Prepubertal exposure to cow's milk reduces susceptibility to carcinogen-induced mammary tumorigenesis in rats

Abstract

Cow's milk contains high levels of estrogens, progesterone and insulin-like growth factor 1 (IGF-1), all of which are associated with breast cancer. We investigated whether prepubertal milk exposure affects mammary gland development and carcinogenesis in rats. Sprague-Dawley rats were given either whole milk or tap water to drink from postnatal day (PND) 14 to PND 35, and thereafter normal tap water. Mammary tumorigenesis was induced by administering 7,12-dimethylbenz[a]anthracene on PND 50. Milk exposure increased circulating E2 levels on PND 25 by 10-fold (p < 0.001) and accelerated vaginal opening, which marks puberty onset, by 2.5 days (p < 0.001). However, rats exposed to milk before puberty exhibited reduced carcinogen-induced mammary carcinogenesis; that is, their tumor latency was longer (p < 0.03) and incidence was lower (p < 0.05) than in the controls. On PND 25 and 50, mammary glands of the milk-exposed rats had significantly less terminal end buds (TEBs) than the tap water-exposed controls (p < 0.019). ER-α protein levels were elevated in the TEBs and lobules of milk rats, compared to rats given tap water (p < 0.019), but no changes in cyclin D1 expression, cell proliferation or apoptosis were seen. IGF-1 mRNA levels were reduced on PND 50 in the mammary glands of rats exposed to milk at puberty. Our results suggest that drinking milk before puberty reduces later risk of developing mammary cancer in rats. This might be mediated by a reduction in the number of TEBs and lower expression of IGF-1 mRNA in the mammary glands of milk-exposed animals.

FIGURE 1

(a) Serum estradiol levels on postnatal day (PND) 25 (n = 5–8/group). Significantly different from each other: *p<0.05. (b) Body weight on PND 50 and 75 (n = 4–6/group, means ± SEM shown). Control and milk-exposed rats do not differ from each other.

FIGURE 2

Effects of milk exposure on vaginal opening (n =60 per group). Rats were exposed to milk or tap water between PND 14 to 35, and then all shifted to tap water. Vaginal opening occurred significantly earlier in the milk group: p<0.001

FIGURE 3

Effects of prepubertal milk exposure on tumor incidence (n = 49 in the control and n=24 in the milk group). Milk-exposed rats exhibited significantly lower mammary tumor incidence: p<0.05.

FIGURE 4

Effects of prepubertal milk exposure on mammary gland morphology on PND 25 and 50. (a) Total number of terminal end buds (TEBs), and (b) density of alveolar buds and (c) lobules assessed visually using a scale between 0–5, are shown. Milk exposed rats had less TEBs (p<0.019) and lower lobular density (p<0.060) than the controls. In addition, the number of TEBs was lower (p<0.001) and the density of lobules was higher (p<0.001) on PND 50 than on PND 25. Values are expressed as mean ± SEM, n = 5–6 per group.

FIGURE 5

Effect of prepubertal milk exposure on mammary gland (a) cell proliferation (PCNA staining), assessed on a visual scale from 0–5, and (b) the number of apoptotic cells per 1000 cells in terminal end buds (TEB), lobulo-alveolar structures (Lobule) and ducts. Each value represents the mean ± SEM, n = 5–6 rats/group. Milk group did not differ from the tap water controls, but when compared to ducts, TEBs contained more proliferating cells (p<0.008) and cells which underwent apoptosis (p<0.002).

FIGURE 6

Effects of prepubertal milk exposure on (a) ER-α and (b) cyclin D1 expression determined using immunohistochemistry assessed in terminal end buds (TEB’s), lobulo-alveolar structures (Lobules) and ducts. Each value represents the mean ± SEM, n = 5–6 rats/group. Compared to tap water controls, milk-exposed rats expressed significantly higher levels of ER-α (p<0.001). In addition, ducts of control rats expressed significantly more ER-α than the lobules or TEBs (p<0.01); this was not seen in the milk group (interaction: p<0.038).

FIGURE 7

IGF-1 mRNA levels in the mammary glands of 50 day-old rats measured by RT-PCR. Values are expressed as mean ± SEM, n = 5–6 per group, significantly different from the control group: * p<0.05.