Commercial cows’ milk has estrogenic activity as revealed by the hypertrophic effects on the uteri of young ovariectomized rats and immature rats
Ganmaa D, Tezuka H, Enkhmaa D, Hoshi K, Sato A.
Commercial cows’ milk has uterotrophic activity on the uteri of young
International Journal of Cancer 2006;118:2363-65.
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, two
series of uterotrophic tests were performed, one with young ovariectomized rats and the other
with 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 three
drinking 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 and
blotted 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 group
were 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 that
of 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.
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, are
elevated (2). The hormone levels in milk exceed those in blood, probably owing to hormone
The major estrogen in milk is estrone sulfate (4), which when consumed can be
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) was
used 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.
Female Wistar Galas Hannover rats, ovariectomized at 6 weeks, were purchased
from 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) and
water. 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.
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 rats
were housed, one per polycarbonate cage, in the same air-conditioned animal room described
above and were provided with powdered chow and water. When the baby animals reached 17
days 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 treated
essentially as described above for the ovariectomized rats, excepting that the vaginal opening
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 from
adhering fats, and the wet uterine weights were recorded to the nearest 0.1 mg. Then, the tip
of each uterus was cut, and the uterus was place on filter paper and gently pressed to blot the
fluid. 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).
All data were analyzed by ANOVA using SPSS (SPSS Inc., Chicago, IL), followed
by Dunnett’s multiple comparisons test, when significant differences existed among groups.
The 0.05 level of probability was used as the criterion of significance.
Body weights were comparable among the three groups (Table 1). Both wet and
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 those
in Low-Fat Milk group, but the difference between two groups was not significantly
The thickness of the uterine epithelia (mean±SD in µm) was significantly greater in
the 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 cell
heights between the Low-Fat Milk and Estrone Sulfate groups was not significantly
No significant difference in body weight was noted among the three groups (Low-Fat
immature rats (Table 2).
significantly 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
None of the immature animals used had an open vagina during the uterotrophic assay
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 premature
thelarche (21). Although endocrine-disrupting agents in the environment were blamed for
these 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 that
The uterotrophic assay is considered the “gold standard” and is an essential
component when testing for estrogenicity, as it incorporates the effects of metabolism and
pharmacokinetics (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,
conjugated) (9) (Table 3). The uterotrophic
(Table 1). However, the
more pronounced in the immature rats than that in the young matured rats (Table 1 vs. Table 2). The uteri of immature rats may be more sensitive than the uteri of young but sexually
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 of
premature vaginal opening appears to be a less sensitive marker of estrogenic activity than is
the 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 the
safety of milk and dairy products, particularly concerning their hormonal effects.
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