Health Effects of Cows’ Milk
http://www.eps1.comlink.ne.jp/~mayus/eng/

Commercial Low-Fat Milk Markedly Stimulates
Mammary Carcinogenesis in Rats
Adapted from:Qin LQ, Xu JY, Wang PY, Ganmaa D, Li J, Wang J, Kaneko T, Hoshi K, Shirai
T, Sato A.
Low-Fat Milk Promotes the Development of 7,12-Dimethylbenz(a)anthracene(DMBA)-Induced Mammary Tumors in Rats
International Journal of Cancer 2004;110:491-6.
Abstract
Commercial cows’ milk contains considerable amounts of estrogens. This study assessed theeffect of commercial low-fat milk on the development of 7,12-dimethylbenz(a)anthracene(DMBA)-induced mammary tumors in rats. Eighty six-week-old female Sprague-Dawley rats
received a single oral dose of 5 mg DMBA. Twenty-four hours later, the animals were divided
into 4 groups of 20 animals each and given one of four test solutions for 20 weeks as theirdrinking liquid: low-fat (1%) milk (M), artificial milk (A), estrone sulfate solution (0.1 µg/ml,
E), or tap water (W). The artificial milk was formulated to supply essentially the same
calories as the milk. The low-fat milk contained 378 pg/ml estrone sulfate. Tumor incidence,the cumulative number of tumors, and the sum of tumor diameters were higher in the M and E
groups than in the A or W groups. Overall, the development of mammary tumors was in theorder: M = E > A = W. Whereas the plasma 17β-estradiol concentration in the M group was
the second highest after the E group, the plasma level of insulin-like growth factor (IGF-I)
was significantly higher in the M group than in the other three groups. In conclusion,commercially available low-fat milk promotes the development of DMBA-induced mammary
tumors in rats. The degree of the promotion is almost comparable to that of 0.1 µg/ml estrone
sulfate. The high estrogen content in the milk may be responsible for the promotional effects,
acting in concert with other hormones such as IGF-I.

Introduction
Breast cancer is the most common female cancer in the world, and is the number one cause ofcancer-related death in non-smoking women (1). International differences in the rates ofbreast cancer incidence and mortality suggest that environmental factors, perhaps dietary ones,
may influence risk for this disease (2,3). An epidemiological analysis revealed that theincidence of breast cancer in 42 countries is highly correlated (r = 0.79) with the consumption1

of milk and dairy products (4). However, the association between milk and breast cancer has
been inconsistent in case-control studies; positive association (5-7), no association (8,9), andinverse association (7,10) have been observed. Cohort studies also have yielded conflicting
findings (11-16). In a meta-analysis, a small increase in breast-cancer risk with high milk
consumption was observed (17). However, in a pooled analysis of cohort studies, nosignificant association was found between dairy products consumption and the risk of breast
cancer (18). These contradicting results may indicate that different components in milk playcomplex roles in the development of breast cancer.
Breast cancer is typically hormone dependent. Known risk factors for this cancer
involve endogenous hormone exposure-- early menarche, late first full-term pregnancy, andlate menopause, all of which increase exposure to hormones, especially estrogens (19).
Exogenous estrogens, derived from oral contraceptives and hormone replacement therapy(HRT), have been positively associated with the risk for breast cancer (20,21). In addition tothese sources, estrogens, both natural (e.g., estrone) and synthetic (e.g., ethinyl estradiol),have been found in water (22) and in foods (23,24). Remesar et al. (23) calculated estrone
intake in a standard human diet, and found that 46.6% of estrone came from dairy products.
Hartmann et al. (24) estimated that dairy products provided 60-80% of dietary estrogens andprogesterone, the other female sex hormone in the Western diet. The concentration of estrone
sulfate in cows’ milk increases greatly during pregnancy (25). Since commercial cows’ milk
in developed countries is produced mainly from pregnant cows, it contains considerable
quantities of estrogens (26).
As components of milk, estrogens have not been addressed as breast-cancer risk
factors in epidemiological studies. It is difficult to evaluate the risk of milk estrogens in suchstudies, as estrogen levels are affected by both endogenous and exogenous factors in women.
Chemically induced mammary tumor models with laboratory animals have been
extensively studied, with 7,12-dimethylbenz(a)anthracene (DMBA) being used mostfrequently (27-29). Although the tumors are not identical across species, hormone dependency
is an important similarity between human mammary tumors and those induced by DMBA inrats (30). The DMBA model is useful for the study of human breast cancer, especially in the
postmenopausal period (29).
The present study was performed to assess the effects of commercial low-fat milk on
the development of DMBA-induced mammary tumors. In order to confirm the role ofestrogens, an aqueous estrone sulfate solution was used as a positive control.

Materials and Methods
Animals and Diets
Eighty female Sprague-Dawley rats were obtained at five weeks of age from
Shizuoka Laboratory Animal Center (Shizuoka, Japan). They were housed individually instainless-steel wire-bottomed cages in an air-conditioned room (22 ± 2 ºC with 55 ± 10%2

relative humidity) with 12/12 hours light/dark cycle. The care and use of laboratory animals
followed the Guidelines for Animal Experiments of the Medical University of Yamanashi.
After one week of acclimation to commercial powder chow (Clea CE-2, Nippon Clea,
Tokyo, Japan) and water, all animals were given a single dose of 5 mg DMBA
(Sigma-Aldrich, St. Louis, MO, USA) dissolved in 0.5 ml corn oil by intragastric intubation
between 14:00 and 16:00 hours. Twenty-four hours after DMBA administration, the rats wereassigned at random to one of four groups, each with a different dietary regimen.
Each rat was maintained on the powder chow (dispensed in a glass container)
throughout the experiment. Each group of rats was given low-fat milk (M), artificial milk (A),estrone sulfate solution (E), or tap water (W) as a drinking liquid in a separate glass containerof the same kind as used for the powder chow. Commercial, nationally available low-fat milk,
the fat content of which was 1%, was purchased locally; the number of the production lot wasrecorded. The artificial milk was formulated to balance the major nutrients contained in the
low-fat milk, and consisted of gluten fortified with lysine, DL-methionine, threonine, andvaline as protein, coconut oil as fat, and dextrin-maltose as carbohydrate, supplemented withcalcium carbonate (Table 1). Estrone sulfate (Sigma-Aldrich) was dissolved in tap water at aconcentration of 0.1 µg/ml. The food and drinking solutions were renewed daily at 16:00
hours.
Chow andliquid consumption wasmonitored daily bysubtracting from what
was provided anything
not consumed by 10:00
hours the following day.
Calculated net intake ofenergy and body weight
was recorded weekly
throughout theexperiment.

Tumorigenesis assessment.
Rats were palpated weekly to monitor tumor development. The two largest
perpendicular diameters of each tumor were measured with calipers (Mitsutoyo CD-15 CP,
Kanagawa, Japan) and the mean of the two measures was used to estimate the tumor size. The
location of each tumor was also recorded at every palpation.
3

Autopsy and histopathological examinations
During the 20th week after DMBA administration, rats were decapitated between
14:00 and 16:00, during proestrus, as determined by vaginal smears (31). Trunk blood fromthe neck was immediately centrifuged at –4 ºC, and the plasma stored at –80 ºC until analysis.
All organs were examined for gross abnormalities, and the location and weight ofeach tumor was recorded. The visible mammary tumors, ovaries and uteri were rapidly
excised and weighed, as was the peritoneal adipose tissue (excepting mesenteric). Mammary
tumors were fixed and stored in 10% neutral buffered formalin and embedded in paraffin.
Sections were cut at 3 µm and stained with hematoxylin and eosin for histopathologicalexaminations. Histological diagnosis was performed according to the classification as
described by Konitowski et al. (32)
Measurement of plasma hormone concentrations
Because estrogen peaks before ovulation, the period of proestrus was selected toobserve the trend of plasma estradiol levels during the experiment. Before DMBA
administration, the blood of 20 randomly selected rats was sampled from tail veins between
14:00 and 16:00 hours, using heparinized capillary tubes, to measure the basal 17β-estradiol
concentration. Every four weeks, blood from each rat was also collected from the tail between
14:00 and 16:00 hours at proestrus. Plasma 17β-estradiol concentration was measured using acommercial ELISA kit (Immuno-Biological Laboratories, Hamburg, Germany), after
extraction with diethyl ether.
The blood plasma collected at autopsy was used to measure the concentrations of
17β-estradiol, estrone, insulin-like growth factor (IGF)-I, and prolactin. Plasma estrone and
IGF-I concentrations were measured using an estrone RIA kit (Diagnostic Systems
Laboratories, Webster, TX, USA) and a rat IGF-I EIA kit (Diagnostic Systems Laboratories),
respectively. Plasma prolactin concentration was measured using a rat prolactin EIA kit(SPI-BIO, Massy Cedex, France).
Measurement of estrogens in low-fat milk
A sample of the low-fat milk was stored at -80º C every four weeks, and was later
assayed for estrone, 17β-estradiol and estriol concentrations. For the analysis of each freeestrogen, 8 ml of diethyl ether was added to each 2-ml milk sample (prepared in duplicate),and shaken vigorously. After centrifugation at 3,000 rpm for 10 min, 7 ml of the ether layer
was recovered and evaporated to dryness in a vacuum centrifuge. The residue was dissolvedin 200 µl of methanol, and 10 µl of this solution was injected into an HPLC system with acoulometric electrode array detector (eight channels, ESA, Chelmsford, MA, USA) (33,34).
For determination of total estrogen (free + conjugated), 2-ml milk samples(prepared in duplicate) were mixed with 200 µl of buffer containing 10 mg sulphatase4

(Sigma-Aldrich) and incubated at 37 ºC for 3 hours before extraction with diethyl ether (33).
The concentration of conjugated estrogen was calculated by subtracting the free from the total
estrogen concentration.
Statistical analysis
Data were subjected to analysis of variance (ANOVA) using StatView 4.0 (Abacus
Concepts, Berkeley, CA, USA), followed by a t test when significant differences existed
among groups. Tumor incidence was analyzed by Fisher’s exact probability test. The number,size and weight of tumors (at autopsy) were analyzed by a Mann-Whitney U-test. P-values
less than 0.05 were considered significant.

Results
Body weight and food intake
No significant difference was observed in body weight between the M and A groups(Fig. 1). Rats in the E groupoccasionally weighed less than rats
in the W group. In general, rats in
the M and A groups were heavier
than rats in the E and W groups.

Fig. 1. Body weights of rats in the four groups during the experimental period. No
significant difference was observed between Milk and Artificial Milk or between Estrone
and Water.

5During the first three
weeks, the energy intake of the rats
increased in every group, andremained at a similar level
thereafter (Fig. 2A). The averageenergy intake (kcal/day) throughout
the experiment was 59.7 ± 3.3, 59.1
± 2.9, 57.8 ± 2.6, and 57.8 ± 3.3 forthe M, A, E, and W groups,
respectively (no significant
difference between M and A or
between E and W).
No significant difference
was observed in the consumption ofdrinking liquid (g/day) between the
M and A groups or between the E
and W groups (Fig. 2B). However,consumption (g/day) was muchgreater in the M (38.2 ± 1.5) and A(37.6 ± 1.1) groups than in the E(23.4 ± 1.8) or W (23.9 ± 1.9)
groups.
Fig.2. Daily intake of food and liquids after DMBA administration. A, Total energy
intake; B, Intake of liquid. No significant difference was observed between Milk andArtificial Milk or between Estrone and Water.
The body weights (g) of the M (332.8 ± 27.9)- and A (325.8 ± 44.9)-group rats at
autopsy were significantly higher than those of the E (293.2 ± 24.7)- or W (301.8 ±32.2)-group rats. The weight of peritoneal adipose tissue (g) was significantly higher in both
the M (25.3 ± 5.6) and A (26.2 ± 7.1) groups than in the E (15.4 ± 4.7) or W (18.6 ± 5.6)
groups. There was no significant difference in the adipose tissue weight between the M and A
groups or between the E and W groups. However, the weight of adipose tissue as a fraction ofbody weight (%) was found to be significantly lower in the E group (5.2 ± 1.3) than in the W(6.1 ± 1.3) group, whereas no significant difference was noted between the M (7.8 ± 1.3) andA (7.9 ± 1.5) groups. The weights of uteri and ovaries were not significantly different among
the four groups.

6Mammary tumorigenesis
Figure 3 shows the tumor
incidence (A), the number oftumors (B) and the size of tumors(C) in the experimental groups, asdetected by palpation. With each of
the three indices of tumorigenesis,
the M and E groups could begrouped together separately fromthe A and W groups, the former
having much higher values than thelatter.
At week 6 after DMBA
administration, the first tumors were
palpated in the M, A, and E groups(Fig. 3A). Palpable tumors in the Wgroup did not appear until week 9
after DMBA administration. The
incidence increased with time, more
rapidly in the M and E groups thanin the A and W groups. Incidence
reached or surpassed 50% at weeks
11, 12, and 16 for the M, E, and A
groups, respectively. The incidence
in the W group never reached 50%.
A significant difference in incidence
between the M and A groups wasnoted consistently from week 14
onward. The incidence in M-grouprats was higher than in W-group rats,
also from week 14 onward. The
difference between the E and W
groups was not statisticallysignificant at the end of theexperiment (P = 0.0528).
Fig. 3. Time course of palpable mammary tumor appearance. A, Tumor incidence; B,
Cumulative number of tumors; C, Sum of tumor diameters.

7

At the final palpation (week 20), tumor incidence was 85% (17/20), 50% (10/20), 75%(15/20), and 45% (9/20) for the M, A, E, and W groups, respectively.
More than twice as many tumors were seen in the M group as in the A group, as ofweek 11 after DMBA administration (Fig. 3B). Similarly, rats in the E group had almost twice
as many tumors as did rats in the W group. No significant difference in number was observed
between the M and E groups or between the A and W groups. At the final palpation (week 20),the cumulative number of tumors in each group was 49, 20, 43, and 17 in the M, A, E, and W
groups, respectively.
The total size of tumors in the M group, estimated by the sum of tumor diameters,
was more than twice that of tumors in the A group (Fig. 3C). The tumors that developed in the
E group were also almost twice as large as those in the W group. No significant difference intumor size was found between the M and E groups or between the A and W groups.
Altogether, the palpation data show that the mammary tumors developed in the order: M = E> A = W.
Autopsy data supported the palpation data described above, except that some small,
impalpable tumors were found at autopsy (Table 2).
The tumor incidence was
17/20, 10/20, 15/20, and9/20 in the M, A, E, andW groups, respectively.
The respective total
numbers of tumors were73, 33, 64, and 25 in thefour groups. The sums oftumor weight were 49.2,
24.3, 47.3, and 23.1 g in
the respective groups. It
is noteworthy that boththe total number oftumors and the total
weight of tumors in the M group were more than double the respective values in the A group.
The same relationship was seen between tumors of the E and A groups.
Histologically, almost all the tumors that developed in each group wereadenocarcinomas. Overall, the four groups could be ranked by the development of mammary
tumors as follows: M = E > A = W.

8

Plasma hormone concentrations
The time courses of plasma 17β-estradiol concentrations in the four groups during
the experiment are shown in Fig. 4.
From week eight onward, theconcentration in the E group was
significantly higher than that in theother three groups. The
concentration in the M groupincreased slightly over time, withno significant difference from thatin the A group throughout theexperiment. At weeks 16 and 20,
the M group had a significantly
higher estradiol concentration in theplasma than did the W group.
Fig. 4. Time course of 17b-estradiol concentration in the plasma. A, Significantly
different from Milk; b, from Artificial Milk; c, from Water.
Plasma concentrations of estrone, 17β-estradiol, IGF-I and prolactin, measured at
autopsy, are shown in Table 3. Although the estrone level in the M group was not
significantly different
from that of the A group(p = 0.064), it wassignificantly higher thanthat in the W group. The
IGF-I concentration was
significantly higher inthe M group than thosein the other three groups,
while no significant
differences were foundamong the A, E and W
groups. The plasma
prolactin concentrations
were significantly higher in the M and A groups than in the E and W groups, although theintra-group variation of the concentrations was considerable. There were no significant
differences in the plasma prolactin concentration between the M and A groups or between the
E and W groups.
9Within each group, there were no significant differences in 17β-estradiol and estrone
concentrations between tumor-bearing and non-tumor-bearing rats (data not shown). However,in both the M and E groups, the plasma IGF-I concentration (mean ± SD in µg/ml) was higher
in the tumor-bearing rats (0.789 ± 0.100 [n = 17] and 0.698 ± 0.117 [n = 15], respectively)
than in the non-tumor-bearing rats (0.586 ± 0.065 [n = 3] and 0.511 ± 0.166 [n = 5],
respectively). Although plasma prolactin concentration (ng/ml) in the M group was muchhigher in rats with tumors (1.94 ± 1.68, n = 17) than in tumor-free rats (0.51 ± 0.19, n = 3),
the difference was not statistically significant.
When data from rats in all groups were pooled, no significant difference wasobserved in 17β-estradiol and estrone concentrations in the plasma (mean ± SD in pg/ml)
between tumor-bearing (n = 51; 81.3 ± 25.3 and 68.3 ± 29.6, respectively) and non-tumor
bearing rats (n = 29; 72.4 ± 21.7 and 54.0 ± 21.2, respectively). However, both IGF-I (µg/ml)
and prolactin (ng/ml) concentrations in the plasma were significantly higher in tumor-bearing
rats (0.716 ± 0.113 and 1.324 ± 0.258, respectively) than in non-tumor-bearing rats (0.600 ±
0.104 and 0.505 ± 0.228, respectively).
Estrogen concentrations in milk
The levels of estrogens in the milk used in the present study are shown in Table 4.
Total estrogen content in
the commercial low-fat
milk was 703 pg/ml, of
which estrone
conjugates comprised
378 pg/ml. The
conjugated form
accounted for 87% of
total estrone. The
17β-estradiol
concentration wasapproximately half thatof estrone.

Discussion
In most of the epidemiological studies that have identified milk consumption as a risk factor
for breast cancer, special attention has been given to the fat content of the milk (2,5,7,11,17).
Milk fat cannot, however, explain all of the breast cancer risk related to milk consumption. In
fact, consumption of whole milk has declined steadily since the 1950s, and been replaced by10fat-reduced milk (35), yet the incidence of breast cancer has increased worldwide over the last
50 years (3).
In the present study, we examined the effect of low-fat milk (with a fat content of1%) on the development of DMBA-induced mammary tumors, excluding the effect of milk
fat. As the development of mammary tumors is influenced by energy intake (27), we included,
as a negative control, a group of rats that consumed artificial milk (A group). The protein, fat
and carbohydrate contents of the artificial milk were the same as those of the commercial
low-fat milk used. We used gluten, fortified with some essential amino acids (Table 1) as thesource of protein, rather than casein (which is produced from milk), to avoid the potential forcarry-over of other milk components. As we expected, rats in the M and A groups consumed
almost the same number of calories (Fig. 2). Body weights in the two groups were similar
throughout the experiment (Fig. 1); at autopsy, both body weights and ratios of adipose tissue
weight to body weight were similar.
Rats in the E group consumed almost the same energy as the W group (Fig. 2). The
volume of drinking solution consumed was also comparable between the two groups. Thissuggests that estrone sulfate (0.1 µg/ml) had no influence on food and water consumption.
However, the adipose tissue weight and the adipose tissue weight/body weight ratio werelower in the E group than in the W group. It is possible that estrone sulfate at a concentrationof 0.1 µg/ml inhibited energy storage.
Since fat and energy intake were balanced between the M and A groups, thesignificantly higher incidence, greater number, and greater growth of mammary tumors in the
M group (Fig. 3) may be attributed to an undetermined component or components in milk,other than fat or calories.
In the present study, the incidence, number, and size of tumors in the M and E
groups were similar. The number and weight of mammary tumors that developed weresignificantly higher in the E group than in the W group, although the difference in incidence
between the two groups (75% vs. 45%) was not statistically significant. Special attention
should be paid to the role of estrone sulfate in the development of DMBA-induced mammary
tumors.
The findings at autopsy (Table 2) and the measurement of palpable tumors (Fig. 3)
indicate that the potential of estrone sulfate to promote the development of DMBA-induced
mammary tumors in rats is comparable to that of commercially available low-fat milk.
The conjugated estrone concentration in the low-fat milk used was about 380 pg/ml(Table 4). In addition to the long plasma half-life of estrone sulfate, it is absorbed in theintestinal mucosa virtually unchanged (36,37). Although the oral bioactivity of free estradiol
and estrone may be relatively low, estrone sulphate, which is a major estrogen in milk, hashigh oral bioactivity (26). Once inside the body it can be readily converted to estrone andestradiol. The entero-hepatic circulation also plays a role in the absorption of estrone
11

conjugates. In the present study, plasma 17β-estradiol concentration increased gradually in theE-group rats, followed by that in the M-group rats (Fig. 4).
Some human studies have also demonstrated that milk consumption increased thelevel of estrogen in circulation. South African black males switched from a vegetarian to aWestern diet--with milk, butter and meat--showed an increase in circulating estrogen levels(38). The relationship between milk consumption and plasma estrogen concentrations is also
supported by the fact that Asian women, whose consumption of milk and dairy products islow, have lower plasma estrogen concentration than do Caucasian women, whose dairy
product consumption is high (39).
Estrogens in Silastic implants have been documented to increase mammary tumor
development in rats (28,29,40). Moreover, increasing evidence has implicated 17β-estradiol
as a cause of breast cancer in postmenopausal women (41-43). Indirect effects of estrogens
may occur through the stimulation of prolactin secretion, which is linked with mammary
cancer (19). In the present study, however, the E group, with a high incidence of DMBA
mammary tumors, had a prolactin concentration similar to the W group, with a low incidenceof tumors (Table 3). By contrast, plasma prolactin was about three-fold higher in the M group,and two-fold higher in the A group than in the E and W groups. Calories or fat intake, whichwere slightly higher in both the M and A groups than in the E or W groups, is likely to havemediated the increase in prolactin level, as suggested by Clinton et al. (44)Another component in cows’ milk may play an additive or synergistic role withestrogens in the development of DMBA-induced mammary tumors. IGF-I has recently
attracted increasing attention, as studies have demonstrated a link between breast cancer risk
and high levels of this growth factor (45,46). IGF-I concentrations in cows’ milk have beenreported to range from 6 to 162 ng/ml (47). In a human study, plasma IGF-I concentration
increased by 10% when healthy subjects consumed cows’ milk (48). In the present study,
plasma IGF-I concentration was significantly higher in the M group than in the other threegroups (Table 3). In addition, tumor-bearing rats in the M and E group had a higher IGF-I
concentration than did non-tumor-bearing rats. It is possible that IGF-I from cows’ milk is
active in rats, because IGF-Is in mammals are highly similar in the structure (49). Estrogens
and other hormones in milk may have been involved in the promotion of DMBA-induced
mammary tumors in the M group.
In conclusion, commercially available low-fat milk promotes the development of
DMBA-induced mammary tumors in rats. The degree of the promotion is almost comparable
to that of 0.1 mg/ml estrone sulfate. The high estrogen content of the milk may be responsible
for the promotional effects, acting in concert with other hormones such as IGF-I. Further
epidemiological and mechanistic studies are needed to verify the hypothesis that milk
consumption is a factor responsible for the development of breast cancer.

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