New insights into fetal mammary gland morphogenesis : differential effects of natural and environmental estrogens

An increased breast cancer risk during adulthood has been linked to estrogen exposure during fetal life. However, the impossibility of removing estrogens from the feto-maternal unit has hindered the testing of estrogen's direct effect on mammary gland organogenesis. To overcome this limitation, we developed an ex vivo culture method of the mammary gland where the direct action of estrogens can be tested during embryonic days (E) 14 to 19. Mouse mammary buds dissected at E14 and cultured for 5 days showed that estrogens directly altered fetal mammary gland development. Exposure to 0.1 pM, 10 pM, and 1 nM 17 beta-estradiol (E2) resulted in monotonic inhibition of mammary buds ductal growth. In contrast, Bisphenol-A (BPA) elicited a non-monotonic response. At environmentally relevant doses (1 mu M), BPA significantly increased ductal growth, as previously observed in vivo, while 1 mu M BPA significantly inhibited ductal growth. Ductal branching followed the same pattern. This effect of BPA was blocked by Fulvestrant, a full estrogen antagonist, while the effect of estradiol was not. This method may be used to study the hormonal regulation of mammary gland development, and to test newly synthesized chemicals that are released into the environment without proper assessment of their hormonal action on critical targets like the mammary gland.Peer reviewe

Perinatal Bisphenol A Exposure Increases Estrogen Sensitivity of the Mammary Gland in Diverse Mouse Strains

BACKGROUND: Studies of low-dose effects of xenoestrogens have yielded conflicting results that may be attributed to differences in estrogen sensitivity between the rodent strains examined. Perinatal exposure of CD-1 mice to low doses of the xenoestrogen bisphenol A (BPA) alters peripubertal mammary gland development. Future studies to assess the role of estrogen receptors as mediators of BPA action require estrogen receptor knock-out mice that were generated on a C57Bl6 background. The sensitivity of the C57Bl6 strain to estradiol and BPA is unknown. OBJECTIVES: In the present study we examined whether the mammary glands of CD-1 and C57Bl6 mice exhibited similar responses to 17β-estradiol (E(2)) and whether perinatal exposure to BPA equally enhanced sensitivity of the mammary glands to E(2) at puberty. METHODS: Immature mice were ovariectomized and treated for 10 days with one of eight doses of E(2). Morphological mammary gland parameters were examined to identify doses producing half-maximal effects. Mice were exposed perinatally to 0 or 250 ng BPA/kg body weight (bw)/day from gestational day 8 until postnatal day (PND) 2. On PND25, female offspring were ovariectomized and given an estrogen challenge of 0, 0.5, or 1 μg E(2)/kg bw/day for 10 days. Morphometric parameters of the mammary gland were compared between strains. RESULTS: Both strains exhibited similar responses to E(2). Perinatal BPA exposure altered responses to E(2) at puberty for several parameters in both strains, although the effect in CD-1 was slightly more pronounced. CONCLUSION: Both mouse strains provide adequate models for the study of perinatal exposure to xenoestrogens

Perinatal Exposure to Environmentally Relevant Levels of Bisphenol A Decreases Fertility and Fecundity in CD-1 Mice

Bac k g r o u n d: Perinatal exposure to low-doses of bisphenol A (BPA) results in alterations in the ovary, uterus, and mammary glands and in a sexually dimorphic region of the brain known to be important for estrous cyclicity. Objectives: We aimed to determine whether perinatal exposure to environmentally relevant doses of BPA alters reproductive capacity. Met h o d s: Female CD-1 mice that were exposed to BPA at 0, 25 ng, 250 ng, or 25 µg/kg body weight (BW)/day or diethylstilbestrol (DES) at 10 ng/kg BW/day (positive control) from gestational day 8 through day 16 of lactation were continuously housed with proven breeder males for 32 weeks starting at 2 months of age. At each delivery, pups born to these mating pairs were removed. The cumulative number of pups, number of deliveries, and litter size were recorded. The purity of the BPA used in this and our previous studies was assessed using HPLC, mass spectrometry, and nuclear magnetic resonance. Res u l t s: The forced breeding experiment revealed a decrease in the cumulative number of pups, observed as a nonmonotonic dose–response effect, and a decline in fertility and fecundity over time in female mice exposed perinatally to BPA. The BPA was 97 % pure, with no evidence of contaminatio

Evidence of Altered Brain Sexual Differentiation in Mice Exposed Perinatally to Low, Environmentally Relevant Levels of Bisphenol A

Humans are routinely exposed to bisphenol A (BPA), an estrogenic chemical present in food and beverage containers, dental composites, and many products in the home and workplace. BPA binds both classical nuclear estrogen receptors and facilitates membrane-initiated estrogenic effects. Here we explore the ability of environmentally relevant exposure to BPA to affect anatomical and functional measures of brain development and sexual differentiation. Anatomical evidence of alterations in brain sexual differentiation were examined in male and female offspring born to mouse dams exposed to 0, 25, or 250 ng BPA/kg body weight per day from the evening of d 8 of gestation through d 16 of lactation. These studies examined the sexually dimorphic population of tyrosine hydroxylase (TH) neurons in the rostral periventricular preoptic area, an important brain region for estrous cyclicity and estrogen-positive feedback. The significant sex differences in TH neuron number observed in control offspring were diminished or obliterated in offspring exposed to BPA primarily because of a decline in TH neuron number in BPA-exposed females. As a functional endpoint of BPA action on brain sexual differentiation, we examined the effects of perinatal BPA exposure on sexually dimorphic behaviors in the open field. Data from these studies revealed significant sex differences in the vehicle-exposed offspring that were not observed in the BPA-exposed offspring. These data indicate that BPA may be capable of altering important events during critical periods of brain development

Evidence of Absence: Estrogenicity Assessment of a New Food-Contact Coating and the Bisphenol Used in Its Synthesis

Consumer concerns about exposure to substances found in food contact materials with estrogenic activity (EA) have created substantial demand for alternatives. We assessed the potential EA of both a new bisphenol monomer used to synthesize polymeric coatings for metal food-contact applications and the nonintentionally added substances (NIAS) that may migrate into food. We evaluated tetramethyl bisphenol F (TMBPF) using <i>in vitro</i> and <i>in vivo</i> assays. We extracted the polymeric coating using food simulants ethanol (50% v/v) and acetic acid (3% w/v) and measured migration using tandem liquid chromatography (LC)/mass spectrometry (MS) and LC time-of-flight MS for TMBPF and NIAS, respectively. We also tested migrants for EA using the E-SCREEN assay. TMBPF did not show estrogenic activity in the uterotrophic assay and did not alter puberty in male and female rats or mammary gland development in female rats. Neither TMBPF nor the migrants from the final polymeric coating increased proliferation of estrogen-sensitive MCF7 cells. TMBPF did not show estrogen-agonist or antagonist activity in the estrogen receptor-transactivation assay. TMBPF migration was below the 0.2 parts per billion detection limit. Our findings provide compelling evidence for the absence of EA by TMBPF and the polymeric coating derived from it and that human exposure to TMBPF would be negligible

Dynamic metabolic disruption in rats perinatally exposed to low doses of bisphenol-A

Along with the well-established effects on fertility and fecundity, perinatal exposure to endocrine disrupting chemicals, and notably to xeno-estrogens, is strongly suspected of modulating general metabolism. The metabolism of a perinatally exposed individual may be durably altered leading to a higher susceptibility of developing metabolic disorders such as obesity and diabetes; however, experimental designs involving the long term study of these dynamic changes in the metabolome raise novel challenges. H-1-NMR-based metabolomics was applied to study the effects of bisphenol-A (BPA, 0; 0.25; 2.5, 25 and 250 mu g/kg BW/day) in rats exposed perinatally. Serum and liver samples of exposed animals were analyzed on days 21, 50, 90, 140 and 200 in order to explore whether maternal exposure to BPA alters metabolism. Partial Least Squares-Discriminant Analysis (PLS-DA) was independently applied to each time point, demonstrating a significant pair-wise discrimination for liver as well as serum samples at all time-points, and highlighting unequivocal metabolic shifts in rats perinatally exposed to BPA, including those exposed to lower doses. In BPA exposed animals, metabolism of glucose, lactate and fatty acids was modified over time. To further explore dynamic variation, ANOVA-Simultaneous Component Analysis (A-SCA) was used to separate data into blocks corresponding to the different sources of variation (Time, Dose and Time*Dose interaction). A-SCA enabled the demonstration of a dynamic, time/age dependent shift of serum metabolome throughout the rats' lifetimes. Variables responsible for the discrimination between groups clearly indicate that BPA modulates energy metabolism, and suggest alterations of neurotransmitter signaling, the latter finding being compatible with the neurodevelopmental effect of this xenoestrogen. In conclusion, long lasting metabolic effects of BPA could be characterized over 200 days, despite physiological (and thus metabolic) changes connected with sexual maturation and aging

Pairwise PLS-DA model parameter comparisons.

<p>^a number of observations in the dataset</p><p>^<i>b</i> number of latent variables included in the PLS-DA model</p><p>^<i>c</i> % of variance explained by the PLS-DA model</p><p>^<i>d</i> predictive ability of the PLS-DA model calculated by 7-fold cross-validation</p><p>^<i>e</i> Intercept values (R2/Q2) of permutation test. The intercept values (R2/Q2) represent the values of R2 and Q2 of a purely random model. A robust model has a Q² intercept value < 0.</p><p>Pairwise PLS-DA model parameter comparisons.</p

A-SCA of serum samples ("Reduced" dataset).

<p>A) One-dimensional PCA score plot of the Time * Dose interaction. The horizontal axis represents the time course, and the vertical axis represents the score value for the first principal component (86.1% of explained variability). Each dot represents the average of animals euthanized at the time point mentioned on the horizontal axis. The solid line corresponds to BPA0.25 and the dashed line corresponds to BPA25. B) Metabolic pathways modulated by concentration (0.25 <i>Vs</i> 25μg /kg BW/day) over time focusing on the biochemical pathways including the metabolites in which concentrations were found to be increased.</p