Bisphenol A increases mammary cancer risk in two distinct mouse models of breast cancer

Abstract

Bisphenol A (BPA) is an industrial plasticizer that leaches from food containers during normal usage, leading to human exposure. Early and chronic exposure to endocrine-disrupting environmental contaminants such as BPA elevates the potential for long-term health consequences. We examined the impact of BPA exposure on fetal programming of mammary tumor susceptibility as well as its growth promoting effects on transformed breast cancer cells in vivo. Fetal mice were exposed to 0, 25, or 250 μg/kg BPA by oral gavage of pregnant dams. Offspring were subsequently treated with the known mammary carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA). While no significant differences in postnatal mammary development were observed, both low- and high-dose BPA cohorts had a statistically significant increase in susceptibility to DMBA-induced tumors compared to vehicle-treated controls. To determine if BPA also promotes established tumor growth, MCF-7 human breast cancer cells were subcutaneously injected into flanks of ovariectomized NCR nu/nu female mice treated with BPA, 17beta-estradiol, or placebo alone or combined with tamoxifen. Both estradiol- and BPA-treated cohorts formed tumors by 7 wk post-transplantation, while no tumors were detected in the placebo cohort. Tamoxifen reversed the effects of estradiol and BPA. We conclude that BPA may increase mammary tumorigenesis through at least two mechanisms: molecular alteration of fetal glands without associated morphological changes and direct promotion of estrogen-dependent tumor cell growth. Both results indicate that exposure to BPA during various biological states increases the risk of developing mammary cancer in mice.

FIG. 1.

Fetal exposure to BPA leads to early vaginal opening in FVB/N female mice. Pregnant FVB/N females were treated with 25 μg kg⁻¹ day⁻¹ bisphenol A or vehicle control by oral gavage beginning on Postcoital Day 8. Female offspring were observed for vaginal opening beginning on Postnatal Day 16. Bars are means ± SD (*P < 6 E-05). Each experimental group contained a minimum of five mice from at least three litters.

FIG. 2.

Fetal exposure to bisphenol A (25 μg kg⁻¹ day⁻¹) by oral gavage has no detectable effect on morphological development of the mammary gland in FVB/N female mice. Pregnant FVB/N females were treated with 25 μg kg⁻¹ day⁻¹ bisphenol A or vehicle control by oral gavage beginning on Postcoital Day 8. Mammary glands were collected from female offspring at 3, 5, and 8 wk of age as well as involution on Day 5 following delivery and nursing of one litter. A) Histology of Carmine Alum-stained whole mounts. B) The distance from mid-lymph node to terminal end bud was quantified. At least five mice from two independent litters were measured for each treatment group. Negative values occur in the 3-wk cohorts because the ducts have not yet reached the midpoint of the lymph node.

FIG. 3.

Fetal exposure to bisphenol A or vehicle control results in increased susceptibility to mammary carcinogenesis after exposure to 7,12-dimethylbenz[a]anthracene (DMBA). Pregnant FVB/N females were treated with 25 μg kg⁻¹ day⁻¹ or 250 μg kg⁻¹ day⁻¹ bisphenol A or vehicle control by oral gavage beginning on Postcoital Day 8. Prenatally exposed female offspring were then treated with DMBA (1 mg/mouse) by oral gavage one dose each at 5 wk and again at 6 wk of age. Female mice were palpated weekly to monitor the formation of mammary tumors. Kaplan-Meier survival analysis is shown. At least 10 mice from each treatment group represent at least three independent litters.

FIG. 4.

Histology of 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumors from FVB/N female mice. Tumors were collected from FVB/N females 1 wk after detection of a palpable tumor, fixed in 4% paraformaldehyde, and stained with hematoxylin and eosin. The only tumor that developed in the vehicle treated mice is shown with representative images of tumors from BPA exposed animals. Magnification ×20.

FIG. 5.

17β-estradiol (solid line) or bisphenol A (dashed line) promote growth of MCF-7 tumors in NCR nu/nu mice. Eight-week-old NCR nu/nu females were ovariectomized and implanted with a 17β-estradiol (1.7 mg/60-day release), bisphenol A (37.5 mg pellet/60-day release), or placebo pellet (37.5 mg pellet/60-day release). One week later, 1 × 10⁶ MCF-7 cells were injected subcutaneously into the flanks of each female (placebo and 17β-estradiol, n = 7; BPA, n = 6). Mice were palpated for tumor formation, and mammary tumor size was measured weekly. Average tumor volume is statistically different between 17β-estradiol and bisphenol A treated cohorts, as indicated: *P < 0.001.

FIG. 6.

A) MCF-7 tumors from mice treated with 17β-estradiol (1.7 mg/60-day release) or BPA (37.5 mg/60-day release) are histologically identical. Xenograft tumor sections from each cohort were stained with hematoxylin and eosin. Representative sections are shown. B) MCF-7 cell xenograft tumors from the 17β-estradiol cohort have a higher percentage of proliferating cells as revealed with MKI67 immunostaining. Xenograft tumor sections were immunostained with an anti-MKI67 antibody. Representative sections are shown. Magnification ×20. C) The percentage of MKI67 positively stained cells was quantified in three mice from each treatment group. Bars are means ± SD (*P < 1.2 E-6).

FIG. 7.

Tamoxifen treatment abrogates estrogen (solid line) or bisphenol A (dashed line) induced growth of MCF-7 tumors. Eight-week-old NCR nu/nu females were ovariectomized and implanted with either 17β-estradiol (1.7 mg/60-day release), bisphenol A (37.5 mg pellet/60-day release), or placebo pellets (37.5 mg pellet/60-day release). One week later, 1 × 10⁶ MCF-7 cells were injected subcutaneously into the flanks of each female. Seven weeks after tumor formation was first detected, females were treated daily with tamoxifen (1 mg mouse⁻¹ day⁻¹) by oral gavage. At 60 days post tumor cell injection, the mice were given a second 17β-estradiol, BPA, or placebo pellet to maintain levels throughout the remainder of the experiment. Mice were palpated and mammary tumors measured weekly (n = 3 for estradiol and BPA).