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Endocrinology Vol. 138, No. 12 5605-5617
Copyright © 1997 by The Endocrine Society

ARTICLES

Morphological Changes Induced by 6-Month Treatment of Intact and Ovariectomized Mice with Tamoxifen and the Pure Antiestrogen EM-800

Antigone Sourla, Shouqi Luo, Claude Labrie, Alain Bélanger and Fernand Labrie

Laboratory of Molecular Endocrinology, Le Centre Hospitalier de l’Université Laval Research Center and Laval University, Québec, G1V 4G2, Canada

Address all correspondence and requests for reprints to: Professor Fernand Labrie, MRC Group in Molecular Endocrinology, CHUL Research Center, 2705 Laurier Boulevard, Québec, G1V 4G2, Canada.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study compares the effects of tamoxifen and EM-800, both administered at the oral daily dose of 100 µg for 6 months, on the uterus, vagina, and mammary gland in the mouse at histopathological examination. Treatment of intact animals with EM-800 resulted in uterine and vaginal atrophy even greater than that achieved after ovariectomy, while the developmental growth of the mammary gland was completely blocked and serum LH was increased. In ovariectomized animals, treatment with EM-800 decreased uterine and vaginal wt below the values observed in control ovariectomized mice while no significant change was observed on serum LH, thus indicating the lack of estrogenic activity of EM-800.

Tamoxifen, on the other hand, showed a stimulatory estrogenic-like action on the mouse uterus in both intact and ovariectomized animals, thus resulting in moderate to severe endometrial hyperplasia. These morphological changes were accompanied by a marked stimulation of both the estrogenic and androgenic 17ß-hydroxysteroid dehydrogenase as well as 5{alpha}-reductase uterine activities. The histological atrophic changes observed in the vagina after tamoxifen treatment were less pronounced than those seen after treatment with EM-800. The agonistic estrogen-like action of tamoxifen was also illustrated by the suppression of serum LH levels in ovariectomized animals.

A marked stimulation of the ovarian stroma, accompanied by a significant reduction in folliculogenic activity, was observed after EM-800 or tamoxifen administration, although the interstitial ovarian hyperplasia was more pronounced after EM-800 treatment. While both antiestrogens blocked the developmental growth of the mammary gland, EM-800 showed more potent antiestrogenic activity than tamoxifen. The highly potent and specific antiestrogenic activity of EM-800 suggests that this compound could improve the therapy of breast cancer while avoiding the undesirable stimulation of the endometrium.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE incidence of breast cancer, as well as the mortality due to this disease, has been rising in most Western countries for decades, except for a recent small decrease in the United States (1, 2, 3). Estrogens are well recognized as being a predominant factor involved in both the development and growth of breast cancer (4, 5, 6, 7, 8, 9).

The therapeutic approaches for estrogen-sensitive breast cancer have focused on the use of antiestrogens, especially tamoxifen, that block the interaction of estrogens with their specific receptor in the mammary gland (10, 11, 12). Although tamoxifen has shown clear beneficial effects for the treatment of breast cancer, especially when given as an adjuvant to surgery (10, 13), this compound is known to possess mixed agonistic/antagonistic activities that are species-, cell-, and gene-specific (14, 15, 16, 17, 18, 19).

The need for compounds having pure antiestrogenic action and thus lacking partial estrogenic action in the human mammary gland and endometrium has led to the development of drugs, such as 7{alpha}-alkyl derivatives of estradiol, that possess pure and potent antiestrogenic activity, both in vivo and in vitro (20, 21, 22, 23, 24, 25).

The present study compares the effects of long-term administration of tamoxifen in intact and ovariectomized mice, in comparison with those seen after treatment with EM-800, a new orally active pure antiestrogen developed in our laboratory. This orally active compound is the most potent and pure antiestrogen in human breast and uterine cancer cells (26, 27, 28, 29). We have also investigated possible alterations of the activity of steroidogenic enzymes, especially in the uterus, after administration of the two compounds.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female BALB/cAnNCrlBR) mice approximately 50 days old and weighing 19–20 g were obtained from Charles-River, Inc. (St. Constant, Québec, Canada) and housed four to five 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 rodent chow and tap water ad libitum. Animals were randomly divided into groups of 20 mice each. Animals of the appropriate groups were bilaterally ovariectomized under general anesthesia (Avertin) while intact mice were used in the other groups.

Chemicals
EM-800 ((+)-7-pivaloyloxy-3-(4'-pivaloyloxyphenyl)-4-methyl-2-(4''-(2'''-piperidinoethoxy)phenyl)-2H-benzopyran) (Fig. 1Go) was synthesized in the medicinal chemistry division of our laboratory while tamoxifen was from Sigma Chemical Co. (St. Louis, MO). EM-800 and tamoxifen, prepared in 4% ethanol, 4% polyethylene glycol 600 (PEG-600), 1% gelatin, 0,9% NaCl, were administered orally, once daily for 24 weeks, in a total volume of 0.2 ml.



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  		Figure 1. Chemical structures of antiestrogens.

 
Treatment
The animals were randomly assigned to the following groups: 1) intact received the vehicle alone; intact animals treated with either 2) EM-800 or 3) tamoxifen at the daily oral dose of 100 µg; 4) ovariectomized animals receiving the vehicle alone; and ovariectomized animals treated with either 5) EM-800 or 6) tamoxifen, at the same daily dose of 100 µg. In a preliminary experiment, increasing oral doses of EM-800 and tamoxifen were given daily for 28 days. The effect of such treatment was measured on uterine and vaginal wt to determine the optimal inhibitory dose.

EM-800 and tamoxifen were administered daily for 24 weeks orally (by gavage) using the vehicle 1% gelatin (wt/vol) and 4% (wt/vol) PEG-600. In ovariectomized animals, treatment was started 4 days after ovariectomy. Body wt was measured once a week. Control animals received the same volume of the vehicle. The animals were killed by decapitation under basal conditions in a longitudinal fashion.

Histology
The tissues (ovaries, uterus, and vagina) 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 previously described (30). Sections of 5–6 µm were cut and stained with hematoxylin-eosin. Examination of the tissue slides was performed by light microscopy.

5{alpha}-Reductase and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) assays
Tissues were homogenized with a Polytron PT-10 homogenizer (Brinkman Instruments, Canada), at a setting of 8 for 10 sec, in 1.0 ml phosphate buffer (20 mM KH2PO4, 0.25 M sucrose, 1 mM EDTA, pH 7.5) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride and 5 mg/ml each of pepstatin A, antipain, and leupeptin) and then centrifuged for 30 min at 1000 x g to remove cell debris. Protein content of tissue homogenates was measured by the method of Bradford using BSA as standard (31). Aliquots of homogenate were incubated at 37 C in 0.5 ml phosphate buffer (12.5 mM KH2PO4, 1 mM EDTA, pH 7.5) containing 0.6 µM 14C-labeled substrates obtained from New England Nuclear (Boston, MA): [14C]testosterone (57.3 mCi/mmol), [14C]estrone (E1) (51.7 mCi/mmol), and [14C]androstenedione (4-dione) (53.9 mCi/mmol). NADP+ was used as cofactor for 5{alpha}-reductase while NADH and NADPH were used as cofactors for the reductive form of 17ß-HSD, and NAD+ and NADP+ were used as cofactors for the oxidative form of 17ß-HSD. Cofactors were present at the final concentration of 1 mM.

Duration of the assay was chosen to maintain the final conversion of the 14C-labeled substrate below 20%. The enzymatic reaction was stopped by chilling the incubation mixtures in an ice water slurry. After 3 ml diethyl ether were added and vortexed for 1 min, the tubes were centrifuged at 2000 x g for 10 min to separate the aqueous and organic phases and then placed in an ethanol-dry ice bath. The organic phase was decanted while the aqueous phase was extracted once more. The two organic phases were pooled and evaporated to dryness under a stream of nitrogen. Next, 20 µg each of unlabeled 4-dione, testosterone, progesterone, and 11-deoxy-cortisol were added as carriers to facilitate identification of the spots on the TLC plates. The residue obtained was suspended in 70 µl dichloromethane (CH2CL2) and chromatographed on 60 f254 silica gel plates with a mixture of toluene-acetone (4:1, vol/vol). Visualization of the carrier steroids was achieved by UV light, and autoradiography was then performed for 24 h. The spots corresponding to substrate and metabolites as revealed by autoradiography of the TLC plates were cut out and transferred into scintillation vials containing 0.5 ml ethanol and 10 ml scintillation fluid for radioactivity counting. Enzymatic activity was expressed as picomoles of product formed per mg protein/min.

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

Statistical analysis
Statistical significance was measured according to the multiple-range test of Duncan-Kramer (32). Data are expressed as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Daily treatment of intact mice with the daily 3, 10, 30, and 100 µg of EM-800 and tamoxifen led to progressive inhibition of uterine and vaginal wt (Table 1Go). While EM-800 led to a 62% (P < 0.01) maximal inhibition of uterine wt at the daily oral dose of 100 µg, the inhibition achieved with the same dose of tamoxifen was only 24% (P < 0.01). On vaginal wt, the 100 µg dose of EM-800 and tamoxifen gave inhibitions of 68% and 48%, respectively (P < 0.01 for both groups vs. intact control). Since a near-plateau was reached at the daily 100 µg oral dose for both compounds, this dose was chosen for the 6-month study.


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  		Table 1. Effect of treatment with increasing doses of the antiestrogen EM-800 or tamoxifen (TAM) on uterine and vaginal wts in intact mice

 
Treatment of intact animals for 6 months with the antiestrogen EM-800 caused a marked reduction in uterine wt from 79.6 ± 6.64 mg to 46.8 ± 1.9 mg (P < 0.01) (Fig. 2Go). In ovariectomized animals, treatment with EM-800 decreased uterine wt below the value found in control ovariectomized animals, namely from 64.0 ± 3.88 mg to 37.2 ± 2.4 mg, respectively (P < 0.01) (Fig. 2Go). On the other hand, as can be seen in the same figure, no statistically significant inhibitory effect on uterine wt was observed after treatment with tamoxifen in either intact or ovariectomized animals.



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  		Figure 2. Effect of 6-month treatment with the daily oral dose of 100 µg EM-800 or tamoxifen on uterine wt in intact and ovariectomized mice (20 animals per group).

 
Similarly, treatment with EM-800 caused a marked inhibition of vaginal wt in both intact and ovariectomized treated animals, which reached values significantly lower from those achieved by ovariectomy (Fig. 3Go). More specifically, EM-800 caused a 58.3% decrease in vaginal wt in intact animals from 63.3 ± 3.89 mg to 26.4 ± 1.04 mg (P < 0.01). On the other hand, in ovariectomized animals, treatment with EM-800 inhibited vaginal wt to a value 32.9% below that found in control ovariectomized mice, namely from 33.8 ± 1.15 mg in control mice to 22.7 ± 0.82 mg, P < 0.01. Tamoxifen, on the other hand, decreased vaginal wt by 69.2% in intact mice (from 63.3 ± 3.89 to 19.5 ± 0.75 mg, P < 0.01) and by 35% (from 33.8 ± 1.15 to 22.0 ± 0.63 mg, P < 0.01), in ovariectomized animals, respectively, in comparison with the corresponding vehicle-treated controls (Fig. 3Go).



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  		Figure 3. Effect of 6-month treatment with the daily oral dose of 100 µg EM-800 or tamoxifen on vaginal wt in intact and ovariectomized mice (20 animals per group).

 
A 91.2% increase in ovarian wt was observed after 24 weeks of treatment with EM-800 from a value of 8.76 ± 0.47 mg in intact control mice to 16.7 ± 0.81 mg in those who received EM-800, while a 41% decrease in ovarian wt to 5.07 ± 0.28 mg resulted from tamoxifen administration during the same time interval in intact animals (data not shown).

At histopathological examination of the tissues, a moderate uterine atrophy was observed 24 weeks after ovariectomy. These changes were characterized by decreased endometrial and myometrial thickness as well as by condensation of the endometrial stroma (Fig. 4BGo compared with 4A). The atrophic glandular epithelial cells had an inactive appearance with a reduced amount of cytoplasm and a cytoplasmic/nuclear ratio <1. The myometrium in ovariectomized animals was composed of basophilic cells with a decreased amount of cytoplasm. Treatment of intact animals with EM-800 at the daily dose of 100 µg, for 6 months, resulted in a moderate to marked uterine atrophy that involved both the endometrial and myometrial layers and was more pronounced than that observed in ovariectomized-control animals. Such reduced thickness of both the endometrial and myometrial layers was accompanied by a decrease in the endometrial to myometrial volume ratio. The endometrial atrophy was characterized by a reduced number of endometrial glands, which were lined by atrophic glandular cells having a cytoplasmic/nuclear ratio <1. On the other hand, the condensed stroma was composed of apparently inactive stromal cells while, in some areas, a mild edema of the stroma was observed. Myometrial cells of EM-800-treated animals had increased basophilia and a decreased amount of cytoplasm.



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  		Figure 4. Uterine histology in (A) intact mice, (B) ovariectomized mice 6 months after castration, and in (C) intact or (D) ovariectomized mice treated with EM-800 at the oral daily dose of 100 µg for 6 months. Treatment with EM-800 was accompanied by a marked uterine atrophy in both intact (C) and ovariectomized (D) animals, an effect that was greater than that achieved by ovariectomy alone (B). Note the decreased thickness of both the endometrial (E) and myometrial (M) layers. One can also observe the atrophic glandular epithelium (GE) as well as the condensed stroma in intact (C) and OVX (D) animals treated with EM-800 (insets). Hematoxylin-eosin (magnification, x80; inset, x500). The micrographs are representative of patterns seen in samples from individual animals.

 
On the other hand, 6-month treatment of intact female mice with tamoxifen, at the daily dose of 100 µg, resulted in histological changes showing moderate to severe cystic endometrial hyperplasia. These changes were characterized by an increased number and crowding of the endometrial glands, thus resulting in a distorted glandular architecture (Fig. 5CGo). A marked, often cystic, dilatation of the endometrial glands was also observed. These changes were accompanied by the accumulation of secretory, eosinophilic material in the glandular and endometrial lumen as well as a regional moderate edema of the endometrial stroma. A polypoid structure, as well as an increased height of the luminal epithelium, was seen in intact animals treated with tamoxifen (Fig. 5CGo). A moderate hypertrophy and stratification of the glandular epithelium was also observed in tamoxifen-treated intact animals, with the presence of mild focal nuclear atypia, characterized by enlargement and pleomorphism of the nuclei containing prominent nucleoli (Fig. 5Go, E and F). In the myometrial layer, treatment with tamoxifen led to a mild to moderate atrophy, this change being accompanied by a reduced amount of cytoplasm in the myometrial cells, thus resulting in decreased myometrial thickness (Fig. 5CGo).



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  		Figure 5. Uterine histology in (A) intact mice, (B) ovariectomized mice 6 months after castration, and in (C) intact or (D) ovariectomized mice treated with tamoxifen at the oral daily dose of 100 µg for 6 months. Tamoxifen administration resulted in a severe cystic endometrial hyperplasia characterized by marked cystic dilatation of the endometrial glands (EG), in both intact (Fig. 5CGo) and ovariectomized (Fig. 5DGo) animals. These changes were accompanied by the accumulation of secretory material(s) in the lumen of the endometrial gland and areas of polypoid conformation of the luminal epithelium (P). Foci of stratification accompanied by mild atypia of the glandular epithelium (GE) were seen in intact and ovariectomized animals treated with tamoxifen (E, F, arrows). Compare with intact (A) and ovariectomized (B) controls. Hematoxylin-eosin (magnification, x80; panel E, x800).

 
As shown in Fig. 4Go, when EM-800 was administered to ovariectomized animals at the daily dose of 100 µg, a moderate to marked uterine atrophy was observed which, interestingly, was greater than that achieved after ovariectomy. On the contrary, treatment of ovariectomized female mice with tamoxifen, at the same dose, caused the appearance of a cystic endometrial hyperplasia analogous to that seen in intact animals treated with the same dose of tamoxifen. An increased endometrial thickness was also observed (Fig. 5Go, D and F).

A severe vaginal atrophy was found 6 months after ovariectomy, the vaginal epithelium being composed of only one to three layers of atrophic germinal epithelial cells (Figs. 6BGo and 7BGo). After treatment with EM-800, a severe vaginal atrophy was seen in intact animals analogous to that seen in ovariectomized controls. These changes were characterized by a marked decrease in the thickness of the vaginal epithelium, which was composed of only one to three layers of germinal epithelial cells (Fig. 6CGo). In ovariectomized animals treated with EM-800, the observed vaginal atrophy illustrated by one to three layers of germinal epithelial cells did not differ from that seen in ovariectomized controls (Fig. 6Go, panels D and B, respectively).



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  		Figure 6. Vaginal histology in (A) intact mice, (B) ovariectomized mice 6 months after castration, and in (C) intact or (D) ovariectomized mice treated with EM-800 at the oral daily dose of 100 µg for 6 months. EM-800 administration to intact animals resulted in a severe vaginal atrophy (C), which was comparable to that caused by ovariectomy 6 months after castration (B). The vaginal epithelium consisted of one to three layers of germinal epithelial cells, with areas of mucification (m) of the superficial layer. A severe vaginal atrophy was also seen in ovariectomized animals treated with EM-800 (D), with no additional histological change compared with those seen in ovariectomized controls (B). Hematoxylin-eosin (magnification, x200).

 


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  		Figure 7. Vaginal histology in (A) intact mice, (B) ovariectomized mice 6 months after castration, and in (C) intact or (D) ovariectomized mice treated with tamoxifen at the oral daily dose of 100 µg for 6 months. Tamoxifen administration to both intact (C) and ovariectomized (D) animals resulted in a moderate vaginal atrophy. The vaginal epithelium consisted of three to five layers of germinal epithelial cells. Compare with ovariectomized controls (B). Hematoxylin-eosin (magnification, x200).

 
In intact animals treated with tamoxifen, a moderate to severe vaginal atrophy was observed, the vaginal epithelium consisting of three to five layers of epithelial cells (Fig. 7CGo). Similarly, in ovariectomized animals treated with the same dose of tamoxifen, the vaginal epithelium consisted of three to five layers of epithelial cells (Fig. 7DGo). Interestingly, a more severe atrophy of the vaginum characterized by one to three epithelial layers of atrophic germinal cells was observed after ovariectomy and treatment with EM-800 of intact and ovariectomized animals (Figs. 7BGo and 6DGo, respectively).

Treatment with 100 µg EM-800, for 6 months, resulted in a moderate hypertrophy as well as in a moderate to marked hyperplasia of the interstitial cells of the ovarian stroma. The interstitial cells were enlarged and contained abundant, pale eosinophilic and finely grandular cytoplasm with a centrally located or laterally displaced nucleus (Fig. 8DGo1). These cells were arranged in nests, causing expansion of the volume of the stromal ovarian tissue. Concomitantly, a significant reduction in folliculogenic activity was seen. The above indicated change was accompanied by a moderate to marked decrease in the number of developing follicles and corpora lutea, while a mild to moderate increase in the number of atretic-anovulatory follicles was also present (Fig. 8BGo). After treatment with tamoxifen, a moderate to marked decrease in the number of developing follicles and corpora lutea was seen. This effect was accompanied by the presence of multiple foci of moderate interstitial cell hypertrophy (Fig. 8CGo). Similar to the changes observed after EM-800 treatment, the interstitial cells contained abundant eosinophilic and finely granular cytoplasm (Fig. 8DGo2).



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  		Figure 8. Ovarian histology in (A) intact mice who received control vehicle and in intact mice treated with (B) EM-800 or (C) tamoxifen at the oral daily dose of 100 µg for 6 months. Treatment with EM-800 (B) resulted in a moderate hypertrophy and a marked hyperplasia of the interstitial cells (IC) as well as a moderate to marked decrease in the number of developing follicles (F) and corpora lutea (CL). This effect was accompanied by a mild increase in the number of atretic follicles (AF). Administration of tamoxifen (C) also led to a marked decrease to absence of developing follicles (F) and corpora lutea (CL). A focal moderate hyperplasia and hypertrophy of the interstitial cells (IC) was also observed in animals treated with tamoxifen. Note the abundant, finely granular, and eosinophilic cytoplasm of the interstitial cells (IC) after treatment with EM-800 (D1) and tamoxifen (D2). Hematoxylin-eosin (magnification, x80; D1-D2 magnification, x800).

 
With respect to the effects on the mammary gland, the two antiestrogens administered to intact animals completely inhibited the growth and tubuloalveolar development of the mammary gland. In fact, the mouse mammary gland normally reaches its histological and full functional differentiation at the age of 3 months. After treatment with the antiestrogens, the mammary gland was composed of a few atrophic ducts, with inactive atrophic epithelial cells and with no lobuloalveolar structures (Figs. 9CGo and 10CGo). In addition, no additional effects on the mammary gland histomorphology were observed after EM-800 (Fig. 9DGo) or tamoxifen (Fig. 10DGo) administration in ovariectomized animals, the mammary gland showing atrophic changes identical to those seen in ovariectomized controls (Figs. 9BGo and 10BGo).



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  		Figure 9. Mammary gland histology in (A) intact mice, (B) ovariectomized mice 6 months after castration, and in (C) intact or (D) ovariectomized mice treated with EM-800 at the oral daily dose of 100 µg for 6 months. In these animals, the mammary gland is composed of only a few ducts (d) showing an atrophic epithelium. Note the absence of lobuloalveolar (L) structures in both intact (C) and ovariectomized (D) animals treated with EM-800. Compare with ovariectomized controls (B). Hematoxylin-eosin (magnification, x200; insets, x800).

 


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  		Figure 10. Mammary gland histology in (A) intact mice, (B) ovariectomized mice 6 months after castration, and in (C) intact or (D) ovariectomized mice treated with tamoxifen at the oral daily dose of 100 µg for 6 months. Note the atrophic mammary gland in both intact (C) and ovariectomized (D) treated animals and the presence of atrophic ducts (d), with atrophic epithelial cells (e). Hematoxylin-eosin (magnification, x200; insets, x800).

 
As illustrated in Fig. 11Go, a significant stimulation of reductive estrogenic 17ß-HSD activity, responsible for the conversion of estrone to estradiol, was observed after tamoxifen administration in both intact (from 1.67 ± 0.09 to 6.30 ± 0.72 pmol/mg/min, P < 0.01) and ovariectomized (from 1.08 ± 0.22 to 7.31 ± 0.67 pmol/mg/min, P < 0.01), animals compared with intact and ovariectomized controls, respectively. On the contrary, it can be seen that the 24-week treatment with EM-800 had no significant effect on the rate of conversion of estrone to estradiol in uterine homogenate obtained from either both intact or ovariectomized-treated animals.



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  		Figure 11. Estrogenic 17ß-HSD activity (estrone to estradiol) in uterine homogenate obtained from intact or ovariectomized mice 6 months after castration and in intact or ovariectomized mice treated with EM-800 or tamoxifen at the oral daily dose of 100 µg for 6 months (20 animals per group).

 
It can be seen in Fig. 12Go that when [14C]androstenedione was used as substrate, treatment with tamoxifen caused a highly significant increase in uterine androgenic reductive 17ß-HSD activity, in both intact (from 3.09 ± 0.44 to 6.44 pmol/mg/min, P < 0.01) and ovariectomized animals (from 4.20 ± 1 to 8.44 ± 1.13 pmol/mg/min, P < 0.01). EM-800 administration, on the other hand, caused no statistically significant change in uterine androgenic reductive 17ß-HSD activity in both intact and ovariectomized mice (Fig 12Go).



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  		Figure 12. Androgenic 17ß-HSD activity (4-dione to testosterone) in uterine homogenate obtained from intact or ovariectomized mice 6 months after castration and in intact or ovariectomized mice treated with EM-800 or tamoxifen at the oral daily dose of 100 µg for 6 months (20 animals per group).

 
Tamoxifen administration for 24 weeks in ovariectomized mice led to a 4-fold (P < 0.01) increase in the activity of uterine 5{alpha}-reductase, which catalyzes the conversion of testosterone to dihydrotestosterone (from 0.40 ± 0.05 to 1.62 ± 0.22 pmol/mg/min, P < 0.01), while no significant change in uterine 5{alpha}-reductase was observed after tamoxifen treatment in intact animals. EM-800 had no significant effect in either intact or ovariectomized animals on this parameter (Fig. 13Go).



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  		Figure 13. Five {alpha}-reductase activity (testosterone to dihydrotestosterone) in uterine homogenate obtained from intact or ovariectomized mice 6 months after castration and in intact or ovariectomized mice, treated with EM-800 or tamoxifen at the oral daily dose of 100 µg for 6 months (20 animals per group).

 
An approximately 4.7-fold increase in serum LH levels was observed after EM-800 administration in intact animals compared with intact controls, from 0.22 ± 0.04 to 1.04 ± 0.40 mg, respectively, P < 0.01) (Fig. 14Go) while no significant effect of EM-800 was observed on serum LH levels in ovariectomized animals where serum LH was increased 30-fold compared with intact animals. On the other hand, tamoxifen administration resulted in a highly significant suppression of serum LH levels in both intact and ovariectomized animals (from 0.22 ± 0.04 to 0.13 ng/ml, P < 0.01; and from 6.37 ± 1.21 to 0.75 ± 0.03 ng/ml in intact and ovariectomized animals, respectively, P < 0.01) (Fig. 14Go).



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  		Figure 14. Serum LH levels in intact mice, ovariectomized mice 6 months after castration, and in intact or ovariectomized mice treated with EM-800 or tamoxifen at the oral daily dose of 100 µg for 6 months (20 animals per group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Women receiving long-term treatment with tamoxifen have shown an increase in the incidence of endometrial hyperplasia, including carcinoma (14, 33, 34). Moreover, an association has been found between the development of endometrial lesions and the duration of treatment as well as the dose of tamoxifen administered (35, 36, 37). The development of a specific antiestrogen thus offers the possibility of providing not only a more efficient therapy for breast cancer but also a reduced incidence of side effects, especially stimulation of the endometrium.

Interestingly, the uterine wt measured after EM-800 treatment of ovariectomized animals was smaller than that found after ovariectomy alone. Moreover, the histopathological changes reflected the changes in uterine wt. Thus, the marked EM-800-induced uterine atrophy, involving both the endometrial and myometrial layers, was accompanied by an apparently inactive endometrium. The atrophic endometrium was composed of a few atrophic glands and a low cuboidal glandular and luminal epithelium, with a cytoplasmic-nuclear ratio lower than 1. It is also noteworthy that the endometrial atrophy achieved with EM-800 in ovariectomized animals was significantly greater than that observed 6 months after castration alone. This dramatic inhibition of uterine morphology achieved with EM-800 appears to be superior to that reported for other recently developed, specific antiestrogens (20) and certainly very different from that of tamoxifen.

In fact, no significant reduction in uterine wt was observed after 24 weeks of treatment of intact animals with tamoxifen, while uterine wt in ovariectomized animals treated with tamoxifen was not significantly different from that of control ovariectomized rats. In fact, a marked stimulation of the endometrial layer was observed in both intact and ovariectomized animals treated with tamoxifen. This stimulatory effect of tamoxifen on the endometrium was characterized by severe cystic endometrial hyperplasia, luminal fluid retention, and hypertrophy of the luminal and glandular epithelium with focal cellular atypia. Most of the histological changes observed in the endometrium after tamoxifen treatment represent typical estrogenic-like uterotrophic effects accompanying increased circulating estrogen levels (38). Such changes, as illustrated in Fig 5Go, are also seen after estradiol treatment in laboratory animals (39, 40, 41).

Although evidence for an estrogenic action of tamoxifen in the human myometrium has been reported (42, 43), we have not observed such an action in the rat myometrium in the present study. On the contrary, treatment of intact animals with tamoxifen resulted in an atrophic myometrium, while no additional effects were seen in the myometrium of ovariectomized animals that received this antiestrogen compared with ovariectomized control animals.

The stimulatory effect of tamoxifen on the uterus, due to its partial estrogenic activity, has been described previously in rats and mice (39, 40, 41, 44, 45, 46, 47, 48) as well as in women (35, 42, 49, 50, 51, 52). A preferential activity of tamoxifen for the glandular uterine epithelium has also been demonstrated, when compared with other antiestrogens, which are also partial agonists for the estrogen receptor (39).

Both EM-800 and tamoxifen treatments led to a decrease in vaginal wt in intact and ovariectomized treated animals, an effect that was significantly greater than the effect of ovariectomy alone. Despite this global inhibition of vaginal wt, the vaginal epithelium was less atrophic after tamoxifen treatment, compared with intact animals treated with EM-800 in whom a severe vaginal atrophy, identical to that seen after ovariectomy, was observed. Due to the relatively small volume of the vaginal epithelial cells of the mucosa, the inhibition by tamoxifen of the volume of the underlying muscular and stromal layer led to an overall inhibition of vaginal wt in ovariectomized animals treated for 6 months with tamoxifen compared with ovariectomy alone. On the other hand, many studies have reported that tamoxifen administration leads to increased maturation and estrogenization of the vaginal mucosa in rats and mice as well as in postmenopausal women (48, 53, 54, 55, 56, 57, 58).

EM-800 treatment led to a significant increase in ovarian wt, which was associated with a corresponding hypertrophy and hyperplasia of the interstitial cells, thus leading to a marked expansion of the interstitial stroma. These changes were accompanied by a significant reduction in folliculogenic activity, suppression of ovulation, absence of new corpora lutea, and development of cystic atretic follicles.

Stromal cell hyperplasia often accompanies ovarian atrophic changes including lack of normally developing follicles and absence of corpora lutea, these two changes being signs of inhibited ovulation and loss of normal ovarian cyclicity (59). On the other hand, the elevation in serum LH levels from 0.22 ± 0.04 to 1.17 ± 0.32 (P < 0.01) in intact animals treated with EM-800 is likely to account, at least in part, for the hyperstimulation of the ovarian stroma seen after treatment with EM-800. In addition, analogous histological ovarian changes have been described by Korach (60) in transgenic estrogen receptor knockout mice having a high circulating level of gonadotropins. An elevation of serum LH levels and an inhibition of ovulation have also been reported by Dukes et al. (61) after treatment with other pure antiestrogens, although at a lower magnitude and after a shorter duration of treatment.

Estrogens are well known to exert a global inhibitory effect at the hypothalamo-pituitary level in the rat (62). It is noteworthy that although serum LH increased in intact animals treated with EM-800, the levels achieved were much lower than those measured after ovariectomy. Since EM-800 achieves a complete blockade of uterine and vaginal wt, one explanation for the relatively smaller increase in serum LH could be the partial access of EM-800 at the hypothalamic site of control of LHRH secretion in the rat. Access to the feedback mechanisms controlling LHRH secretion is limited by the blood-brain barrier, and marked species differences are known to exist.

Analogous, but of lower magnitude, histological changes indicative of suppression of ovulation and ovarian stromal hypertrophy, were seen after tamoxifen treatment. A significant decrease in ovarian wt was also observed in the animals treated with tamoxifen. It is noteworthy that after treatment with tamoxifen, the expansion of the ovarian stroma was mainly due to interstitial cell hypertrophy, and neither significant hyperplasia nor a large number of cystic follicles were seen, as observed in animals treated with EM-800. In addition, the suppression of serum LH levels with tamoxifen administration clearly demonstrates its estrogenic activity at the hypothalamo-pituitary level, as opposed to the pure antiestrogenic activity of EM-800 illustrated by the increases in both serum LH levels and ovarian wt.

Each sex steroid-sensitive tissue, depending upon the type(s) as well as the relative activities of the steroidogenic enzymes expressed locally, can regulate and modulate the formation, metabolism, and action of steroid hormones, according to local requirements, in a tissue-specific manner (63, 64). Thus, compounds having antiestrogenic and/or estrogenic action can potentially modulate the activities of the steroidogenic enzymes. Such an action could provide an additional mechanism for controlling the estrogenic response in target tissues. In this context, the enzyme 17ß-HSD, responsible for the interconversion of estradiol to estrone, is likely to play an important role in the physiology of the uterus, vagina, and mammary gland (63, 65, 66, 67).

A significant increase in the uterine reductive estrogenic as well as androgenic 17ß-HSD activities, with a subsequent expected increase in the formation of estradiol and testosterone from estrone and androstenedione, respectively, was observed in the uterus of both intact and ovariectomized animals treated with tamoxifen. In addition, a significant increase in 5{alpha}-reductase activity, responsible for the formation of dihydrotestosterone from testosterone, was observed in ovariectomized animals treated with tamoxifen. The biological consequences of such effects remain to be determined. In the present study, although the tissue content of active steroids and their metabolites have not been measured, the possibility exists that changes in intracellular levels of androgens and/or estrogens could be responsible up to an unknown extent for the uterotrophic effects of tamoxifen observed in our study in intact animals. On the other hand, treatment with EM-800 did not have any effect on the activities of 17ß-HSD or 5{alpha}-reductase in uterine tissues of both ovariectomized and intact animals treated with this compound.

With respect to the effects on the mammary gland, the two antiestrogens completely inhibited the growth and tubuloalveolar development of the mouse mammary gland. Full development of the mouse mammary gland occurs at the age of 3 months in the mouse, whereas treatment of the animals used in our study started at approximately 2 months. Although tamoxifen administration has been shown to promote the growth of the mammary gland in the immature female rat (68), no such action has been observed under the experimental conditions used in our study in the mouse.

The present data demonstrate the highly potent and pure antiestrogenic activity of EM-800. When compared with tamoxifen, the improved inhibition of estrogen action was observed on all the estrogen-sensitive parameters studied in the mouse, namely the uterus, vagina, mammary gland, and hypothalamo-pituitary-ovarian feedback system. The pure antiestrogenic activity of EM-800, which leads to a more complete blockade of estrogen action in all the estrogen target tissues examined, suggests that this compound could offer the possibility of a more efficient treatment of breast cancer as well as of other estrogen-sensitive conditions, such as endometriosis, leiomyomata, and benign breast disease as well as other estrogen-responsive conditions in men and women.

Received April 17, 1997.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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