Altered Mammary Gland Differentiation and Progesterone Receptor Expression in Rats Fed Soy and Whey Proteins

J. Craig Rowlands1, Reza Hakkak, Martin J. J. Ronis and Thomas M. Badger

Arkansas Children’s Nutrition Center and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202

Received June 7, 2002; accepted August 1, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There are suspected links between an animal’s diet, differentiation status of a target tissue, and sensitivity to chemically induced cancer. We have demonstrated that rats fed AIN93G diets made with soy protein isolate (SPI) or whey protein hydrolysate (WPH) had a lower incidence of 7,12-dimethylbenz(a)anthracene (DMBA)-induced adenocarcinoma than rats fed the same diet made with casein (CAS). The current study was conducted to determine the differentiation status of the mammary glands during development. Offspring of rats (n = 5–10/group) were fed diets made with SPI, WPH, or CAS throughout life (beginning on gestation day 4) and were sacrificed on postnatal day (PND) 21, PND 33, PND 50 or on metaestrous between PND 48 and PND 51. There were no significant differences between the numbers of mammary terminal end buds (TEBs) or lobuloalveoli (LOB) between any of the diets groups at PND 21 or PND 33, but at PND 50 there was an 75% decrease in the mean numbers of TEBs/mm2 in the SPI- or WPH-fed rats, compared with the CAS-fed rats (p = 0.09 and p = 0.06, respectively). In rats sacrificed in metaestrous, there were no significant differences in the proliferation index (PI) in the TEBs or LOB between any of the diet groups. In metaestrous rats, there were twice as many cells expressing estrogen receptor ß (ERß; ~60%) compared with estrogen receptor {alpha} (ER{alpha}; ~30%) in the LOB and 1.5 times more ERß (~60%) compared with estrogen receptor {alpha} (ER{alpha}, ~40%) in the TEBs. There were no diet-dependent differences in expression of ER{alpha} and ERß. Similarly, there were no differences between the diet groups in progesterone receptor (PR) expressing LOB cells. However, in the TEBs there was a diet-dependent difference in PR positive cells with a 34% increase (p < 0.05) in the SPI-fed rats and a 38% increase (p < 0.05) in the WPH-fed rats compared with the CAS-fed rats. These results show that the type of dietary protein alters the phenotype of mammary epithelia in the TEBs. The SPI- and WPH-dependent changes in mammary differentiation may contribute to the reduced sensitivity to DMBA-induced mammary cancer in rats fed these proteins.

Key Words: rats; soy protein isolate; whey protein hydrolysate; casein; diet; protein; terminal end bud; lobuloalveoli; progesterone receptor; estrogen receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The concept that components of the diet can modulate carcinogenesis is well established. Results from epidemiological studies demonstrate that the Western diet is a major factor associated with the high incidence of "Western diseases," including the major hormone-dependent cancers such as breast cancer (Rose, 1986Go; Trowell and Birkitt, 1981Go). Low breast cancer incidence in Asian women and in Japanese women in Hawaii consuming a traditional Japanese diet has been linked to a high intake of soybean products (Adlercreutz et al., 1991Go; Chu et al., 1995Go; Lee et al., 1991Go; Messina, 1995Go; Messina and Barnes, 1991Go; Muir et al., 1987Go; Nomura et al., 1978Go; Setchell et al., 1984Go). The breast cancer preventing effects of soy have been reinforced by studies demonstrating that soybean diets reduced chemically induced mammary gland tumors (Baggott et al., 1990Go). Similar results have been reported for radiation-induced mammary tumors (Troll et al., 1980Go). Experimental studies have also reported that the consumption of bovine whey milk proteins may also afford cancer prevention (Bounous et al., 1991Go). In addition, dietary whey protein hydrolysate (WPH) and soy protein isolate (SPI) administered throughout development prevent 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary cancers in adult female Sprague-Dawley rats (Hakkak et al., 2000Go). The mechanisms by which soy or whey act to reduce cancer incidence are not known, but soy contains high levels of isoflavones such as genistin that can be estrogenic in their aglycone form (genistein), and some studies have reported that genistein can stimulate mammary gland differentiation that is resistant to cancer (Murrill et al., 1996Go). We are not aware of any reported studies on the effects of whey on mammary gland development.

Mammary gland development in prepubertal mammals is under the regulation of estrogens that stimulate formation of ductal branches that end in highly proliferative terminal end buds (TEBs; Russo and Russo, 1994Go, 1995Go). Maximum TEB density in the female rat occurs at PND 21 and decreases steadily as a result of the actions of increased progesterone and estrogens from the onset of puberty. The TEBs differentiate into less proliferative alveolar buds (ABs) and ultimately into the least proliferative lobuloalveoli (LOB; Russo and Russo, 1978aGo, 1996Go). Studies have shown that the highest numbers of chemically induced mammary tumors occurs in rats between PND 40 to PND 60 when there is a high density of TEBs that are rapidly differentiating into ABs and LOB, a process that requires increased cell proliferation (Russo and Russo, 1996Go). In addition, the incidence of mammary carcinomas is positively correlated with the number of TEBs in the mammary gland of the young virgin rat at the time of carcinogen exposure (Russo and Russo, 1978bGo). Therefore, increased differentiation from TEBs to ABs and LOB would result in a net decrease in proliferating cells and might decrease the sensitivity to carcinogens.

The objective of these experiments was to investigate the effects of dietary SPI and WPH on the development of the mammary gland. To this end, we have measured the mammary gland area, densities of mammary gland structures, and several markers important in mammary differentiation including: (1) cell proliferation index (PI), (2) estrogen receptor (ER{alpha} and ERß) expression, and (3) progesterone receptor (PR) expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal treatments.
All animals were housed in an AAALAC approved animal facility with a 12-h light cycle and constant humidity. Studies were approved by the Institutional Animal Care and Use Committee.

Experiment 1.
Time impregnated (gestation day 4; GD 4) female Sprague-Dawley rats were purchased from Harlan Industries (Indianapolis, IN). Rats were randomly assigned to three groups and fed one of three isocaloric semipurified diets made according to the AIN-93G diet formula (Reeves et al., 1993Go), except that corn oil replaced soy oil and the protein source was either casein (CAS), whey protein hydrolysate (WPH; New Zealand Milk Products, Santa Rosa, CA) or soy protein isolate (SPI; a gift from Protein Technologies International Inc., St. Louis, MO). The protein content of each diet was the same. Amino acids were added to each diet to equalize the essential amino acids among the diets. The offspring were weaned to the same diet as their mothers and were fed the same diets throughout the study. At PNDs 21, 33, and 50, 10 female pups (one per litter) from each diet group (n = 10) were sacrificed and their abdominal mammary gland pair no. 4 removed for whole mounts and paraffin embedding.

Experiment 2.
Time impregnated (GD 4) female Sprague-Dawley rats were purchased from Harlan Industries (Indianapolis, IN). Rats were randomly assigned to three groups and fed either CAS-, SPI-, or WPH-diets prepared as described in Experiment 1 above. The offspring, randomly selected from 5–7 litters, were weaned to the same diet as their mothers and were fed the same diets throughout the study starting at PND 32 rats were checked daily at 0700 h for estrous cycle stage. Starting at PND 47, rats were orally gavaged with sesame oil, sacrificed 24 h later at metaestrous and their abdominal mammary gland pair no. 4 removed for paraffin embedding. The median age for each group was PND 48, 48, and 51 for CAS, SPI, and WPH, respectively.

Mammary structure densities and area.
Mammary whole mounts were prepared using the abdominal mammary gland no. 4. Glands were removed and spread on a microscope slide and placed in neutral buffered formalin overnight, and then defatted in acetone overnight and hydrated in 70% alcohol for 30 min followed by water for 15 min. Epithelium was stained in alum carmine overnight. After staining, the mammary glands were dehydrated by incubating in four sequential steps of alcohol (35–95%, for 30 min each and 100% alcohol overnight). Glands were cleared in xylene for 24 h and compressed by sandwiching the gland between a second glass slide held together by paper clips for 24 h in xylene. Glands were allowed to decompress for 24 h and coverslipped using Permount (Fisher Scientific). Mammary gland area was measured using a MCID video microscopy system (Imaging Research, Inc., St. Catharines, Ontario, Canada). Measurements of the area occupied by mammary epithelium into the fat pad from the whole gland (P21) or the ductal extension of the mammary gland into the fat pad between the superior lymph node and the mammary branch border (PND 33, PND 50) were quantified on the digitized images using MCID5+ software (Imaging Research, Inc., St. Catharines, Ontario, Canada ). Mammary structures were counted from the distal portions of the mammary gland whole mounts examined under a stereomicroscope at 3X magnification. Whole mounts were evaluated for TEB, AB, and LOB structures according to the criteria of Russo (Russo et al., 1990Go). The numbers of TEB, AB, or LOB were determined in 10 randomly chosen 1 mm2 areas in the distal portion of the mammary gland, 1 mm from the edge. This is the area that normally contains most of the terminal duct structures in the gland.

General histology.
The abdominal mammary gland no. 4 was removed and fixed in 10% neutral buffered formalin for 24 h and processed through graded concentrations of alcohols and toluene to paraffin. Paraffin-embedded mammary gland was cut into 5-µm-thick sections, placed on glass microscope slides, and dried overnight at 40°C. Slides were deparaffinzed in xylene, rehydrated, prior to immunnohistochemical assays.

Cell proliferation.
Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. For antigen retrieval, sections were incubated in 0.1% calcium chloride for 15 min at 37°C in a water bath. Sections were blocked for 20 min in Cas Block (Zymed) followed by incubations at room temp with (1) anti-PCNA primary antibody (Daco) for 1 h, (2) biotinylated goat anti-mouse IgG secondary antibody (Rockland) for 30 min, (3) peroxidase conjugated streptavidin (Jackson) for 30 min. Color was developed with the chromagen 3,3‘-diaminobenzidine tetrahydrochloride (DAB; Daco) and the cells counterstained with Mayer’s hematoxylin. The cells in which the nuclei stained brown with DAB were scored as being in S-phase, while those that stained blue with hematoxylin were scored as not in the S-phase.

ER{alpha}, ERß, and PR.
Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. For antigen retrieval, sections were incubated in 10 mM citric acid buffer (pH 6.0) for 40 min at 95°C in a water bath. Sections were blocked for 20 min in Cas Block (Zymed) followed by incubations at room temperature with: (1) anti-PR primary antibody (no. A0098; Daco), anti-ER{alpha} primary antibody (MC-20, Santa Cruz), anti- ERß primary antibody (no. 06–629), Upstate Biotechnology (Lake Placid, NY), for 16.5 h; (2) biotinylated goat anti-rabbit IgG secondary antibody (Roche) for 30 min; (3) peroxidase conjugated streptavidin (Jackson) for 30 min. Color was developed with the chromagen 3,3‘-diaminobenzidine tetrahydrochloride (DAB; Daco) and the cells counterstained with Mayer’s hematoxylin. Cells nuclei staining brown with DAB were scored as expressing ER or PR, while those staining blue with hematoxylin were scored as not expressing receptor. Control reactions in which the primary antibody was omitted served as negative controls and did not stain brown (not shown).

Statistics.
Measurement of the percent PI, ER{alpha}, ERß, and PR expressing cells were determined in one female pup/litter from 5–7 litters with the litter used as the unit of measure to account for litter effects. The ratio of brown cells to total cells (brown plus blue) was determined for 10 ductile structures per section. The structures were both TEBs and LOB (Russo and Russo, 1978aGo) that were chosen at random near a lymph node in order to maintain regional congruity between glands. The means were determined for the 5–7 structures and used to calculate the means for the 5–7 glands/group (i.e., means of the means). Statistical analysis was measured between the means using ANOVA and Student-Newman-Keuls Multiple Comparison where appropriate with significance set at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammary Gland Morphology
In all of the diet groups, the density of TEBs decreased with age and there was a corresponding increase in the density of the more differentiated LOB and there was no change in AB densities (Table 1Go, Fig. 1Go). However, there was no statistical difference between the diet groups in mammary structure densities, even though for the terminal end buds, there was a four-fold decrease in densities in the SPI- (p = 0.09) or WPH-fed (p = 0.06) rats, compared with the CAS-fed rats. No differences in the mean area of the mammary glands were observed in 21- or 33-day-old rats between diet groups, but at day 50, the mammary glands in the SPI-fed rats were 38 or 36% larger (p < 0.05) than in the CAS- or WPH-fed rats, respectively. There were no differences between mammary gland areas in the CAS- or WPH-fed rats at day 50.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Mammary Gland Area and Structure Densities from 21-, 33-, and 50-Day-Old Rats Fed CAS, SPI, or WPH Diets
 


View larger version (169K):
[in this window]
[in a new window]
 
FIG. 1. Mammary whole mount from a CAS-fed rat showing a terminal end bud (TEB), alveolar bud (AB), and lobuloalveoli (LOB). Original magnification x3.

 
Mammary Gland Cell Proliferation and Hormone Receptor Expression
The effects of lifetime feeding of SPI-, WPH- or CAS containing diets on mammary cell proliferation, ER{alpha}, ERß, and PR expression in mammary gland terminal end buds and lobuloalveoli were measured (Table 2Go, Fig. 2Go). There were no differences between diet groups in the number of proliferating mammary epithelial cells or in cells expressing either ER{alpha} or ERß. In the TEBs, PR expressing cells were increased by 34% (p < 0.05) in the SPI-fed rats and 38% (p < 0.05) in the WPH-fed rats compared with the CAS-fed rats. There was no difference in PR expression between diet groups in LOB cells.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Proliferating, Estrogen Receptors (ER{alpha} and ERß) and Progesterone Receptor (PR) Positive Mammary Terminal End Bud (TEB) or Lobuloalveoli (LOB) Cells in Mammary Glands from CAS-, SPI-, or WPH-Fed Rats at Age 48–51 Days
 


View larger version (98K):
[in this window]
[in a new window]
 
FIG. 2. Immunohistochemical detection of proliferating TEB cells (PCNA; A, B, C), LOB expressing ER{alpha} (D, E, F) or ERß (G, H, I), and TEBs expressing PR (J, K, L) protein in rats fed CAS-diets (A, D, G, J), SPI-diets (B, E, H, K), or WPH-diets (C, F, I, L). Immunohistochemical staining was performed as described in the Materials and Methods. Original magnification x200.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously reported that in female rats orally gavaged with DMBA at PND 50, there was a significant reduction in mammary tumors in SPI- or WPH-fed rats, compared with CAS-fed rats (Hakkak et al., 2000Go). We studied mammary gland morphology at age 50 days because it is important to understand the developmental status at the time when DMBA was administered. It is also important to determine which cellular systems are affected by these diets in order to ascertain the mechanisms underlying dietary protection against breast cancer. The decreased TEB density coupled with the increased expression of PR in rats fed SPI- or WPH- diets suggests these dietary factors are important stimulators of mammary gland differentiation. In PR gene knockout experiments there was no TEB to LOB differentiation (Lydon et al., 1999Go) implicating the PR as an important regulator for the conversion of the undifferentiated TEB into the more differentiated LOB. Increased numbers of cells expressing PR in the TEBs of rats fed SPI- or WPH-diets suggests that these structures may respond with increased sensitivity to the mammogenic effects of progesterone (Shyamala, 1999Go).

The differentiation status of the mammary gland has been proposed to affect tumor incidence since it is the undifferentiated highly proliferative TEBs that are the target of DMBA-induced tumors (Huggins and Yang, 1962Go; Russo and Russo, 1996Go); thus, lower TEB densities may result in lower tumor incidence. The reduced TEB density in the SPI- and WPH-fed rats suggests that this would be cancer protective. These effects are similar to those of rats fed the soy isoflavone, genistein (Lamartiniere et al., 1995Go). Female rats administered genistein during the prepubertal or neonatal stage had reduced TEB densities and treatment of these rats with DMBA at PND 50 resulted in fewer tumors and increased latency to tumor development (Lamartiniere et al., 1995Go; Murrill et al., 1996Go). In the present study, there was a four-fold decrease in mean TEB density in both SPI and WPH groups, but this did not reach statistical significance. Nonetheless, it suggests that this may be a contributing factor in lower incidence and multiplicity. Interestingly, the increased size of the mammary gland in the SPI-fed rats means that the absolute number of TEBs in these rats is not significantly different than in the CAS-fed rats; whereas the absolute number of TEBs in the WPH-fed rats is lower than in the CAS-fed rats, thus the reduced cancers in the SPI-fed rats appears to be the result of more than just SPI-induced increased gland differentiation.

In the SPI- or WPH-fed rats compared with CAS-fed rats there was reduced DMBA-induced expression of mammary CYP1A1, CYP1A2, and CYP1B1 mRNA and protein (Rowlands et al., 2001Go). CYP1A1 and CYP1B1 are capable of activation of chemically diverse procarcinogens including DMBA (Shimada et al., 1996Go) and CYP1B1 of 4-hydroxylation of estradiol yielding a putative endogenous mutagen (Hayes et al., 1996Go; Liehr et al., 1995Go; Spink et al., 1994Go). Thus, the reduction of CYP1A1 and CYP1B1 expression in the SPI- and WPH-fed rats is consistent with reduced mammary cancer incidence measured in rats fed these diets. Moreover, the reduced CYP1A1 and CYP1B1 expression in the face of increased mammary differentiation may explain the greater reduction of DMBA-induced mammary tumors in the WPH-fed rats compared with the SPI-fed rats where there is functionally only a reduction of DMBA-induced CYP1A1 and CYP1B1 expression measured.

Increases in mammary gland size and PR expression are consistent with an estrogenic activity in the SPI-fed rats. Estrogen is a well-characterized inducer of PR expression (Mohla et al., 1981Go) and estrogens have been reported to induce the growth and area of rat mammary glands (Fendrick et al., 1998Go; Russo and Russo, 1996Go). Numerous estrogenic chemicals have been identified in soy (phytoestrogens) including genistein, daidzein, and sapogenol A (Adlercreutz and Mazur, 1997Go; Rowlands et al., in pressGo). The increased PR expression in the WPH-fed rats is most likely not an estrogenic effect since WPH has not been shown to contain estrogens and there was no observed change in estrogen target tissues such as an increase in mammary gland size or uterine hypertrophy (Badger et al., 2001Go). Instead, the increased PR in the WPH-fed rats may have resulted from the actions of peptides generated in processing, or growth factors found in whey. For example, whey contains IGF-1 (Grosvenor et al., 1993Go; Guimont et al., 1997Go) and PR was shown to be induced by IGF-I in vitro (Cho et al., 1994Go).

In summary, the results reported here have revealed that in rats fed SPI- or WPH-based diets there is increased PR expression and mammary gland differentiation compared with rats fed a CAS-diets. The mechanisms for the SPI- and WPH-induced changes are not known, but for SPI they may in part be due to the phytoestrogens since there were also increases in mammary gland size and estrogens can cause this effect. The WPH-induced mammary gland differentiation may be due to the mammogenic peptides such as IGF-1 in whey or may be due to peptides generated from processing or digestion. The increased differentiation is a potential mechanism for the decreased mammary cancer measured in SPI- or WPH-fed rats compared with CAS-fed rats.


    ACKNOWLEDGMENTS
 
We thank our laboratory colleagues Trudy Brewer, Renée Till, Ying Chen, Cynthia Mercado, and Terry Fletcher for their valuable assistance. Funding has been provided by the U.S. Department of Agriculture-Agricultural Research Service CRIS6251-51000-002-02S.


    NOTES
 
Portions of this data were presented at Experimental Biology, March 2001, Orlando, FL.

1 To whom correspondence should be addressed at present address: Burdock Group, 780 US Highway One, Suite 300, Vero Beach, FL 32962. Fax: (772) 562-3908. E-mail: crowlands{at}burdockgroup.com Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adlercreutz, H., Honjo, H., Higashi, A., Fotsis, T., Hamalainen, E., Hasegawa, T., and Okada, H. (1991). Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am. J. Clin. Nutr. 54, 1093–1100.[Abstract]

Adlercreutz, H., and Mazur, W. (1997). Phyto-oestrogens and Western diseases. Ann. Med. 29, 95–120.[ISI][Medline]

Badger, T. M., Ronis, M. J., and Hakkak, R. (2001). Developmental effects and health aspects of soy protein isolate, casein, and whey in male and female rats. Int. J. Toxicol. 20, 165–174.[ISI][Medline]

Baggott, J. E., Ha, T., Vaughn, W. H., Juliana, M. M., Hardin, J. M., and Grubbs, C. J. (1990). Effect of miso (Japanese soybean paste) and NaCl on DMBA-induced rat mammary tumors. Nutr. Cancer 14, 103–109.[ISI][Medline]

Bounous, G., Batist, G., and Gold, P. (1991). Whey proteins in cancer prevention. Cancer Lett. 57, 91–94.[ISI][Medline]

Cho, H., Aronica, S. M., and Katzenellenbogen, B. S. (1994). Regulation of progesterone receptor gene expression in MCF-7 breast cancer cells: A comparison of the effects of cyclic adenosine 3‘,5‘-monophosphate, estradiol, insulin-like growth factor-I, and serum factors. Endocrinology 134, 658–664.[Abstract]

Chu, B., Anantharam, V., and Treistman, S. N. (1995). Ethanol inhibition of recombinant heteromeric NMDA channels in the presence and absence of modulators. J. Neurochem. 65, 140–148.[ISI][Medline]

Fendrick, J. L., Raafat, A. M., and Haslam, S. Z. (1998). Mammary gland growth and development from the postnatal period to postmenopause: Ovarian steroid receptor ontogeny and regulation in the mouse. J. Mammary Gland Biol. Neoplasia 3, 7–22.[ISI][Medline]

Grosvenor, C. E., Picciano, M. F., and Baumrucker, C. R. (1993). Hormones and growth factors in milk. Endocr. Rev. 14, 710–728.[Abstract]

Guimont, C., Marchall, E., Girardet, J. M., and Linden, G. (1997). Biologically active factors in bovine milk and dairy byproducts: Influence on cell culture. Crit. Rev. Food Sci. Nutr. 37, 393–410.[ISI][Medline]

Hakkak, R., Korourian, S., Shelnutt, S. R., Lensing, S., Ronis, M. J., and Badger, T. M. (2000). Diets containing whey proteins or soy protein isolate protect against 7,12-dimethylbenz(a)anthracene-induced mammary tumors in female rats. Cancer Epidemiol. Biomarkers Prev. 9, 113–117.[Abstract/Free Full Text]

Hayes, C. L., Spink, D. C., Spink, B. C., Cao, J. Q., Walker, N. J., and Sutter, T. R. (1996). 17ß-estradiol hydroxylation catalyzed by human cytochrome P450 1B1. Proc. Natl. Acad. Sci. U.S.A. 93, 9776–9781.[Abstract/Free Full Text]

Huggins, C., and Yang, N. C. (1962). Induction and instinction of mammary cancer. Science 137, 257–262.[ISI][Medline]

Lamartiniere, C. A., Moore, J., Holland, M., and Barnes, S. (1995). Neonatal genistein chemoprevents mammary cancer. Proc. Soc. Exp. Biol. Med. 208, 120–123.[Abstract]

Lee, H. P., Gourley, L., Duffy, S. W., Esteve, J., Lee, J., and Day, N. E. (1991). Dietary effects on breast-cancer risk in Singapore. Lancet 337, 1197–1200.[ISI][Medline]

Liehr, J. G., Ricci, M. J., Jefcoate, C. R., Hannigan, E. V., Hokanson, J. A., and Zhu, B. T. (1995). 4-Hydroxylation of estradiol by human uterine myometrium and myoma microsomes: Implications for the mechanism of uterine tumorigenesis. Proc. Natl. Acad. Sci. U.S.A. 92, 9220–9224.[Abstract]

Lydon, J. P., Ge, G., Kittrell, F. S., Medina, D., and O’Malley, B. W. (1999). Murine mammary gland carcinogenesis is critically dependent on progesterone receptor function. Cancer Res. 59, 4276–4284.[Abstract/Free Full Text]

Messina, M. (1995). Isoflavone intakes by Japanese were overestimated. Am. J. Clin. Nutr. 62, 645.

Messina, M., and Barnes, S. (1991). The role of soy products in reducing risk of cancer. J. Natl. Cancer Inst. 83, 541–546.[ISI][Medline]

Mohla, S., Clem-Jackson, N., and Hunter, J. B. (1981). Estrogen receptors and estrogen-induced gene expression in the rat mammary glands and uteri during pregnancy and lactation: Changes in progesterone receptor and RNA polymerase activity. J. Steroid Biochem. 14, 501–508.[ISI][Medline]

Muir, C., Waterhouse, J., Powell, M. T., and Whelan, S. (1987). Cancer incidence in five continents. International Agency for Research on Cancer, Lyon.

Murrill, W. B., Brown, N. M., Zhang, J. X., Manzolillo, P. A., Barnes, S., and Lamartiniere, C. A. (1996). Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis 17, 1451–1457.[Abstract]

Nomura, A., Henderson, B. E., and Lee, J. (1978). Breast cancer and diet among the Japanese in Hawaii. Am. J. Clin. Nutr. 31, 2020–2025.[Abstract]

Reeves, P. G., Nielsen, F. H., and Fahey, G. C., Jr. (1993). AIN-93 purified diets for laboratory rodents: Final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123, 1939–1951.[ISI][Medline]

Rose, D. P. (1986). Dietary factors and breast cancer. Cancer Surv. 5, 671–687.[ISI][Medline]

Rowlands, J. C., Berhow, M. A., and Badger, T. M. (in press). Estrogenic and antiproliferative properties of soy sapogenols in human breast cancer cells in vitro. Food Chem. Toxicol.

Rowlands, J. C., He, L., Hakkak, R., Ronis, M. J., and Badger, T. M. (2001). Soy and whey proteins down-regulate DMBA-induced liver and mammary gland CYP1 expression in female rats. J. Nutr. 131, 3281–3287.[Abstract/Free Full Text]

Russo, I. H., and Russo, J. (1978a). Developmental stage of the rat mammary gland as determinant of its susceptibility to 7,12-dimethylbenz[a]anthracene. J. Natl. Cancer Inst. 61, 1439–1449.[ISI][Medline]

Russo, I. H., and Russo, J. (1996). Mammary gland neoplasia in long-term rodent studies. Environ. Health Perspect. 104, 938–967.[ISI][Medline]

Russo, J., Gusterson, B. A., Rogers, A. E., Russo, I. H., Wellings, S. R., and van Zwieten, M. J. (1990). Comparative study of human and rat mammary tumorigenesis. Lab. Invest. 62, 244–278.[ISI][Medline]

Russo, J., and Russo, I. H. (1978b). DNA labeling index and structure of the rat mammary gland as determinants of its susceptibility to carcinogenesis. J. Natl. Cancer Inst. 61, 1451–1459.[ISI][Medline]

Russo, J., and Russo, I. H. (1994). Toward a physiological approach to breast cancer prevention. Cancer Epidemiol. Biomarkers Prev. 3, 353–364.[Abstract]

Russo, J., and Russo, I. H. (1995). Hormonally induced differentiation: A novel approach to breast cancer prevention. J. Cell. Biochem. Suppl. 22, 58–64.[Medline]

Setchell, K. D., Borriello, S. P., Hulme, P., Kirk, D. N., and Axelson, M. (1984). Nonsteroidal estrogens of dietary origin: Possible roles in hormone-dependent disease. Am. J. Clin. Nutr. 40, 569–578.[Abstract]

Shimada, T., Hayes, C. L., Yamazaki, H., Amin, S., Hecht, S. S., Guengerich, F. P., and Sutter, T. R. (1996). Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1. Cancer Res. 56, 2979–2984.[Abstract]

Shyamala, G. (1999). Progesterone signaling and mammary gland morphogenesis. J. Mammary Gland Biol. Neoplasia 4, 89–104.[ISI][Medline]

Spink, D. C., Hayes, C. L., Young, N. R., Christou, M., Sutter, T. R., Jefcoate, C. R., and Gierthy, J. F. (1994). The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a novel 17ß-estradiol 4-hydroxylase. J. Steroid Biochem. Mol. Biol. 51, 251–258.[ISI][Medline]

Troll, W., Wiesner, R., Shellabarger, C. J., Holtzman, S., and Stone, J. P. (1980). Soybean diet lowers breast tumor incidence in irradiated rats. Carcinogenesis 1, 469–472.[Abstract]

Trowell, H. C. and Birkitt, D. P. (1981). Western diseases: Their emergence and prevention. Edward Arnold, London.