Arkansas Childrens Nutrition Center and Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202
Received June 7, 2002; accepted August 1, 2002
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ABSTRACT |
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Key Words: rats; soy protein isolate; whey protein hydrolysate; casein; diet; protein; terminal end bud; lobuloalveoli; progesterone receptor; estrogen receptor.
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INTRODUCTION |
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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, 1994, 1995
). 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, 1978a
, 1996
). 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, 1996
). 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, 1978b
). 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 and ERß) expression, and (3) progesterone receptor (PR) expression.
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MATERIALS AND METHODS |
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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., 1993), 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 57 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 (3595%, 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., 1990). 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 Mayers 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, 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 primary antibody (MC-20, Santa Cruz), anti- ERß primary antibody (no. 06629), 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 Mayers 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, ERß, and PR expressing cells were determined in one female pup/litter from 57 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, 1978a
) that were chosen at random near a lymph node in order to maintain regional congruity between glands. The means were determined for the 57 structures and used to calculate the means for the 57 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.
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RESULTS |
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DISCUSSION |
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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, 1962; Russo and Russo, 1996
); 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., 1995
). 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., 1995
; Murrill et al., 1996
). 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., 2001). CYP1A1 and CYP1B1 are capable of activation of chemically diverse procarcinogens including DMBA (Shimada et al., 1996
) and CYP1B1 of 4-hydroxylation of estradiol yielding a putative endogenous mutagen (Hayes et al., 1996
; Liehr et al., 1995
; Spink et al., 1994
). 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., 1981) and estrogens have been reported to induce the growth and area of rat mammary glands (Fendrick et al., 1998
; Russo and Russo, 1996
). Numerous estrogenic chemicals have been identified in soy (phytoestrogens) including genistein, daidzein, and sapogenol A (Adlercreutz and Mazur, 1997
; Rowlands et al., in press
). 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., 2001
). 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., 1993
; Guimont et al., 1997
) and PR was shown to be induced by IGF-I in vitro (Cho et al., 1994
).
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.
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ACKNOWLEDGMENTS |
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NOTES |
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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
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