induced with testosterone was subjected to polyacrylamide gel electrophoresis, six bands that stained for 17 beta-hydroxysteroid dehydrogenase activity was observed. A protein fraction containing the enzyme corresponding to the fastest migrating band and devoid of the other hydroxysteroid dehydrogenase activities has been obtained. This preparation appears to be distinct from the previously isolated 3(17) beta-hydroxysteroid dehydrogenase (EC 1.1.1.51) in its chromatography properties on DEAE-cellulose, substrate and cofactor specificity, immunological properties and heat stability. The preparation appears devoid of 3alpha-, 3beta-, 11beta-, 17alpha-, 20alpha-, and 20beta-hydroxysteroid dehydrogenase activities. The enzyme transfers th 4-pro-S-hydrogen of NADH from estradiol-17beta (1,3,5(10)estratriene-3,17beta-diol) to estrone (3-hydroxy-1,3,5(10)-estratriene-17-one).     

Development of a monoclonal antibody for use in radioimmunoassay of ethynylestradiol

Production of monoclonal antibodies (MABs) to a 17 alpha-ethynyl-1,3,5 (10)-estratriene-3,17 beta-diol (ethynylestradiol, EE2) hapten is described. Culture antibodies generated by hybridized cells tested in enzyme-linked immunosorbent assay (ELISA) bound the 6-oxoethynyl-estradiol-6-(0-carboxymethyl) oxime-bovine serum albumin conjugate (EE2-6-0 CMO-BSA) but not BSA. On radioimmunoassay (RIA) with tritiated ethynylestradiol (3H-EE2), some of the antibodies demonstrated high binding affinity (Ka = 2.8 X 10(10) M-1) to EE2. Estradiol-17 beta, estrone and estriol did not show any displacement of 3H-EE2 from the MABs even at high concentration (1 X 10(4) ng/mL). Among the widely used progestins, norethynodrel and norethisterone exhibited considerable cross-reactivity (25.7-100% and 0.3-54.1%, respectively) but not levonor-gestrel with the MABs. The high affinity demonstrated by the MABs to EE2 3-sulfate but not to EE2 17-sulfate and EE2 3,17-disulfate suggests the dominant role of the 17 beta-hydroxyl group in immunogenicity.     

Conjugates of steroids and anti-cancer agents. III. The synthesis of estrynamine and certain derivatives

Propargyl amine was protected by condensing it with 2,5-hexane-dione to give 2,5-dimethyl-N-(2'-propyn-1'-yl)pyrrole (2). The latter was converted to the corresponding Grignard reagent with ethylmagnesium bromide, and then condensed with estrone tetrahydropyranyl ether to give 17 alpha-[3'-(2',5'-dimethyl-1'-pyrryl)-1'-propyn-1'-yl)-1,3 ,5( 10)- estratriene-3,17 beta-diol 3-tetrahydropyranyl ether (3), in 85% yield. Acetic acid and methanol cleaved the tetrahydropyranyl ether group, and hydroxylamine and sodium bicarbonate cleaved the pyrrole ring to give 17 alpha-(3'-amino-1'-propyn-1'-yl)-1,3,5(10)-estratriene-3,17 beta-diol (1), estrynamine. Several derivatives and analogs of 1 were also synthesized. Estrynamine binds to estrogen receptor with an RBA of 0.0045 (estradiol = 1.0). Several of the compounds, including estrynamine, are weak estrogens (stimulation of prolactin synthesis).     

Biliary metabolites of estriol in the rat

Following the subcutaneous administration of estriol-6,7-³H to rats, biliary metabolites were identified and quantitated. Approximately 70% of the metabolites were excreted in the form of “glucosiduronate” conjugates. 3, 17β-Dihydroxy-2-methoxy-1,3,5(10)-estratrien-16-one was the major metabolite in this conjugate fraction. Significant amounts of 3,17β-dihydroxy-1,3,5(10)-estratrien-16-one and 2,3,17β-trihydroxy-1,3,5(10)-estratrien-16-one, as well as smaller quantities of 1,3,5(10)-estratriene-2,3,16α,17β-tetrol and 2-methoxy-1,3,5(10)-estratriene-3,16α, 17β-triol, were also found. In 17α-ethinylestradiol - treated animals, the rate of excretion of radioactivity and the proportion of 16-oxo-17β-ol metabolites found in the “glucosiduronate” fraction were reduced.     

Comparison of the effect of 4-hydroxy-4-androstene-3,17-dione on aromatase activity in granulosa cells from preovulatory follicles of rats, rabbits, and humans

The effect of the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the synthesis of estradiol (1,3,5 (10)-estratriene-3,17 beta-diol) by granulosa cells from preovulatory follicles of rats, rabbits and humans was examined. Granulosa cells from all three species were incubated for 4 h without treatment (control) or in the presence of androstenedione (4-androstene-3,17-dione, 0.5 microM), 4-OH-A (5 microM), or both compounds together. Estradiol levels were determined in the medium and cells by radioimmunoassay. In all three species, estradiol synthesis was markedly increased by androstenedione and this increase was blocked by 4-OH-A. In the rabbit, however, 4-OH-A alone caused a small but significant increase in radioimmunoassayable estradiol. The apparent increase seen with 4-OH-A alone may be due to a metabolite of 4-OH-A that cross-reacts in the estradiol radioimmunoassay. With granulosa cells from humans, in which 4-OH-A is of potential therapeutic importance, no similar effect of 4-OH-A alone was observed.     

The preparation of 2,4-dibromoestra-1,3,5(10),6-tetraene-3,17 beta-diol diacetate.

[2,4,6,7-3H4]Estradiol is used for the quantitative determination of estradiol receptors. The synthesis of 2,4-dibromoestra-1,3,5(10),6-tetraene-3,17 beta-diol 3,17-diacetate, the convenient precursor of [2,4,6,7-3H4]estradiol, is described. 6-Alkoxy compounds were also prepared and investigated.     

Iodoestrogens, syntheses, and interaction with uterine receptors.

The rationale for undertaking the present study was to evaluate the utility of iodoestradiol analogs made highly radioactive with iodine isotopes in (a) the non-invasive differentiation of estrogen-dependent from estrogen-independent breast tumors, (b) spread of metastases containing estrogen receptors, and (c) potential application in therapeutic irradiation of target tissues. In the present paper, the model syntheses of a number of nonradioactive 127I-estrogen analogs are described. The analogs were tested for their ability to displace (compete with) [3H]estradiol from receptor sites. The most active compounds, 16beta-iodoestra-1,3,5(10)-triene-3,17 beta-diol (17) and 6-iodoestra-1,3,5(10),6-tetraene-3,17 beta-diol (10b), showed a relative binding affinity of 0.57 and 0.49, respectively.     

Precursor role of 15alpha-hydroxyestradiol and 15alpha-hydroxyandrostenedione in the formation of estetrol

Studies were designed to elucidate the origin of estetrol (15alpha-hydroxyestriol (estra-1,3,5(10)triene-3,15alpha,17beta-tetrol) or E4) during late human pregnancy. 3H-Labelled 15alpha-hydroxyestradiol (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E2) and 14C-labelled 17beta-estradiol (estra-1,3,5(10)-triene-3,17beta-diol or E2) were infused into the fetus during transfusion in utero for erythroblastosis fetalis, and in another study the same substrates were injected intravenously into the maternal circulation. In a third study, 3H-labelled 15alpha-hydroxyandrostenedion (15alpha-hydroxyandrost-4-ene-3,17-dione or 15delta4) and 14C-labelled E2 were infused into the fetus. Maternal urine was collected for 5--6 days, and after Glusulase hydrolysis, the following metabolites were isolated: estriol (estra-1,3,5(10)-triene-3,16alpha,17beta-triol or E3) containing 14C only and 15alpha-hydroxyestrone (3,15alpha-dihydroxyestra-1,3,5(10)-trien-17-one or 15E1), 15E2, and E4, all containing both labels. From the isotope content of these metabolites, it was concluded that E4 was derived from both fetal E2 and 15delta4 and only partially via 15E2. When administered to the fetus E2 and 15delta4 contributed approximately equal amounts to urinary E4. The yield of 15alpha-hydroxylated estrogens from E2 injected into the mother was very low indicating the predominantly fetal origin of the 15alpha-hydroxylase. 15delta4 was a better precursor than E2 for urinary 15E2.     

Biological activities of 4-fluoro estrogen analogues

As part of a study on the biological activity of gamma-emitting estradiol analogues, a series of fluorinated and/or brominated analogues of estradiol were synthesized. 4-Fluoro-1,3,5(10-estratrien-3,17 beta-diol (4-fluoro-estradiol) and 16 alpha-bromo-1,3,5(10)-estratrien-3,17 beta-diol (16 alpha-bromo-estradiol) had relative binding affinities (RBA) for the rabbit uterine cytosol receptor which were comparable to those of estradiol. However, while 4-fluoro-estradiol stimulated the increase in uterine weight of immature mice to the same amount as estradiol, 16 alpha-bromo-estradiol was less effective in this assay indicating that its metabolism is probably more rapid than that of estradiol. 16 beta-Bromo-1,3,5(10)-estratrien-3,17 beta-diol had a low RBA and was ineffective in stimulating uterine weight at the doses used. 4-Fluoro-16 alpha-bromo-estradiol had a lower RBA than either the 4-fluoro- or 16 alpha-bromo-estradiol and all the 4-fluoro-seleno-estrogens tested possessed low RBA's.     

Synthesis and biologic activities of 11 beta-substituted estradiol as potential antiestrogens

The effect of attachment of a dimethylaminoethoxy or a dimethylaminopropoxy group at the 11 beta-position of estradiol (E2) on its relative binding affinity (RBA) to estrogen receptor (ER) and intrinsic biologic activity is described. The binding of 11 beta-[2-(N,N-dimethylamino) ethoxy]estra-1,3,5(10)-triene-3,17 beta-diol (4) and 11 beta-[3-(N,N- dimethylamino)propoxy]estra-1,3,5(10)-triene-3,17 beta-diol (5) to the ER from immature rat uterine tissue was measured relative to that of [3H]E2 by a competitive binding assay. It was found that the 11 beta-substituted E2 analogs have considerably lower RBA to ER than the corresponding parent compound. The intrinsic activity of compounds 4 and 5 were studied in terms of uterotrophic and antiuterotrophic activity. It was found that the uterotrophic activity of these compounds was drastically reduced compared with E2. However, no antiuterotrophic activity was observed in these compounds at dosages ranging from 1 to 100 micrograms/rat/d.     

Role of estrogen in controlling the genital microflora of female rats

Plate counts of viable bacteria recovered by lavage from rat vaginae demonstrated that the number of bacteria associated with the vaginal epithelium varied cyclically and that this pattern was abolished by ovariectomy. After ovariectomy, vaginal bacterial counts remained relatively stable at low levels. The estrogen 17beta-estradiol (1,3,5(10)-estratriene-3,17beta-diol cypionate) administered to ovariectomized rats caused a significant increase in vaginal bacterial counts on day 3 post-treatment. A similar effect was seen in non-ovariectomized rats, but a larger dose of estrogen antagonist may have been present in non-ovariectomized animals. Progesterone (4-pregnene-3,20-dione) given with estradiol diminished the effect of the estrogen on vaginal bacterial counts, but did not abolish it. Progesterone administered without estradiol had no detectable effect on vaginal bacterial counts. These findings suggested that the cyclic variation in bacterial content of rat vaginae could be explained primarily as the effect of the secretory pattern of ovarian estrogen.     

Role of estrogen in controlling the genital microflora of female rats.

Plate counts of viable bacteria recovered by lavage from rat vaginae demonstrated that the number of bacteria associated with the vaginal epithelium varied cyclically and that this pattern was abolished by ovariectomy. After ovariectomy, vaginal bacterial counts remained relatively stable at low levels. The estrogen 17beta-estradiol (1,3,5(10)-estratriene-3,17beta-diol cypionate) administered to ovariectomized rats caused a significant increase in vaginal bacterial counts on day 3 post-treatment. A similar effect was seen in non-ovariectomized rats, but a larger dose of estrogen antagonist may have been present in non-ovariectomized animals. Progesterone (4-pregnene-3,20-dione) given with estradiol diminished the effect of the estrogen on vaginal bacterial counts, but did not abolish it. Progesterone administered without estradiol had no detectable effect on vaginal bacterial counts. These findings suggested that the cyclic variation in bacterial content of rat vaginae could be explained primarily as the effect of the secretory pattern of ovarian estrogen.     

Synthetic approach to analogues of 19-norsteroids with an acyclic side chain

Racemic 14 beta-hydroxy-3-methoxy-8 alpha,9 alpha-1,3,5(10)-estratriene-17-one (I), obtained by total synthesis, was converted into a derivative with alkoxycarbonyl-ethylenic side chain, rac-(20E)-21-methoxycarbonyl-19-nor-8 alpha,9 alpha-pregna- 1,3,5(10),20-tetraene-3,14 beta-diol 3-methyl ether (XII) using two Wittig reactions. Analogous derivatives of 5 alpha-androstane were prepared as synthetic models. In the estrane series the stereochemistry of attachement of the side chain in position 17, biological activity of some compounds, and their chromatographic properties were investigated.     

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.     

Mechanism of inactivation of 3-oxosteroid delta 5-isomerase by 17 beta-oxiranes

The affinity label (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) inactivates 3-oxosteroid delta 5-isomerase from Pseudomonas testosteroni by formation of a covalent bond between Asp-38 of the enzyme and the steroid. High-performance liquid chromatography (HPLC) analysis of tryptic digests of inactivated enzyme shows that two isomeric steroid-containing peptides are formed in a ratio of 9:1 at pH 7 (TPS1 and TPS2). Hydrolysis of each of these peptides produces a different steroid: TPS1 releases 17 alpha-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 beta-diol (S1) whereas TPS2 yields 17 beta-(hydroxymethyl)estra-1,3,5(10),6,8-pentaene-3,17 alpha-diol (S2). Inactivation of the enzyme by (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran-18O]-3-ol, followed by mass spectral analysis of the diacetate of the steroid released upon hydrolysis of the enzyme-inhibitor bond, reveals that TPS1 is formed by attack of Asp-38 at the methylene carbon of the oxirane. In contrast, TPS2 is produced by Asp-38 attack at the tertiary carbon. These results imply that inactivation occurs through concurrent SN1 and SN2 reactions of Asp-38 with the protonated inhibitor and that Asp-38 is located on the alpha face of the steroid when it is bound to the active site in the correct manner to react for both the SN1 and SN2 processes.     

Inactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase by 16-methylene estrone, an affinity alkylator enzymatically generated from 16-methylene estradiol-17 beta

The substrate 16-methylene estra-1,3,5(10)-triene-3,17 beta-diol (16-methylene estradiol-17 beta) and its enzyme-generated alkylating product, 3-hydroxy-16-methylene estra-1,3,5(10)-triene-17-one (16-methylene estrone), were synthesized to study the 17 beta- and 20 alpha-hydroxysteroid dehydrogenase activities which coexist in homogeneous enzyme purified from human placental cytosol. 16-Methylene estradiol, an excellent substrate (Km = 8.0 microM; Vmax = 2.8 mumol/mg/min) when enzymatically oxidized to 16-methylene estrone in the presence of NAD+ (256 microM), inactivates simultaneously the 17 beta- and 20 alpha-activities in a time-dependent and irreversible manner following pseudo-first order kinetics (t1/2 = 1.0 h, 100 microM, pH 9.2). 16-Methylene estradiol does not inactivate the enzyme in the absence of NAD+. 16-Methylene estrone (Km = 2.7 microM; Vmax = 2.9 mumol/mg/min) is an affinity alkylator (biomolecular rate constant k'3 = 63.3 liters/mol-s, pH 9.2; KI = 261 microM; k3 = 8.0 X 10(-4) S-1, pH 7.0) which also simultaneously inhibits both activities in an irreversible time-dependent manner (at 25 microM; t1/2 = 7.2 min, pH 9.2; t1/2 = 2.7 h, pH 7.0). Substrates (estradiol-17 beta, estrone, and progesterone) protect against inhibition of enzyme activity by 16-methylene estrone and 16-methylene estradiol. Affinity radioalkylation studies using 16-methylene [6,7-3H]estrone demonstrate that 1 mol of alkylator binds per mol of inactivated enzyme dimer. Thus, 16-methylene estradiol functions as a unique substrate for the enzymatic generation of a powerful affinity alkylator of 17 beta,20 alpha-hydroxysteroid dehydrogenase and should be a useful pharmacological tool.     

Spatial relationship of the vaginal microflora to the vaginal epithelium of female rats: scanning electron microscopy.

The vaginal mucosa of ovariectomized female rats has been examined by scanning electron microscopy before and after estrogen [1,3,5(10)-estratriene-3,17beta-diol] treatment. Without estrogen stimulation vaginal colonization is minimal and the epithelium is characterized by a layer of epithelial cells covered with small microvillous-like projections. Progressive changes that were consistent with estrogenic cytoproliferative effects were seen after estrogen treatment. By post-treatment day 3 bacterial colonization was maximal and the epithelium was comprised of flat squamous cells that tended to become detached from the underlying tissue layers. Bacteria were seen in association with the intercellular borders of this tissue and occurred singly or as microcolonies. No distinct physical attachment structures were identified, although an amorphous extracellular material that may serve to attach the bacteria to the squamous epithelial cells was frequently seen.     

Early events in the steroidal regulation of alpha2mu globulin in rat liver. Evidence for both androgenic and estrogenic induction.

1. A double-antibody radioimmunoassay for alpha2mu globulin has been developed. With the help of this highly sensitive radoimmunoassay the early effects of both androgen and estrogen treatments on the hepatic synthesis of alpha2mu globulin in the rat have been investigated. 2. Results show that the earlier observation of the long lag period in the androgenic induction of alpha2mu globulin is more apparent than real. 3. Single injections of either 5alpha-dihydrotestosterone(5alpha-dihydro-17beta-hydroxy-4-androsten-3-one) or its physiological antagonist estradiol-17beta (1,3,5(10)-estratriene-3,17beta-idol) to castrated female rats resulted in the induction of alphamu globulin reaching maximum hepatic level of the protein between 6--9 h after the hormone administration. Administration of cycloheximide 15 prior to hormone treatment blocked both androgenic andestrogenic induction of alpha2mu globulin. 4. Daily pretreatments with 5alphamu-dihydrotestosterone increased the sensitivity of subsequent androgenic response to alpha2muglobulin synthesis. On the other hand, daily pretreatments with estradiol 17beta decreased and ultimately abolished the estrogenic induction of alpha2mu globulin. 5. The possible mechanism of both androgenic and estrogenic induction of alpha2mu globulin in rat liver mediated through a sex-hormone-binding protein with dual affinity for both dihydrotestosterone and estradiol has been suggested.