Perinatal exposure to bisphenol a increases adult mammary gland progesterone response and cell number

Abstract

Bisphenol A [BPA, 2,2,-bis (hydroxyphenyl) propane] is one of the highest-volume chemicals produced worldwide. It is detected in body fluids of more than 90% of the human population. Originally synthesized as an estrogenic compound, it is currently utilized to manufacture food and beverage containers resulting in uptake with food and drinks. There is concern that exposure to low doses of BPA, defined as less than or equal to 5 mg/kg body weight /d, may have developmental effects on various hormone-responsive organs including the mammary gland. Here, we asked whether perinatal exposure to a range of low doses of BPA is sufficient to alter mammary gland hormone response later on in life, with a possible impact on breast cancer risk. To mimic human exposure, we added BPA to the drinking water of C57/Bl6 breeding pairs. Analysis of the mammary glands of their daughters at puberty showed that estrogen-dependent transcriptional events were perturbed and the number of terminal end buds, estrogen-induced proliferative structures, was altered in a dose-dependent fashion. Importantly, adult females showed an increase in mammary epithelial cell numbers comparable to that seen in females exposed to diethylbestrol, a compound exposure to which was previously linked to increased breast cancer risk. Molecularly, the mRNAs encoding Wnt-4 and receptor activator of nuclear factor κB ligand, two key mediators of hormone function implicated in control of mammary stem cell proliferation and carcinogenesis, showed increased induction by progesterone in the mammary tissue of exposed mice. Thus, perinatal exposure to environmentally relevant doses of BPA alters long-term hormone response that may increase the propensity to develop breast cancer.

Fig. 1.

Perinatal BPA exposure and pubertal mammary gland development. A, Range of BPA doses used in the present study expressed as daily uptake (μg/kg-bw). Regulatory benchmarks and estimated human exposure are indicated. B–D, Quantitative RT-PCR analysis of relative mRNA expression of the estrogen-regulated genes PR, SLPI, and amphiregulin as well as ERα in mammary glands of females exposed perinatally to BPA or DES. Values were normalized to cytokeratin 18 (n =18–20 per condition). E and F, Stereomicrograph of a whole-mounted inguinal mammary gland of 30-d-old, unexposed female showing epithelial structures with TEB. E, Scale bar, 1 cm. Higher magnification of growing ductal tips; arrowheads point to TEB. F, Scale bar: 1 mm. G, Effect of BPA and DES on adjusted TEB numbers. The black curve represents median smooth. Note, daughters of mothers with a daily intake of 3 μg BPA/kg-bw show an increase in numbers of TEB, which is statistically significant. To evaluate statistical significance, t values were determined because the denominator degrees of freedom used to penalize certainty when computing F statistics were unknown given the multilevel data (* indicates statistical significance). NOAEL, No observed adverse effect level.

Fig. 2.

Perinatal BPA exposure and mammary cell populations in adulthood. A, Relative cell numbers in mammary glands from 3-month-old BPA- or DES-exposed or control animals. For each count, two to three animals were pooled per condition. Number of independent experiments is n = 5 f for DES exposure, n = 20 and n = 6, for 6/12 and 600/1200 μg/kg-bw BPA exposure, respectively. B, FACS analysis of single cells derived from mammary glands of exposed and unexposed females. The proportions of distinct cell populations characterized by distinct cell surface antigens are plotted over BPA and DES exposure doses (indicated values are in mg/kg-bw/d). Mammary epithelial cell compartments, luminal (CD24 high) and basal (CD24 low) and the percentage of fibroblasts (CD140a) and combined population of immune and endothelial cells (cocktail: CD45 and CD31) were not significantly altered with P > 0.06 for all conditions (from left to right: luminal, P = 0.34/0.67/0.44; basal, P = 0.07/0.42/0.94; fibroblasts, P = 0.29/0.58/0.34; immune/endothelial, P = 0.12/0.17/0.68). Each dot represents one mouse; experiments were performed in triplicate. Bars represent median values. ctrl, Control.

Fig. 3.

Perinatal BPA exposure alters response to progesterone. A and B, Histological sections of right inguinal mammary glands from control (panel A), or BPA-exposed (6 μg/kg-bw/d) (panel B), 12-month-old females, stained with antibody against PR. Scale bar, 50 mm. C, Bar graphs showing percentage of PR-positive luminal epithelial cells in mammary glands of unexposed and BPA-exposed females at 6 and 12 months of age (n = 5–7 per condition). More than 500 cells were counted in three distinct sectors. D, Basal levels of Wnt-4 mRNA are increased in the mammary glands of BPA-exposed (6 μg/kg-bw/d) females vs. their age-matched unexposed controls (n = 12). E and F, Bar graphs showing induction by R5020 of RANK (panel E) and Wnt-4 mRNA (panel F) in unexposed females (solid bars) and in daughters of parents with a daily intake of 6 μg/kg-bw BPA (open bars). Four independent experiments are shown. Ratios of RANKL and Wnt-4 mRNA induction in response to R5020 treatment between control and BPA-exposed animals are shown in right panels. Note, BPA exposure results in a significant increase in Wnt4 and RANKL induction. Bars represent mean values ± sd of triplicate RT-PCR. ctrl, Control.