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Endocrinology Vol. 139, No. 5 2645-2656
Copyright © 1998 by The Endocrine Society

ARTICLES

Effect of Twenty-Four-Week Treatment with the Antiestrogen EM-800 on Estrogen-Sensitive Parameters in Intact and Ovariectomized Mice

Shouqi Luo, Antigone Sourla, Claude Labrie, Sylvain Gauthier, Yves Merand, Alain Belanger and Fernand Labrie

Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center, Centre Hospitalier Universitaire de Québec et Université Laval, Québec, G1V 4G2, Canada

Address all correspondence and requests for reprints to: Prof. Fernand Labrie, Laboratory of Molecular Endocrinology, Centre Hospitalier de l’Université Laval Research Center, 2705 Laurier Boulevard, Québec, G1V 4G2, Canada. E-mail: Fernand.Labrie{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Treatment with the antiestrogen EM-800, at the daily oral dose of 3 µg, 10 µg, 30 µg, or 100 µg for 24 weeks, caused a marked inhibition of uterine and vaginal weight in both intact and ovariectomized mice. Maximal 64% and 41% inhibitions of uterine weight were achieved in intact and ovariectomized animals, respectively. Similar inhibitory effects of EM-800 were observed on vaginal weight with maximal inhibitions of 71% and 35%, in intact and ovariectomized animals, respectively. The pure antiestrogenic activity of EM-800 on the hypothalamo-pituitary-ovarian axis is illustrated by the 76–91% increases in ovarian weight observed in intact animals treated with the 10–100 µg doses of the antiestrogen. Serum 17ß-estradiol was 93% increased at the 100 µg daily dose of EM-800, whereas serum androstenedione, testosterone, and dihydrotestosterone were 141–713% increased over control at the same dose of the antiestrogen. Serum LH was increased by treatment with EM-800 in intact animals, whereas no effect was observed on the elevated gonadotropin levels in ovariectomized animals. At all doses used in intact animals, the antiestrogen caused a complete disappearance of the glandular elements of the mammary gland, the atrophy being comparable with that observed in ovariectomized mice. The mammary gland of EM-800-treated animals was exclusively composed of an atrophied ductal system lined by atrophied epithelial cells with an absence of lobulo-glandular elements. No effect of the compound was observed on the histology of the mammary gland in ovariectomized animals, thus showing the pure antiestrogenic effect of EM-800 on the mammary gland, as shown also for the uterus, vagina, and hypothalamo-pituitary axis. At histopathology, all doses of EM-800 in intact animals led to a moderate to severe uterine and vaginal atrophy. The uterine atrophy affected both the myometrium and the endometrium. Interestingly, the uterine atrophy achieved in intact animals treated with EM-800 was greater than that observed after ovariectomy alone, thus clearly demonstrating the pure antiestrogenic activity of EM-800. The present data show the highly potent and pure antiestrogenic activity of EM-800 on all parameters measured after 6 months of treatment in both intact and ovariectomized mice, a maximal effect being reached at the daily 10 µg dose of the antiestrogen in intact animals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS are recognized to play the predominant role in breast cancer development and growth (1, 2, 3, 4, 5, 6). Because the first step in the action of estrogens in target tissues is binding to the estrogen receptor (7, 8), a logical approach for the treatment of estrogen-sensitive breast cancer is the use of antiestrogens, or compounds that block the interaction of estrogens of all sources with the estrogen receptor.

Unfortunately, until very recently, no compound having pure antiestrogenic activity has been available. In fact, Tamoxifen, the only antiestrogen used for the treatment of breast cancer in women, behaves as a mixed agonist/antagonist of estrogen action (9, 10, 11), thus limiting its therapeutic potential and possibly explaining the limited success obtained with this compound in the treatment of breast cancer in women (1, 2, 3).

Recently, 7{alpha}-alkyl derivatives of estradiol (12, 13, 14, 15), 11ß-amidoalkoxyphenyl estradiols (16), or estradiol 7{alpha}-alkyl derivatives possessing additional changes designed to increase their affinity for the estrogen receptor and/or increase their bioavailability (17, 18, 19, 20, 21) have been synthesized and shown to possess pure and potent antiestrogenic activity in the most rigorous in vitro and in vivo systems (12, 17), including human breast cancer cells in vitro (12, 19, 20, 21) and in vivo in nude mice (13, 15, 18).

These 7{alpha}-alkyl and 11ß-amidoalkoxyphenyl derivatives of estradiol, however, have low oral bioavailability, thus limiting their acceptability for the treatment of breast cancer. We have thus developed a series of even more potent estrogen antagonists that possess high oral bioavailability in the mouse, rat, monkey, and human. The present study describes the characteristics of long-term treatment with the pure antiestrogen EM-800 on a series of biological parameters in the intact and ovariectomized female mouse, thus demonstrating the high potency of the compound by the oral route and its pure antiestrogenic activity in the uterus, vagina, mammary gland, and hypothalamo-pituitary axis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female BALB/c mice (BALB/cAnNCrlBR) approximately 50 days old and weighing 19–20 g were obtained from Charles-River, Inc. (St-Constant, Québec, Canada) and housed 4–5 per cage in a temperature (23 ± 1 C)- and light (12 h light/day, lights on at 0715 h)-controlled environment. The mice were fed rat chow and tap water ad libitum. Animals were randomly divided into groups, each group containing 20 mice. Animals of the appropriate groups were bilaterally ovariectomized under general anesthesia (Avertin), whereas intact mice were used in the other groups. Treatment was initiated 4 days after ovariectomy.

Chemicals
EM-800 ((S)-(+)-4-[7-(2,2-dimethyl-1-oxopropoxy)-4-methyl-2-[4-[2-(1-piperidinyl)-ethoxy]phenyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-dimethylpropanoate was synthesized in the medicinal chemistry division of our laboratory. The structure of this antiestrogen is illustrated in Fig. 1Go. The antiestrogen was administered orally by gastric gavage, once daily for 24 weeks in a total volume of 0.2 ml of 4% ethanol, 4% polyethylene glycol 600 (PEG-600), 1% gelatin, and 0.9% NaCl aqueous suspension. Control intact and ovariectomized animals received the same volume of the vehicle. The compound was administered at the daily oral dose of 3, 10, 30, or 100 µg/animal or approximately 0.15, 0.50, 1.50, or 5.0 mg/kg, respectively, at start of treatment. The same dose was administered during the course of the study with no adjustment for increased body weight.



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  		Figure 1. Chemical structure of EM-800.

 
Estrogen receptor assay
Preparation of cytosol. Uterine tissue was homogenized in 20 volumes of buffer A (25 mM Tris-HCl, 1.5 mM EDTA disodium salt, 10 mM {alpha}-monothioglycerol, 10% glycerol, and 10 mM sodium molybdate, pH 7.4). The homogenates were then centrifuged at 105,000 x g for 60 min at 4 C. The steroid binding assay was performed with freshly prepared cytosol. Protein concentration was determined with the method of Bradford (22) using BSA as standard.

Assay. [2,4,6,7-3H]Estradiol (S.A. 115 Ci/mmol) was purchased from New England Nuclear (Boston, MA), whereas Diethylstilbestrol (DES) and 17ß-estradiol were obtained from Sigma Chemical Co. (St. Louis, MO). [3H]estradiol binding was measured using the dextran-coated charcoal absorption technique, essentially as described (6, 23); in brief, 0.2 ml aliquots of the cytosol preparation were incubated with 0.1 ml [3H]E2 (90,000 cpm, final concentration 3 nM) in the presence or absence of a 100-fold excess of DES for 3 h at room temperature. Unbound steroids were separated by incubation for 15 min at 4 C with 0.3 ml of 0.5% Norit A, 0.05% Dextran T-70 (DCC) in buffer B (1.5 mM EDTA disodium salt, 10 mM {alpha}-monothioglycerol and 10 mM Tris-HCl, pH 7.4) and centrifugation at 1,000 x g for 15 min. Aliquots of the supernatant (0.3 ml) were then taken for radioactivity measurement after addition of 10 ml liquid scintillation cocktail.

RIAs
Serum steroid levels were measured by RIAs following methanol and diethyl ether extraction and chromatography on LH-20 columns as described in detail elsewhere (24, 25). EM-800 does not interfere in the steroid assays. All the samples were chromatographed and underwent RIA simultaneously.

Serum LH was measured by double-antibody RIA using rat hormones (LH-I-6 for iodination and rat LH-RP-2 as standard), and the rabbit anti-r-LH-S-8 antiserum, generously supplied by the National Pituitary Program (Baltimore, MD).

Histology
The ovaries, uterus, vagina, and mammary gland from each animal were weighed, immersed in a solution of 10% buffered formalin for 48 h, routinely processed in a tissue processor, and embedded in paraffin as described (26). Sections of 5–6 microns were cut and stained with hematoxylin-eosin. Examination of the tissue slides was performed by light microscopy.

Statistical analysis
Statistical significance was measured according to the multiple-range test of Duncan-Kramer (27). Data are expressed as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Daily oral treatment with EM-800 for 24 weeks caused a marked inhibition of uterine weight in both intact and ovariectomized mice (Fig. 2Go). In intact animals, the low oral dose of 3 µg already caused a 30% decrease in uterine weight from 79.6 ± 6.6 mg to 56.0 ± 3.3 mg (P < 0.01). A maximal inhibition was achieved with the dose of 10 µg that decreased uterine weight by 64% to 29.0 ± 1.8 mg (P < 0.01). At the 30 µg and 100 µg doses, uterine weight was measured at 33.1 ± 1.7 (59% inhibition) and 46.8 ± 1.9 mg (41% decrease) (P < 0.01), respectively. It is of interest to see in the same figure that in ovariectomized animals, treatment with the same increasing doses of EM-800 caused changes in uterine weight from 64.0 ± 3.9 mg (control ovariectomized) to 67.6 ± 3.8 (N.S.), 50.7 ± 4.1 (P < 0.05), 37.8 ± 3.3 (P < 0.01), and 37.2 ± 2.4 (P < 0.01) mg, respectively, whereas ovariectomy itself caused a 19.6% inhibition (P < 0.05) compared with intact animals. In ovariectomized animals, a maximal inhibition of 41% was achieved at the daily 100 µg oral dose of the antiestrogen.



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  		Figure 2. Effect of 24-week treatment with increasing doses of the antiestrogen EM-800 on uterine weight in intact and ovariectomized mice. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg, or 100 µg. *, P < 0.05; **, P < 0.01 vs. respective controls; +, P < 0.05, OVX vs. intact.

 
Similar effects were observed on vaginal weight (Fig. 3Go). In intact mice, EM-800 decreased vaginal weight from 63.4 ± 3.9 to 46.1 ± 2.7 mg (27% inhibition, P < 0.01) and 18.4 ± 0.7 mg (71% inhibition, P < 0.01) at the doses of 3 and 10 µg, respectively. The 30 µg and 100 µg doses of EM-800 led to vaginal weights of 19.0 ± 0.6 mg (70% inhibition, P < 0.01) and 26.4 ± 1.0 mg (58% decrease, P < 0.01), respectively. Administration of the same doses of EM-800 to ovariectomized mice led to a progressive decrease in vaginal weight from 33.8 ± 1.2 mg to 31.4 ± 1.2 (N.S.), 25.2 ± 1.4 mg (P < 0.01), 21.8 ± 0.9 mg (P < 0.01) and 22.7 ± 0.8 mg (P < 0.01), whereas ovariectomy itself decreased vaginal weight by 46.7% (P < 0.01) compared with intact animals.



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  		Figure 3. Effect of 24-week treatment with increasing doses of the antiestrogen EM-800 on vaginal weight in intact and ovariectomized mice. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg, or 100 µg. **, P < 0.01 vs. respective controls; ++, P < 0.01, OVX vs. intact.

 
Ovarian weight, on the other hand, increased from 8.76 ± 0.47 mg in intact control animals to 15.42 ± 0.82 mg (P < 0.01), 16.75 ± 0.85 mg (P < 0.01) and 16.75 ± 0.81 mg (P < 0.01) at the doses of 10, 30, and 100 µg, respectively, whereas the lowest dose used (3 µg) had no significant effect on this parameter (Fig. 4Go). Treatment of intact animals with the daily doses of 10 µg, 30 µg, and 100 µg of EM-800 led to an increase in serum LH from 0.22 ± 0.04 in controls to 0.24 ± 0.05 (NS), 0.60 ± 0.03 (P < 0.01), and 1.17 ± 0.32 (P < 0.01) ng/ml. Serum LH levels were elevated at 6.37 ± 1.21 ng/ml (P < 0.01) 24 weeks after OVX, and no significant effect was observed on serum LH levels in ovariectomized animals at any of the doses of EM-800 used (Fig. 5Go).



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  		Figure 4. Effect of 24-week treatment with increasing doses of the antiestrogen EM-800 on ovarian weight in intact mice. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg, or 100 µg. **, P < 0.01 vs. control.

 


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  		Figure 5. Effect of 24-week treatment with increasing doses of the antiestrogen EM-800 on serum LH levels in intact and ovariectomized mice. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg, or 100 µg. **, P < 0.01 vs. respective controls.

 
As illustrated in Fig. 6Go, treatment with the highest dose of EM-800 caused a 93% (P < 0.01) increase in serum 17ß-estradiol (E2) levels, whereas the lower doses had no significant effect. Serum estrone concentration, on the other hand, was not affected by treatment at any of the doses used of the antiestrogen. Serum androstenedione (4-dione) concentration increased from 0.24 ± 0.03 nM in intact control mice to 1.58 ± 0.42 nM (558% over control) (P < 0.01) and 1.95 ± 0.89 nM (713% over control) (P < 0.01) in animals treated with the 30 µg and 100 µg doses of EM-800, respectively (Fig. 7Go). Serum dehydroepiandrosterone and androstenediol (5-diol) concentrations were unchanged by treatment with EM-800 (Fig. 7Go). Serum testosterone (testo), on the other hand, increased from 0.47 ± 0.05 nM in control intact mice to 2.44 ± 0.41 nM (419% over control) (P < 0.01) and 2.86 ± 0.24 nM (509% over control) (P < 0.01) in animals treated with the 30 µg and 100 µg doses of EM-800 (Fig. 8Go), respectively. It can be seen in the same figure that serum dihydrotestosterone (DHT) increased by 76% (P < 0.05) and 141% (P < 0.05) over control at the two highest doses of EM-800 with no significant effect at the lower doses (Fig. 8Go). Although there was a tendency for decreased serum levels of progesterone at all doses of EM-800, the decrease did not reach statistical significance (data not shown).



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  		Figure 6. Effect of 24-week treatment with increasing doses of the antiestrogen EM-800 on serum estrone (E1) and 17ß-estradiol (E2) levels in intact mice. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg, or 100 µg. **, P < 0.01 vs. control.

 


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  		Figure 7. Effect of 24-week treatment of intact mice with increasing doses of the antiestrogen EM-800 on serum dehydroepiandrosterone (DHEA), androstenedione (4-dione), and androst-5-ene-3ß,17ß-diol (5-diol) levels. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg or 100 µg. **, P < 0.01 vs. respective controls.

 


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  		Figure 8. Effect of 24-week treatment of intact mice with increasing doses of the antiestrogen EM-800 on serum testosterone (testo) and dihydrotestosterone (DHT) levels. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg, or 100 µg. *, P < 0.05; **, P < 0.01 vs. respective controls.

 
Treatment with EM-800 caused a dramatic fall in uterine [3H]E2 binding to the uterine estrogen receptor (ER) in both intact and ovariectomized animals (Fig. 9Go). Treatment with the lowest dose used, namely 3 µg of EM-800, decreased uterine ER levels in intact animals from 570 ± 40 fmol/mg protein to 250 ± 5 fmol/mg protein (P < 0.01) (56% inhibition), whereas treatment with the three higher doses caused 88%, 97%, and 99% inhibitions of uterine ER levels. Ovariectomy itself increased uterine ER levels to 961 ± 112 fmol/mg protein (P < 0.01), whereas inhibitions of 46%, 87%, 97%, and 99.5% were seen with the respective daily doses of 3 µg, 10 µg, 30 µg, and 100 µg of EM-800 administered to ovariectomized animals.



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  		Figure 9. Effect of 24-week treatment with increasing doses of the antiestrogen EM-800 on uterine estrogen receptor (ER) levels in intact and ovariectomized mice. The antiestrogen was administered daily by the oral route at the doses of 3 µg, 10 µg, 30 µg, or 100 µg. **, P < 0.01 vs. respective controls.

 
At all doses given for 24 weeks to mature mice (2 months old at start of treatment and 8 months old at the end of the experiment), EM-800 led to a complete absence of glandular elements in the mammary gland. In fact, the mammary gland was then exclusively composed of a ductal system, with small ducts lined by atrophic epithelial cells, having the same histologic appearance as the atrophic glands seen in ovariectomized control mice with an absence of lobulo-glandular elements (Fig. 10Go). In intact animals, normal histologic appearance of the mammary gland was observed, with a fully developed ductal system and lobular structures (Fig. 10AGo). On the other hand, treatment of ovariectomized mice with EM-800 had no effect on the histopathology of the mammary gland in addition to the changes caused by ovariectomy (Fig. 11Go).



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  		Figure 10. Histology of the mammary gland in (A) control intact mice and in intact mice treated with EM-800 at the oral daily doses of (B) 3 µg, (C) 10 µg, (D) 30 µg, or (E) 100 µg, for 24 weeks. Treatment with all doses of EM-800 resulted in a complete inhibition of mammary gland maturation and development. The mammary gland of EM-800-treated animals is composed of only atrophic ducts (d) with the absence of lobulo-glandular elements (L). Hematoxylin-eosin (magnification, x80). Higher magnification of a mammary duct (d) showing the atrophic ductal epithelium (magnification, x800) (F).

 


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  		Figure 11. Histology of the mammary gland in (A) ovariectomized mice 6 months after castration and in ovariectomized animals treated with EM-800 at the daily oral doses of (B) 3 µg, (C) 10 µg, (D) 30 µg, or (E) 100 µg, for 24 weeks. Treatment with EM-800 had no effect on mammary gland histology in addition to the changes already induced by ovariectomy. The mammary gland is essentially composed of an atrophied ductal system (d) without evidence of normal development and maturation of the glandular component. Hematoxylin-eosin (magnification, x80). Higher magnification of a mammary duct (d) showing the atrophic ductal epithelium (magnification, x800) (F).

 
Treatment of intact mice with all doses of EM-800 resulted in a moderate hypertrophy as well as in a moderate to marked hyperplasia of the interstitial cells of the ovaries (Fig. 12Go). Interstitial cells were enlarged, with abundant, pale, eosinophilic and finely granular cytoplasm and a centrally located nucleus. These cells arranged in nests cause a marked expansion of the stromal ovarian tissue. EM-800 at the daily oral doses of 10, 30, and 100 µg also led to a significant reduction in folliculogenic activity characterized by a moderate to marked decrease in the number of developing follicles and corpora lutea, whereas a mild increase in the number of atretic-anovulatory follicles was also present.



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  		Figure 12. Ovarian histology in (A) intact mice who received control vehicle and in intact mice treated with EM-800 at the daily oral doses of (B) 3 µg, (C) 10 µg, (D) 30 µg, or (E) 100 µg, for 24 weeks. Treatment with all doses of EM-800 resulted in a moderate hypertrophy and a moderate to marked hyperplasia of interstitial cells (IC), which was more accentuated in the higher dose groups (C–E). A moderate to marked decrease in the number of developing follicles (F) and corpora lutea (CL) was also observed beginning at the dose of 10 µg (C–E), this effect being accompagnied by a mild increase in the number of atretic follicles (AF). Hematoxylin-eosin (magnification, x80.)

 
All doses of EM-800 caused a moderate to severe atrophy of the uterus that involved both endometrial and myometrial layers, although the atrophic changes were more pronounced in the myometrium. The uterine atrophy is characterized by the presence of atrophic endometrial glands with epithelial cells having a decreased cytoplasmic/nuclear ratio. The condensed stroma is composed of inactive, atrophied stromal cells, whereas the myometrial cells show increased basophilia and a decreased amount of cytoplasm. A mild edema of the stroma is also observed in same areas. All changes were dose dependent, the doses of 10 and 30 µg of EM-800 being the most effective. Treatment with EM-800 of both intact (Fig. 13Go) and ovariectomized (Fig. 14Go) animals resulted in a moderate to marked uterine atrophy that, interestingly, was greater than that achieved after 24 weeks of castration alone.



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  		Figure 13. Uterine histology in (A) intact mice who received control vehicle, (B) mice ovariectomized 24 weeks previously and in intact mice treated with EM-800 at the daily oral doses of (C) 3 µg, (D) 10 µg, (E) 30 µg, or (F) 100 µg, for 6 months. Treatment with all doses of EM-800 resulted in a moderate (C) to marked (D–F) atrophy of the uterus characterized by decreased thickness of both myometrial (M) and endometrial (E) layers, atrophic inactive glandular epithelium (GE), and a condensed, regionally edematous stroma (S). EM-800 treatment caused a greater uterine atrophy than that observed after ovariectomy alone (B). Hematoxylin-eosin (magnification, x80, inset, x800).

 


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  		Figure 14. Uterine histology in (A) intact mice who received control vehicle, in (B) mice ovariectomized 24 weeks previously and in ovariectomized mice treated with EM-800 at the daily oral doses of (C) 3 µg, (D) 10 µg, (E) 30 µg, or (F) 100 µg, for 6 months. Treatment with all doses of EM-800 resulted in a marked uterine atrophy (C–F), which was much greater as compared with ovariectomized controls (B). Note the decreased thickness of both endometrial (E) and myometrial (M) layers, the atrophic glandular epithelium (GE) as well as the condensed stroma (S). Hematoxylin-eosin (magnification, x80, inset, x800).

 
Treatment with all doses of EM-800 was accompanied by a moderate to severe vaginal atrophy in both intact (Fig. 15Go) and ovariectomized animals (Fig. 16Go). The vaginal atrophy is characterized by a marked decrease in the thickness of the vaginal epithelium, which decreased from two to six layers (intact treated) to one to three layers (ovariectomized treated) of germinal epithelial cells. Areas of mucification of the vaginal epithelium with the presence of columnar, mucus-containing cells were already observed in intact animals treated with EM-800 at the dose of 3 µg. Treatment of ovariectomized animals with EM-800 had no histologically significant effect on the atrophy achieved by ovariectomy, although, as shown in Fig. 3Go, vaginal weight was further inhibited in ovariectomized animals receiving EM-800.



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  		Figure 15. Vaginal histology in (A) intact mice who received control vehicle, in (B) mice ovariectomized 24 weeks previously or intact mice treated with EM-800 at the daily oral doses of (C) 3 µg, (D) 10 µg, (E) 30 µg, or (F) 100 µg, for 6 months. All doses of EM-800 resulted in a moderate (C) to severe (D–F) vaginal atrophy which was comparable to that caused by ovariectomy (B), especially at the doses of 10, 30, and 100 µg. The vaginal epithelium consisted of two to six layers of germinal epithelial cells, with areas of mucification (m) of the superficial layer (D). Hematoxylin-eosin (magnification, x200).

 


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  		Figure 16. Vaginal histology in (A) intact mice who received control vehicle, in (B) mice ovariectomized 24 weeks previously and in ovariectomized mice treated with EM-800 at the daily oral doses of (C) 3 µg, (D) 10 µg, (E) 30 µg, or (F) 100 µg, for 6 months. All doses of EM-800 resulted in severe (C–F) vaginal atrophy comparable with that caused by ovariectomy alone. The vaginal epithelium was markedly decreased in thickness, consisting only of one to three layers of germinal atrophic epithelial cells with areas of mucification (m) of superficial layer (c). Hematoxylin-eosin (magnification, x200).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present data show the highly potent and pure antiestrogenic activity of EM-800 on all the parameters measured in both intact and ovariectomized mice after daily treatment with the antiestrogen for 24 weeks. In fact, the pure antiestrogenic activity of the new compound is demonstrated on the uterus, vagina, mammary gland, and hypothalamo-pituitary-gonadal axis.

The pure antiestrogenic activity of EM-800 on the hypothalamo-pituitary-ovarian axis is illustrated by the 76% increase in ovarian weight at the 10 µg dose. A comparable hyperstimulation of the ovaries by excess gonadotropin has been observed in homozygous estrogen receptor (ER) mutant mice following disruption of the estrogen negative feedback by a different mechanism, namely the absence of ER{alpha} (28), whereas, in the present study, the ER has been down-regulated and its action blocked by EM-800. In fact, the morphological changes observed in the ovary after chronic treatment with EM-800 show strong similarities with those observed in transgenic ER knockout mice treated with gonadotropins (28). In such homozygous ER mutant mice, granulosa and theca cells are present, although follicle development appears to proceed through primary and secondary stages but arrests before formation of ovulatory follicles. In those transgenic animals, exogenous gonadotropins led to the formation of a few antral or ovulatory follicles although most were unviable (cystic atretic) follicles.

Stromal cell hyperplasia is known to accompany ovarian atrophic changes characterized by the lack of normal development of follicles and corpora lutea, both being signs of inhibited ovulation and loss of normal ovarian cyclicity. Interstitial cells are steroidogenically active, secreting especially androstenedione and testosterone, thus explaining the elevated blood levels of these steroids in intact animals treated with EM-800 (29).

In the present study, the hyperstimulation of the ovaries by excess endogenous gonadotropin secretion is further illustrated by the increased circulating levels of androstenedione, testosterone, dihydrotestosterone, and estradiol. These changes in serum steroid levels were seen, however, at different doses of the antiestrogen; significant increases in serum testosterone, dihydrotestosterone, and androstenedione were observed at the 30 µg dose, whereas the 100 µg dose was required to cause a significant increase in serum estradiol. Because the interstitial elements of the ovary were hypertrophied, it is likely that androstenedione as well as testosterone and dihydrotestosterone were secreted directly by the interstitial ovarian cells, whereas estradiol was synthesized up to an unknown extent in peripheral tissues from androstenedione and testosterone before being released into the general circulation. The intracrine formation of estradiol in peripheral tissues from the precursors androstenedione and testosterone is the most likely explanation for the observation that increased serum estradiol was seen at a higher dose of EM-800 than the changes in the serum levels of the precursor steroids androstenedione and testosterone (30, 31). In fact, all the enzymes required to transform androstenedione and testosterone into E2, namely 17ß-hydroxysteroid dehydrogenase and aromatase, are present in a large series of peripheral intracrine tissues (31, 32, 33, 34). Another possibility is a blockade by EM-800 of the transformation of 4-dione and testosterone into estrogens in the ovary itself.

The mouse mammary gland reaches its histological and functional differentiation and maturity at the age of three months. The presence of an atrophic mammary gland with no glandular elements, in the intact animals receiving EM-800 for 24 weeks, further demonstrates the highly potent antiestrogenic activity of EM-800 in this tissue. In fact, even at the lowest dose of EM-800 used, namely 3 µg, the development of the mammary gland was completely prevented. Moreover, in ovariectomized animals, a complete lack of effect was observed, thus supporting the pure antiestrogenic characteristics of EM-800 in the mammary gland.

The biphasic effect of EM-800 on uterine and vaginal weight seen at high doses of EM-800 in intact animals is most likely due to the well-known androgenic stimulatory effect of high serum levels of androstenedione, testosterone, and dihydrotestosterone in these two tissues. However, the slightly greater maximal inhibition of uterine and vaginal weight achieved in intact compared with ovariectomized animals cannot be explained with our current knowledge of estrogenic and antiestrogenic action, but it could result from so far undetermined protein-ER interactions that could be modulated in yet unknown fashions by estradiol and EM-800.


   Acknowledgments
 
We wish to thank Mr. Roger Lachance and Mr. Simon Caron for their skillful technical assistance.

Received September 30, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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