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The Journal of Clinical Endocrinology & Metabolism 89(5):2319 –2325 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-031728

Adrenocortical Effects of Oral Estrogens and Soy Isoflavones in Female Monkeys CHARLES E. WOOD, J. MARK CLINE, MARY S. ANTHONY, THOMAS C. REGISTER, JAY R. KAPLAN

AND

Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1040 The goal of this study was to evaluate the long-term adrenocortical effects of premenopausal oral contraceptives (OC) and postmenopausal conjugated equine estrogens (CEE) and soy isoflavones in a female cynomolgus monkey model. Half of the animals received a triphasic OC for a period of 26 months, after which all monkeys were ovariectomized and randomized to one of three diet groups for 36 months: 1) isoflavonedepleted soy protein (control) (n ⴝ 54); 2) soy protein with isoflavones (129 mg/d equivalent) (SPIⴙ) (n ⴝ 56); or 3) isoflavone-depleted soy protein with CEE (0.625 mg/d equivalent) (n ⴝ 59). In the premenopausal phase, OC treatment resulted in significantly higher cortisol (F) and lower dehydroepiandrosterone sulfate, androstenedione, and testosterone rel-

I

N POSTMENOPAUSAL WOMEN, adrenal androgens have an important role in maintaining sexual function (1–3), bone density (3– 4), psychological well-being (5– 6), muscle mass (6 –7), and cognitive function (8). Aging is associated with a progressive and marked decline in these androgens (9 –10), and the postmenopausal state is thus characterized by lower ovarian and adrenal sex hormones. Recently, there has been increased interest in the concept of androgen supplementation for older women (11), particularly those with a history of ovariectomy (12). The syndrome of androgen deficiency is poorly defined clinically, however, and factors contributing to adrenal androgen suppression have not been adequately examined. Of particular interest is the role of exogenous estrogens in regulating postmenopausal adrenal androgen production. The use of oral contraceptives (OC) to treat various symptoms associated with the perimenopause is becoming commonplace in clinical practice (13), although there is a lack of information about hormonal effects that may carry over into the menopause and interact with postmenopausal treatments. In postmenopausal women, studies of estrogen therapy and serum androgens have provided inconsistent results (14 –26), likely due to the small size, short duration, and/or variation in subject reproductive histories within and among the studies. The effects

Abbreviations: A4, Androstenedione; BMI, body mass index; BW, body weight; CEE, conjugated equine estrogen(s); DHEA-S, dehydroepiandrosterone sulfate; E2, 17␤-estradiol; ER, estrogen receptor(s); F, cortisol; H&E, hematoxylin and eosin; OC, oral contraceptive(s); SPI⫺, soy protein depleted of isoflavones; SPI⫹, soy protein with isoflavones; T, testosterone. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

ative to intact controls. In the postmenopausal phase, CEE treatment resulted in significantly higher basal F and lower dehydroepiandrosterone sulfate, androstenedione, and testosterone when compared with control and SPIⴙ diets. Serum F and androgens in the SPIⴙ group did not differ significantly from the control group. The SPIⴙ group had significantly lower adrenal weight than either control or CEE groups, and this effect was localized primarily to the zona fasciculata region of the adrenal cortex. These findings suggest that longterm estrogen treatment may contribute to an androgendeficient and hypercortisolemic state. (J Clin Endocrinol Metab 89: 2319 –2325, 2004)

of soy isoflavones on adrenal androgens are similarly unclear. Soy isoflavones are phytoestrogenic compounds widely marketed and used by postmenopausal women as a natural alternative to traditional estrogen therapies (27). Isoflavones competitively bind estrogen receptors (ER) (28 –29), induce expression of estrogen markers (30), and may potentially lower androgen concentrations (31–34). However, the steroidogenic targets of isoflavones in vivo are unclear, and direct adrenocortical effects have not been evaluated. In the present study, we used a female monkey model to evaluate adrenocortical effects of premenopausal OC treatment and subsequent interactions with postmenopausal conjugated equine estrogen (CEE) or soy isoflavone treatment. We hypothesized that OC, CEE, and soy isoflavones would suppress adrenal androgen production and that these effects would be reflected in adrenal morphology. Materials and Methods Animals One hundred eighty-nine adult female premenopausal cynomolgus monkeys (Macaca fascicularis) were obtained from the Institute Pertanian Bogor, Bogor, Indonesia. The reproductive endocrinology of female macaques is similar to that of women in many aspects, including a 28-d menstrual cycle, menopause, distribution of steroidogenic enzymes, and response to postmenopausal hormone therapies (35–37). Macaques are also similar in producing large amounts of adrenal C19 androgens that decline with age as in women (38). In this study, all animals were housed in stable social units of four to six animals per group for the entire experiment. One hundred seventy-seven monkeys completed the experiment, and adrenal glands were obtained at necropsy from 169 of these animals. Average age at the time of import was 6.0 yr, as estimated from dentition. All procedures involving animals were conducted in compliance with State and Federal laws, standards of the United States Department of Health and Human Services, and guidelines established by the Wake Forest University Animal Care and Use Committee.

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Study design This investigation was part of a large randomized controlled experiment designed to evaluate multisystem effects of premenopausal OC and postmenopausal CEE or soy isoflavones. Other aspects of this study have been reported elsewhere (39 – 46). This study incorporated premenopausal and postmenopausal treatment phases spanning a total of 5.5 yr (Fig. 1) (40 – 41). During the 26-month premenopausal period, half the animals received, in the diet, a triphasic OC (Triphasil, Wyeth-Ayerst Laboratories, Inc., Philadelphia, PA) containing varying proportions of ethinyl estradiol (0.03– 0.04 mg/1800 kcal) and levonorgestrel (0.05– 0.125 mg/1800 kcal) for the initial 21 d of each cycle, with a placebo given for the final 7 d, as in women. Monkeys receiving OC were sampled on several occasions for plasma concentrations of ethinyl estradiol and levonorgestrel (d 21 of the pill cycle), revealing blood concentrations comparable with those observed in women using the same regimen. All monkeys consumed a moderately atherogenic diet containing 17% of calories from protein, 45% from fat, 38% from carbohydrates, and 0.28 mg cholesterol/kcal. After the premenopausal phase, all animals were ovariectomized to make them surgically menopausal. The postmenopausal study followed a three-group, parallel arm design and lasted for 36 months. Using a stratified randomization scheme based on premenopausal social group and OC treatment, monkeys were assigned to one of three postmenopausal treatment groups: 1) alcohol-washed soy protein depleted of isoflavones (SPI⫺ control); 2) soy protein with isoflavones (SPI⫹); and 3) SPI⫺ with CEE. For this randomization, all animals remained in their original social groups, and approximately equal numbers of OC⫹ and OC⫺ animals were assigned to each of the three postmenopausal groups. The control SPI⫺ diet contained isolated soy protein that had been alcohol-washed to remove the isoflavones. The SPI⫹ treatment group received soy protein isolate with intact soy isoflavones at a dose approximately equivalent to 129 mg/d for women, expressed as aglycone units (approximately 91 mg genistein, 31 mg daidzein, and 7 mg glycitein). The CEE group received alcohol-washed soy protein isolate plus CEE (Premarin, Wyeth-Ayerst Laboratories, Inc.) at a dose comparable with 0.625 mg/d for women. CEE and isoflavone doses were scaled to 1800 kcal of diet (the estimated daily intake for U.S. women) so that monkeys fed 120 kcal of diet/kg BW consumed approximately 0.042 mg CEE/kg BW or 8.6 mg isoflavones/kg BW. This caloric adjustment of dose should account for differences in metabolic rates between the monkeys and the human subjects. The isolated soy proteins used for this study were generously provided by Solae, a division of DuPont (St. Louis, MO). The unextracted soy protein (SUPRO 670-HG) contained, on the average, 1.105 mg genistein, 0.365 mg daidzein, and 0.08 mg glycitein/g soy protein isolate, whereas the alcohol-extracted soy protein (SUPRO 670-IF) contained 0.04 mg genistein, 0.01 mg daid-

Wood et al. • Adrenal Effects of Estrogens and Isoflavones

zein, and 0.01 mg glycitein/g isolate (expressed in aglycone units). Monkeys were fed approximately 120 kcal/kg BW/d split into two feedings (one third in the morning, two thirds in the afternoon). Diets were formulated to be isocaloric and equivalent for the macronutrients, cholesterol (0.28 mg/kcal), calcium (830 mg/1800 kcal), and phosphorus (820 mg/1800 kcal). Dietary intake was confirmed by measurement of isoflavone and 17␤-estradiol (E2) concentrations in blood samples collected 4 h after feeding the morning meal. E2 concentrations, measured by RIA (double antibody RIA, Diagnostic Products Corp., Los Angeles, CA) on unextracted serum during month 6 of the postmenopausal period, were 17.9 ⫾ 3.2, 11.5 ⫾ 3.9, and 192.9 ⫾ 11.4 pg/ml for the SPI-, SPI⫹, and CEE groups, respectively. Serum isoflavone concentrations were measured at month 34 of the postmenopausal period by HPLCmass spectrometry (47) in Dr. Stephen Barnes’ laboratory (University of Alabama, Birmingham, AL), as described previously (41). The mean total isoflavone concentration of the SPI⫹ group was 805 ⫾ 88 nmol/liter aglycone equivalents. Isoflavone metabolite concentrations (nmol/liter) were as follows: equol, 473.1 ⫾ 47.0; genistein, 169.8 ⫾ 33.7; daidzein, 118.1 ⫾ 18.4; dihydrodaidzein, 27.9 ⫾ 4.5; and o-desmethylangolensin, 17.3 ⫾ 2.3. Total serum isoflavone concentrations from all SPI⫺ animals were less than 80 nmol/liter.

Social status To assess and adjust for the potential influence of social stress on adrenocortical measures, the dominance status of each animal, relative to the others in her social group, was determined by methods described previously (40). Briefly, social rank was assigned based on data collected during weekly, 30-min observations beginning after social group formation and before provision of contraceptive steroids. Dominance and subordination were determined by the outcomes of conflicts, which are highly asymmetric in this species and yield clear winners and losers as judged by specific facial expressions, postures, and vocalizations (48). For purposes of analysis, animals ranking first or second in groups of five were considered dominant, as were animals ranking 1, 2, or 3 in groups of six; the remainder of monkeys were subordinate.

Necropsy and histomorphometry At the end of the postmenopausal phase, monkeys were sedated with ketamine and killed using sodium pentobarbital (100 mg/kg, iv) as recommended by the Panel on Euthanasia of the American Veterinary Medical Association. At necropsy, pituitary and adrenal glands were carefully dissected away from surrounding tissues, removed, weighed, placed in 4% paraformaldehyde for 24 h, and stored in 70% ethanol. After fixation, adrenal glands were cut longitudinally, embedded in paraffin, and sectioned at 5 ␮m for routine hematoxylin and eosin (H&E) staining. Histomorphometric analysis was used to quantitate cortical and medullary area and thickness of the cortical layers. H&E-stained slides were digitized using a Sony DXC-390 camera (Scion Corp., Frederick, MD), and measurements were taken with public domain software [National Institutes of Health (NIH) Image v1.60]. To minimize error due to tangential sectioning artifact, cortical thickness measurements were taken at a standardized location in the lower left quadrant of the adrenal image immediately adjacent to a rounded margin. The thickness of the zona glomerulosa layer was too small (⬍0.1 mm) for accurate and repeatable measurements, and these values were excluded. All measurements were made blinded to animal group. H&E-stained adrenal glands were also evaluated qualitatively for microscopic changes by a board-certified veterinary pathologist (J.M.C.).

Endocrine assays

FIG. 1. Randomized controlled trial design with premenopausal and postmenopausal treatment phases. At the time of ovariectomy, animals were stratified by OC treatment and then randomized so that each postmenopausal group contained approximately equal numbers of OC-treated and OC-untreated animals.

Blood collections were performed 4 h after the morning meal (between 1030 h and 1200 h) via femoral venipuncture after the animals had been sedated with ketamine HCl (10 mg/ml, im). Previous studies indicate that ketamine does not significantly alter cortisol (F) concentrations in nonhuman primates (49 –50). Premenopausal blood samples were collected at month 17 for androgens and month 22 for F. Postmenopausal blood samples were collected at a single timepoint 27 months into the postmenopausal period. Blood collections were staggered so that all animals in a single pen were sampled on the same day and all animals were sampled at the same time from the start of treat-

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ment. All postmenopausal hormone assays were run from a subset of animals based on serum availability at month 27. Concentrations of dehydroepiandrosterone sulfate (DHEA-S), androstenedione (A4), and total testosterone (T) were quantitated by RIA, with commercially available kits, using antibody-coated tubes (Coat-A-Count, Diagnostic Products Corp.). LH was determined via bioassay using reagents from the National Hormone and Pituitary Program, as previously described (51). F concentrations were assayed using a commercial kit from Diagnostic Systems Laboratories (Webster, TX). Assays were performed at either the Yerkes Regional Primate Research Center Endocrinology Laboratory (DHEA-S, LH, F) or the Clinical Chemistry and Endocrinology Laboratory at the Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine (A4, T). Hormone assays were modified as needed for use in monkeys, using charcoal-stripped monkey serum as a diluent for low standards. Intraassay coefficients of variation were less than 10% for all assays.

Statistics Premenopausal hormone data were analyzed by one-way ANOVA. Postmenopausal hormone and adrenal morphology data were subjected to three-way ANOVA using the following between-subject factors: premenopausal treatment (OC, no OC); postmenopausal treatment (control, SPI⫹, CEE); and social status (dominant, subordinate). The final model for postmenopausal data adjusted for premenopausal treatment (OC, no OC) but not social status because it was not a significant covariate. All variables were evaluated for their distribution and equality of variances between groups. Log10 transformations were performed for variables that violated the Levene’s test of equal variances (premenopausal and postmenopausal DHEA-S, A4, T, F); all hormone data were retransformed using the inverse log10 for presentation in tables. Right and left adrenal gland measurements were averaged for each individual animal. Adrenal morphology and pituitary gland values were analyzed using BW (body weight) as a covariate. Animals with missing adrenal gland weights were excluded from analysis. A two-tailed significance level of 0.05 was chosen for all comparisons. The SAS statistical package (SAS Institute, Cary, NC) was used for all analyses.

Results Age, BW, and body mass index (BMI)

No significant differences in age, mean BW, or BMI (BW/ trunk length2) were observed among treatment groups during either the premenopausal or postmenopausal phases. Mean age at death for control, SPI⫹, and CEE groups was 12.19 ⫾ 0.15, 12.06 ⫾ 0.15, and 11.85 ⫾ 0.14 yr, respectively (by ANOVA, P ⫽ 0.26). After the premenopausal phase, mean BW and BMI in the OC⫹ group were 2.95 ⫾ 0.04 kg and 39.9 ⫾ 0.3 kg/m2, respectively, vs. 2.92 ⫾ 0.04 kg and 39.8 ⫾ 0.3 kg/m2 in the OC⫺ group (by ANOVA, P ⫽ 0.48 for BW; P ⫽ 0.76 for BMI). At the end of the postmenopausal phase, BW for the control group averaged 3.34 ⫾ 0.10 kg compared with 3.40 ⫾ 0.10 kg for the SPI⫹ group and 3.29 ⫾ 0.10 kg for the CEE group (by ANOVA, P ⫽ 0.72). BMI for control, SPI⫹, and CEE groups was 44.2 ⫾ 1.2, 44.3 ⫾ 1.1, and 42.4 ⫾ 1.1 kg/m2, respectively (by ANOVA, P ⫽ 0.39). All

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animals had evidence of previous pregnancy as indicated by adventitial expansion and accumulation of extracellular matrix around the myometrial veins (52). Serum hormones and isoflavones

Premenopausal and postmenopausal androgen concentrations were comparable with those found in respective populations of women (11). In the premenopausal phase, OC treatment resulted in significantly higher basal F (⫹34.4%) and lower DHEA-S (⫺27.0%), A4 (⫺52.9%), and total T (⫺50.0%) (Table 1). In the postmenopausal phase, treatment with CEE resulted in significantly higher basal F (⫹19% vs. control, ⫹36% vs. SPI⫹) and lower DHEA-S (⫺29% vs. control, ⫺35%, vs. SPI⫹), A4 (⫺34% vs. control, ⫺26% vs. SPI⫹), and total T (⫺52% vs. control, ⫺41% vs. SPI⫹) when compared with the control and SPI⫹ groups (Table 2). The SPI⫹ group did not differ significantly from the control group in serum F, DHEA-S, A4, or total T. CEE, but not SPI⫹, treatment was associated with significantly lower LH (⫺81% vs. control, ⫺80% vs. SPI⫹; Table 2) and pituitary weight (⫺18% vs. control, ⫺17% vs. SPI⫹; Table 3). Adjusting for age and BW had negligible effects on hormone analyses. Postmenopausal androgens in the OC⫺ and OC⫹ subgroups did not differ significantly within each postmenopausal group or in combined analysis of the three postmenopausal groups (Table 4). Previously reported androgen values from SPI⫺, SPI⫹, and CEE groups at month 10 of the postmenopausal phase are as follows: DHEA-S (␮g/dl), 5.81 ⫾ 0.82, 6.85 ⫾ 0.97, 6.02 ⫾ 0.82; A4 (ng/dl), 66.4 ⫾ 9.2, 117.7 ⫾ 16.0, 48.7 ⫾ 6.4; T (ng/dl), 6.8 ⫾ 0.7, 7.7 ⫾ 0.6, 6.4 ⫾ 0.9. A4 was higher in the SPI⫹ group (P ⬍ 0.01), but no significant differences were found between SPI⫺ and CEE groups. Postmenopausal F was significantly higher in animals with a history of OC treatment when postmenopausal groups were combined (Table 4); this effect was limited to the SPI⫺ (P ⬍ 0.001) and CEE⫹ (P ⫽ 0.02) groups and not the SPI⫹ group (P ⫽ 0.8) (postmenopausal*OC treatment interaction, P ⫽ 0.03) (Table 4). Social status was not associated with significant differences in serum androgens or basal F in the premenopausal or postmenopausal phases. Adrenal morphology

The SPI⫹ group had significantly lower adrenal weight compared with both control (⫺13%) and CEE (⫺6%) groups (Table 3). Soy isoflavone effects on adrenal weight were reflected in lower cortical thickness and area and localized primarily to the zona fasciculata (Table 3), the major F producing region of the adrenal gland. A history of OC treat-

TABLE 1. Effects of OC on serum cortisol and androgens in the premenopausal perioda

DHEA-S (␮g/dl) Androstenedione (ng/dl) Total testosterone (ng/dl) Cortisol (␮g/dl) Cortisol/DHEA-S ratio

OC⫺ (n ⫽ 86)

OC⫹ (n ⫽ 83)

P values

14.25 (⫹0.98, ⫺0.94) 126.7 (⫹6.1, ⫺5.9) 9.3 (⫹0.6, ⫺0.5) 14.45 (⫹0.41, ⫺0.40) 1.00 (⫹0.07, ⫺0.06)

10.41 (⫹0.84, ⫺0.81) 59.6 (⫹3.7, ⫺3.6) 4.5 (⫹0.3, ⫺0.3) 19.42 (⫹0.56, ⫺0.55) 1.75 (⫹0.17, ⫺0.14

0.003 ⬍0.0001 ⬍0.0001 ⬍0.0001 ⬍0.0001

P values are from two-way ANOVA. For conversion to SI units (nmol/liter), divide by the following conversion factors: 0.039 for DHEA-S, 28.64 for androstenedione, 28.84 for total testosterone, and 3.625 for cortisol. a Values are retransformed means ⫾ SE.

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Wood et al. • Adrenal Effects of Estrogens and Isoflavones

TABLE 2. Effects of conjugated equine estrogens and soy isoflavones on serum cortisol and androgens in the postmenopausal perioda

DHEA-S (␮g/dl) Androstenedione (ng/dl) Total testosterone (ng/dl) Cortisol (␮g/dl) Cortisol/DHEA-S ratio LH (ng/ml)

SPI⫺ control (n ⫽ 45)

SPI⫹ (n ⫽ 44)

CEE (n ⫽ 59)

5.57 (⫹0.74, ⫺0.67) 57.5 (⫹0.5, ⫺0.4) 7.9 (⫹1.2, ⫺1.1) 28.66 (⫹1.54, ⫺1.48) 4.73 (⫹0.57, ⫺0.49) 5.36 (⫹0.31, ⫺0.31) (n ⫽ 28)

6.08 (⫹0.81, ⫺0.73) 51.2 (⫹0.5, ⫺0.4) 6.5 (⫹1.0, ⫺0.9) 25.08 (⫹1.42, ⫺1.36) 3.95 (⫹0.45, ⫺0.39) 5.04 (⫹0.29, ⫺0.28) (n ⫽ 32)

3.94 (⫹0.48, ⫺0.44) 38.1 (⫹0.4, ⫺0.4) 3.7 (⫹0.5, ⫺0.4) 34.17 (⫹1.54, ⫺1.48) 8.01 (⫹1.04, ⫺0.87) 1.01 (⫹0.20, ⫺0.19) (n ⫽ 36)

P values ANCOVA

Control vs. SPI⫹

Control vs. CEE

SPI⫹ vs. CEE

0.02

0.62

0.05

0.01

0.003

0.33

0.001

0.02

0.0002

0.34

0.0001

0.004

⬍0.0001

0.08

0.01

⬍0.0001

⬍0.0001

0.24

0.001

⬍0.0001

⬍0.0001

0.44

⬍0.0001

⬍0.0001

ANCOVA, Analysis of covariance. ANCOVA included prior OC treatment as a covariate. SPI⫹, Soy protein with 129 mg/d equivalent of isoflavones; SPI⫺, soy protein without isoflavones; CEE, SPI⫺ with conjugated equine estrogens at 0.625 mg/d equivalent. Approximately half of the animals in each group (⫾1) were treated previously with OC (SPI⫺, n ⫽ 23; SPI⫹, n ⫽ 23; CEE, n ⫽ 29). For conversion to SI units (nmol/liter), divide by the following conversion factors: 0.039 for DHEA-S, 28.64 for androstenedione, 28.84 for total testosterone, and 3.625 for cortisol. a Values are retransformed means ⫾ SE. TABLE 3. Effects of conjugated equine estrogens and soy isoflavones on adrenal morphology at the end of the postmenopausal perioda

Adrenal weight (mg) Left Right Average Cortical area (mm2) Medullary area (mm2) Cortical thickness (␮m) Zona fasciculata (␮m) Zona reticularis (␮m) Pituitary weight (mg)

P values

SPI⫺ control (n ⫽ 54)

SPI⫹ (n ⫽ 56)

CEE (n ⫽ 59)

ANCOVA

Control vs. SPI⫹

Control vs. CEE

340 ⫾ 14 261 ⫾ 9 299 ⫾ 8 11.89 ⫾ 0.39 6.54 ⫾ 0.33 729 ⫾ 15 421 ⫾ 10 199 ⫾ 7 76 ⫾ 3

296 ⫾ 9 229 ⫾ 9 261 ⫾ 8 10.51 ⫾ 0.39 6.31 ⫾ 0.33 662 ⫾ 14 384 ⫾ 9 177 ⫾ 7 75 ⫾ 3

310 ⫾ 8 242 ⫾ 7 277 ⫾ 7 11.00 ⫾ 0.38 5.88 ⫾ 0.31 718 ⫾ 14 424 ⫾ 10 186 ⫾ 7 62 ⫾ 2

0.01 0.01 0.003 0.04 0.32 0.002 0.008 0.06 ⬍0.0001

0.002b 0.003b 0.0006b 0.01

0.31 0.30 0.13 0.10

0.06 0.03 0.04 0.38

0.001 0.01

0.62 0.67

0.004 0.003

0.75

⬍0.0001

0.0003

SPI⫹ vs. CEE

ANCOVA, Analysis of covariance. ANCOVA included prior OC treatment and BW as covariates. SPI⫹, Soy protein with 129 mg/d equivalent of isoflavones; SPI⫺, soy protein without isoflavones; CEE, SPI⫺ with conjugated equine estrogens at 0.625 mg/d equivalent. Left and right adrenal measures were averaged for each individual animal. Approximately one half of the animals in each group (⫾2) were treated previously with OC (SPI⫺, n ⫽ 25; SPI⫹, n ⫽ 29; CEE, n ⫽ 29). a Values are means ⫾ SE. b Postmenopausal*OC treatment interaction.

ment abolished the effect of SPI⫹ on average adrenal weight (postmenopausal*OC treatment interaction, P ⬍ 0.01). When postmenopausal groups were subdivided based on prior OC treatment, the non-OC SPI⫹ group had significantly lower adrenal weight (P ⬍ 0.0001), cortical area (P ⬍ 0.01), cortical thickness (P ⬍ 0.02), and ZF thickness (P ⫽ 0.05) compared with the non-OC SPI⫺ control group. In contrast, none of these differences were significant between SPI⫹ and control groups within the OC-treated subset of animals. Total cortical thickness (r ⫽ 0.55), cortical area (r ⫽ 0.64), and zona fasciculata thickness (r ⫽ 0.53) correlated significantly with adrenal weight after adjusting for BW (P ⬍ 0.0001 for all measures). No significant differences in adrenal morphology were detected between control and CEE groups. Serum F and DHEA-S did not correlate significantly with mean thickness of the zona fasciculata (r ⫽ 0.10, P ⫽ 0.24) and zona reticularis (r ⫽ 0.15, P ⫽ 0.08) layers, respectively. Social subordinates had greater adrenal weight, cortical area, and zona fasciculata thickness, although these differences were not statistically significant.

Discussion

This study is the first direct evaluation of exogenous estrogen and soy isoflavone effects on adrenocortical parameters in a primate model. Results from this 5.5-yr randomized trial indicate that extended treatment with premenopausal OC and postmenopausal conjugated estrogens may have dissociative effects on adrenocortical function, suppressing adrenal androgens and elevating basal F levels. In contrast, soy isoflavones did not significantly alter adrenal androgen concentrations but resulted in diminished corticotropic regions of the adrenal cortex in animals without prior OC treatment. The ratio of F to DHEA-S increases with advanced age (53), and this dissociation in older adults is considered to be an underlying risk factor for various chronic diseases (54 –58). The present study suggests that exogenous estrogens in the form of OC or CEE may exacerbate this dissociation by diverting adrenocortical steroidogenesis toward F and away from androgen synthesis. This finding is consistent with several studies of women taking OC (19, 59 – 61) or meno-

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TABLE 4. Carryover effects of premenopausal OC treatment on postmenopausal androgens, cortisol, and adrenal morphologya

DHEA-S (␮g/dl) Androstenedione (ng/dl) Total testosterone (ng/dl) Cortisol (␮g/dl) Cortisol/DHEA-S ratio LH (ng/ml)

Adrenal weight (mg) Cortical area (mm2) Medullary area (mm2) Cortical thickness (␮m) Zona fasciculata (␮m) Zona reticularis (␮m) Pituitary weight (mg)

OC⫺ (n ⫽ 73)

OC⫹ (n ⫽ 75)

P values

4.87 (⫹0.52, ⫺0.47) 46.8 (⫹3.4, ⫺3.3) 4.9 (⫹0.6, ⫺0.5) 26.57 (⫹1.16, ⫺1.12) 5.01 (⫹0.48, ⫺0.42) 3.73 (⫹0.21, ⫺0.21) (n ⫽ 48)

5.40 (⫹0.55, ⫺0.51) 50.7 (⫹3.5, ⫺3.4) 6.6 (⫹0.8, ⫺0.7) 31.87 (⫹1.30, ⫺1.26) 5.45 (⫹0.53, ⫺0.47) 3.56 (⫹0.21, ⫺0.21) (n ⫽ 48)

0.46 0.48 0.07 0.0002b 0.51 0.57

(n ⫽ 86) 281 ⫾ 6 11.13 ⫾ 0.30 6.14 ⫾ 0.25 709 ⫾ 11 411 ⫾ 9 191 ⫾ 5 69.1 ⫾ 2

(n ⫽ 83) 277 ⫾ 6 11.05 ⫾ 0.33 6.35 ⫾ 0.28 697 ⫾ 12 413 ⫾ 9 184 ⫾ 6 72.7 ⫾ 2

0.64 0.98 0.53 0.49 0.96 0.24 0.31

All values were adjusted for postmenopausal treatment. Adrenal and pituitary measures were covaried by BW. For conversion to SI units (nmol/liter), divide by the following conversion factors: 0.039 for DHEA-S, 28.64 for androstenedione, 28.84 for total testosterone, and 3.625 for cortisol. a Values are means ⫾ SE using combined data from all postmenopausal groups; hormone values have been retransformed. b Postmenopausal*OC treatment interaction.

pausal estrogen therapy (18 –22) as well as epidemiologic data showing lower DHEA-S and higher basal F in older women compared with men (53). Contradictory findings in cell culture studies (62) and other studies of postmenopausal women may relate to differences in acute vs. long-term effects. All of the studies reporting elevated androgens (14 –17) or no effect (23–26) with estrogen treatment were 4 months or less in duration, whereas longer-term intervention studies tend to show significantly lower androgens (18, 21). Previously published androgen data from this study also show no significant CEE effects on androgens after 10 months of postmenopausal treatment (41). Estrogens have previously been shown to block androgen production in Leydig and ovarian thecal cells by inhibiting, via the ER, the expression and activity of the enzyme P450 17␣-hydroxylase/17,20-lyase (P45017␣) (63– 64), which regulates a key branchpoint in the flow of precursors into androgen and glucocorticoid pools. ER-␣ knockout mice also display elevated serum T and increased androgen biosynthesis and P45017␣ activity in Leydig cells (65). Given that ER-␣ may be highly expressed in adrenocortical cells, it is plausible that estrogens may exert a similar negative feedback on androgenic pathways in the adrenal cortex. It should be noted that in this study, we did not measure conjugated androgen metabolites such as androsterone-G and androstane-3␣, 17␤-diol, which may provide a more complete indication of the total androgen status (66). Previous studies in women support our findings that exogenous estrogens may increase baseline F when taken either as OC (67) or postmenopausal therapy (15–16, 19). Similar findings have also been described recently in ovariectomized monkeys receiving oral estradiol (68). In this study, OCtreated animals in the SPI⫺ and CEE groups had significantly higher F concentrations 27 months into the postmenopausal period. Previously reported data on a subset of animals from this study also show significantly higher basal F in the CEE animals and those with a history of OC treatment at month 12 of the postmenopausal period (43). These data suggest that a history of OC treatment may contribute

to resting hypercortisolemia in some women. However, Fbinding globulin, F clearance, and F response to ACTHchallenge were not evaluated in the present study, and further investigation is needed to determine whether this increase in basal F is clinically relevant. Soy isoflavones did not reduce circulating adrenal androgens or exert a significant estrogen-like effect on the pituitary gland. The lack of androgen suppression with dietary isoflavones in this study contrasts with previous studies in men showing an inverse association between soy intake and androgens (32–33). This antiandrogenic effect of soy in men is not a consistent finding (69 –70), however, and has only been evaluated in cross-sectional or short-term intervention studies. It should also be noted that the present study controlled for soy protein (i.e. all animals received soy protein); and so, potential androgen-lowering effects of soy protein were not evaluated. In premenopausal women, production and excretion of equol, a soy isoflavone metabolite, has been associated with significantly lower serum androgens and F (71). In the present study, no androgen suppression was observed even though equol was the primary isoflavone metabolite, comprising over half of the total serum isoflavones. The lack of an isoflavone effect on LH is consistent with previous studies in both premenopausal (71–72) and postmenopausal women (73–74). In this report, the SPI⫹ animals without prior OC treatment had significantly lower adrenal weight and zona fasciculata thickness, suggesting that soy isoflavones may reduce glucocorticoid activity. This finding is supported by two studies in H295R cells, both of which showed significant inhibition of cAMP-stimulated F production by the soy isoflavones genistein and daidzein at low micromolar concentrations (75–76). The enzyme 3␤-HSD II is highly expressed in the zona fascicularis and is considered a key enzyme in F production. Isoflavones have been shown in culture to inhibit 3␤-HSD II, resulting in a decreased F to DHEA-S ratio (76 –77). In this study, SPI⫹ treatment did not significantly lower the F to DHEA-S ratio, suggesting that adrenal 3␤-HSD II is not strongly inhibited by isoflavones in

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vivo. The observed interaction between SPI⫹ and prior OC treatment for adrenal weight was not present for other adrenal measures, and further study is needed to confirm this finding. Previous studies have suggested that prolonged social stress may reduce circulating androgens through either gonadal or adrenocortical suppression (78 –79). Data from this study suggest that chronic stress in females, as indicated by social subordination, does not significantly affect adrenal androgens or basal F in the premenopausal or postmenopausal state. We previously reported in this group of animals that premenopausal hypoestrogenism associated with subordinate status conferred increased risk for cardiovascular disease, and that this risk was ameliorated by OC (39) and CEE treatment (40). Findings presented herein suggest that any such effects of chronic stress on circulating sex steroids are specific to the premenopausal hypothalamic-pituitaryovary axis and do not necessarily involve the adrenal gland. Although the direct physiologic actions of adrenal androgens are not clearly defined, recent studies point to potential osteoprotective, antidepressive, antidiabetic, and immunoenhancing properties (80). This evidence has contributed to intense interest in the use of androgens as an alternative or complementary therapy to estrogens (11). In this report, we identify the adrenal gland as an important target of exogenous estrogens and suggest that extended use of oral postmenopausal estrogen therapy may contribute to an androgen-deficient state in healthy women. These findings support a physiologic rationale for the addition of an androgenic component to current postmenopausal estrogen therapies. Acknowledgments The authors thank Jean Gardin, Dianna Swaim, Tim Vest, Matt Dwyer, and Dewayne Cairnes for their technical contributions; Prof. Thomas B. Clarkson for guidance and assistance; and Prof. Stephen Barnes for isoflavone measurements. For hormone measurements, we thank the staff of the Endocrine Core Laboratory at Yerkes Primate Research Center and the Clinical Chemistry and Endocrinology Laboratory of the Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine. Soy products were generously provided by Solae. Received October 3, 2003. Accepted February 3, 2004. Address all correspondence and requests for reprints to: Charles E. Wood, D.V.M., Comparative Medicine Clinical Research Center, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157-1040. E-mail: chwood@wfubmc. edu. This work was supported, in part, by Program Project Grant HL45666 from the NIH/National Heart, Lung, and Blood Institute, Bethesda, Maryland (to J.R.K.), by the NIH/National Center for Complementary and Alternative Medicine R01-AT00639 (to J.M.C.), and by the NIH/National Center for Research Resources T32 RR 07009 (to C.E.W.).

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