Tissue-selective actions of estrogen analogs

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TISSUE-SELECTIVE ACTIONS OF ESTROGEN ANALOGS

G.L. Evans and R.T. Turner Departments of Orthopedics and Biochemistry and Molecular Biology Mayo Clinic, Rochester, MN 55905

ABSTRACT The biological actions of estrogen analogs have frequently confounded prevailing views regarding the mechanism of estrogen action. Agents originally intended as antifertility drugs are now used clinically to promote ovulation. The early work with antiestrogens as antifertility agents lead to the realization that triphenylethylene antiestrogens suppressed the growth of breast tumors. The subsequent ubiquitous clinical use of tamoxifen for adjuvant breast cancer therapy is a direct result of this research. Basic studies using tamoxifen and related antiestrogens revealed the previously unsuspected tissue selective actions of these compounds. This peculiar property is being taken advantage of to gain new insight into the molecular mechanisms of estrogen action. This property also forms the basis for an exciting new approach to hormone replacement therapy to prevent postmenopausal osteoporosis and cardiovascular disease. The laboratory rat played an integral and essential part in each of these developments. The success of the ovariectomized rat model in predicting the tissue selective effects of tamoxifen in women greatly increases confidence that this animal model will be useful in development of a new generation of estrogen analogs designed specifically for postmenopausal hormone replacement. INTRODUCTION Estrogen is important for normal growth of bone as well as maintenance of the skeleton in adults. The hormone also has profound effects on mineral homeostasis (49). The cellular response of the skeleton to estrogen is reasonably well characterized, but the molecular mechanisms by which the hormone acts on bone and mineral homeostasis are obscure (46,47). Extrapolation from reproductive tissues suggests that most of estrogen's action on bone cells are mediated through specific receptors for the hormone (30). Synthetic analogs of estrogen can interact with these receptors and have estrogen agonist and/or antagonist activities. The recent realization that estrogen analogs also can have cell, tissue and gene specificity has generated considerable excitement and is the subject of this review (9,40,50). The principal focus will be the physiological actions of estrogen analogs in laboratory animals, principally rats. We will also briefly discuss the potential use of antiestrogens for postmenopausal hormone replacement therapy. Skeletal Effects of Estro2ens

There are substantial species differences in estrogen's role in bone physiology (48). It is therefore important to critically evaluate the relevance of laboratory animal models used in studies designed to evaluate the clinical and toxic potential of estrogen analogs. The immature rat is an excellent laboratory animal model for evaluating the effects of estrogen analogs on reproductive tissues and on bone growth. Agonistic activity is measured in ovariectomized rats whereas antagonistic activity is determined in either Address for correspondence and reprints." Dr. G. Evans, Mayo Clinic, Department of Orthopedic Research, 200 First Street SW, Rochester, MN 55905.

© 1995 by Elsevier Science Inc.

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ovary-intact or estrogen treated ovariectomized animals. The ovariectomized growing rat is also the most frequently used laboratory animal model for postmenopausal osteoporosis and has been used extensively for characterizing the effects of estrogen analogs on bone turnover (26). Table I compares the skeletal changes in the animal model with those in postmenopausal women. The rat model is excellent for replicating the increased bone turnover which occurs in estrogen deficient women and is believed to lead to the bone loss. The limitations of the animal model include growth changes following ovariectomy which increase bone strength and obscure alterations in bone remodeling, the failure of the model to reproduce the atraumatic fractures associated with postmenopausal osteoporosis and a pattern of bone loss which may differ from humans (48,56). TABLE I Comparison of Skeletal Effects of Ovariectomy in Rats with Postmenopausal Bone Loss in Women Similarities - Increased serum and urine markers for bone turnover - Decreased bone mineral density - Increased endocortical resorption in long bones - Cancellous osteopenia in long bones and vertebrae due to a net increase in bone resorption - Histological evidence for elevated cancellous bone turnover Differences - Humans, but not ovariectomized rats, at risk for atraumatic fractures - Cancellous osteopenia in humans is due to a combination of decreased trabecular number and thickness whereas decreased trabecular number predominates in ovariectomized growing rats - Estrogen deficiency results in cancellous osteopenia in humans due to altered bone remodeling; whereas in rats it is due to increased resorption by chondroclasts and altered bone remodeling* - Ovariectomy results in increased strength of long bones in growing rats* - Ovariectomy results in increased longitudinal growth of long bones in growing rats* - Ovariectomy results in increased radial growth of long bones in growing rats* *These differences decrease as age of rat at ovariectomy is increased Role of Estrogen Receptors

in M e d i a t i n g

the Skeletal Effect of Estrogen

The reader is directed to recent publications reviewing the respective roles of estrogen receptors and cytokines in mediating the skeletal effects of estrogen (19,49,57). Briefly, estrogen receptor mRNA and/or peptide was reported in primary cultures of cells from human bone, osteosarcoma cell lines and skeletal tissues from laboratory animals (49,57). The hormone was also reported to have effects on bone cell proliferation and differentiation in monoculture (49). These findings support the potential for direct estrogen receptor mediated actions of the hormone on bone cells. Although estrogen receptors have been detected in skeletal tissues there is little information regarding cellular distribution of the receptor. There is also no compelling evidence that the major physiological actions of estrogen on bone growth and turnover are mediated by estrogen receptors located in differentiated bone cells (57). Some of the actions of the hormone in the reproductive tissues are on estrogen receptor negative cells and have been shown to be mediated by paracrine factors released by receptor positive cells (5,10). This mechanism could be relevant to bone because estrogen may regulate release of cytokines which influence generation of osteoclasts, including IL-1, IL-6, and TNF-a, by marrow stromal cells and peripheral monocytes (19). Regardless of their precise cellular locations, studies with estrogen analogs clearly indicate that altered receptor-ligand interactions can profoundly influence bone metabolism. Classes of Estrogen Analoes

The chemical structures for representative estrogen analogs known to influence bone and mineral metabolism are shown in Fig. 1.

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G . L . Evans and R. T. Turner Estrogen analogs /

OH

CH=

~

N

~

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d CA-168794B-OSA

FIG. 1: The chemical structure of 17[3-estradiol and selected estrogen analogs with activity on bone and mineral metabolism.

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17 I~-Estradiol is a natural estrogen which preferentially binds with high affinity to estrogen receptors (45). Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen analog which binds to the estrogen receptor more tightly than 171~-estradiol and has more potent estrogenic activity. DES cannot be metabolized to other classes of sex steroids and binds to their receptors more weakly than 17[I-estradiol. As a result, DES is used in studies in which it is critical to establish hormone specificity (45). DDT is an example of a polychlorinated biphenyl wide spectrum insecticide. It is also a weak estrogen agonist. Because of it's inherent stability DDT continues to represent a significant environmental contaminant. D D T results in disturbed mineral homeostasis in birds resulting in eggshell thinning and reproductive failure (6). It also induces uterine hypertrophy in rats (54). DDT displaces estrogen from the estrogen receptors (46) suggesting that the detrimental effects of the insecticide on mineral homeostasis are estrogen receptor mediated. Thus, even weak agonists can have important biological consequences. Tamoxifen, centchroman and clomiphene are examples of substituted triphenylethylene antiestrogens. These compounds are referred to as antiestrogens because they antagonize uterine growth and vaginal cornification induced by 171~-estradiol in rats (22). These compounds were originally developed as postcoital contraceptives. Paradoxically, these antiestrogens induce ovulation in selected anovulatory women by inducing increased output of pituitary gonadotropins. Antiestrogens are of great clinical importance because they antagonize the growth of estrogen receptor positive breast cancer cells. The successful introduction of tamoxifen as an adjuvant therapy for breast cancer (23) has led to the examination of related compounds. These include the benzothiophene derivative raloxifene (formally called keoxifene) and the hydroxylated triphenylethylene droloxifene, compounds which have high binding affinities for the estrogen receptor (7,22). Although tamoxifen is the only antiestrogen approved for treating breast cancer in the United States, the tissue selective activities of the more potent newer antiestrogens has suggested the possible use of this class of estrogen analogs for postmenopausal hormone replacement. This interest has prompted structure function studies designed to test the hypothesis that tissue selectivity is due to an unique ligand-induced estrogen receptor conformation. 3-[4-(1,2Diphenylbut-l-enyl)phenyl] acrylic acid is a new triphenylethylene estrogen analog with functional selectivity for bone that was synthesized as a result of these studies (59). The intrinsic estrogenic activity of tamoxifen may be responsible, in part, for escape of tumor cells from the tumorstatic action of the drug during long-term therapy. This possibility has led to the development of antiestrogens (ICI 182, 780 and ZM 189, 154) which have little or no estrogen agonist activities (53). Not surprisingly, these "pure" antiestrogens have detrimental effects on the rat skeleton (11,15). Estrogen replacement therapy is very effective in reducing postmenopausal bone loss as well as the incidence of osteoporotic fractures (34). A positive side effect is the reduction in risk for cardiovascular disease (37). Unfortunately, estrogen replacement therapy results in many undesirable side effects (34). The side effects of estrogen greatly reduce the acceptance of this therapy for hormone replacement. The reluctance to take estrogen is justified considering some of the side effects are life threatening. Furthermore, to be most effective the therapy must be administered to asymptomatic women. An unfortunate consequence is that estrogen replacement therapy undoubtedly results in serious side effects in some women who would never develop an osteoporotic fracture. The attractiveness of estrogen replacement therapy to prevent osteoporotic fractures could be greatly improved by reducing the incidence and severity of the side effects. Many of the undesirable side effects of estrogen replacement therapy are the result of the hormones stimulatory actions on reproductive tissues. The uterine hyperplasia and attendant risk for uterine cancer can be reduced in women by combination therapy with progesterone. Unfortunately, including progesterone also reduces the beneficial effects of estrogen on lipid chemistry (1). In contrast, studies of the effects of tamoxifen in women and

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rats suggest that tissue selective estrogen analogs can maintain the beneficial effects of estrogen on the skeleton and cardiovascular system and reduce undesirable side effects on reproductive tissues. Limitations of Estrogen Replacement Therapy The principal benefits and undesirable side effects of estrogen replacement therapy for postmenopausal osteoporosis are tabulated in Table II.

TABLE II The Benefits and Risks of Unopposed Postmenopausal Estrogen Replacement Therapy Benefits Improvement of calcium balance Halting the loss of height Reduction of bone loss Decrease in fracture rate Decrease in serum cholesterol Increase in serum HDL Decrease in serum LDL Decrease in cardiovascular disease Decrease in overall mortality

Risks Breast soreness Increased risk of breast cancer Breakthrough bleeding Cystic mastitis Endometrial hyperplasia Increased risk of endometrial cancer Gallstones Return of menses Weight gain

Tissue Selective Actions of TAM in Rats Researchers initially anticipated a detrimental effect of tamoxifen on the skeleton. However, doses of tamoxifen which do not stimulate uterine growth in ovariectomized rats completely antagonized the skeletal effects of ovariectomy and had no detrimental effects on bone in ovary-intact rats (25,50).

The differential effect of tamoxifen on the uterus and bone is illustrated in Fig. 2. Ten-week-old rats were ovariectomized or sham-operated and treated with tamoxifen or placebo for 3 weeks using controlled release pellets designed to deliver a total dose of 3 mg of the antiestrogen (35). Ovariectomy resulted in severe cancellous osteopenia in the proximal tibia metaphysis which was prevented by tamoxifen treatment. Under these conditions, tamoxifen had a very weak uterotrophic effect. Additionally, administration of tamoxifen to ovary-intact rats had no detrimental effects on cancellous bone volume but was very effective in inducing uterine atrophy (35). Tamoxifen consistently prevented the increases in body weight, endocortical bone resorption, radial bone growth, longitudinal bone growth, and cancellous bone resorption which occurred during the first month following ovariectomy in growing rats (29,35,50). Also, Jordan, et al. demonstrated in long-term studies that tamoxifen antagonized the decrease in ash weight/bone volume in mature ovariectomized rats. Dose response studies in ovariectomized rats revealed that tamoxifen antagonized the skeletal effects of estrogen over a wide range in dose (35,58). The effects of tamoxifen on the skeleton were not limited to estrogen deficient animals. Submaximal concentrations of estrogen and tamoxifen had additive effects to decrease radial bone growth and increase cancellous bone volume (29,35). One of the major skeletal effects of tamoxifen is prevention of production of osteoclasts by inhibition of fusion of osteoclast precursors (47,50,51). Tamoxifen also prevented the increase in osteoclast number in the immobilized limb of rats following unilateral sciatic neurotomy (54). Tamoxifen had only a modest effect to reduce osteoclast number in the weight bearing limb suggesting that the antiestrogen does not reduce the lifespan of mature osteoclasts (54). Tamoxifen reduced ash weight/bone volume in ovary-intact animals (13). The significance of this latter finding is difficult to interpret in growing rats because estrogens influence bone volume as well as ash weight. However, tamoxifen is a partial estrogen agonist on the skeleton; its potency and efficacy are

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Values

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less than 1713-estradiol (58). As a consequence, high doses of tamoxifen may reduce the maximal response to added estrogen (29,58). Although tamoxifen maintains bone growth and turnover at normal levels in estrogen deficient rats in short term studies, high doses of the estrogen analog may antagonize bone turnover in rats with intact ovaries (35). Tamoxifen is a partial estrogen agonist on liver. The net result of treatment with the analog include decreases in serum levels of IGF-1 (18,23) and cholesterol (16). The tumorstatic action of tamoxifen on breast tumor cells with high levels of estrogen receptor are well established. As previously discussed, tamoxifen prevents uterine growth in ovary-intact rats and antagonizes estrogen induced uterine growth in ovariectomized animals. The uterus is, however, heterogeneous. Tamoxifen induces luminal epithelium to undergo hypertrophy comparable to 1713estradiol stimulation (9,22). However, unlike treatment with estrogen there is no increase in cell number (36). Tamoxifen stimulates stromal and myometrial cells to a lesser extent than 1713-estradiol but, interestingly, luminal epithelium hypertrophy to a greater extent (22). The effects of tamoxifen are species specific. As discussed, tamoxifen is a partial estrogen agonist/antagonist with target cell and tissue selectivity in rats. The estrogen analog appears to be a pure estrogen antagonist on the avian oviduct (42). Additionally, tamoxifen inhibits estrogen induced avian hepatic and skeletal synthesis of egg yolk proteins and medullary bone, respectively (44). In contrast, tamoxifen has substantial estrogenic activity on the mouse uterus (22). The mechanisms for these species differences are unknown but might yield important clues toward understanding the molecular mechanisms of estrogen action. Other Tissue Selective Estro~,en Analol~s

The interesting effects of tamoxifen on the skeleton has stimulated research on related compounds with more potent antiestrogenic activities. Raloxifene has received the most attention but animal studies have also been performed with droloxifene, centchroman, clomiphene, and 3-[4-(1,2-diphenylbut-1enyl)phenyl] acrylic acid. The actions of raloxifene on ovary-intact and ovariectomized rats are superficially similar to tamoxifen. Raloxifene antagonizes the changes in cortical and cancellous bone histomorphometry (12), bone densitometry (8,38), ash weight/bone volume (25), and bone strength (43) which follow ovariectomy. Raloxifene also reduces serum cholesterol (8,12). However, in unpublished long term studies the authors have found that raloxifene was more effective than tamoxifen in maintaining cancellous bone volume. Limited studies indicate that droloxifene, centchroman, and clomiphene (2) have skeletal activities in ovariectomized rats similar but not identical to tamoxifen. Human Studies

There is extensive clinic experience with long-term tamoxifen treatment. Its use for breast cancer has become wide spread because of the low incidence of side effects. Formerly, there was a concern that tamoxifen treatment would result in accelerated postmenopausal bone loss. Fortunately, tamoxifen has consistently been found to reduce bone loss in women with breast cancer (14,17,31-33,60). In addition, the estrogen analog has beneficial effects similar to estrogen on blood lipid chemistry (3,20,39). The success of tamoxifen for adjuvant therapy to prevent tumor recurrence has prompted investigation to determine whether this analog will reduce the risk for breast cancer in disease-free women (18,41). Many of the women will be premenopausal. It will be important to insure by careful monitoring that tamoxifen does not have detrimental effects on bone and mineral homeostasis in these young women with normal ovarian function. Mechanisms of Action An understanding of the mechanisms for the tissue, target cell and gene specific effects of tamoxifen would promote the rationale design of new antiestrogens with improved efficacy for postmenopausal

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hormone replacement therapy. A detailed discussion of the possible mechanisms is beyond the scope of this review. In general, research in this area has focused on the interaction of estrogen analogs with activating domains on the estrogen receptor (4,52,59), regulation of specific genes (40), nongenomic effects of estrogen analogs (22,24), the role of metabolites with greater biological activity than the parent compounds (22), and indirect effects mediated by changes in systemic and locally produced growth factors (24). The recent demonstration that the estrogen receptor contains two distinct transactivating sites is of special interest (52). Antiestrogens with tissue selective activity exhibit differential regulation of these sites compared to estrogen which is cell type and promotor context dependent (3). This differential regulation may be an important mechanism for the tissue and species specific actions of estrogen analogs. ACKNOWLEDGMENTS

The authors thank Ms. Lori M. Rolbiecki for typing this manuscript. This work was supported by NIH grant AR41418 and the Mayo Foundation. REFERENCES

1. Anonymous. Effects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. JAMA 273:199-208; 1995. 2. Beall, P.T., Misra, L.IC, Young, R.L., Spjut, H.J., Evans, H.J., LeBlanc, A. Clomiphene protects against osteoporosis in the mature ovariectomized rat. Calcif Tissue Int 36:123-125; 1984. 3. Bagdade, J.D., Wolter, J., Subbaiah, P.V., Ryan, W. Effects of tamoxifen treatment on plasma lipids and lipoprotein lipid composition. J Clin Endocrinol Metab 70:1132-1135; 1990. 4. Berry, M., Metzger, D., Chambon, P. Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4hydroxytamoxifen. EMBO J 9:2811-2818; 1990. 5. Bigsby, R.M., Cunha, G.R. Estrogen stimulation of deoxyriboneclic acid synthesis in uterine epithelial cells which lack estrogen receptors. Endocrinology 119:390-396; 1986. 6. Bitman, J., Cecil, H.C., Harris, S.J., Fries, G.F. DDT induces a decrease in eggshell calcium. Nature 224:44-46; 1969. 7. Black, L.J., Jones, C.D., Falcone, J.F. Antagonism of estrogen action with a new benzothiophene derived antiestrogen. Life Sci 32:1031-1036; 1983. 8. Black, L.J., Sato, M., Rowley, E.R., Magee, D.E., Bekele, A., Williams, D.C., Cullinan, G.J., Bendele, R., Kauffman, R.F., Bensch, W.R., Frolik, C.A., Termine, J.D., Bryant, H.U. Raloxifene (LY139481 HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats. J Clin Invest 93:63-69; 1994. 9. Branham, W.S., Zehr, D.R., Sheehan, D.M. Differential sensitivity of rat uterine growth and epithelium hypertrophy to estrogens and antiestrogens. Proc Soc Exp Biol Med 203:297-303; 1993. 10. Cunha, G.R., Chung, L.W.tC, Shannon, J.M., Reese, B.A. Stromal-epithelial interactions in sex differentiation. Biol Reprod 22:19-43; 1980. 11. Dukes, M., Chester, R., Yarwood, L., Wakeling, A.E. Effects of non-steroidal pure antioestrogen, ZM 189,154, on oestrogen target organs of the rat including bones. J Endocrinol 141:335-341; 1994. 12. Evans, G.L., Bryant, H.U., Magee, D., Sato, M., Turner, R.T. The effects of raloxifene on tibia histomorphometry in ovariectomized rats. Endocrinology 134:2283-2288; 1994. 13. Feldman, S., Minne, H.W., Parviz, S., Pfeifer, M., Lempert, V.G., Bauss, F., Zeigler, R. Antiestrogen and antiandrogen administration reduce bone mass in the rat. Bone Min 7:245-254; 1989. 14. Fentiman, C.M., Rodin, A., Murby, A., Fogelman, I. Bone mineral content of women receiving tamoxifen for mastalgia. Br J Cancer 60:262-264; 1989. 15. Gallagher, A., Chambers, T.J., Tobias, J.H. The estrogen antagonist ICI 182,780 reduces cancellous bone volume in female rats. Endocrinology 133:2787-2791; 1993. 16. Gold, E., Stapley, S., Goulding, A. Tamoxifen and norethisterone: Effects on plasma cholesterol and total body calcium content in the estrogen-deficient rat. Horm Metab Res 26:100-103; 1994.

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17. Gotfredson, A., Christiansen, C., Palshof, T. The effect of tamoxifen on bone mineral content in premenopausal women with breast cancer. Cancer 53:853-857; 1984. 18. Grady, D., Rubin, S.M., Petitti, D.B., Fox, C.S., Black, D., Ettinger, B., Ernster, V.L., Cummings, S.R. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann Intern Med 117:1016-1037; 1992. 19. Horowitz, M.C. Cytokines and estrogens in bone: anti-osteoporotic effects. Science 260:626-627; 1993. 20. Ingram, D. Tamoxifen use, oestrogen binding and serum lipids in postmenopausal women with breast cancer. Aust NZ J Surg 60:673-675; 1990. 21. Isserow, J.A., Rucinski, B., Romero, D.F., Mann, G.N., Liu, C.C., Epstein, S. The effect of medroxyprogesterone acetate on bone metabolism in the oophorectomized, tamoxifen-treated rat. Endocrinology 136:713-719; 1995. 22. Jordan, V.C. Biochemical pharmacology of antiestrogen action. Pharmacol Rev 36:245-269; 1984. 23. Jordan, V.C. Long-term adjuvant tamoxifen therapy for breast cancer. Breast Cancer Res Treat 15:125-136; 1990. 24. Jordan, V.C., Murphy, C.S. Endocrine pharmacology of antiestrogens as antitumor agents. Endocr Rev 11:578-610; 1990. 25. Jordan, V.C., Phelps, E., Lindgren, J.U. Effects of anti-estrogens on bone in castrated and intact female rats. Breast Cancer Res Treat 10:31-35; 1987. 26. Kalu, D.N. The ovariectomized rat model for postmenopausal bone loss. Bone Miner 15:175-191; 1991. 27. Kalu, D.N., Arjmandi, B.H., Liu, C.C., Salih, M.A., Birnbaum, R.S. Effects of ovariectomy and estrogen on the serum levels of insulin-like growth factor-I and insulin-like growth factor binding protein3. Bone Miner 25:135-148; 1994. 28. Kalu, D.N., Liu, C.C., Solerno, E., Hollis, B., Echon, R., Ray, M. Skeletal response of ovariectomized rats to low and high doses of 1713-estradiol. Bone Miner 14:175-187; 1991. 29. Kalu, D.N., Salerno, E., Liu, C.C., Echon, R., Ray, M., Garza-Zapata, M. A comparative study of the actions of tamoxifen, estrogen, and progesterone in the ovariectomized rat. Bone Miner 15:109-123; 1991. 30. Katzenellenbogan, B.S. Dynamics of steroid hormone receptor in action. Annu Rev Physiol 42:1735; 1980. 31. Kristensen, B., Moridsen, H.T., Holmegaard, S.N., Transbol, I. Amelioration of postmenopausal primary hyperparathyroidism during adjuvant tamoxifen for breast cancer. Cancer 64:1965-1967; 1989. 32. Love, R.R., Barden, H.S., Mazess, R.B., Epstein, S., Chappell, R.J. Effect of tamoxifen on lumbar spine bone mineral density in postmenopausal women after 5 years. Arch Intern Med 154:2585-2588; 1994. 33. Love, R.R., Mazess, R.B., Barden, H.S., Epstein, S., Newcomb, P.A., Jordan, V.C., Carbone, P.P., DeMets, D.L. Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 326:852-856; 1992. 34. Mack, T.M., Ross, R.IC Risks and benefits of long-term treatment with estrogens. Schweiz Med Wochenschr 119:1811-1820; 1989. 35. Moon, L.Y., Wakley, G.K., Turner, R.T. Dose-dependent effects of tamoxifen on long bones in growing rats: Influence of ovarian status. Endocrinology 129:1568-1574; 1991. 36. Mukku, V.R., Kirkland, J.L., Hardy, M., Stancel, G.M. Stimulatory and inhibitory effects of estrogen and antiestrogen on uterine cell division. Endocrinology 109:1005-1010; 1981. 37. Nabulsi, A.A., Folsom, A.R., White, A., Patsch, W., Heiss, G., Wu, ICIC, Szklo, M. Association of hormone-replacement therapy with various cardiovascular risk factors in postmenopausal women. N Engl J Med 328:1069-1075; 1993. 38. Sato, M., McClintock, C., Kim, J., Turner, C.H., Bryant, H.U., Magee, D., Slemenda, C.W. Dualenergy x-ray absorptiometry of raloxifene effects on the lumbar vertebrae and femora of ovariectomized rats. J Bone Miner Res 9:715-724; 1994. 39. Schapira, D.V., Kumar, N.B., Lyman, G.H. Serum cholesterol reduction with tamoxifen. Breast Cancer Res Treat 17:3-7; 1990.

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40. Shull, J.D., Beams, F.E., Baldwin, T.M., Gilchrist, C.A., Hrbek, M.J. The estrogenic and antiestrogenic properties of tamoxifen in GHnC 1 pituitary tumor cells are gene specific. Mol Endocrinol 6:529-535; 1992. 41. Spicer, D.V., Pike, M.C., Henderson, B.E., Groshen, S. Tamoxifen and estrogen replacement therapy as agent of disease prevention. J Natl Cancer Inst 83:63-64; 1991. 42. Sutherland, R., Mester, J., Baulieu, E.E. Tamoxifen is a potent "pure" anti-estrogen in chick oviduct. Nature 267:434-435; 1977. 43. Turner, C.H., Sato, M., Bryant, H.U. Raloxifene preserves bone strength and bone mass in ovariectomized rats. Endocrinology 135:2001-2005; 1994. 44. Turner, R.T., Bell, N.H., Gay, C.V. Evidence that estrogen binding sites are present in bone cells and mediate medullary bone formation in Japanese quail. Poult Sci 72:728-740; 1993. 45. Turner, R.T., Eliel, L.P. Nuclear estrogen receptors in the reproductive tract of laying Japanese quail. Gen Comp Endocrinol 34:141-148; 1978. 46. Turner, R.T., Eliel, L.P. o,p'-DDT as an estrogen: An evaluation of its ability to compete with 3Hestradiol for nuclear estrogen receptor sites in the quail oviduct. Bull Environ Contam Toxicol 19:139142; 1978. 47. Turner, R.T., Evans, G.L., Wakley, G.K. Mechanism of action of estrogen on cancellous bone balance in tibiae of ovariectomized growing rats: Inhibition of indices of formation and resorption. J Bone Miner Res 8:359-366; 1993. 48. Turner, R.T., Evans, G.L., Wakley, G.K. Reduced chondroclast differentiation results in increased cancellous bone volume in estrogen-treated growing rats. Endocrinology 134:461-466; 1994. 49. Turner, R.T., Riggs, B.L., Spelsberg, T.C. Skeletal effects of estrogen. Endocr Rev 15:275-300; 1994. 50. Turner, R.T., Wakley, G.K., Hannon, ICS., Bell, N.H. Tamoxifen prevents the skeletal effects of ovarian hormone deficiency in rats. J Bone Miner Res 2:449-456; 1987. 51. Turner, R.T., Wakley, G.IC, Hannon, ICS., Bell, N.H. Tamoxifen inhibits osteoclast-mediated resorption of trabecular bone in ovarian hormone-deficient rats. Endocrinology 122:1146-1150; 1988. 52. Tzukerman, M.T., Esty, A~, Santiso-Mere, D., Danielian, P., Parker, M.G., Stein, R.B., Pike, J.W., McDonnell, D.P. Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:2130; 1994. 53. Wakeling, A~E. Therapeutic potential of pure antiestrogens in the treatment of breast cancer. J Steroid Biochem Mol Biol 37:771-775; 1990. 54. Wakley, G.IC, Baum, R.L., Hannon, ICS., Turner, R.T. Tamoxifen treatment reduces osteopenia induced by immobilization in the rat. Calcif Tissue Int 43:383-388; 1988. 55. Welch, R.M., Levin, W., Conney, A.H. Estrogenic action of DDT and its analogs. Toxicol Appl Pharmacol 14:358-367; 1969. 56. Westerlind, ICC., Wakley, G.IC, Evans, G.L., Turner, R.T. Estrogen does not increase bone formation in growing rats. Endocrinology 133:2924-2934; 1993. 57. Westerlind, ICC., Sarkar, G., Bolander, M.E., Turner, R.T. Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum. Steroids, In Press. 58. Williams, D.C., Paul, D.C., Black, L.J. Effects of estrogen and tamoxifen on serum osteocalcin levels in ovariectomized rats. Bone Miner 14:205-220; 1991. 59. Willson, T.M., Henke, B.R., Momtahen, T.M., Charifson, P.S., Batchelor, K.W., Lubahn, D.B., Moore, L.B., Oliver, B.B., Sauls, H.R., Triantafillou, J.A., Wolfe, S.G., Baer, P.G. 3-[4-(1,2-Diphenylbut1-enyl)phenyi]acrylic acid: A non-steroidal estrogen with functional selectivity for bone over uterus in rats. J Med Chem 37:1550-1552; 1994. 60. Wright, C.D., Garrahan, N.J., Stanton, M., Gazet, J.C., Mansell, R.E., Compston, J.E. Effect of long-term tamoxifen therapy on cancellous bone remodeling and structure in women with breast cancer. J Bone Miner Res 9:153-159; 1994. 61. Wronski, T.J., Cintron, M., Doherty, A.L., Dann, L.M. Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats. Endocrinology 123:681-686; 1988.