In Utero exposure to low-dose alcohol induces reprogramming of mammary development and tumor risk in MMTV-erbB-2 transgenic mice

Abstract

There is increasing evidence that prenatal exposure to environmental factors may modify breast cancer risk later in life. This study aimed to investigate the effects of in utero exposure to low-dose alcohol on mammary development and tumor risk. Pregnant MMTV-erbB-2 mice were exposed to alcohol (6 g/kg/day) between day 13 and day 19 of gestation, and the female offspring were examined for tumor risk. Whole mount analysis indicated that in utero exposure to low-dose alcohol induced significant increases in ductal extension at 10 weeks of age. Molecular analysis showed that in utero alcohol exposure induced upregulation of ERα signaling and activation of Akt and Erk1/2 in pubertal mammary glands. However, enhanced signaling in the EGFR/erbB-2 pathway appeared to be more prominent in 10-week-old glands than did signaling in the other pathways. Interestingly, tumor development in mice with in utero exposure to low-dose alcohol was slightly delayed compared to control mice, but tumor multiplicity was increased. The results indicate that in utero exposure to low-dose alcohol induces the reprogramming of mammary development by mechanisms that include altered signaling in the estrogen receptor (ER) and erbB-2 pathways. The intriguing tumor development pattern might be related to alcohol dose and exposure conditions, and warrants further investigation.

Figure 1

Plasma E2 levels on day 18 of gestation in MMTV-erbB-2 mice with alcohol exposure. The mice were exposed to alcohol via oral feeding of 0.5 mL of 10% (days 10–12) or 20% (days 13–19) alcohol/water twice a day. n = 5 animals per group. p = 0.09.

Figure 2

In utero exposure to alcohol results in lower body weight. MMTV-erbB-2 mice were exposed to 3 g/kg/day alcohol between day 10 and day 12 and 6 g/kg/day alcohol between day 13 and day 19 of gestation by feeding. The body weight of the pups was measured weekly for the first 4 weeks. * p < 0.05; ** p < 0.01.

Figure 3

In utero exposure to alcohol induces prolonged mammary ductal extension. Mammary whole mounts were harvested from control and alcohol-exposed mice at 5 and 10 weeks of age and stained with carmine red (A1,A2,B1,B2). The number of terminal end buds (TEBs) at 5 weeks (A3) and the ductal extension beyond the lymph node (LN) at 10 weeks (B3) were quantified. Six glands from each group were examined. ** p < 0.01.

Figure 4

In utero exposure to low-dose alcohol induces activation of the ER pathway in pubertal mammary glands. Protein lysates were prepared from mammary tissue from five-week-old mice after in utero treatments. Protein levels of the indicated markers of the ER and erbB-2 pathways were detected using Western blotting (A,B). Each group contained three samples from different mice that had undergone the same treatment.

Figure 5

In utero exposure to alcohol induced transcriptional regulation of ER target genes and growth factor receptors and cognate ligands. Total RNA was extracted from mammary tissue from five-week-old MMTV-erbB-2 mice after different in utero treatments. Equal amounts of RNA from the third pair of glands from four different mice were pooled for further analysis. The relative mRNA levels of individual genes were detected by qRT-PCR in triplicate. Bars represent standard error of triplicate measurements in the qRT-PCR assay.

Figure 6

In utero exposure to alcohol induces the activation of the ER pathway at 10 weeks of age. Mammary tissues from control and alcohol-exposed mice were harvested at 10 weeks of age. Protein and mRNA levels of the indicated markers were detected using Western blot (A,B) and real time RT-PCR (C), respectively. Bars in C represent standard error of triplicate measurements in the qRT-PCR assay.

Figure 7

In utero exposure to alcohol modifies mammary tumor development in MMTV-erbB-2 transgenic mice. (A) Kaplan–Meier curves for tumor-free animals were calculated based on the tumor latency of the control (circle) and alcohol-exposed (square) mice. p > 0.05; and (B) Tumor multiplicity in MMTV-erbB-2 mice after different in utero treatments. Tumor multiplicity was based on the number of tumors in each animal by euthanization, when the first tumor reached 1.8 cm³ in volume. * p < 0.05.