Effects of Soybean Glyceollins and Estradiol in Postmenopausal Female Monkeys Charles E. Wood, Thomas B. Clarkson, Susan E. Appt, Adrian A. Franke, Stephen M. Boue, Matthew E. Burow, Thomas McCoy, and J. Mark Cline Abstract: Glyceollins are a novel class of soybean phyto- alexins with potential cancer-protective antiestrogenic ef- fects. The purpose of this study was to evaluate the estro- gen-antagonist effects of glyceollin-enriched soy protein on biomarkers for breast cancer risk. Thirty female postmen- opausal cynomolgus macaques were randomized to one of three dietary treatments for 3 wk: 1) estradiol (E2, 1 mg/day) + casein/lactalbumin (control); 2) E2 + soy protein isolate (SPI) containing 194 mg/day isoflavonoids; and 3) E2 + glyceollin-enriched soy protein (GLY) containing 189 mg/ day isoflavonoids + 134 mg/day glyceollins. Doses are ex- pressed in calorically scaled human equivalents. Mean se- rum glyceollin concentrations at 4 h postfeeding were 134.2 ± 34.6 nmol/L in the GLY group and negligible in the SPI group (P = 0.0007). Breast proliferation was significantly in- creased in the control group (+237%, P = 0.01) but not in the SPI group (+198%, P = 0.08) or GLY group (+36%, P = 0.18). Gene expression of trefoil factor 1 and progesterone receptor, two markers of estrogen receptor activity in breast epithelium, were also significantly higher in the control (P < 0.05 for both) but not in the GLY group. These preliminary findings suggest that soybean glyceollins are natural com- pounds with potential estrogen-modulating properties in the breast. Introduction Estrogen exposure is an important determinant of breast cancer risk (1,2). Prospective epidemiologic studies demon- strate a strong association between endogenous estrogens and breast cancer risk (3), and recent clinical trials indicate that pharmacologic antiestrogenic agents such as raloxifene may prevent a significant proportion of breast cancer cases (4). Dietary factors that reduce estrogen exposure may thus have an important role in breast cancer prevention. Isoflavonoids are a class of natural phytochemicals with structural similarities to mammalian estrogens. The major source of dietary isoflavonoids is soy (5,6), which is rich in the glycosylated forms of genistein and daidzein. Regular con- sumption of soy isoflavonoids has been associated with re- duced risk of breast and uterine cancer (7–9), particularly in women with higher estradiol exposure (10,11). Soy isofla- vonoids competitively bind estrogen receptors and, in the pres- ence of estradiol, may attenuate estrogenic effects (12–14). This evidence suggests that any potential chemopreventive benefits of dietary soy may relate to the estrogen-antagonist properties of its component phytoestrogens. Nevertheless, the role of specific isoflavonoids and their derivatives in modulat- ing estrogen effects remains poorly understood. Glyceollins are a class of phytoalexins produced in soy- beans under the influence of stressors such as trauma or in- fection (15). Three major forms of glyceollins have been identified (I–III), all of which are derived from the parent isoflavone daidzein through a series of pterocarpan interme- diates (Figure 1). Compared with genistein and daidzein, pu- rified glyceollins show greater inhibition of estradiol effects on proliferation and estrogen receptor signaling in breast cancer cells (16). Glyceollins also have enhanced antago- nism toward the estrogen receptor alpha relative to beta and lack any of the estrogen agonist activity of genistein and daidzein seen in low-estrogen conditions (16). These find- ings suggest that soy protein enriched in glyceollins may have distinct estrogen-modulating properties compared with standard soy protein. To test this idea, we used a post- menopausal primate model to evaluate the short-term effects of glyceollin-enriched soy protein and standard soy protein isolate in combination with estrogen. NUTRITION AND CANCER, 56(1), 74–81 Copyright © 2006, Lawrence Erlbaum Associates, Inc. C.E. Wood, T. B. Clarkson, S. E. Appt, and J. M. Cline are affiliated with the Department of Pathology/Section on Comparative Medicine, Wake Forest Uni- versity School of Medicine, Winston-Salem, NC 27157. A. A. Franke is affiliated with the Cancer Research Center of Hawaii, Honolulu, HI. S. M. Boue is affili- ated with the Southern Regional Research Center, United States Department of Agriculture, New Orleans, LA. M. E. Burow is affiliated with the Tulane Univer- sity School of Medicine, New Orleans, LA. T. McCoy is affiliated with the Department of Biostatistical Sciences, Wake Forest University School of Medicine, Winston-Salem, NC 27157.