Theoretical basis for the benefit of postmenopausal estrogen substitution

Experimental Gerontology 34 (1999) 587– 604

Perspective

Theoretical basis for the benefit of postmenopausal estrogen substitution M.M. Millera,*, K.B.J. Franklinb a

Departments of Obstetrics and Gynecology, Anatomy, Experimental Medicine, and Centre for Studies on Aging, Royal Victoria Hospital, McGill University, Montreal, Quebec H3A 1A1, Canada b Department of Psychology, McGill University, Montreal, Quebec H3A 1A1, Canada

Received 11 January 1999; received in revised form 26 March 1999; accepted 29 March 1999

Abstract Women are being presented with an increasing number of choices for health care management as they move through the aging process. Estrogen has positive effects on mood, sexual function, target end organs and cognitive function, and may play an important role in the etiology of Alzheimer’s Disease by acting to prevent amyloid plaque formation, oxidative stress, or deterioration of the cholinergic neurotransmitter system. The benefits of estrogen therapy for osteoporosis, the cardiovascular system, and lipid metabolism are far reaching, but the possibility of developing breast cancer later in life is also relevant. Understanding the mechanisms for the action of the estrogens, anti-estrogens, and the selective estrogen receptor modulators, and possible alternative routes of symptom management for some menopausal events is important to make appropriate decisions on choice of therapy. This review discusses the theoretical basis for estrogen’s actions in the management of the postmenopausal stage of the life cycle. © 1999 Elsevier Science Inc. All rights reserved.

1. Introduction Women are now living one third or more of their lives in a postmenopausal state. The ability of estrogen, either alone or with progestin to prevent menopause-associated symptoms and diseases speaks to the focal position of estrogen in the pathophysiology of these disease states (Bryant and Dere, 1998). Important chronic diseases such as heart disease, osteoporosis, and Alzheimer’s Disease have been associated with or attributed to long-standing estrogen deprivation. The endocrine physiology of the menopausal woman

* Corresponding author. Tel.: ⫹001-514-842-1231; fax: ⫹001-514-843-1678. E-mail address: [email protected] (M. Miller) 0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 3 2 - 7

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who wants to maintain healthy aging supports a rationale for hormone replacement therapy (HRT). The choice of whether not to take such therapy will have a major effect on their health and quality of life many years down the line. In choosing replacement therapy, use of estrogen alone may be appropriate for those who have had a hysterectomy, but the use of progestin is necessary in those women with a uterus. For the purposes of this discussion we will define the use of estrogen alone as estrogen replacement therapy (ERT) and the combination of estrogen with one of several progestins as hormone replacement therapy (HRT). In females, endocrine physiology changes dramatically with increasing age. The “perimenopausal period” is the phase of aging in women that marks the transition from the reproductive phase to the nonreproductive phase. “Menopause” is marked as being one year after the final menstrual period. This occurs during the perimenopausal period at an average age of 51 in North American women. Women over the age of 40 experience changes that include lengthened or shortened cycles attributable to decreased length of the follicular phase, increased plasma follicle-stimulating hormone, but not luteinizing hormone levels, lowered mid-cycle estradiol levels (Utian, 1994), and decreased progestin pulsatility (Longcope et al., 1986). Perimenopausal women demonstrate increased follicular phase estrone excretion compared with younger cycling women. Periods of persistently low estrone excretion are reported in perimenopausal women and these become more common with proximity to menopause (Santoro et al., 1996). There are important differences in the sex steroid profiles between pre- and postmenopausal women including decreased estrogen production, inability to produce estrogen in a cyclical fashion, and increased plasma androgens relative to estrogens. In addition, the major free estrogen becomes estrone, and androstendione becomes its major precursor (Utian, 1994). Perimenopausal period symptoms are a result of decreased ovarian activity and hormonal deficiency and include specific symptoms such as hot flashes, perspiration, and atrophic vaginitis, and nonspecific symptoms such as insomnia, nervousness, depression, and perceived changes in mental function. The latter include decreased concentration and memory function (Utian, 1994; Bryant and Dere, 1998). There are also associated psychological factors including mood swings and sleep disorders (Bryant and Dere, 1998). The disagreeable nature of these symptoms and the possible deleterious consequences of estrogen loss may lead many women to consider estrogen replacement. Others, influenced by the notion that menopause is a “natural” event, or by fears of possible risks, do not accept the idea of estrogen replacement. In the end, only women facing menopause and their physicians can make a rational choice by considering the balance of benefit and risk.

2. Classification of estrogens Estrogens include several groups of compounds that differ markedly In their chemical structure and general properties, but share a single biologic activity: the stimulation of growth and maintenance of female sexual characteristics. Among hormones, the diversity of compounds that have estrogenic action makes them unique. The effects of these compounds are equally as diverse, ranging from growth and function of the female sexual organs, sexual development, and proliferation of breast tissue to extra-genital effects such as distribution of body fat, hepatic synthesis of some proteins, and mineral metabolism. Estrogens can be classified by their chemical structure (steroidal versus nonsteroidal) and

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by their occurrence (natural versus synthetic). Natural steroid estrogens are the “classic” estrogens including estradiol 17␤, estrone, and estriol; the “equine” estrogens including equilin, and equilenin; and some of the metabolites of these estrogens called the “conjugated” estrogens. Synthetic estrogens include esters and 17␣-ethinyl derivatives of natural estrogens. Nonsteroidal estrogens include the stilbenes, such as diethylstilbesterol and some therapeutic agents that can display estrogenic capabilities under certain circumstances, such as the cardiac glycosides (Henzl, 1986). It has also been unequivocally demonstratated that environmental estrogens, including phytoestrogens can act as functional, albeit less efficacious, ligands (Hyder et al., 1998). The means by which estrogen delivers its effects are complex and have required more than two decades to delineate.

3. Mechanisms of estrogenⴕs action in target tissues Estrogen diffuses through the plasma membrane of cells. The subcellular distribution of the receptor is largely nuclear, even without hormone (Katzenellenbogen et al., 1997). There are at least two receptors upon which estrogen can act, designated ␣ (the classic receptor) and ␤. ER␣ and ER␤ share many functional similarities in terms of substrate and antagonist binding affinities, but there are differences in the mechanisms regulation their transcriptional activities and localization (Gustafsson, 1997).

Fig. 1. A diagrammatic representation of the human estrgoen receptor illustrating the six different functional domains. The mid-region (domain C) is the DNA binding domain (DBD). Domain E is the hormone binding domain (HBD). Contained within domain F is the region modulating the magnitude of gene transcription by estrogens and anti-estrogens. Please see text for specific details.

In addition to the receptor and the hormone, other factors regulate receptor activity in target tissues. Steroid receptors act as ligand-activated transcription factors. The ER protein has six different functional domains (Fig. 1). The A/B domain contains one of two transcriptional activation factors (AF). AF-1, and AF-2 in the carboxyl terminal, activate transcription in a cell and promotor-specific manner. AF-1 is constitutively active. AF-2 is induced upon binding of hormone to the receptor. Two domains are highly conserved

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regions: one in the mid-region (domain C) important for DNA binding, and a functionally complex one in the carboxyl terminal region (domain E/F) that binds hormone. The DNA binding domain (DBD) contains two zinc fingers that mediate receptor binding to estrogen response elements (EREs) in the promotors of hormone-responsive genes. Region E in the carboxyl terminus is defined as the hormone binding domain (HBD), which contains two regions of sequence homology with other hormone receptors and bestows hormone specificity and selectivity (MacGregor and Jordan, 1998). The 3⬘ located F domain modulates the magnitude of gene transcription by estrogen and anti-estrogens (Montano et al., 1995). Upon binding of estrogen to the HBD, phosphorylation and dimerization increase the affinity of ER binding to DNA (Nardulli et al., 1996). By interacting with the ERE, the ER changes cell function to modulate transcription of estrogen-responsive genes (Nardulli et al., 1996). Estrogen responsive genes typically contain one or more copies of an ERE, the consensus of which is a palidromic sequence. Usually located 5⬘ to the promoter of the estrogen-regulated gene, it increases transcription initiation efficiency. Estrogen-responsive genes that contain imperfectly palindromic EREs either in isolation or in multiple arrays are being found in endogenous estrogenresponsive genes. The affinity of the ER for the ERE is an important indicator of the ability of an individual ERE to activate transcription. Affinity of the ER for the ERE, location of an ERE within a promoter, and magnitude and orientation of DNA bends induced by binding of ER or other proteins are all important in transcription of estrogenresponsive genes (Nardulli et al., 1996). Once estrogen has bound to the receptor changes in conformation and homodimerization occurs. Phosphorylation of the receptor by ER kinase, DNA-dependent-kinase, protein kinase C, protein kinase A, and/or casein kinase II allows the receptor to become transcriptionally active (MacGregor and Jordan, 1998). The estrogen-occupied receptor interacts with an array of other components of the transcription complex that function as signaling intermediates and modulate the general transcription machinery. Several coregulatory factors that interact with steroid receptors have been described including basal transcription factors, coactivators, corepressors, enhancers, and cointegrators (Katzenellenbogen, 1996). Other cell signalling pathways affect the bioactivity of the ER, including growth factors, neurotransmitters, and second messengers (Katzenellenbogen et al., 1997). The action of this hormone may also be via mechanisms independent of the classic genomic pathway of steroid action, because estrogen has been shown in several cell systems to bind to membranes and induce rapid cellular events within seconds or minutes of application. These are exemplified by rapid electrical changes on neuronal membranes (Schumaker, 1990).

4. Anti-estrogens and selective estrogen receptor modulators Estrogens promote ligand-dependent interaction with activation function (AF) regions that allow for optimal transcriptional activation. Anti-estrogens, by contrast, bind to the receptor and the anti-estrogen-receptor complex shows conformational changes that are different from those seen with estrogen. Therefore, anti-estrogens usually fail to activate gene transcription or do so less effectively than estrogens. Anti-estrogens are very effective in the treatment of hormone-responsive breast cancer. Tamoxifen is the most commonly used agent because of its effectiveness and ease of use. It has also been shown to be of benefit in prevention of tumor development in high-risk patients, to enhance bone

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Table 1 Activity of estrogens, anti-estrogens, and selective estrogen receptor modulators on target tissues

Ligand

Action in uterus

Action in breast

Action on bone

Action on cholesterol metabolism

Natural, equine, and synthetic estrogens Anti-estrogen (e.g., tamoxifen) Selective estrogen receptor modulators (e.g., raloxifene)

Agonist

Agonist

Agonist

Agonist

Partial agonist

Antagonist

Agonist

Agonist

Antagonist

Antagonist

Agonist

Agonist

maintenance, and preserve a favorable lipid profile. About 25% of women have vasomotor symptoms while taking tamoxifen; those who already have hot flushes have worse flushing. Agents like tamoxifen are referred to as “mixed” agonists and antagonists. Unfortunately, tamoxifen has estrogen-like stimulatory effects on uterus and liver. This may underlie an increased incidence of endometrial hyperplasia leading to cancer, and alterations in liver function. By altering the chemical structure of anti-estrogens, the estrogen-like action on bone and cardiovascular tissue can be preserved, without stimulating breast and uterus or disturbing liver function (Katzenellenbogen et al., 1997). Because estrogen therapy has been implicated in increased breast and uterine cancer, and the use of estrogen causes periodic bleeding and mood swings, a group of pharmacologic agents called selective estrogen receptor modulators (SERMS), has been developed. SERMS fully antagonize the effects of estrogen on uterus and mammary tissue, while mimicking the effects of estrogen on bone and the cardiovascular system. These substances are now being intensively investigated as ERT alternatives (Bryant and Dere, 1998). One such compound, raloxifene, has positive effects on bone and serum lipids, but does not adversely affect the reproductive system (Frolik et al., 1996). The mechanism for the selectivity of agonists versus antagonist actions of SERMS is under intense investigation. The ER is a nuclear transcription factor that is activated by ligand to assume a conformation that allows binding to a specific DNA sequence and subsequent activation or inhibition of gene expression. Raloxifene can compete with estrogen for binding to the ER, but raloxifene prevents transcriptional activation of EREcontaining genes (Bryant and Dere, 1998). Various ligand-induced receptor conformations may be responsible for the pharmacological effects reported for SERMS, including raloxifene (McDonnell et al., 1995). The estrogen receptor-raloxifene complex binds to DNA sequences that are distinct from the ERE in tissues where raloxifen has estrogenagonist effects (Bryant and Dere, 1998). Thus, it prevents stimulation by estrogen, accounting for its antagonist profile in uterus and mammary tissues. A novel pathway for ER mediated activation of cell function, called the raloxifene response element (RRE), has been identified. The unique conformation induced by the raloxifene:ER complex recruits other transcription factors for DNA binding. Several estrogen metabolites are activators of the RRE pathway, which may be a natural pathway for mediating the effects of estrogen in bone, distinct from the pathway that mediates stimulatory effects of estrogen on reproductive tissue (Bryant and Dere, 1998).

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5. The benefits of estrogen therapy for osteoporosis Osteoporosis affects more than 20 million women and the cost of treating its complications exceeds $11 billion (Kulak and Bilezikian, 1998). Bone loss after skeletal maturity is a universal phenomenon (Avioli, 1994). The largest change in bone mass occurs in the first 5 years after menopause (Avioli, 1994) or other causes of ovarian failure (Christiansen, 1995). As soon as estrogen production drops, bone turnover increases rapidly (Christiansen, 1995). An imbalance appears between bone resorption and formation with resorption being greater (Christiansen, 1995). Cortical bone loss during the immediate postmenopausal period may be as much as 2% per year (Avioli, 1994), resulting in net bone mass loss due to excessive osteoclast bone resorption (Christiansen, 1995). The rate of bone loss after menopause in women with ovaries varies significantly from one woman to another (Avioli, 1994). Estrogen deficiency after ovarian failure can result in a twofold increase in bone turnover, an increased loss of vertebral trabecular bone of 2– 4% per year during the early menopausal period, and 0.6 – 0.8% per year at the end of the first decade after menopause (Christiansen, 1995). Analyses of age-related bone loss reveal several patterns. First, a progressive loss of axial bone mass that usually begins before clinical menopause and accelerates in 20 – 40% of females during the immediate postmenopausal years. Second, a more gradual, but progressive decrease in cortical bone mass beginning in the immediate postmenopausal years, and proceeding at an accelerated rate for the next 4 – 8 years (Avioli, 1994). Although peak bone mass is determined by genetic influences, other factors (e.g. dietary calcium intake, exercise, and hormone sufficiency) are determined by “life style” factors (Kulak and Bilezikian, 1998). ERT at menopause is beneficial for osteoporosis because it arrests bone loss (Lindsay, 1996). The protective effect lasts as long as estrogen is used, and it occurs regardless of the route of administration or the concomitant use of progestins (Lindsay, 1996). When estrogen is discontinued, bone loss progresses at the same accelerated pace that would have occurred if menopause were entered without estrogen (Kulak and Bilezikian, 1998). In fact, evaluation of the skeletal status of women older than 70 who had taken estrogen for less than 5 years revealed no remaining beneficial effect of the intervention (Lindsay, 1996). Thus estrogens may need to be prescribed for long periods. Estrogen is presently considered the best therapy for the prevention of postmenopausal osteoporosis (The Writing Group for the PEPI Trial, 1996). For women who cannot or will not take estrogens, estrogen analogs and nonhormonal bisphosphonates, analogs to pyrophosphate, have become available recently and are very effective inhibitors of bone resorption (Kulak and Bilezikian, 1998). A major epidemiologic study on the effects of HRT on the bone mineral density was the Postmenopausal Estrogen/Progestin Interventions (PEPI) trial, a three year, multicenter, randomized, double-blind, placebo-controlled trial. A total of 875 participants aged 45– 64 with no known contraindication to hormone therapy were assessed. Differences between unopposed estrogen (0.625 mg/day conjugated equine estrogen), three estrogen/ progestin treatments (0.625 mg/day conjugated equine estrogens plus cyclic medroxyprogesterone acetate 10 mg/day for 12 days/month; or 0.625 mg/day conjugated equine estrogens plus consecutive 2.5 mg/day medroxyprogesterone acetate 10 mg/day for 12 days/month; or 0.625 conjugated equine estrogens plus cyclic micronized progesterone 200 mg/day for 12 days/month), and placebo were studied in healthy postmenopausal women. The major conclusion of the portion of the study on bone mineral density was that ERT increases bone mineral density at clinically important sites. Bone mineral density at

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the spine and hip was decreased in postmenopausal women given placebo, whereas women assigned to estrogen therapy increased bone mass density during the 36-month period. As expected, the effects of estrogen on bone mineral density were greater in the spine than hip, because the spine has a greater remodeling rate than hip. There were no significant differences between treatment groups in the total number of fractures, or the total number of women who had fractures. These data also suggest that estrogen may be of use to delay accelerated bone loss in early menopause as well as to enhance bone density in older women a decade or more past menopause (The Writing Group for the PEPI Trial, 1996). Other recent studies have shown that postmenopausal women with undetectable serum estradiol concentrations and high serum concentrations of sex steroid binding globulin have a higher risk for vertebral fracture than those with detectable serum estradiol levels (Cummings et al., 1998). Prospective studies have shown that doses of estrogen equivalent to conjugated equine estrogens of 0.625 mg/day or higher are needed to produced significant increase in lumbar spine bone mineral density. However, a recent study showed that esterified estrogens at doses as low as 0.3 to 1.25 mg/day administered unopposed by progestin produce positive changes on bone (Genant et al., 1997). The exact mechanism of estrogen’s action on the skeleton has not been determined. Since ERs have been localized to bone (DeCherney, 1993), estrogen may act through a receptor-mediated mechanism. Another possible mechanism occurs in normal bone remodeling. Interleukin-1 (IL-1) stimulates the activity of mature osteoclasts and promotes osteoclast differentiation. Estradiol inhibits the production of IL-1 and other cytokines. Thus, the loss of estrogen would increase production of IL-1 in bone, which subsequently may increase bone turnover (Avioli, 1994). Type I osteoporosis is a form of estrogen dependent bone loss resulting from accelerated and disproportionate loss of trabecular bone immediately after menopause. Type II osteoporosis occurs later and is associated with both trabecular and cortical bone loss. In younger postmenopausal women prone to Type I osteoporosis, an inverse correlation between circulating parathyroid hormone and estrogen levels suggests estrogens are essential to maintain the normal homeostasis of the parathyroid gland to circulating levels of ionized calcium (Avioli, 1994). Our understanding of how the hormonal milieu affects age-related bone loss is based largely on cross sectional studies. Several endocrine components are important. First, decreased ovarian function with concomitant alterations in the balance of estradiol and estrone and with secondary increases in bone resorption by cytokines (IL-1, TFN␣, and IL-6). Second, a lower calcitonin reserve in the immediate post menopausal period. Third, decreased vitamin D intake and less exposure to sunlight with parallel decreases in circulating 25(OH)D3. Fourth, an altered biologic set point for parathyroid hormone release as a result of estrogen deficiency and a gradual increase in parathyroid secretion with age. At least two of these endocrine components are estrogen dependent. Although the progressive and accelerated loss of skeletal tissues in the post menopausal period can be substantially ameliorated by estrogen treatment, compliance to continued and prolonged ERT is essential for the hormone’s therapeutic affect (Avioli, 1994).

6. The benefits of estrogen therapy for the cardiovascular system and lipid metabolism Heart disease is the leading cause of death in postmenopausal women in the western hemisphere. There is also increased risk of coronary heart disease in young women with

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bilateral oophorectomy, and a beneficial effect of ERT in postmenopausal women. In the PEPI trial, four biologic endpoints were assessed: high-density lipoprotein (HDL) cholesterol, systolic blood pressure, serum insulin, and fibrinogen. This study demonstrated that estrogen alone or in combination with a progestin improves lipoproteins and lowers fibrinogen levels without detectable effects on postchallenge insulin or blood pressure. Unopposed estrogen was the optimal regimen for elevated HDL cholesterol. However, the rate of endometrial hyperplasia restricts its use to women who have had a hysterectomy. In women with a uterus, conjugated equine estrogens with cyclic micronized progesterone had the most favorable effect on HDL cholesterol with no excess risk of endometrial hyperplasia (The Writing Group for the PEPI Trial, 1995). The Heart and Estrogen/progestin Replacement Study (HERS) examined postmenopausal women with a uterus and with preexisting coronary heart disease (CHD) with bypass surgery, percutaneous transluminal coronary angioplasty, or other mechanical revascularization or at least 50% occlusion of a major coronary artery. This randomized, double blind, placebo-controlled trial done at 20 HERS North American centers was designed to test the efficacy and safety of estrogen plus progestin therapy for prevention of CHD. Participants ranged in age from 44 to 79 years (mean, 66.7 ⫾ 6.7). Most were white, married, and had completed high school. The prevalence of coronary risk is high: 62% were past or current smokers, 59% had hypertension, 90% had serum low-density lipoprotein (LDL)-cholesterol of 100 mg./dL or higher, and 23% had diabetes. Women were randomly assigned to receive either 0.625 mg conjugated estrogens plus 2.5 mg medroxyprogesterone daily, or placebo. The null hypothesis was that the incidence of CHD events in women randomly assigned to receive estrogen plus progesterone does not differ from those receiving placebo. For the 2763 HERS participants studies were done over 4.1 years initiated in 1993 and 1994 (Grady et al., 1998). Data indicated that oral estrogen plus progestin did not reduce the overall rate of CHD events in postmenopausal women who already have coronary disease. In fact, treatment increased thromboembolic events and gall bladder disease. Therefore this treatment regime is presently not recommended for the purpose of secondary prevention of CHD. However, there is a favorable pattern of CHD events after several years of therapy, and it may be appropriate for women already receiving the treatment to continue (Hulley et al., 1998). The beneficial effect of estrogen is thought to depend, in part, on its ability to increase HDL, and decrease LDL, cholesterol, and LDL oxidation (Skafar et al., 1997). However, whereas ERT improves lipoprotein profiles in more than 40% of postmenopausal women, this effect does not account for all of estrogen’s protective effect on the cardiovascular system (Skafar et al., 1997). Atheroprotection is partially accounted for by the hypolipidemic effects of the hormone (Selzman et al., 1998). Estrogen also has effects on lipid and carbohydrate metabolism, coagulation factors, and blood pressure (Barrett-Connor and Bush, 1991). Progestins which resemble androgens may attenuate the HDL cholestrolelevating properties of estrogens, but the addition of progesterone apparently does not significantly attenuate estrogen’s effects (Skafar et al., 1997). Estrogen’s ability to inhibit vascular smooth muscle cell growth, proliferation, neointimal formation, and extracellular matrix formation is also protective (Skafar et al., 1997). Estrogen inhibits increase in vascular media surface area after vascular injury in mice lacking ER ␣, suggesting an important role for ER ␤ (Iafrate et al., 1997). Some of estrogen’s physiological effects on smooth muscle have been demonstrated in tissue culture where the steroid hyperpolarizes muscle resting membrane potential (Lin et al., 1987) and attenuates voltage-dependent calcium channel currents. The latter effect may

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contribute to hyperpolarization in vivo and attenuation of myocardial and vascular contractility (Skafar et al., 1997). A rapid nongenomic component involving membrane phenomena in the vessel wall has been proposed to involve alteration of membrane ionic permeability (Farhat et al., 1996). Estrogen also attenuates responses to agents that increase calcium release from intracellular storage suggesting it has multiple simultaneous effects on vascular smooth muscle cell function (Skafar et al., 1997). Estrogen also has effects on endothelial cells. It is well documented that estradiol mediates the production of nitrous oxide (NO). This leads to the production of cyclic GMP and subseqeunt activation of protein kinase G. This in turn phosphorylates and stimulates specific calcium channel subtypes (Skafar et al., 1997). Additionally, estradiol stimulates endothelial NO secretion and both pregnancy and estradiol increase calcium dependent endothelial NO synthase activity and mRNA levels (Skafar et al., 1997). Estradiol also affects endothelial cell differentiation, regeneration, and angiogenesis by promoting neovascularization, proliferation, and migration (Morales et al., 1995). Other effects are on monocyte adhesion, platelet aggegation, enhancement of transcription of endothelial growth factors, tumor necrosis factor ␣, endothelial cells, leukocyte adhesion molecules, and integrans (Skafar et al., 1997), An antioxidant property of estrogen is also proposed. Equine estrogens are better at inhibiting the peroxidation of both fatty acids and cholesterol than estradiol (Subbiah, 1998). This is important because peroxidation of lipoproteins is key in atherogenesis. LDL oxidation results in the production of aldehydes and toxic oxysterols. Estrogen may inhibit production of reactive oxygen species. Estrogen also protects against DNA damage by either hydrogen peroxide or arachidonic acid (Subbiah, 1998). Further, estrogen promotes vasodilation, in part by stimulating prostacyclin and prostaglandin synthase (Farhat et al., 1996) and increases levels of prostaglandin metabolites, which are antithrombotic (Selzman et al., 1998). Estrogen’s action may be via receptor activation and gene transcription with direct action on endothelium. Estrogen receptors have been identified in myocardial, vascular smooth muscle cells, and endothelial cells in humans and animals; either ER␣ or ER ␤ may exert effects (Skafar et al., 1997). Changes in serum estrogen levels regulate ER concentration, because binding of estradiol is higher in coronary arteries of mature female animals than in males with gonadectomy (Vargas et al., 1993) and uterine artery cytosol ER levels are highest in the late follicular phase of the menstrual cycle and in uterine arteries of pregnant versus nonpregnant women (Leibermann et al., 1990). Changes in plasma estrogen may activate the receptor by causing receptor redistribution to the nuclear compartment (Farhat et al., 1996). Estrogen has a number of actions that may protect against CDH. The action of ERT on the cardiovascular system of the postmenopausal woman is multifaceted and requires intensive study to link these diverse effects into a composite picture of the beneficial effects of the steroid.

7. Conundrum: estrogen therapy and breast cancer Estrogen’s protection against cardiovascular disease is so great that current long-term use is now revealing another problem that tends to occur later in life. There is serious concern that exogenous estrogen may increase the incidence of breast cancer (Subbiah, 1998). About one in eight women will develop breast cancer in their lifetime. The incidence of breast disease is increasing due entirely to the increasing numbers of women

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over age 40 (Speroff, 1996). ERs have been found in breast tissue suggesting a classic mechanism of steroid action on this tissue. Although estrogens promote breast cancer in experimental animals, results in humans are inconsistent (Subbiah, 1998). Some studies have shown that use of HRT for 5 years is associated with a 35– 40% increase in risk of developing breast cancer (Colditz, 1998), but not of dying of breast cancer (Faiz and Fentiman, 1998). Results of a recent meta analysis provided two main conclusions: first, while women are taking combined oral contraceptives, and in the 10 years after stopping, there is a small increase in the relative risk of having breast cancer diagnosed; and second, there is no significant excess risk of having breast cancer diagnosed 10 or more years after stopping use (Collaborative Group on Hormonal Factors in Breast Cancer, 1996). Present evidence suggests that a daily dose of 0.625 mg conjugated estrogens taken for several years does not appreciably increase the risk of breast cancer, but a dose of 1.25 mg or higher may do so (Speroff, 1996). There is considerable interest in whether women who have been treated for breast cancer can be safely administered HRT. Whereas the available data are reassuring (Vessey, 1997), most women and physicians err on the side of caution (DiSaia et al., 1996). This view is strengthened by the finding that mammography is less sensitive and less specific in women receiving HRT than in other women as a consequence of the increase in breast density in the former group (Beral et al., 1997). With the exception of one small randomized trial, all of the reported studies are of the observational type. Two large-scale randomized controlled trials of the long-term effects of HRT are in progress: the Women’s Health Initiative in the U.S., and a separate study by the Medical Research Council of the United Kingdom. Unfortunately, useful information about breast cancer, menopause, and estrogen use from these analyses is unlikely to emerge until the middle of next decade, but there are some helpful findings available now. A study by Colditz and coworkers of the Nurses’ Health Study Group in the U.S. was a meta-analysis covering only epidemiologic studies to mid-1991 (Colditz et al., 1995). Data for estrogen alone and combined therapy were combined because no difference was found between these two therapy choices. Ten features of HRT use were examined including ever-use versus never-use, duration of use, recency of use, time since first use, dosage, type of therapy (estrogen alone or combined with progestin), effect of modification by family history, history of benign breast disease, type of menopause, and place of study (North America vs elsewhere) (Vessey, 1997). Most of the data were generated in 31 studies and provide little evidence of effect of HRT on breast cancer risk. There was an indication of a small increase in risk with long duration of HRT use (10 years or more, relative risk 1.23; 15 years or more, relative risk 1.29; 20 years or more 1.51). The risk increases in current users (relative risk 1.40) and in European based studies (relative risk 1.31). In Europe HRT is more likely to be of the combined type and to contain estradiol than in North America (Vessey, 1997; Colditz et al., 1995). For women taking hormones less than 5 years, the relative risk was 1.0, but for 5 or more years of use, it was 1.4 (Vessey, 1997). The 1996 report of the Collaborative Group on Hormonal Factors In Breast Cancer has provided our most current data on the risks of estrogen use. Most of these data are from North America. Data on 53,297 women with breast cancer and 100,239 women without breast cancer from 54 studies in 25 countries were analyzed. While women are taking oral contraceptives and in the 10 years thereafter, there is a small increase in the relative risk of having breast cancer. The study also found that there is no significant excess risk of

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having breast cancer diagnosed 10 or more years after stopping use (Collaborative Group on Hormonal Factors in Breast Cancer, 1996). The results of the meta-analysis of Collaborative Group on studies specific to menopause are not yet complete. Age at menopause is an important confounding variable in data analysis. Women with early menopause (natural or surgical) are more likely to use HRT, especially for extended periods. These women are at reduced risk for breast cancer compared to women with late menopause. Analyses failing to take age at menopause into account may, therefore, not detect an adverse effect of HRT. This caveat aside, findings to date are summarized as follows: First, conjugated equine estrogens are the most common estrogen, and medroxy progesterone acetate is the most common progestin. Second, any effect of HRT on breast cancer carries a small relative risk. Breast cancer is common in postmenopausal women. Therefore small increases in relative risk are important in terms of absolute risk. Third, although data vary, there is probably a modest increase in risk of breast cancer with increasing duration of HRT during menopause; this risk may be concentrated in current users. Fourth, although data sets are small, findings indicate there is little difference between findings for the use of estrogen alone, or when combined with progesterone. Fifth, any effect of HRT on breast cancer risk may be greater for carcinoma in situ, possibly attributable to detection bias in HRT use. This correlates with previous data indicating that carcinomas are more favorably staged in HRT users than non-users. Finally, data on use of HRT in women with previously reported but inactive breast cancer, and data on potential adverse effects of HRT on the interpretation of mammograms will require further study (Vessey, 1997). Large doses and prolonged administration of estrogen are required to induce clinical breast cancer (Subbiah, 1998). Blood levels of estradiol produced by the usual doses of postmenopausal estrogen are relatively low, but may be near the threshold for producing breast cancer-promoting effects. Therefore the tumour response will vary depending on genetic susceptibility factors, such as a history of premenopausal breast cancer in a first-degree relative, the presence of abnormal BRCA1, BRCA2, and p53 genes, and a history of smoking. Consumption of 5 grams or more daily of alcohol along with estrogen administration results in elevation of blood estradiol levels to values equivalent to those of the menstrual cycle’s periovulatory peak. This may exceed the breast-cancer-promoting threshold. Increased risk of breast cancer from postmenopasual estrogen administration can be eliminated by limiting alcohol consumption and diminishing the capacity to 16 ␣-hydroxylate estradiol. This can be accomplished with either pharmacological agents (indol-3-carbinol) or by increasing consumption of cruciferous vegetables (Zumoff, 1998).

8. The benefits of estrogen therapy for mood and cognitive function Natural or surgical menopause is associated with an increase in complaints of psychological distress and cognitive deficits. Some of the most frequent complaints are of sleep disturbance, depression, and difficulty in remembering things. It is not established that all of these symptoms can be directly attributed to loss of estrogen, but estrogen treatment does reduce the frequency of these complaints and their severity. Sleep disturbances including difficulty falling asleep, restless sleep and frequent awaking are common complaints, but these difficulties are also often reported by males as well as they get older. A 7-month prospective randomized, double-blind crossover study found

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that estrogen treatment reduced awakenings, improved subjective sleep quality and reduced daytime tiredness (Polo-Kantola et al., 1998). However a study of the effect of 12 days out of 28 estrogen plus norgestrel on sleep in the laboratory did not find any effect of treatment on polysomnographic sleep stage measures, even though subjects reported their psychological well-being was improved (Purdie et al., 1995). Because subjects’ estimates of their sleep time are notoriously unreliable, more laboratory studies are necessary to determine whether sleep architecture is altered by estrogen treatment. Women have a higher risk of depression than men, but there seems to be no increase in the rate of depression at menopause. Nevertheless, women with a history of affective or mood disorder associated with reproductive events may have an increased risk of depression at menopause (Pearlstein et al., 1997). Many studies have reported improved mood with estrogen therapy, and the effect is more prominent when estrogen is combined with androgen (Sherwin, 1994). A recent meta-analysis of the literature (Zweifel and O’Brien, 1997) found that the average estrogen-treated patient had lower levels of depressed mood than 76% of the controls. Progesterone in combination with estrogen produced a smaller reduction in depressed mood than estrogen alone, whereas androgen plus estrogen produced a larger improvement in mood. The mood enhancing effect of estrogen may be associated with its ability to augment serotonergic activity (Halbreich, 1997). This notion is consistent with recent evidence that in elderly women the antidepressant effect of the specific serotonergic uptake inhibitor, fluoxetine, is significantly improved by concurrent estrogen treatment (Schneider and Farlow, 1997). There are several reasons to suspect that estrogens might be beneficial for cognitive function. First, the hippocampus, which is important to memory function, contains ERs. Estrogen is reported to enhance synaptic function in the hippocampus (Gould et al., 1998). Second, estrogen has a major effect on the forebrain cholinergic system (Toran-Allerand et al., 1992; Miller et al., 1998) that is implicated in memory and cognitive function (Muir, 1997). Thirdly, estrogen has significant effects on neuronal growth and morphology, electrical activity, neurotransmitter turnover, enzyme regulation, neurosecretion, neurotransmitter uptake, and neurotransmitter receptors (McEwen and Parsons, 1982). It is not presently clear whether estrogen levels do influence cognitive ability. Androgen effects are thought to account for male superiority on tests of spatial ability, and consistent with this, women perform more poorly on tests of spatial performance during the mid-cycle estrogen surge than at other points of the cycle (Haskell et al., 1997). On the other hand, estrogen is thought to account for women’s superior verbal ability, and there is evidence that it enhances or maintains verbal abilities and verbal memory function in healthy postmenopausal estrogen treated women (Sherwin, 1998). It can be inferred that the likelihood of detecting a beneficial (or deleterious) effect of estrogen will depend on the particular cognitive ability examined, so that negative effects in one test cannot be taken as evidence against positive effects found in another test. Furthermore the decision to take estrogen is correlated with cognitive ability, education and social class, so that results of surveys cannot be taken at face value. Nevertheless, a number of studies have examined memory or cognitive function in postmenopausal women and found that women taking estrogen have superior performance to women not taking estrogen when the two groups are matched for age and education (Sherwin, 1998). A recent meta-analysis concluded that estrogen produced a small improvement of cognitive function in menopausal women who experienced significant symptoms, and that estrogen treatment seemed to reduce the risk of AD by 29% (Yaffe et al., 1998). The reviewers comment that controlled clinical trials are necessary to establish whether estrogen treatment is advan-

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tageous for cognitive function. However, the only double-blind, randomized, crossover study to date (Polo-Kantola et al., 1998) found no difference between the effects of estrogen and placebo on cognitive speed, accuracy, attention and memory after a 3-month treatment. In contrast recent brain imaging studies suggest that estrogen has effects on the brain that would be expected to influence cognitive performance. One study used PET to measure regional cerebral blood flow while women performed the Wisconsin Card Sorting Test, or a control task. Ovarian function was suppressed by a GnRH agonist, and estradiol or progesterone were administered in a double-blind, crossover design (Berman et al., 1997). The GnRH agonist attenuated the normal frontal cortex activation pattern, and either estradiol or progesterone normalized it. A magnetic resonance imaging study of postmenopausal women found that estrogen users performed better on almost all neuropsychological tests, and that MRI data indicated that they had a lower rate of clinically unsuspected brain damage (Schmidt et al., 1996).

9. The benefits of estrogen therapy for Alzheimer’s Disease (AD) Several lines of evidence support this notion that ERT has the potential to alleviate the effect of AD. Being female confers an additional risk to having AD because women live into the risk period. Even if adjustments are made for survival, women are at greater risk than men (Gao et al., 1998), but women who receive ERT perform at a higher cognitive level than those who do not. This risk is decreased with increasing estrogen dosage, and with an earlier age of menarche. The relative risk for AD in women who have used ERT at some point is reduced 50 – 60% compared to those who have never used ERT, and the age of onset of women who become demented is significantly higher for women who use ERT than for non-users. In several trials of estrogen for AD, women with AD treated with ERT did show significant improvement in performance scores for cognitive function (reviewed in Yaffe et al., 1998; Tang et al., 1996). However, to date very few women have been tested. Also the studies have not included controls for age, duration of therapy, education and depression, and most studies have not been randomized or placebocontrolled. There are plausible mechanisms whereby estrogens might retard degenerative changes in AD. Extracellular amyloid deposits in the parenchyma of amygdala, hippocampus, and neocortex are major histopathological markers for AD (Muller et al., 1997; Price et al., 1995) and increased amyloid deposition (amyloid load) is tightly linked to the decline in memory function. Amyloid precursor protein (APP) is the source of an insoluble form of amyloid ␤, which accumulates in the brains of AD patients and may damage or kill neurons (Mattson, 1997). An alternative APP processing yields soluble APP (Jaffe et al., 1994), which does not form amyloid plaques. Secretion of the soluble form of APP is stimulated by protein kinase C (PKC) activation (Koo, 1997; Slack et al., 1997) which can be upregulated by estrogen (Jaffe et al., 1994). Another potential contributor to AD pathology is oxidative stress and associated free radical mediated oxidative damage. Amyloid ␤ induces lipid peroxidation, and hydrogen peroxide may mediate amyloid ␤ toxicity. Estrogens, in contrast with all other natural steroids, are antioxidants of membrane phospholipid peroxidation because of their phenolic structure. Under some circumstances 17␤ estradiol and 17␣ estradiol can prevent intracellular peroxide accumulation, and this could lessen neuronal degeneration caused by free radicals. The neuroprotectant antioxidant activity of estrogens is dependent on the presence of the hydroxyl group in the

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C3 position on the A ring of the steroid molecule, but interestingly is independent of an activation of the estrogen receptor (Behl et al., 1997). Another means by which estrogen may help AD symptoms may be via action through the cholinergic system. The basal forebrain cholinergic neurons are the primary source of cholinergic innervation to the hippocampal formation and cortex. Currently the only clinically approved therapies for dementias associated with aging are cholinergic. The best-developed approach to cholinergic therapy is cholinesterase inhibition, which improves cognitive symptoms in some AD patients (Farlow and Evans, 1998). When tacrine (cholinesterase inhibitor) and estrogen are given together, cognitive function is significantly improved, suggesting an interaction of estrogen and the cholinergic system in AD (Schneider et al., 1996). Estrogen influences the amount of the synthetic enzyme for acetylcholine (Gibbs, 1996), and the number of cholinergic neurons (Miller et al., 1999). The mechanism of this effect is not known, but it may involve both direct and indirect effects of estrogen on cholinergic neurons (see Miller et al., 1999).

10. Alternative treatments: phytoestrogens Many women are now choosing dietary supplements as a source of estrogen at menopause. All environmental estrogens compete with estradiol 17␤ for binding to both ER subtypes with similar preference and degree. In most cases the relative binding affinities are about 1000-fold lower that of estrogen. Some phytoestrogens compete more efficaciously for estrogen binding to ER ␤ than ER ␣. Phytoestrogens stimulate the transcriptional activity of both ER subtypes and antiestrogenic activity is not reported for these compounds (Kuipper et al., 1998). One alternative treatment is the use of dietary phytoestrogens with soya (isoflavones) and linseed (lignins) (Seidl and Stewart, 1998). The relative potency of isoflavinoid phytoestrogen is at most 2% that of estradiol 17␤ (Yaffe et al., 1998; Kuipper et al., 1998). In post menopausal women with low endogenous circulating estrogen, isoflavinoids can occupy ERs efficaciously and have some estrogen action (Seidl and Stewart, 1998). Societies with this diet have statistically less cardiovascular disease. The effects also extend to physiological parameters. For example, women in Japan do not complain of hot flashes. This may be because there is no word in the Japanese language for hot flash, or the result of cultural inappropriateness for complaint, or their traditional diet which is rich in phytoestrogens such as soy products (tofu)(Kessel, 1998; Lock, 1994; Lock, 1998). In Indonesia perimenopausal women increase dietary intake of phytoestrogen-rich papaya and have only a 30% incidence of hot flashes (Kessel, 1998). Interestingly, a clinical trial of phytoestrogen rich diets revealed increased sex hormone binding globulin levels and decreased hot flush scores. Clinical studies are now underway to assess the effects of these foods on bone and lipid profiles (Kessel, 1998). Whereas the estrogenic potency of industrially derived estrogenic compounds such as DDT and alkyphenols is limited, the estrogenic potency of phytoestrogens is significant, especially for ER␤. Phytoestrogens may trigger many of the biologic responses that are evoked by physiological estrogens in ovary, uterus, pituitary, hypothalamic/preoptic area, and brain cortex (Kuipper et al., 1998). Although epidemiologic data are inconclusive, several putative mechanisms could account for chemoprotective effects of phytoestrogens. In particular, phytoestrogens may exert anti-estrogenic effects thereby preventing development of hormone-related cancers. Alternatively, alteration of cancer cell differentiaion, inhibition of protein tyrosine ki-

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nases, suppression of angiogenesis, and direct anti-oxidant effects may also be factors. Because of the major potential beneficial effects of increased food intake of phytoestrogens in the prevention of postmenopausal osteoporosis and cardiovascular disease, a great deal of research is needed into these naturally occurring compounds (Kuipper et al., 1998).

11. Conclusion At the end of the 20th century a large proportion of women are living longer than men, and longer than anyone ever lived in previous centuries. For the first time in recorded history, and probably in the history of the species, the majority of women are living many years beyond the end of their reproductive period. Historically, menopause meant freedom from the dangers of childbearing. Now the risk of childbearing is quite low, the loss of estrogen associated with the termination of the reproductive cycle appears to have many deleterious consequences and few benefits. Experimental and clinical evidence suggests that estrogen replacement is effective in reducing the negative consequences of the menopause. Both basic and clinical evidence continues to grow that estrogen has positive effects on mood, sexual function, target end organs, and cognitive function. Despite the mounting evidence on the benefits of ERT more research is needed on the mechanisms of the effects of estrogen, selective estrogen receptor modulators, antiestrogens, and phytoestrogens to determine their long term effectiveness and risks.

Acknowledgments The authors wish to thank Drs. D. Stephen Snyder and Roger Gosden for their helpful commentary and discussions in the preparation of this manuscript. We also wish to thank the Alzheimer Society of Canada for its support in the publication of this manuscript.

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