In utero exposure to bisphenol A shifts the window of susceptibility for mammary carcinogenesis in the rat

Abstract

Background: Bisphenol A (BPA) is a ubiquitous environmental chemical with reported endocrine-disrupting properties.

Objective: Our goal in this study was to determine whether prenatal exposure to BPA predisposes the adult rat mammary gland to carcinogenesis.

Methods: Pregnant rats were treated orally with 0, 25, or 250 microg BPA/kg body weight (BW) from gestation day (GD) 10 to GD21. For tumorigenesis experiments, prenatally exposed female offspring received a single gavage of 7,12-dimethylbenz(a)anthracene (DMBA; 30 mg/kg BW) on postnatal day (PND) 50, or PND100.

Results: Prenatal exposure of the dam to 250 microg BPA/kg BW combined with a single exposure of female offspring to DMBA on PND100, but not on PND50, significantly increased tumor incidence while decreasing tumor latency compared with the control group. Prenatal exposure of the dam to 250 microg BPA/kg BW, in the absence of DMBA to the female offspring, increased cell proliferation and elicited differential effects at the protein level at PND100 compared with PND50. Differentially regulated proteins in the mammary gland included estrogen receptor-alpha, progesterone receptor-A, Bcl-2, steroid receptor coactivators, epidermal growth factor receptor, phospho-insulinlike growth factor 1 receptor, and phospho-Raf.

Conclusions: Our study demonstrates that oral prenatal exposure to BPA increases mammary cancer susceptibility in offspring and shifts the window of susceptibility for DMBA-induced tumorigenesis in the rat mammary gland from PND50 to PND100. These changes are accompanied by differential effects of prenatal BPA exposure on the expression of key proteins involved in cell proliferation.

Figure 1

Western blot analysis of ER-α, PR-A, and Bcl-2 in mammary glands of (A) 50-day-old and (B) 100-day-old rats exposed prenatally to 250 μg BPA/kg BW or an equal volume of sesame oil (controls). Values represent mean density ± SE as a percentage of the control, with densitometry values for controls set to 100; n = 6–8 samples per group. Insets are representative immunoblots for each protein per treatment. *p < 0.05 compared with corresponding controls.

Figure 2

Western blot analysis of SRC-1, SRC-2, and SRC-3 in mammary glands of (A) 50-day-old and (B) 100-day-old rats exposed prenatally to 250 μg BPA/kg BW or an equal volume of sesame oil (controls). Values represent mean density ± SE as a percentage of the control, with densitometry values for controls set to 100; n = 6–8 samples per group. Insets are representative immunoblots for each protein per treatment. *p < 0.05 compared with corresponding controls.

Figure 3

Western blot analysis of EGFR, phospho-IGF-1R, phospho-c-Raf, phospho-ERK 1/2, phospho-ErbB2, and phospho-Akt in mammary glands of (A) 50-day-old and (B) 100-day-old rats exposed prenatally to 250 μg BPA/kg BW or an equal volume of sesame oil (controls). Values represent mean density ± SE as a percentage of the control, with densitometry values for controls set to 100; n = 6–8 samples per group. Insets are representative immunoblots for each protein per treatment. *p < 0.05 compared with corresponding controls.

Figure 4

Cell proliferation in mammary glands of 100-day-old rats prenatally exposed to 250 μg BPA/kg BW or an equal volume of sesame oil (controls). (A) Ki-67 expression as an indicator of cell proliferation. Ducts from six biologically distinct samples (n = 6) were analyzed per treatment; magnification, 40×; bar = 100 μm. (B) Contingency table of cell proliferation data. (C) Ki-67 labeling index values (mean ± SE) as a percentage of the control group. *p < 0.05 compared with controls.

Figure 5

Tumor incidence (proportion of rats that developed at least one tumor; A), tumor multiplicity (number of tumors per rat; B), and Kaplan-Meier survival curve with median time (days) to first tumor (C) in female offspring prenatally exposed to 250 μg BPA/kg BW or an equal volume of sesame oil (controls) and gavaged with a single dose of 30 mg DMBA/kg BW on PND100. Statistical analyses for tumorigenesis are described in “Statistical Methods.” **p = 0.022, and ^#p = 0.070 compared with the control group.