Intratesticular steroid levels and their hormonal control

Litter-mate adult male rats were treated with daily intramuscular injections of ACTH (10.5 micrograms), dexamethasone (2.0 mg), ethynyl estradiol (1.7 micrograms) and hCG (5 IU) for three consecutive days. The animals were sacrificed on the fourth day and the intratesticular and peripheral plasma steroid levels were analyzed. The steroids measured by radioimmunoassay included pregnenolone, 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone, 17-hydroxyprogesterone, androstenedione, testosterone and dihydrotestosterone. In addition, the sulphoconjugated forms of pregnenolone, dehydroepiandrosterone, testosterone and dihydrotestosterone were estimated in the peripheral blood. The administration of ACTH diminished the intratesticular levels of all steroids studied. Also dexamethasone and ethynyl estradiol treatment suppressed all intratesticular steroid levels, except that of pregnenolone (the former) and of 17-hydroxyprogesterone (the latter). The suppressive effect of ethynyl estradiol was strongest on the levels of the delta 5-steroids and that of dexamethasone on the delta 4-steroids; the latter was significantly stronger than the effect of ACTH. The stimulatory effect of hCG was limited to the metabolism of progesterone and was restricted to the sequence: 17-hydroxyprogesterone----androstenedione----testosterone---- dihydrotestosterone. Dexamethasone-suppression, and hCG-stimulation of the intratesticular levels of delta 4-steroids, was mirrored by corresponding changes in the peripheral plasma levels, with the exception of the plasma levels of androstenedione which were not influenced by any of the treatments studied. Also the suppression of intratesticular testosterone and dihydrotestosterone levels by ACTH, dexamethasone, or ethynyl estradiol was closely reflected by their plasma levels both in the unconjugated and sulphoconjugated forms. On the hand, the administration of ACTH diminished the intratesticular levels of pregnenolone and progesterone but significantly increased those in the plasma. Moreover, both ACTH and ethynyl estradiol reduced the levels of all delta 5-steroids in testicular tissue, but not in the peripheral plasma, although they decreased the circulating levels of pregnenolone sulphate and dehydroepiandrosterone sulphate. The data are interpreted as suggesting that the hormonal agents studied interfere with testicular steroidogenesis through different mechanisms.     

Medroxyprogesterone acetate in human serum

Conjugates of medroxyprogesterone acetate (MPA) in human serum are investigated using chromatography and techniques (equilibrium dialysis, gel filtration, and polyacrylamide gel electrophoresis) previously described for studying the binding of MPA. 17 serum samples were obtained from 7 women at various times after the intramuscular injection of 150 mg Depo-Provera. Mean concentration of MPA in the unconjugated fraction of serum was 3.9 mg/ml (range 0.8-10.7 ng/ml); in the conjugated fraction, the value was 2.7 ng/ml (range 0.6-11.4 ng/ml), a mean value of 81.7% (range 18.4-286%) of that in the unconjugated fraction. The conjugate appears to be mainly a glucuronide since solvolysis released only small amounts of MPA. MPA metabolites were also detected in blood. The MPA levels in blood measured by radioimmunoassay were generally lower when serum was extracted with an organic solvent rather than when the assay was carried out directly in the serum. This finding suggests the presence in blood of either MPA in a conjugated form or metabolites interacting with the antiserum which were not extracted by the solvents used. Equilibrium dialysis showed that undiluted plasma bound 85.8% of triated hydrogen-MPA; with increasing dilution of the plasma, the amount of bound triated hydrogen-MPA decreased. The apparent association constant calculated according to the method of Vermeulen and Verdonck was 2.6 x 10 4 1/mol. MPA appeared to be loosely bound to albumin in blood but there was no specific binding protein for the steroid. MPA conversion to the glucuronide may be 1 of the factors regulating the level of the unconjugated but presumably biologically active steroid in blood.     

Serum albumin stimulates serotonin uptake into human blood platelets

Active uptake of serotonin (5-hydroxytryptamine, 5-HT) into blood platelets from healthy donors exhibited a lower Vmax value in buffer media than in plasma. Also in plasma ultrafiltrate Vmax was reduced, but it returned to the level measured in plasma upon addition of human serum albumin (40 milligrams) containing fatty acids. Fatty-acid-free albumin was even more stimulatory and when added to platelets in a phosphate-buffered medium, it increased Vmax beyond the value observed in plasma. Km values calculated on the basis of unbound 5-HT were not affected by the media except for a slight decrease in ultrafiltrate as compared to plasma. The fraction of 5-HT (0.5 mumol/l) bound to 40 milligrams albumin was 17% with the preparation containing fatty acids and 22% with fatty-acid-free albumin, while total plasma proteins dissolved in phosphate buffer bound 24%. The uptake of 1 mumol/l 5-HT was enhanced by both albumin preparations already at 1 milligram and near-maximal effects occurred at 10 milligrams.     

Human pharmacokinetics of ethynyl estradiol 3-sulfate and 17-sulfate

Pharmacokinetic parameters of ethynyl estradiol 3-sulfate (EE-3) and 17-sulfate (EE-17) were estimated. Each sulfate was administered orally and intravenously to five ovariectomized volunteer women. Blood samples were taken over a period of 24 h. Radioimmunoassay for free and sulfoconjugated ethynyl estradiol (EE) was performed. The analysis of the plasma concentrations obtained after administration of EE-3 and EE-17 indicates significant differences in their pharmacokinetic profiles. EE-3 is cleared more rapidly from the central compartment (systemic circulation), which may indicate that differences in protein binding, tissue binding, metabolism, and distribution exist between EE-3 and EE-17. It has been suggested that these conjugates are a slow-release reservoir for maintenance of blood levels of free EE itself. However, previous studies in baboons have shown that the half-lives of the free and sulfoconjugated EE are similar (ranging from 8.8 to 11.2 h), which is not consistent with this hypothesis. The t1/2 beta (mean 9.28 h) of the 17-sulfate after IV administration was almost identical in women and baboons, and similar to the t1/2 beta of free EE, confirming the previous observation. Only 3.4% of IV and 11.4% of the orally administered 17-sulfate appeared in the blood as free EE; with the 3-sulfate, the conversions were 13.7 and 20.7%, respectively, suggesting that these sulfates are not important slow-release reservoirs. The similarity of pharmacokinetic parameters between women and baboons suggests that this species of nonhuman primate is, in important respects, a suitable animal model for clinical pharmacology.     

Electrophoretic characterization of purified bovine, porcine, murine, rat, and human uterine estrogen receptors

The calf uterine estrogen receptor (E2R) in the presence of sodium molybdate has been purified, 7,000-fold by a single passage over an estradiol affinity column. A dominant 70,000-dalton band and two minor bands at 50,000 and 30,000 daltons were observed by electrophoretic analysis. These bands had been eluted using estradiol, sodium sulfocyanate, CHAPS, and HEPES (pH 7.4) with insulin as a carrier protein. The identities of the protein bands were initially confirmed by their failure to bind the affinity column when saturated with estradiol. This single step purification procedure was reproducible and rapid, with yields of 10-20%, providing 25% purity. Diffusion blot analysis, with specific 35S- and 125I-labeled monoclonal antibodies to E2R, confirmed that the 70,000-dalton band represented the estrogen receptor. Specificity was demonstrated by inhibition of binding of purified E2R by both estradiol and diethylstilbestrol but not testosterone, progesterone, corticosterone, aldosterone, or hydrocortisone. The relative binding affinity of the purified receptor was: ethynyl estradiol greater than 17 beta estradiol greater than estriol greater than or equal to estrone greater than or equal to 17 alpha-estradiol greater than mestranol. Pig, human, mouse, and rat uterine estrogen receptors were similarly purified with the affinity column. As with the calf uterine preparations, a dominant 70,000-dalton band with minor bands at 50,000 and 30,000 daltons was identified by diffusion blot analysis in all the species examined.     

2-Iodoestradiol binds with high affinity to human sex hormone binding globulin (SHBG)

Human sex hormone binding globulin (SHBG) binds a set of steroids that differ slightly from each other in structure. Dihydrotestosterone and testosterone are bound with high affinity by SHBG whereas estradiol is bound with a lower affinity. In this work we have studied the binding to human SHBG of the derivatives obtained by substituting iodine in the aromatic A-ring of estradiol. Three A-ring iodinated estradiol derivatives, 2-iodoestradiol, 4-iodoestradiol and 2,4-di-iodoestradiol, were obtained by treating 17 beta-estradiol with NaI and Chloramine T and separating the reaction products by HPLC. Their structures were confirmed by mass spectrometry and 1H-NMR. The corresponding radioactive compounds were obtained with use of Na[125I] in the same synthesizing procedure. Incubation of whole serum, serum albumin and purified SHBG with each of the three [125I]iodoestradiols followed by agarose gel electrophoresis showed only 2-iodoestradiol to have a strong binding to SHBG. This steroid was also bound to albumin, but with a lower affinity. Besides SHBG and albumin, there were no other binders of 2-iodoestradiol in human serum. The affinity constant for the binding of 2-iodoestradiol to purified human SHBG at 37 degrees C and physiological pH was determined by a dextran-coated charcoal method to be 2.4 x 10(9) M-1 (i.e. exceeding that of dihydrotestosterone). It was found that 0.9 mol of 2-iodoestradiol was bound per mol of SHBG dimer (93 kDa) at saturation, and that 2-iodoestradiol competed with dihydrotestosterone for the same binding site of SHBG. It was concluded that 2-iodoestradiol has a remarkably high affinity for human SHBG, and that its gamma-emitting 125I-analog is useful for binding studies of human SHBG.     

Comparative survey of blood thyroxine binding proteins in turtles

The nature of plasma thyroxine (T4) binding activity was surveyed in turtles; binding to [125I]T4 was measured on polyacrylamide gel electrophoresis--PAGE--and on minicolumns of Sephadex G-25. An electrophoretically distinct T4 binding protein was identified in all 8 species of Pseudemys studied and in 3 other genera (Chrysemys, Deirochelys, and Emyoidea) of the same family, Emydidae. Levels of this binding activity were highly variable among individuals, but they consistently showed a similar low relative mobility (Rf) compared to albumin, and a relatively low capacity was indicated by displacement with unlabeled T4. Two emydids (Terrapene, Clemmys) showed a similar slow migrating binding peak, but binding activity was low and not as easily displaced by unlabeled T4. T4 binding to albumins was minimal in most of these emydid species, even when binding to the higher affinity, low capacity component was low or displaced by unlabeled T4 (2.5 micrograms/ml). In contrast, there was no clear evidence for a similar high affinity, low capacity binding protein in any of the other 19 species representing 13 genera of 8 families from two suborders. In these species, binding activity on Sephadex G-25 was typically low and binding on PAGE was associated largely with albumin; binding levels for albumins were highly variable. In several nonemydids (from distant lineages), binding activity on Sephadex was elevated and PAGE showed a second binding protein distinct from albumin, but it had high capacity (not readily saturable). Thus, an evolutionary divergence in T4 transport proteins is suggested within Chelonia.     

Binding of the gestagens norethisterone and levonorgestrel in blood of various species

This study investigates the binding of 2 widely used contraceptive steroids, levonorgestrel and norethisterone, by plasma from various animal species and compares the results to those obtained with human plasma. Equilibrium dialysis of plasma samples and polyacrylamide gel electrophoresis were performed as previously described. The plasma samples were diluted with phosphate-buffered saline on the percentage of levonorgestrel and norethisterone bound in comparison to human plasma. The concentration of total protein and albumin was measured colorimetrically in each sample. An ammonium sulphate precipitation technique measured the level of sex-hormone binding globulin (SHBG). Results of the equilibrium dialysis show that binding of levonorgestrel and norethisterone in plasma was similar in adult female rhesus monkeys and baboons to that of humans with both high-affinity and low-affinity classes of binding sites. The dissociation constants of the high-affinity class for levonorgestrel was 4-fold lower than that for norethisterone in all 3 primates, indicating levonorgestrel was more tightly bound. Total protein and albumin concentrations were also the same in all 3 primates. SHBG levels in female monkeys and baboons however were 3-4 times those found in normal human females. Although differences exist in the binding of the 2 gestagens between human, baboon, and rhesus monkey plasma, there are no significant differences in the metabolism of the gestagens in the 3 primates. Overall, the results indicate that in the human, baboon, and rhesus monkey, binding of norgestrel and norethisterone occur mainly to SHBG, which had a greater affinity for norgestrel than for norethisterone, and to a lesser extent, albumin. Differences in the binding of gestagens between human and nonprimate species (rat, dog, rabbit) studied suggest that only baboon and rhesus monkeys may be considered appropriate animal models for extrapolation of results of contraceptive studies to humans.     

17 alpha-allyl estradiol analogues as candidates for development of high-affinity fluorescein-estradiol conjugates

In order to develop stable, high-affinity fluorescein-estradiol conjugates, the fluorescein moiety must be leashed to the estradiol molecule at a point which interferes least with estradiol's binding to the receptor. Because of the high affinity of 17 alpha-substituted estradiol (e.g. ethynyl estradiol), we investigated a series of 17 alpha-substituted estradiol compounds to determine the optimal properties of a leash at this position. Twelve estradiol derivatives bearing a three-carbon 17 alpha side chain with or without a terminal functional group and with varying degrees of unsaturation were synthesized. Initial comparison of the receptor binding affinities of some of these derivatives suggested that three factors might reduce affinity: internal hydrogen bonding of the 17 beta-hydroxyl proton with an oxygen atom of the 17 alpha side chain; hydrophilicity of the ligand; or steric interference of the side chain with receptor binding. Further comparisons were designed to evaluate the relative contribution of these factors. The results suggest that the relative affinities of these 17 alpha-substituted estradiol derivatives are influenced primarily by the steric interference of the side chains and also by their hydrophilicity. Internal hydrogen bonding involving the 17 beta-hydroxyl proton does not seem to have a profound effect.     

Enantioselective binding of mephobarbital to plasma proteins

The enantioselective protein binding of mephobarbital (MPB) was investigated in human plasma and human serum albumin solutions by equilibrium dialysis. A small but statistically significant difference was observed in the in vitro plasma protein binding of the enantiomers; (S)-MPB was approximately 59% bound and (R)-MPB approximately 67% bound. The binding to albumin [(S)-MPB: approximately 29% bound, and (R)-MPB: approximately 41% bound] was less than to plasma proteins but showed somewhat greater enantioselectivity, suggesting that albumin binding is a major source of the enantioselectivity in plasma. The effects of MPB concentration, of varying enantiomeric concentration ratio, and of phenobarbital on the enantioselective binding of MPB were studied. The effect of age was also investigated by measuring the binding in plasma from 8 young (18-25 yr) and 8 elderly (greater than 60 yr) male subjects who took single doses of MPB. The results were in close agreement with the in vitro binding data, and the binding of both enantiomers was marginally but significantly lower in the young compared with the elderly subjects. These differences in binding were consistent with previously observed pharmacokinetic differences between the two subject groups.     

Evidence of sex hormone binding globulin binding sites in the medial preoptic area and hypothalamus.

We have demonstrated a high density of both radiolabeled progesterone and estradiol conjugated to bovine serum albumin binding sites in the medial preoptic area and hypothalamus. Infusions of sex hormone binding globulin into the medial preoptic area of rats increased their female sexual receptivity similarly to the effect of estradiol conjugated to bovine serum albumin, suggesting sex hormone binding globulin acts at binding sites for estradiol conjugated to bovine serum albumin. In this study sex hormone binding globulin was used to displace radiolabeled progesterone conjugated to bovine serum albumin from plasma membrane fractions from the medial preoptic area-anterior hypothalamus and medial basal hypothalamus of ovariectomized rats injected with either 5 microg estradiol benzoate or sesame oil vehicle. We found that sex hormone binding displaced radiolabeled progesterone conjugated to bovine serum albumin in both areas and that in vivo estradiol treatment greatly increased the relative displacement by sex hormone binding globulin in the medial preoptic area-anterior hypothalamus. We interpret these data as indicating the presence of sex hormone binding globulin receptors in brain plasma membranes and further suggest that endogenous steroid conditions may alter these receptors.     

Binding of physostigmine to rat and human plasma and crystalline serum albumins

The binding of 3H-physostigmine (3H-Ph) to human and rat plasma proteins and crystalline serum albumin was studied by ultrafiltration technique. This study showed that the percentage of 3H-Ph bound to rat plasma slightly decreased from 49% to 41% whereas human plasma showed an increase in binding from 29% to 43% over a 50-fold increase in drug concentration. Human plasma samples which were collected in a bag coated with citrate phosphate dextrose adenine-1 solution bound 50% less 3H-Ph than samples collected with EDTA indicating a drug-drug interaction between 3H-Ph and anticoagulants. No significant change in binding was observed if the samples were frozen prior to use. Scatchard plots for binding of 3H-Ph resulted in a positive slope for human plasma and a negative slope for rat plasma; whereas curvilinear Scatchard plots with negative slopes were obtained for binding to human and rat crystalline serum albumin.     

Activation of purified cytoplasmic 17 beta-hydroxysteroid dehydrogenase from human placenta by human albumin and globulin

The cytoplasmic 17 beta-hydroxysteroid dehydrogenase of human placenta, purified more than 2500-fold, was activated by small amounts of human albumin and globulin. This activation was dependent on substrate concentration. At 20 microM estradiol (10 X KM) and two different concentrations of enzyme (0.01 and 2 micrograms/ml), the activation was greatest at albumin or globulin concentrations between 0 and 30 micrograms/ml. At "low" concentrations of estradiol (20 nM = 10(-2) X KM) and enzyme (0.01 microgram/ml), maximal activity occurred at approximately 10 micrograms/ml. Higher concentrations of albumin and globulin led to a decline in activity.     

Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum

Many estrogenic chemicals found in the environment (xenoestrogens) show a lower affinity for plasma estrogen binding proteins relative to the natural estrogens such as estradiol. These binding proteins, which include alphafetoprotein in rats and mice, sex hormone binding globulin in humans, and albumin in all species, regulate estrogen uptake into tissues. Therefore, the in vivo estrogenic potency relative to estradiol of xenoestrogens that show lower binding to these serum proteins will thus be underestimated in assays that compare the potency of xenoestrogens to estradiol and do not take serum binding into account. We have examined the effects of the binding components in serum on the uptake of a number of xenoestrogens into intact MCF-7 human breast cancer cells. Since most estrogenic chemicals are not available in radiolabeled form, their uptake is determined by competition with [³H]estradiol for binding to estrogen receptors (ER) in an 18-h assay. Serum modified access (SMA) of cell uptake of xenoestrogens is calculated as the RBA in serum-free-medium ÷ the RBA in serum, and the bioactive free fraction of xenoestrogen in serum is then also calculated. We predicted the concentration of two xenoestrogens, bisphenol A and octylphenol, required to alter development of the prostate in male mouse fetuses. Whereas octylphenol was predicted to be a more potent estrogen than bisphenol A when tested in serum-free medium, our assay predicted that bisphenol A would be over 500-times more potent than octylphenol in fetal mice. The finding that administration of bisphenol A at a physiologically relevant dose predicted from our in vitro assay to pregnant mice from gestation day 11 to 17 increased adult prostate weight in male offspring relative to controls (similar to the effect of estradiol), while the same doses of octylphenol did not alter prostate development, provided support for our hypothesis.     

Detection of genetic variation with radioactive ligands. I. Electrophoretic screening of plasma proteins with a selected panel of compounds.

To detect new genetic variation in human plasma proteins, a panel of 63 radioactive substances were screened as potential radioligands using polyacrylamide gel electrophoresis (PAGE) and autoradiography. Vitamins, hormones, drugs, amino acids, purines, pyrimidines, sugars and lipids labeled with 14C or other radionuclides were among those substances tested. A majority bound to albumin and a smaller fraction to prealbumins and lipoproteins. Several vitamins and hormones bound to specific alpha and beta globulins. (1) Electrophoretic polymorphisms of vitamin D-binding protein (group-specific component), a vitamin B12-binding protein (transcobalamin II), and thyroxine-binding alpha-globulin are described elsewhere. (2) Testosterone-binding beta-globulin (TeBG) showed an electrophoretic polymorphism in Caucasians and a possible deficiency allele. (3) Transcortin showed an electrophoretic doublet in all persons tested but no electrophoretic variation. (4) A protein binding derivative of norepinephrine or epinephrine was identified as transferrin. (5) A nonpolymorphic protein running cathodal to albumin and binding a derivative of riboflavin was tentatively identified as a fraction of albumin with mobility altered as a result of interaction with the ligand.     

The binding mechanisms of plasma protein to active compounds in Alismaorientale rhizomes (Alismatis Rhizoma)

Ultrafiltration and HPLC were employed to assess binding rates between rat plasma protein and two active compounds with lipid-regulating properties (alisol B 23-acetate and alisol A 24-acetate) from Alismaorientale rhizomes (Alismatis Rhizoma), a traditional Chinese medicine. SDS–PAGE was used for the evaluation of the binding between the alisol acetates and Hb in plasma. The fluorescence spectroscopy and circular dichroism spectroscopy were also combined with molecular modeling to explore binding mechanisms between Hb and the alisol acetates under imitative physiological condition. The ultrafiltration results show that alisol B 23-acetate bound more strongly than alisol A 24-acetate to plasma protein. SDS–PAGE results may suggest that alisols bind to Hb in plasma. The spectroscopy results are consisting with the molecular modeling results, and they indicate that the differences in plasma protein binding strength between the two compounds may be related to their side chains. A folded side chain/parent ring bound more strongly to Hb than an open side chain/parent ring.     

Contraceptive progestins. Various 11-substituents combined with four 17-substituents: 17alpha-ethynyl, five- and six-membered spiromethylene ethers or six-membered spiromethylene lactones

Norethisterone (NET) is a 19-nortestosterone derivative with progestagenic and some androgenic activity, which was used in the first generation of contraceptives. NET was succeeded by levonorgestrel (LNG) and later on by desogestrel (DSG) and gestodene (GSD). Although these latter two progestins had increased potency, there was still androgenicity with gestodene and to a lesser extent with desogestrel. New progestins were synthesized in order to further enhance progestagenic and to reduce androgenic activity. Four different chemical moieties were introduced in position 17 of 19-nortestosterone, viz. 17alpha-ethynyl, five- and six-membered spiromethylene ethers, and a six-membered-spiromethylene lactone. In combination with these structures seven different substituents were added at position 11, i.e. methylene, methyl, ethyl, ethenyl, ethynyl, 2-propenyl and 1-propynyl. All substituents except for methylene occupied the 11beta-position. All these 32 compounds were synthesized and analysed in vitro and in vivo against etonogestrel (ETG, 3-keto-desogestrel), the biologically active metabolite of desogestrel. Their relative binding potency to progesterone (PR), androgen (AR) and estrogen (ER) receptors were determined in cell lysates of human breast tumor MCF-7 cells and to glucocorticoid (GR) receptors in that of human leukemic IM-9 cells. Moreover, their relative agonistic activities were assessed in Chinese hamster ovary cell-based transactivation assays. All in vivo activities were determined in McPhail (progestagenic), ovulation inhibition (progestagenic and estrogenic), Hershberger (androgenic), hormone screening (glucocorticoid and estrogen) and Allen-Doisy (estrogenic) tests after oral and for the McPhail test also after subcutaneous administration. The progestagenic binding and transactivation potencies of all compounds in the three 17-spiro series were higher than those of the corresponding analogues in the 17alpha-ethynyl series. None of the compounds showed estrogenic or clear androgenic binding and transactivation potential except for a six-membered-spiromethylene lactone with a propynyl group. This compound showed strong androgenic binding. The glucocorticoid binding and transactivation were very low for the compounds with the 17alpha-ethynyl and the five-membered-spiromethylene ether groups, whereas both six-membered-spiro series showed, clearly with methyl and ethynyl substituents, and less pronounced with methylene and ethenyl, higher binding and transactivation values. For the 17alpha-ethynyl series, the McPhail test showed high potencies with methylene, methyl and ethenyl substituents after oral treatment or with propenyl after subcutaneous administration. The introduction of the spiro substituents in position 17 led to high potencies for other 11-substituents as well. Besides methyl, also ethyl, ethynyl and propynyl were potent substituents. With ovulation inhibition tests, the ethyl, ethenyl and ethynyl substituents were the more potent compounds in all four series. However, compounds with methyl or ethynyl additions appeared to be glucocorticoidal in the hormone screening test irrespective of the 17-substituent, while with the three spiro series even methylene and ethenyl groups became active. Androgenicity was only observed at dose levels at or above 5 mg/kg, which is 2.5-fold weaker than ETG. Moreover, estrogenicity appeared negligible with the three spiro series, while with the 17alpha-ethynyl series methyl, ethyl, ethenyl and ethynyl substituents, a very high estrogenic potential was assessed. Based on the high efficacy and low side-effects, the following compounds show a high selectivity: 17alpha-ethynyl with ethyl, ethenyl and 2-propenyl substituents, six-membered spiromethylene ether with ethyl and six-membered-spiromethylene lactone with ethyl, 2-propenyl or 1-propynyl substituents. (ABSTRACT TRUNCATED)     

Binding characteristics of estrogen receptor (ER) in Atlantic croaker (Micropogonias undulatus) testis: different affinity for estrogens and xenobiotics from that of hepatic ER.

An estrogen receptor (ER) was identified in cytosolic and nuclear fractions of the testis in a marine teleost, Atlantic croaker (Micropogonias undulatus). A single class of high affinity, low capacity, and displaceable binding sites was identified by saturation analysis, with a Kd of 0.40 nM in cytosolic extracts and a Kd of 0.33 nM in nuclear extracts. Competition studies demonstrated that the receptor was highly specific for estrogens (diethylstilbestrol > estradiol > estriol = estrone) and also bound several antiestrogens. Testosterone and 5alpha-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10(-3) to 10(-5) that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.     

Influence of progestins on serum hormone levels in postmenopausal women with advanced breast cancer--I. General findings

The influence of oral high dose progestin (medroxyprogesterone acetate, MPA and megestrol acetate, MA) treatment on serum hormone levels was studied in ten postmenopausal women with advanced breast cancer. The gonadotropins and ACTH were significantly reduced by greater than 50 and 23%, respectively. Serum cortisol, DHEAS, androstenedione and testosterone were all significantly reduced (mean reduction between 64 and 76%), while serum estrone, estradiol and estrone sulfate were significantly reduced by 20-30%. Sex hormone binding globulin (SHBG) and corticosteroid binding globulin (CGB) were reduced by 68 and 25%, respectively. Although the dose of MA used (160 mg/day) was only 1/6 of the MPA dose (1000 mg/day), the mean serum level of MA was 2-fold higher than the mean serum level of MPA. MPA treatment gave a more pronounced suppression of SHBG than MA treatment, while estrone sulfate levels were more suppressed by MA. These findings suggest a differential effect of MPA and MA on certain plasma hormones, possibly of importance for understanding the mechanism of action of the two drugs. The reduction of estrone sulfate may be beneficial for the action of MA against breast cancer.     

Oxidative metabolism and de-ethynylation of 17alpha-ethynylestradiol by baboon liver microsomes.

Incubations of tritiated 17alpha-ethynylestradiol (EE2) with liver explants of baboon and mouse showed the primate species to be more efficient in the removal of the ethynyl group. Liver microsomes from sexually immature male and female baboons were then incubated with tritiated EE2 and estradiol (E2). Each hormone bound irreversibly to the microsomal pellet. Addition of glutathione reduced the irreversible or covalent association. Incubations with E2 demonstrated significant conversion to estrone (E1). The EE2 experiments demonstrated a conversion to estrone only in the presence of an NADPH-generating system, and the addition of SKF-525A reduced the conversion of EE2 to E1. The cleavage reaction appears to be an oxidative event.