Health Effects of Cows’ Milk
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Commercial cows’ milk has estrogenic activity as revealedby the hypertrophic effects on the uteri of young
ovariectomized rats and immature rats
Adapted from:

Ganmaa D, Tezuka H, Enkhmaa D, Hoshi K, Sato A.
Commercial cows’ milk has uterotrophic activity on the uteri of young
ovariectomized rats and immature rats.
International Journal of Cancer 2006;118:2363-65.
Abstract
Cows’ milk has considerable amounts of estrogens, mainly in the form of estrone sulfate. To
determine whether the commercial milk has any biologically significant hormonal effects, twoseries of uterotrophic tests were performed, one with young ovariectomized rats and the otherwith sexually immature rats. Thirty-six rats were used for each test. They were divided into 3
groups of 12 animals each, and were kept for 7 days on powdered chow with one of threedrinking solutions: low-fat milk (experimental), artificial milk (negative control), or artificial
milk containing estrone sulfate at 100 ng/ml (positive control). At autopsy, both the wet andblotted uterine weights were measured. The cell heights of uterine epithelia in ovariectomized
rats were also determined. In each test, the weights of the uteri in the Low-Fat Milk groupwere significantly greater than those of the respective weights in the Artificial Milk group(p<0.01). Furthermore, in ovariectomized rats, the uterine epithelial-cell height in the
Low-Fat Milk group was significantly greater than that observed in the Artificial Milk group(p<0.01). The uterotrophic effect of 100 ng/ml Estrone Sulfate solution was greater than thatof Low-Fat Milk in immature rats (p<0.01), whereas the effect of the solution was almost
comparable to that of Low-Fat Milk (p>0.05). In conclusion, commercially available low-fat
milk has uterotrophic effects in both young ovariectomized rats and sexually immature rats.
Introduction
Cows’ milk contains considerable amounts of estrogens (estrone, estradiol-17β and estriol)(1). Because of modern dairy practices, 75% of commercial cows’ milk come from cows
during pregnancy, when the estrogen levels in their blood, and hence in their milk, areelevated (2). The hormone levels in milk exceed those in blood, probably owing to hormone
synthesis in the mammary glands (3).
The major estrogen in milk is estrone sulfate (4), which when consumed can be1

readily converted to estrone or estradiol-17β (5). Because of its hydrophilic nature, this main
conjugate can be easily absorbed from the gastrointestinal tract. Quantitatively, estrone
sulfate is the most important blood estrogen (6,7). Exogenously administered estrone sulfate
has been shown to stimulate mammary tumor growth (8,9).
To determine whether the cows’ milk on the market has any biologically significant
hormonal effects, two series of uterotrophic assays were performed, one with ovariectomized
young rats and the other with sexually immature female rats.

Materials and Methods
The low-fat (1%) milk in this study (Holstein milk sterilized at 130 ºC for 2 sec) was the same
one as that was used previously in the mammary carcinogenesis study (9). The artificial milk,
which was used as a negative control solution, contained the same amount of protein (gluten
fortified with lysine, DL-methionine, threonine and valine), fat (coconut oil) and carbohydrate(dextrin maltose) as the low-fat milk. The composition of the artificial milk has been
described elsewhere (9). A solution of estrone sulfate in the artificial milk (100 ng/ml) wasused as a positive control solution. The estrone sulfate (3-hydroxyestra-1,3,5(10)trine-17-one)
was obtained from Sigma Chemical Company (Tokyo, Japan).
The care and use of laboratory animals followed the Guidelines for Animal
Experiments of the Medical University of Yamanashi.
Ovariectomized rats
Female Wistar Galas Hannover rats, ovariectomized at 6 weeks, were purchasedfrom Nippon Clea (Tokyo, Japan). Upon receipt, the rats were housed, 3 per polycarbonate
cage, inside an air-conditioned animal room (22±2 ºC) with artificial lighting from 06:00 to
18:00 hour; the rats were provided with a diet of powdered chow (CE-2, Nippon Clea) andwater. After a week of acclimatization, the rats at 8 weeks of age were weighed, numbered
and randomly assigned to 3 groups of 12 animals each. Each group was then maintained on
the powdered chow plus one of the three test solutions as the only drinking fluid: low-fat milk
(LFM, esperimntal); artificial milk (AM, negative control); or artificial milk containing
estrone sulfate at 100 ng/ml (ES, positive control). Food and liquid solutions were renewed
daily at 10:00 hour. Daily consumption was determined as the difference between that which
was provided and that which remained unconsumed at 10:00 hour the next day. The
consumption was recorded in grams per cage (3 rats) per day. Body weight was measured
every day, starting just prior to the change of dietary regimen.
Immature rats
Thirteen-day-old female Wistar Galas Hannover rats were obtained from Nippon
Clea (Tokyo, Japan) as litters accompanied by the dam or a foster dam. Upon receipt, the rats2

were housed, one per polycarbonate cage, in the same air-conditioned animal room describedabove and were provided with powdered chow and water. When the baby animals reached 17days of age, they were weighed, numbered, and assigned to one of the three groups (LFM,
AM or ES), which each consisted of 12 rats. The immature female rats were then treatedessentially as described above for the ovariectomized rats, excepting that the vaginal opening
in immature rats was checked daily.
Autopsy
After being maintained on the test liquids for 7 days, the animals were killed by ether
inhalation at 16:00 hour, ~24 hours after the last treatment. The uteri were dissected free fromadhering fats, and the wet uterine weights were recorded to the nearest 0.1 mg. Then, the tipof each uterus was cut, and the uterus was place on filter paper and gently pressed to blot thefluid. The blotted weights of the uteri were recorded. The cell heights of uterine epithelia in
ovariectomized rats were determined on HE-stained sections using a microscope (Olympus
BX 50, Tokyo, Japan) with an attached image analyzer (Nippon Digital, Tokyo, Japan),according to Newbold et al. (10).
Statistical analysis
All data were analyzed by ANOVA using SPSS (SPSS Inc., Chicago, IL), followedby Dunnett’s multiple comparisons test, when significant differences existed among groups.
The 0.05 level of probability was used as the criterion of significance.

Results
Ovariectomized rats
Body weights were comparable among the three groups (Table 1). Both wet andblotted weights of theuteri in the Low-Fat
Milk group weresignificantly greater than
the respective weights in
the Artificial Milk group(negative control)(p<0.01). The ratio of
the wet uterine weight tothe body weight wasalso significantly higher
in the Low-Fat Milk
group than in the3Artificial Milk group (p<0.01).
The wet and blotted weights of the uteri in the Estrone Sulfate group (positive
control) were also significantly greater than the respective weights in the Artificial Milk
group (p<0.01). The ratio of the uterine weight to the body weight was higher in the Estrone
Sulfate than that in the Artificial Milk group (p<0.01) (Table 1). In general, both the absolute
ad relative values of the uterine weights were higher in the Estrone Sulfate group than thosein Low-Fat Milk group, but the difference between two groups was not significantly
significant (p>0.05).
The thickness of the uterine epithelia (mean±SD in µm) was significantly greater inthe Low-Fat Milk group (8.4±1.4) than that in the Artificial Milk group (6.8±1.6) (p<0.01).
The uterine epithelial cells of rats in the Estrone Sulfate group (9.4±1.7) were significantly
higher than the cells of rats in the Artificial Milk group (p<0.01). The difference in the cellheights between the Low-Fat Milk and Estrone Sulfate groups was not significantly
significant (p>0.05).
Immature rats
No significant difference in body weight was noted among the three groups (Low-Fat
Milk, Artificial Milk andEstrone Sulfate) ofimmature rats (Table 2).
Both wet and blottedsulfate uterine weightsof rats in the Low-Fat
Milk group weresignificantly greater thanthose of rats in the
Artificial Milk group(p<0.01), respectively.
The ratio of the wetuterine weight to bodyweight was alsosignificantly higher in the Low-Fat Milk group than that in the Artificial Milk group (p<0.05).
All the uterine values of rats in the Estrone Sulfate group were significantly higher
than the respective values of rats in both the Artificial Milk and the Low-Fat Milk groups(p<0.01).
None of the immature animals used had an open vagina during the uterotrophic assay(18-24 days-old).

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Discussion
There is growing concern regarding the decline of reproductive health (11-14), the increased
incidence of hormone-dependent cancers (15-20), and the frequent occurrence of prematurethelarche (21). Although endocrine-disrupting agents in the environment were blamed forthese phenomena (22), the possible role of endogenous estrogens from food has not bee
widely discussed. Indeed, the relative potency of estradiol-17β is 10-fold to 100,000-fold thatof most identified xenoestrogens (23).
The uterotrophic assay is considered the “gold standard” and is an essential
component when testing for estrogenicity, as it incorporates the effects of metabolism andpharmacokinetics (24). The present study clearly indicates that commercially available
low-fat milk has a weak but significant uterotrophic effect on both young ovariectomized rats
and immature rats with intact, undeveloped uteri.
The low-fat milk used in the present study contained about 380 pg/ml estrone sulfate,in addition to 210 pg/ml
estradiol-17β (free +conjugated) and 50
pg/ml estriol (free +conjugated) (9) (Table3). The uterotrophic
effect of the milk was
similar to the effect of
100 ng/ml estrone
sulfate in theovariectomized rats
(Table 1). However, theeffect of estrone sulfate
at the same
concentration was much
more pronounced in the immature rats than that in the young matured rats (Table 1 vs. Table2). The uteri of immature rats may be more sensitive than the uteri of young but sexuallymatured rats (25).
None of the immature animals had an open vagina during the uterotrophic assay.
Nonetheless, milk and estrone sulfate produced a clear uterotrophic effect. The observation ofpremature vaginal opening appears to be a less sensitive marker of estrogenic activity than isthe stimulation of uterine growth, as has been previously reported (26).
In conclusion, commercially available milk has uterotrophic effects in both young
ovariectomized rats and sexually immature rats. Further studies are necessary to ensure thesafety of milk and dairy products, particularly concerning their hormonal effects.
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