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Short Communication |
Arkansas Children’s Nutrition Center and Departments of Pediatrics [R. H., S. R. S., S. L., M. J. J. R., T. M. B.] and Pathology [S. K.], University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202
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Abstract |
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19 rats/group) were p.o. gavaged with 80 mg/kg
7,12-dimethylbenz(a)anthracene, and mammary glands were
evaluated when 100% of the casein-fed group developed at least one
palpatable tumor. Rats grew well on all three diets, but casein-fed
rats gained slightly more body weight than soy- or whey-fed rats
(P < 0.05). Vaginal opening occurred 1 day earlier
in soy-fed rats than in casein- or whey-fed rats, but no other
differences in reproductive and developmental parameters were observed
between groups. When 50% of the casein-fed rats had at least one
mammary tumor, lower tumor incidences (24–34%) were observed in the
soy-fed (P < 0.009) and whey-fed groups
(P < 0.001). When 100% of the casein-fed rats had
at least one tumor, soy-fed rats had a lower tumor incidence (77%) in
experiment B (P < 0.002), but not in experiment A
(P < 0.12), and there were no differences in tumor
multiplicity. Whey-fed rats had lower mammary tumor incidence
(54–62%; P < 0.002) and multiplicity
(P < 0.007) than casein-fed rats in both
experiments. Our results indicate that diets rich in soy reduce the
incidence of chemically induced mammary tumors by approximately 20%.
Furthermore, whey appears to be at least twice as effective as soy in
reducing both tumor incidence and multiplicity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was conducted to determine the possible preventive effects of diets containing soy protein isolate or bovine whey proteins on DMBA3 -induced breast tumors in rats. The experiments were designed to determine the effects of long-term consumption of these proteins in diets that were formulated to meet the allowances recommended by the American Institute of Nutrition for the rat.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rats were allowed to breed, and the offspring were weaned to the same diet as their mothers. Offspring from different parents within a diet group were selected at random and mated to form the F2 generation. Offspring from both F1 and F2 were fed their respective diets throughout their entire lives. Two generations were selected for studies to simulate populations of people consuming the same basic diet for several generations. At 50 days of age, female offspring from the F1 generation (experiment A) and the F2 generation (experiment B) were p.o. gavaged with sesame seed oil (Sigma, St. Louis, MO) containing 80 mg/kg DMBA (Sigma), a chemical procarcinogen used widely to produce mammary adenocarcinoma in rats. This dose was selected based on studies reported by Barnes et al. (5) and Lamartiniere et al. (6) . Rats were weighed weekly, and, beginning 3 weeks after DMBA treatment, each mammary gland was palpated twice weekly. The detection date and location of each mammary mass were recorded. When at least one palpatable mammary mass was present in 100% of the casein-fed rats, all rats were killed, and all mammary glands from each rat were visually examined for tumors. However, rats in which tumor masses exceeded 3 cm in diameter were killed early for humane reasons according to our institutional animal care and use committee-approved animal protocol. All tumors were weighed and measured for volume, and a section of the tumor or the entire mass, depending on the size, was fixed in buffered formalin. Sections (5 µm) of the paraffin-embedded tumors were stained with H&E for histological analysis.
Pathology.
An American College of Pathology-certified pathologist (S. K.)
evaluated tumors in a blinded protocol and classified them as follows:
(a) benign (mammary tissue minimal proliferation);
(b) intraductal proliferation; or (c)
adenocarcinoma (ductal carcinoma in situ or invasive ductal
and lobular carcinoma).
Statistical Analysis.
For each experiment, a mixed model ANOVA (7)
was used to
analyze adult body weight after injection with DMBA. Because there were
multiple measurements per animal over time, day was included in the
model as a with-in-animal effect to account for the correlation of
measurements from the same animal. The effect of diet and the
interaction between diet and day were also included in the model. A
Kaplan-Meier analysis of tumor latency was performed (8)
.
Product limit survival estimates were used to determine the median
tumor latency time and confidence limits. A generalized Wilcoxon test
(9)
was used to compare median tumor-free times. To
examine the prevalence of tumors according to breast location, a
generalized estimating equation logistic regression model
(10)
was used to account for the clustering of tumor
findings on each animal, adjusting for diet. Fisher’s exact test
(8)
was used to compare the percentage of animals with
tumors in each treatment group for each experiment. The median numbers
of tumors per tumor-bearing rat (multiplicity) for each diet were
compared using the nonparametric Kruskal-Wallis test. Statistical
significance was set at P < 0.05, and all
Ps were not adjusted for multiple comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Total body weight gains were similar until approximately 5 weeks of
age, when casein-fed rats began to gain slightly more weight than
either soy-fed or whey-fed rats. By 8 weeks, body weights of casein-fed
rats were slightly greater than those of soy-fed or whey-fed rats
(P < 0.01) in both experiments; however, body weights
did not differ between soy-fed and whey-fed rats. When development and
reproductive parameters of the casein-fed group were compared with
those of the soy-fed or whey-fed groups, a 1-day advancement in the age
(day 37 versus day 38) of the vaginal opening of soy-fed
rats (P 
0.05) was the only significant difference
observed during the two-generation period. No differences were noted in
relative organ weights, estrous cycle, breeding success rate, sex
ratio, or number of offspring/litter (data not shown).
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, and data concerning these tumors are presented in Table 1
. At the time when 100% of the casein-fed rats developed at least one
breast mass, relatively fewer rats fed whey protein had tumors compared
to casein-fed rats in both experiments (P < 0.002);
whereas soy-fed rats had relatively fewer tumors compared to casein-fed
rats (P < 0.008) only in experiment B.
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.
However, another representative measure of tumor latency is the
posttreatment time at which the probability of developing a tumor is
50%. Using the day at which 50% of the rats developed a tumor as a
measure of latency for rats from either the F1 or
F2 experiments, whey-fed rats had a 31–35-day
(60–64%) longer latency period than casein-fed rats
(P < 0.002), and soy-fed rats had a 13–14-day
(25–26%) longer latency period than the casein group
(P < 0.009). Furthermore, whey delayed this onset more
than soy in experiment B (P < 0.05), but not in
experiment A (P < 0.197).
Tumor Characteristics.
Ninety-five percent (experiment A) and 85% (experiment B) of the rats
bearing at least one tumor had an adenocarcinoma in the casein-fed
group, whereas 90% (experiment A) and 79% (experiment B) of
soy-treated rats and 86% (experiment A) and 63% (experiment B) of
whey-treated rats had at least one adenocarcinoma, respectively (Table 1)
. There were no statistically significant differences between groups
in the mean tumor volume (Table 1)
.
Tumor Multiplicity and Location.
Of the tumor-bearing rats, the median number of mammary
tumors/tumor-bearing rat (i.e., multiplicity) was lower
(P < 0.007) in each experiment for whey-fed rats than
for casein-fed rats (Table 1)
, whereas multiplicity for soy-fed rats
did not differ significantly from that of the casein-fed group. Tumor
location was studied, and cervical and thoracic regions were at higher
risk of developing tumors (data not shown).
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Discussion |
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To our knowledge, this is the first demonstration that rats fed whey protein-containing diets develop fewer DMBA-induced breast tumors than rats fed either casein- or soy-based diets. The whey protein diet delayed the age of onset of DMBA-induced tumors, reduced the percentages of rats that develop tumors, and attenuated tumor multiplicity compared with soy protein isolate or casein protein diets.
The mechanisms by which whey protein could alter carcinogenesis are unknown. Speculation has focused primarily on increases in tissue GSH levels observed during whey consumption (2) . Greater GSH concentrations would tend to be protective because of: (a) the well-known xenobiotic detoxification pathway involving GSH and glutathione S-transferases; (b) GSH free radical detoxification; and (c) improved immune responses (13) . Other possible mechanisms could include bioactive peptides that are either fragments of whey hydrolysis or contaminants of whey protein, such as insulin-like growth factor I, which could act on one of many cellular processes to reduce tumor incidence. Studies are currently underway in our laboratory to determine the mechanisms by which whey protein consumption prevents DMBA-induced breast cancer.
Although the mechanisms by which soy protein isolate prevents such cancers are still unknown, factors that are physically bound to or associated with the isolated protein, especially the isoflavones, have been implicated as being important (5) . Genistein is one such isoflavone present in soybeans as genistein glucoside or the 6'-O-malonylglucoside. Lamartiniere et al. (6) demonstrated that rat pups receiving high doses of pure genistein aglycone at ages 2, 4, and 6 days developed fewer DMBA-induced breast tumors. This work has been substantiated in unpublished studies in our laboratory.4 However, it should be noted that isoflavones have not been reported to be uniformly protective against chemically induced mammary tumor models in animals. For example, Hilakivi-Clarke et al. (14) reported a doubling of chemically induced mammary tumors in the offspring of mothers treated with genistein during pregnancy. Hsieh et al. (15) reported that dietary genistein enhanced the growth of MCF-7 tumors that were implanted s.c. in ovariectomized athymic mice. It should be pointed out that these two studies used genistein aglycone rather than diets made with soybean meal, soybean flour, or soy protein isolates. The disparity between laboratories using various animal models may reflect differences in species, developmental timing of genistein treatment, differences in endocrine status (e.g., intact versus ovariectomized athymic females), or the chemical form of the isoflavone (pure aglycone, glucoside, or protein-bound form) and points to the need for further careful research on the conditions under which dietary factors affect cancer risk.
In the current study, soy-fed rats received a diet in which the entire protein source was the soy protein isolate used in the majority of commercially available soy-based infant formulas (Protein Technologies International, Inc.). A 333-g rat in our study consumed approximately 25 g/day of diet formulated with 20% soy protein (w/w). Because the genistein content was 1.36 mg/g protein, the genistein intake was approximately 6.8 mg/day (20.4 mg/kg/day). This is the highest dose of isolated soy protein possible within the AIN-93G diet formula. This genistein intake compares with the approximately 11 mg/day reported for 4-month-old infants who consume soy-based infant formula (16) . Dose-response and time course studies for soy isolated protein effects and its cancer-preventive actions remain to be determined.
Several mechanisms have been proposed for the anticarcinogenic activity
of isoflavones, including: (a) inhibition of proteases;
(b) antioxidant activity; (c) increased synthesis
and decreased degradation of steroid hormone binding globulin
synthesis; (d) weak estrogenic agonist/antagonist activity
through estrogen receptor 
; (e) estrogen receptor
ß-mediated actions; (f) altered hormone production,
metabolism, or action; and (g) induction of Phase II
detoxification systems (for a review, see Ref. 17
).
However, in addition to genistein, there are several other biologically
active factors associated with soy protein. For example, other
isoflavones such as diadzien and other factors such as saponins,
phytosterols, protease inhibitors, and inositol hexaphosphate that have
also been implicated in cancer prevention are present
(1)
.
Although no overt effects of soy or whey diets were observed on sexual development (other than the a 1-day advance in vaginal opening), reproductive organ weights, or reproductive performance, enhanced breast differentiation represents a potential mechanism underlying the protective effects of these diets. This would be more likely in rats fed soy diets because of the well known estrogenic isoflavones than in those fed whey diets, in which no such estrogenic factors have ever been reported. Other potential mechanisms might involve altered DMBA metabolism, DNA repair, or proliferative/apoptotic responses to DMBA-induced damage. These possibilities are currently under investigation in our laboratory.
Studies involving dietary prevention of tumors often use casein as the "control" protein source and compare all dietary effects to the results of groups fed casein. One possibility not often discussed is that casein-fed rats could be at greater risk of developing DMBA-induced mammary tumors than rats fed diets containing whey or soy protein isolate. If that were true, our data would suggest that factors in casein and soy protein isolate could promote tumor formation compared with whey. This is an area requiring further research.
Several studies have demonstrated that calorie restriction (30–40%) in rats resulted in reduced tumor formation (see Ref. 18 for a review). Rats fed either soy or whey in the present study had a tendency to gain weight at a slightly but significantly lower level than rats fed casein diets. Thus, whether the reduced DMBA-induced tumors in soy-fed or whey-fed rats were related to the lower body weight gain could become an issue. However, rats in calorie restriction studies had severe diet restriction (30%) and a much greater reduction in weight gains (30–60%). The situation in the present study differs substantially from calorie restriction studies on several counts. For example, the rats in the present study were not diet-restricted, and although food intake in the current studies was not monitored, it has been monitored in subsequent and similar studies using the same diets and rats of the same age and has been found not to differ significantly between casein, whey, and soy diets. Thus, because food consumption was equivalent in all three dietary groups, neither reduced caloric intake nor lower protein intake or quality could account for the protective effects of soy or whey diets as compared with casein diets. Previously published studies comparing casein-fed and soy protein-fed rats demonstrated similar slight reductions in body weight gains in rats consuming soy-based diets, although food intake was the same (19, 20, 21) . The slightly lower body weight gains of soy-fed rats could be associated with the recently reported estrogenic effects on lowering body weight gains demonstrated by addition of estradiol to rat diets (22) . Furthermore, soy-fed and whey-fed rats had excellent weight gains that were just slightly lower than those in casein-fed rats. Thus, it is not clear why rats gain slightly less weight on diets made with the same formula and components except for the source of protein. However, it is clear that: (a) rats in the present study were not calorie-restricted; (b) the weight gains in each group were excellent; and (c) it is highly unlikely that reduced tumor development was related to this very slight reduction in body weight gains. Furthermore, it should be emphasized that there were no differences between the weight gains of soy-fed rats and whey-fed rats, but there were significant differences between tumor formation, demonstrating clear dietary effects at the same rate of weight gain. This latter point illustrates a disconnection between prevention of DMBA-induced breast tumors and the small differences in body weight gain in the present study.
It should be noted that chemical induction of breast tumors (i.e., the chemical induction model of carcinogenesis) using agents such as DMBA is the most widely used animal model for studying breast cancer. The relevance of this model to human breast cancer undergoes constant scientific debate, including the perspective that human breast cancer may not be caused by a single bolus of a carcinogen such DMBA and/or that inactivation or detoxification of carcinogens such as DMBA may have little application to humans. On the other hand, DMBA is a polycyclic aromatic hydrocarbon. Polycyclic aromatic hydrocarbons generated by cooking and in cigarette smoke are considered to be major human dietary and environmental carcinogens. Increased Phase I and II metabolism (detoxification) of such compounds (exogenous and endogenous) is also considered to result in chemoprotection and reduced cancer risk (23 , 24) . Thus, results presented herein could have significant relevance to prevention of human breast cancer. Further research into the mechanisms discussed above is required to answer these important questions.
DMBA-induced mammary tumors were studied in female rats fed diets formulated with single protein sources. Rats fed diets with the same soy protein isolate used in the majority of soy-based infant formulas had lower mammary tumor incidence than rats fed diets made with casein. Diets formulated with whey protein provided significantly more protection against DMBA-induced mammary tumors than casein- or soy-based diets. These results suggest diets containing these commonly consumed protein sources have great potential in reducing the risks of breast cancer. Our data further suggest that whey protein may be one of the most potent dietary sources of cancer prevention identified to date.
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Acknowledgments |
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Footnotes |
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1 Supported by a United States Department of
Agriculture-Agriculture Research Service Arkansas Children’s Nutrition
Center grant. 
2 To whom requests for reprints should be
addressed, at Arkansas Children’s Hospital Research Institute, 1120
Marshall Street, Little Rock, AR 72202. Phone: (501) 320-2785; Fax:
(501) 320-2818; E-mail: Badgerthomasm{at}exchange.uams.edu
3 The abbreviations used are: DMBA,
7,12-dimethylbenz(a)anthracene; GSH, glutathione.
4 T. M. Badger, unpublished observations.
Received 6/10/99; revised 10/ 1/99; accepted 10/15/99.
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), or whey diet
(n = 24; 
). In experiment B (bottom
panel), F2 rats were fed either casein diets
(n = 39), soy diet (n = 31), or
whey diet (n = 35). Data are presented as
means ± SEM.