Abstract

Cowsﾕ 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.

Introduction

Cowsﾕ milk contains considerable amounts of estrogens (estrone, estradiol-17_ and estriol) (1). Because of modern dairy practices, 75% of commercial cowsﾕ 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 synthesis in the mammary glands (3).

The major estrogen in milk is estrone sulfate (4), which when consumed can be readily converted to estrone or estradiol-17b (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ﾕ 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 ｼC 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 sulfate estrone (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 purchased from Nippon Clea (Tokyo, Japan). Upon receipt, the rats were housed, 3 per polycarbonate cage, inside an air-conditioned animal room (22ｱ2 ｼC) 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.

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 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 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 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).

Statistical analysis
All data were analyzed by ANOVA using SPSS (SPSS Inc., Chicago, IL), followed by Dunnettﾕ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 and blotted weights of the uteri in the Low-Fat Milk group were significantly greater than the respective weights in the Artificial Milk group (negative control) (p<0.01). The ratio of the wet uterine weight to the body weight was also significantly higher in the Low-Fat Milk group than in the 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 significant (p>0.05).

The thickness of the uterine epithelia (meanｱSD in micrometer) was significantly greater in the Low-Fat Milk group (8.4ｱ1.4) than that in the Artificial Milk group (6.8ｱ1.6) (p<0.01). The uterine epithelial cells of rats in the Estrone Sulfate group (9.4ｱ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 significant (p>0.05).

Immature rats

No significant difference in body weight was noted among the three groups (Low-Fat Milk, Artificial Milk and Estrone Sulfate) of immature rats (Table 2). Both wet and blotted uterine weights of rats in the Low-Fat Milk group were significantly greater than those of rats in the Artificial Milk group (p<0.01), respectively. The ratio of the wet uterine weight to body weight was also 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 (p<0.01).

None of the immature animals used had an open vagina during the uterotrophic assay (18-24 days-old).

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 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-17b is 10-fold to 100,000-fold that of most identified xenoestrogens (23).

The uterotrophic assay is considered the ﾒgold standardﾓ 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, in addition to 210 pg/ml estradiol-17b (free + conjugated) and 50 pg/ml estriol (free + conjugated) (9) (Table 3). The uterotrophic effect of the milk was similar to the effect of 100 ng/ml estrone sulfate in the ovariectomized rats (Table 1). However, the effect 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. Table 2). The uteri of immature rats may be more sensitive than the uteri of young but sexually matured 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 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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