Effect of steroidal and nonsteroidal antiestrogens on the growth of a tamoxifen-stimulated human endometrial carcinoma (EnCa101) in athymic mice.

Tamoxifen (TAM), a nonsteroidal antiestrogen, is used in the adjuvant treatment of breast cancer. Previous studies, however, have indicated that some human breast and endometrial tumors are stimulated to grow with TAM in the athymic mouse. One such TAM-stimulated tumor is the EnCa101 human endometrial adenocarcinoma. Our aim was to evaluate the ability of different doses of TAM or other nonsteroidal antiestrogens to stimulate the growth of EnCa101 tumors in athymic mice. Additionally we have evaluated less estrogenic antiestrogens (two steroidal antiestrogens, RU 39,411 and ICI 164,384, and two nonsteroidal antiestrogens, keoxifene and MER-25) for their ability to inhibit TAM-stimulated growth. All experiments were done in ovariectomized athymic mice transplanted in the axillary mammary fat with 1-mm3 pieces of EnCa101 tumor. Sustained release preparations (0.5-2.0-cm Silastic capsule or 5-mg TAM cholesterol pellet) of TAM caused similar tumor growth. The growth rate was not altered by an additional daily i.p. injection of 1 mg TAM in 0.1 ml peanut oil. A 3-mg TAM daily dose was toxic. Four weeks of treatment (100-micrograms s.c. injections, every other day) with nonsteroidal antiestrogens, trioxifene mesylate, enclomiphene, or nafoxidine stimulated tumor growth. However, keoxifene stimulated this tumor to a lesser degree than TAM and partially inhibited TAM-stimulated growth. ICI 164,384 showed no stimulatory activity (1-mg s.c. injections every other day) alone compared to controls but inhibited TAM-stimulated (0.25-cm Silastic capsule) growth. In a parallel experiment, RU 39,411 (1-mg s.c. injections every other day) stimulated EnCa101 to grow. In contrast when RU 39,411 was administered in a sustained release preparation (2.0-cm Silastic capsule) there was no stimulatory growth compared to controls. Additionally RU 39,411 inhibited TAM-stimulated growth, but the low-potency antiestrogen, MER-25, was less effective in this regard. These data suggest that less "estrogenic" antiestrogens can inhibit TAM-stimulated tumor growth in vivo. Thus these compounds or derivatives may prove useful as a second-line endocrine therapy should TAM-stimulated tumor growth occur in the clinic.

Fetal regulation of transplacental cortisol-cortisone metabolism in the baboon.

We determined the role of the fetus and estrogen on transuteroplacental cortisol (F)-cortisone (E) metabolism in the baboon (Papio anubis). The interconversion of F-E at mid-gestation (day 100; term = day 184) was compared with that in animals near term (day 170) in which the fetus, but not the placenta, was removed (fetectomy) on day 100 and that in baboons treated daily between days 140 and 170 of gestation with the antiestrogen ethamoxytriphetol [1-(p-diethylamino-ethoxyphenol)1-phenyl-2-p-methoxyphenolethan ol (MER-25)]. In fetectomized animals at term, transuteroplacental conversion of E to F (30%) exceeded (P less than 0.05) that of the reverse reaction (7%). This pattern of metabolism was significantly different from that measured in intact pregnant animals at term, in which oxidation of F to E (28%) exceeded reduction of E to F (4%). In contrast, placental metabolism in fetectomized baboons at term was similar to that in pregnant animals at mid-gestation, in which conversion of F to E (20%) was lower (P less than 0.05) than reduction of E to F (39%). Treatment of intact pregnant baboons with MER-25 also resulted in a pattern of F-E metabolism across the placenta at term which was similar to that measured at midgestation but different from that in untreated baboons at term. Collectively, our findings show that the striking alteration in F-E interconversion from reduction (E to F) at midgestation to oxidation (F to E) by term, as measured across the placenta in vivo during the second half of baboon pregnancy, does not occur in animals lacking a fetus or in intact baboons in which the action of estrogen was inhibited. Therefore, we suggest that the fetus and/or the hormones of pregnancy that are dependent upon the fetus (i.e. estrogen) regulate transuteroplacental corticosteroid metabolism.

Effect of the antiestrogen ethamoxytriphetol (MER-25) and luteectomy on serum progesterone concentrations in pregnant baboons.

The present study determined whether the reduction in serum progesterone (P4) concentrations which follows the administration of the antiestrogen ethamoxytriphetol [1-(rho-2-diethylaminoethoxyphenyl)1-phenyl-2-rho-methoxyphenyl ethanol (MER-25)] to pregnant baboons reflects a decline in placental and/or luteal function. Maternal saphenous venous blood was collected at 1- to 4-day intervals between day 70 of gestation and term in pregnant baboons. Four females received no other treatment, and eight females received MER-25 (15 mg/kg BW, orally) daily between day 130 of gestation and term. Four of the MER-25-treated baboons received no other treatment, and four had the corpus luteum of pregnancy surgically excised between days 104 and 118 of gestation. Serum P4 concentrations in the untreated baboons fluctuated, but no significant progressive rise or fall in P4 occurred. Administration of antiestrogen to intact pregnant baboons resulted in a 50% decline (P less than 0.001) in serum P4 concentrations from mean pretreatment values of 7.0-25.1 to 4.2-10.8 ng/ml thereafter. Although removal of the corpus luteum alone had no effect on serum P4, administration of MER-25 to luteectomized females resulted in an 80% decrease (P less than 0.001) in serum P4 concentrations from pretreatment means of 10.6-16.6 to 2.5-3.2 ng/ml thereafter. The results indicate that most or all of the P4 that remained in the peripheral circulation after MER-25 administration to intact pregnant baboons originated from the ovary, primarily the corpus luteum. Thus, the major site of action of antiestrogen in reducing P4 production during baboon pregnancy is on the placenta.

Modulation of adrenocorticotropin-stimulated baboon fetal adrenal dehydroepiandrosterone formation in vitro by estrogen at mid- and late gestation.

We have reported that ACTH stimulation of dehydroepiandrosterone (DHA) formation by the baboon fetal adrenal at midgestation was suppressed by estrogen. Because fetal adrenal regulation changes with advancing gestation, the action of estrogen on fetal adrenal steroidogenesis may also be dependent on the degree of fetal adrenal maturation. We examined this possibility in the present study by determining the effects of ACTH and estrogen on DHA formation by adrenal cells of fetuses obtained from baboons at mid- and late gestation and from animals administered the antiestrogen MER-25 throughout late gestation. Because low density lipoprotein (LDL) provides substrate for the fetal adrenal, we also determined whether the effect of estrogen was mediated by LDL uptake. Adrenals were removed from baboon fetuses on day 100 (midgestation; n = 7) and day 170 (late gestation; n = 6; term, day 184) of gestation from untreated animals and on day 170 from fetuses whose mothers were treated with MER-25 on days 140-170 (25 mg/kg BW.day; n = 7). Cells were dispersed with 0.2% collagenase and incubated at 37 C for 3 h in 4 ml medium 199 with 10 nM ACTH, 10(-6) M estradiol and/or 500 micrograms LDL. The secretion of DHA into medium was determined by RIA. At midgestation, mean (+/- SE) basal DHA formation (nanograms per 10(5) cells/3 h) was 5.8 +/- 2.1, and DHA was increased (P less than 0.01) by ACTH to 20.0 +/- 5.9. Although estradiol alone had no effect, estradiol prevented the increase in DHA obtained with ACTH. Basal DHA production by adrenals of late gestation (0.7 +/- 0.3 ng/10(5) cells) was lower (P less than 0.01) than at midgestation. ACTH increased (P less than 0.01) DHA in a comparable manner near term in the presence (2.0 +/- 0.4) or absence (1.7 +/- 0.4) of estradiol. Thus, in contrast to day 100, estrogen did not attenuate the action of ACTH on adrenal cells on day 170. In fetal adrenal cells obtained on day 170 from MER-25-treated baboons, DHA formation (1.4 +/- 0.6 ng/10(5) cells) was comparably increased (P less than 0.05) to 2.4 +/- 0.2 and 3.0 +/- 0.5 ng/10(5) cells by ACTH in the absence or presence of estradiol. Thus, ACTH remained effective in enhancing DHA by adrenal cells of fetuses exposed in utero to antiestrogen. DHA formation by adrenals of midgestation was increased (P less than 0.05) to 15.4 +/- 4.8 and 27.4 +/- 7.5 ng/10(5) cells, respectively, by LDL and ACTH plus LDL.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of the antiestrogen ethamoxytriphetol (MER-25) on placental low density lipoprotein uptake and degradation in baboons.

The present study determined if the decline in placental progesterone (P4) production that results from administration of the antiestrogen ethamoxytriphetol (MER-25) to pregnant baboons results from a change in placental low density lipoprotein (LDL) uptake and/or degradation. Pregnant baboons (Papio anubis) were untreated (n = 10) or received MER-25 (25 mg/kg BW, orally; n = 10) daily on days 140-170 of gestation (term, 184 days). Placentas were removed by cesarean section on day 170 of gestation, and villous tissue was dispersed with 0.1% collagenase at 37 C for 40 min. Placental cells (10(6)) were incubated in medium 199 (pH 7.2) for 12 h at 37 C with increasing amounts (5-100 micrograms) of [125I]LDL, with or without a 100-fold excess of unlabeled baboon LDL. Mean (+/- SE) peripheral serum P4 concentrations on days 140-170 of gestation were 51% lower (P less than 0.01) in MER-25-treated (5.7 +/- 0.3 ng/ml) than in untreated (11.6 +/- 0.5 ng/ml) baboons. The uptake of LDL was 56% lower (P less than 0.01) in placental cells from antiestrogen-treated (6.3 +/- 1.6 ng/micrograms cell protein) than in those from untreated (14.4 +/- 1.9 ng/micrograms cell protein) baboons. The dissociation constants for placental LDL uptake, as assessed by Scatchard analysis, however, were similar in untreated (0.80 microgram/ml) and MER-25-treated (0.76 microgram/ml) animals. The amount of [125I]LDL concomitantly degraded by cells from baboons that received MER-25 was 54% of that degraded by cells from untreated controls. The relative decline in LDL degradation by cells of antiestrogen-treated baboons was proportionate to the decline in overall LDL uptake. The results indicate, therefore, that antiestrogen treatment decreased the amount of placental LDL uptake, but did not change the affinity for the lipoprotein. We suggest that the decline in placental P4 production elicited in pregnant baboons by antiestrogen results, at least in part, from subnormal LDL uptake. We propose that one of the mechanisms by which estrogen regulates the biosynthesis of P4 by the placenta during baboon pregnancy is by increasing receptor-mediated placental cell uptake of cholesterol in the form of LDL. Estrogen, therefore, may regulate LDL uptake by the placenta and thus the availability of cholesterol for P4 biosynthesis via the LDL pathway.

Regulation of progesterone formation by human placental cells in culture.

In the present study, a culture system of human placental cells was established to examine the role of estrogen and androgen in progesterone (P4) formation. Normal human placentae were obtained at term, and cells were dispersed in Hank's Balanced Salt Solution (5 ml/g tissue) containing 0.1% collagenase, 0.1% hyaluronidase, 0.01% deoxyribonuclease, and 1% fetal bovine serum for 2 h at 37 C. Dispersed placental cells (10(6) cells/ml) were placed in medium 199 with modified Earle's salts (pH 7.4) containing 10% fetal bovine serum, 12.5 mM HEPES buffer, 26 mM NaHCO3, and 40 micrograms/ml Gentamycin-SO4 and incubated for 72 h at 37 C and 5% CO2 in air to allow cell attachment. Medium was then changed (time zero), and P4 formation was studied thereafter. Culture of placental cells for 96 h resulted in linear increases in P4 and estradiol (E2) formation, indicating the maintenance of cell viability and steroidogenic function. Mean +/- SE P4 formation at 48 h was 246 +/- 16 pg/micrograms DNA. To assess the role of estrogen on P4 formation, placental cells were incubated for a period of 48 h with various amounts (10(-7)-10(-4)M) of the antiestrogen ethamoxytriphetol (MER-25), the aromatase inhibitor 4-hydroxyandrostenedione (4-OHA), and/or E2. Both MER-25 and 4-OHA resulted in a dose-dependent decline (P less than 0.01) in P4 formation (greater than 80% decline at 10(-4)M MER-25 or 4-OHA). The marked reduction in P4 formation caused by 4-OHA alone was reversed by concomitant addition of E2; however, E2 alone had no effect. To assess the role of androgens on P4 formation, cells were incubated for 48 h with increasing amounts (10(-7)-10(-4)M) of androstenedione, dehydroepiandrosterone (DHA), or dihydrotestosterone. Although the formation of E2 was enhanced by DHA, formation of P4 was not affected by the aromatizable androgens DHA or androstenedione or the nonaromatizable dihydrotestosterone. The decline in P4 formation by human placental cells in culture elicited by MER-25 or 4-OHA supports the hypothesis of a regulatory role for estrogen in placental P4 formation during human pregnancy. The lack of effect of exogenous estrogen suggests that the action of estrogen on P4 formation may be permissive.

Transuterofetoplacental conversion of pregnenolone to progesterone in antiestrogen-treated baboons.

The present study was designed to characterize the dynamics of progestin metabolism peripherally and across the uterus during normal baboon pregnancy and to determine whether the decline in placental progesterone (P4) production which results from administration of the antiestrogen ethamoxytriphetol (MER-25) to baboons reflects a decrease in conversion of pregnenolone (P5) to P4 and thus delta 5-3 beta-hydroxysteroid dehydrogenase activity. To examine this possibility, the conversion of [3H]P5 to [3H]P4 was determined by the constant infusion method on day 100 (midgestation) and day 175 (near term) of gestation in baboons that received MER-25 (25 mg/day X kg BW, po) on days 95-100 or 140-175 (term = 184 days). Baboons were sedated with ketamine HCl, then received a constant iv infusion of [3H]P5 (1.0 mu Ci/0.388 ml X min) and [14C]P4 (0.2 mu Ci/0.388 ml X min for 110 min. Radiolabeled progestins were purified from blood samples withdrawn from saphenous, uterine, and umbilical vessels, and the MCR of P4 and P5, uterine extraction of P5, and transfer constants (rho) for the peripheral, transuterofetoplacental, and transuteroplacental conversion of P5 to P4 were determined. The formation of P4 from P5 by incubates of placental cells obtained on day 175 from untreated and MER-25-treated baboons was also assessed. During normal baboon pregnancy the mean (+/- SE) % P5 extracted (i.e. metabolized) by the uterus was 31.0 +/- 3.3 at midgestation and 45.7 +/- 5.6 late in gestation. Peripheral and transuterofetoplacental rho values of P5 to P4 in untreated baboons were 6.9 +/- 1.8% and 37.3 +/- 7.9%, respectively, at midgestation and 6.1 +/- 0.6% and 46.8 +/- 10.1%, respectively, near term. The transuteroplacental rho of P5 to P4 was only slightly lower than the transuterofetoplacental values, indicating minimal conversion of P5 to P4 by the fetus. The peripheral contribution of P5 production to the total production rate of P4 at term in baboons was 1%. The contribution of uteroplacental conversion of P5 to P4 to the total conversion of P5 to P4 at midgestation was estimated to be 22%. MER-25 caused a 53% decline (P less than 0.01) in serum P4 concentrations from a mean (+/- SE) of 12.5 +/- 2.4 ng/ml during the pretreatment period to 5.4 +/- 0.3 ng/ml between days 140 and 175 of gestation.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of progesterone biosynthesis by estrogen during baboon pregnancy: placental mitochondrial cholesterol side-chain cleavage activity in antiestrogen (ethamoxytriphetol, MER-25)-treated baboons.

We determined whether the reduction in placental progesterone (P4) production observed after administration of the antiestrogen ethamoxytriphetol (MER-25) to pregnant baboons was associated with a decline in activity and/or content of the placental mitochondrial cholesterol side-chain cleavage system (P-450scc). Pregnant baboons (Papio anubis) were untreated (n = 9) or administered MER-25 (25 mg/kg BW, orally; n = 6) daily on days 140-170 of gestation (term = 184 days). Placentas were obtained by cesarean section on day 170 of gestation, and P-450scc activity and cytochrome P-450 content were determined on mitochondria-rich fractions. Administration of MER-25 to pregnant baboons resulted in a 40% reduction (P less than 0.01, by Student's t test) in the mean (+/- SE) peripheral serum P4 concentration (6.3 +/- 0.3 ng/ml) compared to that in untreated (10.4 +/- 0.3 ng/ml) baboons. P-450scc activity, as determined by formation of pregnenolone (P5) and P4 during a 30-min incubation (picomoles per mg protein), was 37% lower (P less than 0.01) in placental mitochondria obtained from MER-25-treated baboons (179.8 +/- 25.0) than in that from untreated (285.4 +/- 13.4) baboons. Mitochondrial cytochrome P-450 content, assessed by spectral analysis, was 28% lower (P less than 0.02) in antiestrogen-treated (46.7 +/- 2.1 pmol/mg protein) than in untreated (64.8 +/- 5.2 pmol/mg protein) baboons. The initial (time zero) free cholesterol content (nanomoles per mg protein) of mitochondrial-rich preparations was not significantly different in antiestrogen-treated (189.3 +/- 13.0) and untreated (225.0 +/- 15.1) animals. Collectively, these results suggest that the decline in placental P4 production observed in baboons in response to MER-25 occurs at least in part as a result of a decrease in cytochrome P-450scc activity. The loss in P-450scc activity appears to be an intramitochondrial event and not a result of depletion of the total mitochondrial cholesterol pool. We propose, therefore, that one mechanism by which estrogen may regulate the production of P4 by the placenta during primate pregnancy is via the maintenance of placental mitochondrial cytochrome P-450, the terminal oxidase of cytochrome P-450scc.

Activation of the baboon fetal pituitary-adrenocortical axis at midgestation by estrogen: adrenal delta 5-3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase-17,20-lyase activity.

We have recently demonstrated that treatment of pregnant baboons with androstenedione (delta 4 A) at midgestation to increase estrogen production induced a pattern of placental cortisol (F) metabolism which was similar to that at term and resulted in de novo F production by the fetus, presumably by activation of the fetal hypothalamic-pituitary-adrenocortical axis. The present study was designed to examine the subcellular events in the fetal adrenal that were apparently stimulated by estrogen-induced alterations in transplacental corticosteroid metabolism. Therefore, we determined the effects of estrogen treatment at midgestation and removal of estrogen action near term on the specific activity of the rate-limiting enzymes delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17-hydroxylase-17,20-lyase (17 alpha-OHase). Fetal adrenals were obtained on day 100 (n = 11) or day 165 (n = 11) of gestation (term = day 184) from untreated animals, on day 100 from animals receiving delta 4 A daily between days 70-100 (n = 9) to increase placental estrogen production, and on day 165 from baboons treated daily between days 130-164 with antiestrogen ethamoxytriphetol (MER-25; n = 7). The activity of 17 alpha-OHase was determined by incubating adrenal microsomes (105,000 x g) with [3H] progesterone, NAD+, and NADH in phosphate buffer. The radiolabeled products 17-hydroxyprogesterone, delta 4 A, and testosterone were purified, and enzyme activity expressed as picograms of product per min/mg tissue. The activity of 3 beta HSD was determined by incubating adrenal microsomes with [3H]pregnenolone and NAD+ in phosphate buffer. The radiolabeled progesterone product was purified, and enzyme activity was expressed as nanograms per min/mg tissue. Treatment with delta 4 A increased estrogen concentration at midgestation 3-fold to levels comparable to those measured near term. Although fetal adrenal weight was greater at term than at midgestation (p less than 0.05), weight was not increased by delta 4 A treatment. The specific activity (mean +/- SE) of fetal adrenal 17 alpha-OHase at midgestation (181 +/- 29) was increased (P less than 0.05) 3-fold by treatment with delta 4 A to levels (591 +/- 105) comparable to those in adrenal microsomes prepared from untreated animals near term (816 +/- 130). Enzyme activity in adrenals of MER-25-treated baboons was 40%, but not significantly lower than that in term controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Antiestrogen action in the uterus: biological ineffectiveness of nuclear bound estradiol after antiestrogen.

These studies attempt to analyze the basis of the estrogenic and antiestrogenic action of three nonsteroidal clomophene-type compounds as monitored by their ability to bind to immature rat uterine cytoplasmic estrogen receptor, transfer receptor sites to the nucleus, and elicit estrogenic responses (increased uterine weight and induction of the synthesis of a specific uterine protein, called induced protein, or "IP"), and by their ability to antagonize the effects of estradiol on these receptor interactions and uterine responses. Both CI-628 (CI) and U-11, 100A (UA) [50 mug] elicit slight IP induction at 1-2 hand give pronounced uterine weight increases at 24 h but feeble increases at 72 h (3 single daily injections). Both bind to cytosol, and effect the transfer of receptor sites to the nucleus, which may account for the estrogenicity of these compounds. Both CI and UA give rapid (by 2-4 h), prolonged (for over 24 h), and complete blockage of estradiol-stimulated treatment abolishes short-term estradiol-stimulated uterine weight increase and antagonizes the 72 h estradiol-stimulated uterine weight response to the level attributable to the antiestrogen alone. MER-25, at the same dose (50 mug), had no estrogenic or antiestrogenic activity. Both CI and UA rapidly deplete the cytoplasmic estrogen binding capacity, reducing it to barely detectable levels for 24-42 h. Although during this period, no IP or uterine wet weight response can be elicited by estradiol, administration of saturating levels of [3H]estradiol in vivo or in vitro results in the appearance of considerable [3H]estradiol in the nucleus, bound to a macromolecule sedimenting identically with that of the nuclear receptor-estradiol complex (5.5S) formed in the absence of prior antiestrogen exposure. Hence, the estradiol which becomes bound in the nucleus after antiestrogen is biologically ineffective. The return of IP responsiveness after antiestrogen correlates well with the level of cytoplasmic receptor capable of translocation to the nucleus, and not with the nuclear estradiol uptake capacity, Presumably, then, the antiestrogenic action of CI and UA results from their depletion of cytoplasmic receptor sites and not from their ability to block specific estradiol-nuclear receptor binding per se. These studies indicate that one should be cautious in assuming that the magnitude of an estrogen response is necessarily related to the level of estrogen receptor complex in the nucleus.

Regulation of immunoreactive inhibin patterns in baboon pregnancy: maternal, placental, and fetal considerations.

To better understand the sources and regulation of circulating inhibin during primate pregnancy, immunoreactive inhibin was measured in sera obtained from the maternal saphenous vein, uterine vein, and the fetus at varying times of baboon pregnancy. In both intact and fetectomized (fetus removed on day 100 of gestation; term = 184 days) animals, maternal serum inhibin concentrations were relatively constant between day 80 (first sampling day) and day 110 of gestation, after which they then steadily increased until days 155-165 (end of sampling). The increase in inhibin concentrations was significantly less in the fetectomized animals than in the intact baboons. Restoration of estrogen levels in the fetectomized animals did not significantly alter the circulating inhibin concentrations. Similarly, administration of the estrogen antagonist MER-25 to intact animals in the last trimester had no effect on maternal serum inhibin concentrations. Inhibin concentrations in uterine venous blood collected on day 100 of gestation were not significantly different from those in the maternal saphenous vein. However, the inhibin concentrations of uterine venous blood collected late in gestation (days 155-165) in either intact or fetectomized animals were significantly higher than the corresponding maternal venous concentrations, suggesting that the uteroplacental tissue becomes a source of circulating inhibin during the third trimester of pregnancy. Consistent with this suggestion was the detection of inhibin alpha-subunit mRNA in the placentae of intact or fetectomized animals obtained late in pregnancy, but its absence at midgestation. Immunoreactive inhibin concentrations were about 16 times higher (6500 +/- 831 mu Leq/mL) in fetal blood than in maternal blood (411 +/- 23 mu Leq/mL) at midgestation. The fetal blood concentrations significantly decreased to about 2800 mu Leq/mL by days 160-165 of gestation, but were still greater than those in the mother (approximately 1000 mu Leq/mL). The umbilical arterial and venous concentrations were the same as the fetal blood concentration of inhibin. The role of the baboon fetal adrenal in inhibin production was studied. Fetal adrenals collected from days 59, 135, and 167 of gestation contained the mRNA for the inhibin alpha-subunit in relatively high abundance. The in utero administration of ACTH for 30 min to five fetuses at midgestation (days 100-110) apparently did not alter the fetal concentration of immunoreactive inhibin. In summary, maternal serum inhibin levels increase during the last trimester of baboon pregnancy. This is suggested to be due to an increasing contribution of placental inhibin secretion, which is regulated not by placental estrogen production but, perhaps, by placental growth.(ABSTRACT TRUNCATED AT 400 WORDS)

The relationship of estrogen to placental steroidogenesis in the baboon.

In previous studies in pregnant baboons, estrogen deprivation produced by the administration of the estrogen antagonist MER-25 resulted in a decline in plasma progesterone. In the present study, pregnant baboons in whom the corpus luteum-bearing ovary was removed early in pregnancy were used to distinguish between a luteal or placental effect of this estrogen deprivation. MER-25, administered from days 35 through 48 of pregnancy, resulted in a significant decline in plasma progesterone, indicating a direct placental action of estrogen deprivation on progesterone production. In a second experiment using intact pregnant baboons, concomitant administration of the nonsteroidal estrogen diethylstilbestrol (DES) prevented the inhibitory effect of MER-25 on placental progesterone, indicating that the effect was due to antiestrogenic action of MER-25 and not to some other pharmacological effect of the antagonist. Since DES administration alone had no effect on plasma progesterone levels, the role of estrogen may be a permissive action. The administration of MER-25 did not inhibit estradiol production in intact pregnant baboons with or without corpora lutea. When DES was administered to intact pregnant baboons, alone or together with MER-25, there was a significant decrease in plasma estradiol concentrations, which was only reversed after the cessation of treatment. These studies indicate that estrogen, perhaps by different mechanisms, may be important in the regulation of placental estradiol and progesterone production in the primate.

Molecular structures and conformational studies of triarylcyclopropyl and related nonsteroidal antiestrogens.

Molecular structures and conformational characteristics of a series of 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), which were reported previously to be distinctly antiestrogenic and inhibitors of the estrogen-receptor-positive MCF-7 human breast cancer cells in culture, are reported. In addition, structural and conformational features of the DTACs were compared to the first-known nonsteroidal antiestrogen, MER25, and the clinically useful antiestrogen Tamoxifen. The molecular structures of four DTAC compounds were determined by X-ray diffraction. Crystallographic structures show that the DTAC molecules have nearly the same relative conformation for the three aryl rings which is designated as a "nonpropeller" conformation in contrast to the observed "propeller" conformation for the three rings in all known triarylethylenes. Systematic conformational searches were performed to find the conformational preferences of DTACs, MER25, and Tamoxifen using idealized model compounds built from their respective crystal structure. Energy-minimization and conformational-search studies demonstrated that all DTAC molecules have a common, single global minimum energy conformer for their central core containing the dichlorotriarylcyclopropyl system, which is similar to that found in their crystal structures. Conformational search of MER25 showed that the molecule can assume a number of low-energy conformers of which two, one anti (A1) and one gauche (G1A), have about the same energy. The anti conformation is similar to the one observed in its crystal structure and resembles the estrogenic E-isomer of Tamoxifen, while the lowest energy gauche conformer of MER25 resembles more closely the antiestrogenic Z-isomer of Tamoxifen. NMR spectroscopic analysis of MER25 showed that the molecule exists predominantly in the anti conformation in solution. A comparative review of the structural features and bioactivities of Tamoxifen, DTACs, and MER25 provides a possible explanation for their low estrogen receptor binding affinity which is common to these compounds together with their antiestrogenic activity.

Oestrogenic, anti-oestrogenic and fertility effects of some triphenylethanes and triphenylethylenes related to ethamoxytriphetol (MER 25).

Five triphenylethylenes, a triphenylethane and a triphenylethanol, carrying methyl substituents ortho to one or both of the ring oxygen functions, have been examined for oestrogenic, and antioestrogenic activity in mice, and three of the compounds, alpha-[4-(beta-diethylaminoethoxy)-3, 5-xylyl]-alpha-phenyl-beta-4-methoxyphenyl-ethanol (IV), alpha'-[4-(beta-diethylaminoethoxy)-3, 5-xylyl]-4-methoxy-bibenzyl (V) and alpha'-[4-(beta-diethylaminoethoxy)-3, 5-xylyl]-4-methoxy-stilbene (VI), were tested for their effects on fertility in mice. 2 Orthomethylation reduces oestrogenic and/or anti-oestrogenic activity compared with the reported activities of non-methylated analogues. 3 The anti-oestrogenic ethamoxytriphetol (MER 25) reduced fertility in mice whereas its inactive dimethylated analogue (IV) was ineffective. The weakly active anti-oestrogens, V and VI, did not affect fertility in mice.

Differential depletion of cytoplasmic high affinity oestrogen receptors after the in vivo administration of the antioestrogens, clomiphene, MER-25 and tamoxifen.

1 The in vivo actions of the oestrogen antagonists, MER-25 and tamoxifen upon the cytosol oestrogen receptors prepared from amygdala, hypothalamus, pituitary and uterus of rats were studied 24 h after drug administration. 2 There was a dose-related depletion of cytosol oestrogen receptors. However, the uterine and pituitary receptors were consistently affected at a lower dose than were those from the brain. 3 The ratios of the combined central ED50 to the combined peripheral ED50 were clomiphene 169 greater than MER-25 19.2 greater than tamoxifen 2.13. 4 The receptor changes were not related to biological activity monitored by serum luteinizing hormone levels and uterotrophic response. 5 The possible role of these drug effects in the induction of ovulation and future developments are discussed.

Suprachiasmatic lesions disrupt the daily rhythmicity in the sexual behaviour of normal male rats and of male rats treated neonatally with antioestrogen.

Male rats were treated daily with oil or 100 micrograms of the antioestrogen, ethamoxytriphetol (MER-25), for the first 10 days of life and, when adult, lesions were made in the suprachiasmatic nuclei (SCN) of the hypothalamus or control lesions were made above the SCN and the rats were tested for sexual behaviour. Treatment with MER-25 enhanced the daily rhythmicity in both mounting and lordosis behaviour and SCN lesions disrupted these behavioural rhythms and the rhythm in the mounting behaviour of oil-treated rats. Rats treated with MER-25 and with SCN lesions showed high levels of mounting and lordosis behaviour throughout the light : darkness cycle. These results support the hypothesis that sexual differentiation by perinatal androgen stimulation uncouples the central rhythm generator from the neural substrates of sexual behaviour in rats.

Neonatal treatment with antioestrogen increases the diurnal rhythmicity in the sexual behaviour of adult male rats.

Male rats were given daily injections of the antioestrogen ethamoxytriphetol (MER-25, 100 microgram/day) or oil during the first 10 days of life. Rats treated with MER-25 showed a more pronounced diurnal rhythm in both mounting behaviour and lordosis behaviour than did oil-treated rats when tested as intact adults and after castration together with treatment with testosterone- or oestradiol-filled constant release implants. Serum levels of androgen varied markedly in samples obtained at four different times of the light : darkness (LD) cycle in both neonatal treatment groups and no significant LD-dependent pattern was obvious. Castration and treatment with testosterone implants produced stable androgen levels which showed little individual variation and did not vary with the LD cycle. The results supported the hypothesis that perinatal androgen stimulation affects the development of sexual behaviour in rats primarily by decreasing the diurnal rhythmicity of the behaviour of the adult.

Effects of anti-oestrogen treatment of neonatal male rats on lordosis behaviour and mounting behaviour in the adult.

Male rats were treated daily with 100 microgram of the anti-oestrogen ethamoxytriphetol (MER-25) or oil during the first 10 days of life and tested for lordosis behaviour and mounting behaviour as intact adults, after castration and after castration and oestradiol benzoate or testosterone propionate treatment. The MER-25-treated rats showed higher levels of lordosis behaviour than oil-treated rats in all four treatment groups. Under each of these endocrine conditions, except after castration alone, the MER-25-treated rats showed a reduced capacity to ejaculate. Treatment of the neonatal rat with MER-25 reduced body weight in adulthood but did not change the weight of the accessory sexual glands, the testes, the number of cornified papillae on the glans penis or plasma testosterone concentrations during development. The response of the accessory sexual glands and cornified papillae on the glans penis to treatment with oestradiol benzoate or testosterone propionate after castration in adulthood was unaffected by treatment with MER-25. It is suggested that formation of oestrogen in the neonatal male rat brain from testosterone in the circulation inhibits the capacity to show lordosis behaviour and facilitates the capacity to ejaculate in response to ejaculate in response to gonadal hormone treatment in adulthood.

Possible mechanisms for the agonist actions of tamoxifen and the antagonist actions of MER-25 (ethamoxytriphetol) in the mouse uterus.

Experiments were conducted to determine why tamoxifen, a non-steroidal antiestrogen, is uterotrophic in mice, whereas MER-25 (ethamoxytriphetol), a structurally related compound, is antiuterotrophic. Initial experiments indicated that the pituitary was not required for a uterotrophic response in mice to either estradiol (E2), tamoxifen (TAM), or 4-hydroxytamoxifen (4-OH-TAM) MER-25 was not uterotrophic in mice but was capable of completely inhibiting the uterotrophic responses of mice to estrogens (E2) as well as antiestrogens (TAM and 4-OH-TAM); this inhibition was reversible by increasing the dose of the antiestrogen (TAM). The relative binding affinities (RBA) of TAM, 4-OH-TAM, and MER-25 to mouse uterus estrogen receptor (ER) and mouse liver antiestrogen binding sites (AEBS) were compared to determine whether either (or both) of these sites mediate the biological effects of these compounds. E2 is arbitrarily assigned an RBA of 100 for ER; similarly, TAM is assigned an RBA of 100 for AEBS. MER-25 bound to AEBS with an RBA of 8.9 and to ER with an RBA of less than 0.06; in contrast, TAM and 4-OH-TAM bound to AEBS with RBAs of 100 and 53, respectively, and to ER with RBAs of 2 and 131, respectively. Five other compounds that had similar RBAs as MER-25 for AEBs (RBAs in the range 4-9) and for ER (RBAs less than 0.06) were tested for their antiuterotrophic activities in vivo against both estrogen (E2) and antiestrogen (TAM) in ovariectomized mice. None of these compounds were antiuterotrophic against either estradiol or tamoxifen (P less than 0.01), nor were any of the compounds uterotrophic in mice. These data suggest that differences in the biological actions of tamoxifen and MER-25 in mice are not mediated through AEBS and are most likely due to differences in their interactions with ER.

In vivo effect of anti-estrogens on the localisation and replenishment of estrogen receptor.

Interactions between estrogen recpetors and triaryl ethylene anti-estrogens (U-11100A, MER 25 and CI 628) were tested on immature rat uteri. The accessible and the total (accessible and occupied) estrogen receptor sites were assayed in the cytosol and nuclear extracts using charcoal adsorption. After in vivo administration of anti-estrogens, the estradiol receptor sites were occupied and subsequently transferred to the nuclear compartment. The nuclear localisation of the receptor induced by the antagonist lasted for several days, during which replenishment of the cytosol receptor occurred. The nuclear receptors transferred by anti-estrogens and labelled in vitro with [3H]estradiol, were similar to the nuclear receptor-estradiol complex formed in vivo as far as their sedimentation constants and extractability from nuclei were concerned. Although these anti-estrogens are capable to translocate the estrogen receptor to the nucleus and to induce the replenishment of the cytosol receptor, the mechanism of their antagonism and of their weak estrogenic activity is still not clear.