Mónica Muñoz-de-Toro

Prenatal bisphenol A exposure induces preneoplastic lesions in the mammary gland in Wistar rats.

Background Humans are routinely exposed to bisphenol A (BPA), an estrogenic compound that leaches from dental materials, food and beverage containers, and other consumer products. Prenatal exposure to BPA has produced long-lasting and profound effects on rodent hormone-dependent tissues that are manifested 1–6 months after the end of exposure. Objective The aim of the present work was to examine whether in utero exposure to BPA alters mammary gland development and increases its susceptibility to the carcinogen N-nitroso-N-methylurea (NMU). Methods Pregnant Wistar rats were exposed to BPA (25 μg/kg body weight per day) or to vehicle. Female offspring were sacrificed on postnatal day (PND) 30, 50, 110, or 180. On PND50 a group of rats received a single subcarcinogenic dose of NMU (25 mg/kg) and they were sacrificed on either PND110 or PND180. Results At puberty, animals exposed prenatally to BPA showed an increased proliferation/apoptosis ratio in both the epithelial and stromal compartments. During adulthood (PND110 and PND180), BPA-exposed animals showed an increased number of hyperplastic ducts and augmented stromal nuclear density. Moreover, the stroma associated with hyperplastic ducts showed signs of desmoplasia and contained an increased number of mast cells, suggesting a heightened risk of neoplastic transformation. Administration of a subcarcinogenic dose of NMU to animals exposed prenatally to BPA increased the percentage of hyperplastic ducts and induced the development of neoplastic lesions. Conclusions Our results demonstrate that the prenatal exposure to low doses of BPA perturbs mammary gland histoarchitecture and increases the carcinogenic susceptibility to a chemical challenge administered 50 days after the end of BPA exposure.

Sex reversal effects on Caiman latirostris exposed to environmentally relevant doses of the xenoestrogen bisphenol A

Exposure to environmental contaminants known as endocrine disruptors (EDs) alters the development and function of reproductive organs in several species. Bisphenol A (BPA) is an estrogenic chemical that leaches from dental materials and plastic food and beverage containers. BPA has been found in sewage, surface and drinking water, and therefore poses a potentially significant risk for human and wildlife. Prenatal exposure of rodents to environmentally relevant doses of BPA alters the development of the reproductive organs of male and female offspring. Species with temperature dependent sex determination (TSD) could act as sentinels of ecosystem health by providing sensitive biomarkers of endocrine disruptors effects. We selected Caiman latirostris as an animal model to study endocrine disruption caused by BPA. The aim of this study was to determine whether exposure in ovum to BPA could cause estrogen-like effects on the reproductive system of C. latirostris. Sex determination and gonadal histoarchitecture were the endpoints evaluated after in ovum exposure to different doses of BPA and 17beta-estradiol (E(2)). We confirmed that C. latirostris is a species with TSD and additionally demonstrated that BPA causes estrogen-like developmental effects by reversing gonadal sex and altering gonadal histoarchitecture. Differences in responses to BPA and E(2) in our in vivo system were on the order of 100-fold. In contrast published in vitro studies have reported differences on the order of 10,000x or more. These results support the utility of C. latirostris, a species in which sex determination is temperature dependent, as a tool in assessing estrogenic activity in vivo and as a sentinel to monitor EDs in aquatic environment.

Neonatal exposure to bisphenol A reduces the pool of primordial follicles in the rat ovary

We evaluated whether exposure to bisphenol A (BPA) disrupts neonatal follicle development in rats. From postnatal day 1 (PND1) to PND7, pups received corn oil (control), diethylstilbestrol (DES20: 20 μg/kg-d, DES0.2: 0.2 μg/kg-d), or BPA (BPA20: 20mg/kg-d, BPA0.05: 0.05 mg/kg-d). We examined follicular dynamics, multioocyte follicles (MOFs) incidence, proliferation and apoptosis rates, expression of steroid receptors (ERα, ERβ, PR, AR) and cyclin-dependent kinase inhibitor 1B (p27) in PND8 ovaries. DES20, DES0.2 and BPA20-ovaries showed fewer primordial follicles and increased growing follicles. DES20-ovaries exhibited increased incidence of MOFs. Oocyte survival, AR, PR and apoptosis were not changed. Primordial and recruited follicles from BPA20-ovaries showed higher p27, whereas ERβ and proliferation were both increased in recruited follicles. ERα positive primary follicles increased in BPA 20-ovaries. Results show that BPA reduces the primordial follicle pool by stimulating the neonatal initial recruitment, associated with an increased proliferation rate likely mediated by an estrogenic pathway.

Neonatal Exposure to Bisphenol A Alters Rat Uterine Implantation-Associated Gene Expression and Reduces the Number of Implantation Sites

Endocrine disrupters have been associated with reproductive pathologies such as infertility and gynecological tumors. Using a rat model of early postnatal exposure to bisphenol A (BPA), we evaluated the long-term effects on 1) female reproductive performance, 2) uterine homeobox A10 (Hoxa10) and Hoxa10-target gene expression, and 3) ovarian steroid levels and uterine estrogen receptor α and progesterone (P) receptor expression. Newborn female rats received vehicle, BPA.05 (0.05 mg/kg · d), BPA20 (20 mg/kg · d), diethylstilbestrol.2 (0.2 μg/kg · d), or diethylstilbestrol 20 (20 μg/kg · d) on postnatal d 1, 3, 5, and 7. A significant decrease in the number of implantation sites was assessed in the xenoestrogen-exposed females. To address the molecular effects of postnatal xenoestrogen exposure on the pregnant uterus, we evaluated the expression of implantation-associated genes on d 5 of pregnancy (preimplantation uterus). All xenoestrogen-treated rats showed a lower expression of Hoxa10. In the same animals, two Hoxa10-downstream genes were misregulated in the uterus. β(3) Integrin, which is up-regulated by Hoxa10 in controls, was decreased, whereas empty spiracles homolog 2, which is down-regulated by Hoxa10, was increased. Furthermore a clear down-regulation of estrogen receptor α and P receptor expression was detected without changes in estradiol and P serum levels. The early exposure to BPA produced a lower number of implantation sites in association with a defective uterine environment during the preimplantation period. Alterations in the endocrine-regulated Hoxa10 gene pathways (steroid receptors--Hoxa10--β(3) integrin/empty spiracles homolog 2) could explain, at least in part, the BPA effects on the implantation process.

Developmental Exposure to Bisphenol A Impairs the Uterine Response to Ovarian Steroids in the Adult

Morphoregulator genes like members of the Hox gene family regulate uterine development and are associated with endocrine-related processes such as endometrial proliferation and differentiation in the adult uterus. Exposure to neonatal endocrine disruptors could affect signaling events governed by Hox genes, altering the developmental trajectory of the uterus with lasting consequences. We investigated whether neonatal exposure to bisphenol A (BPA) alters Hoxa10 and Hoxa11 mRNA uterine expression shortly after treatment as well as in the adult. Moreover, we studied whether xenoestrogen exposure may affect the adult uterine response to hormonal stimuli. Newborn females received vehicle, 0.05 mg/kg.d BPA, 20 mg/kg*d BPA, or diethylstilbestrol (0.2 microg/kg*d) on postnatal d 1, 3, 5, and 7). At postnatal d 8, real time RT-PCR assays showed a decrease in Hoxa10 and Hoxa11 expression in all xenoestrogen-treated groups. To evaluate the long-term effects, we used adult ovariectomized rats with hormonal replacement. The subepithelial stroma in BPA- and diethylstilbestrol-treated animals showed an impaired proliferative response to steroid treatment associated with a silencing of Hoxa10 but not associated with changes in the methylation pattern of the Hoxa10 promoter. BPA animals showed that the Hoxa10 reduction was accompanied by an increased stromal expression of the silencing mediator for retinoic acid and thyroid hormone receptor. The spatial coexpression of steroid receptors Hoxa10 and silencing mediator for retinoic acid and thyroid hormone receptor was established using immunofluorescence. Our data indicate that postnatal BPA exposure affects the steroid hormone-responsiveness of uterine stroma in adulthood. Whether this impaired hormonal response is associated with effects on uterine receptivity and decidualization is currently under investigation.

Neonatal exposure to bisphenol A or diethylstilbestrol alters the ovarian follicular dynamics in the lamb.

We hypothesized that neonatal xenoestrogen exposure affects the ovarian follicular dynamics in lambs. Female lambs were exposed from postnatal day (PND) 1-14 to low doses of diethylstilbestrol (DES) or bisphenol A (BPA). At PND 30, the follicular dynamics and ovarian biomarkers (ERα, ERβ, AR, Ki67, p27) were evaluated. Lambs exposed to DES or BPA showed a decline in the stock of primordial follicles with stimulation of follicular development. BPA reduced ovarian weight and increased the number of multioocyte follicles. BPA promoted proliferation of granulosa/theca cells in antral follicles, and increased both the number of antral atretic follicles and p27 expression. Neonatal exposure to BPA or DES reduced the primordial follicle pool by stimulating their initial recruitment and subsequent follicle development until antral stage. In prepubertal lambs, the accelerated folliculogenesis resulted in increased incidence of atretic follicles. These alterations may affect the ovarian function in the adult.

Cellular Turnover in the Rat Uterine Cervix and Its Relationship to Estrogen and Progesterone Receptor Dynamics

Abstract Histoarchitectural changes of the uterine cervix allow its successful adaptation to different physiological conditions. In this study, we evaluated cell turnover in each cellular compartment of the uterine cervix in association with steroid hormone receptor expression in order to establish the range of physiological changes. Proliferation, apoptosis, and progesterone receptor (PR) and estrogen receptor α (ERα) expression were evaluated in cycling, pregnant, and postpartum rats. In estrus and diestrus II, ERα and PR expression exhibited variations according to the region evaluated. Proliferation and apoptosis showed a reciprocal pattern, the epithelium being the region with higher cell turnover. High apoptotic index (AI) in estrus was associated with the lowest ERα and the highest PR scores. During pregnancy, proliferation of the epithelium was the predominant event and AI was low. On Postpartum Day 1 (PPD1), proliferation decreased while apoptosis increased. As described for the estrous cycle, during pregnancy and PPD1, AI and ERα were negatively correlated. In the fibroblastic stroma, low proliferation was observed throughout pregnancy; however, there was a net increase in cell number because very few cells underwent apoptosis. No difference in ERα was observed in fibroblastic cells during pregnancy and postpartum; however, a great decrease of this receptor in the epithelial compartment was observed after delivery. Unlike cervical epithelium, PR was highly expressed in stromal cells. At term, a dramatic increase in epithelial PR was observed. While epithelial PR remained high on PPD1, a decrease was observed in muscle stroma. These results show that, in all stages studied, 1) ERα and PR have different patterns of expression with differential responses to signals that modulate proliferation and/or apoptosis depending on the cellular compartment, and 2) even though the epithelium is the region with the highest cell turnover, the fibroblastic and muscle stroma are active regions that have their own patterns of behavior.

Developmental exposure to endocrine disruptor chemicals alters follicular dynamics and steroid levels in Caiman latirostris.

Human and wildlife are exposed at critical periods of development to endocrine disruptor chemicals (EDC) that may be responsible for reproductive disorders. To test the hypothesis that in ovum exposure to EDC at a critical period for gonadal organogenesis alters post-hatching folliculogenesis and steroidogenesis in Caiman latirostris, we studied the impact of in ovum exposure to 17 beta-estradiol (E2), bisphenol A (BPA), endosulfan (END) and atrazine (ATZ) on gonadal differentiation, follicular dynamics and circulating levels of steroid hormones in neonatal and juvenile caiman. Since C. latirostris is a species with temperature dependent sex determination, eggs were incubated at male (33 degrees C) or female (30 degrees C) producing temperatures and the effect of EDC was evaluated. Neonatal ovaries exhibited germ cells mainly located in clusters evidencing proliferative activity and type I to III follicles. Juvenile ovaries exhibited germ cells and advanced stages of pre-vitellogenic follicles. Prenatal exposure to the highest doses of E2 (1.4 ppm) or BPA (140 ppm) overrode male temperature effect on sex determination. Neonatal females produced by sex reversion lacked type III follicles, while females prenatally exposed to the lowest doses of E2 (0.014 ppm) and BPA (1.4 ppm) or ATZ (0.2 ppm) showed an increase in type III follicles. Juvenile caiman prenatally exposed to E2 or BPA showed an augmented incidence of multioocyte follicles. Neonatal female caiman exposed in ovum to E2 or BPA had higher estrogen serum levels whereas exposure to E2, BPA, ATZ and END decreased T levels. Present data demonstrates that exposure to EDC during gonadal organogenesis alters follicular dynamics and steroid levels later in life. These effects might have an impact on caiman fertility.

Neonatal exposure to bisphenol A alters estrogen-dependent mechanisms governing sexual behavior in the adult female rat

This study examines the effects of neonatal exposure to the endocrine disruptor bisphenol A (BPA) on the hypothalamic circuitry controlling the female sexual behaviors of adult rats. From postnatal day 1 (PND1) to PND7, pups were injected with corn oil (control) or BPA (BPA20: 20mg/kg-d; BPA.05: 0.05 mg/kg-d) and at PND85 the rats were bilaterally ovariectomized (OVX). At PND100, OVX-rats received estradiol alone or estradiol and progesterone to evaluate estrogen-dependent gene expression in the hypothalamus and sexual behavior. In BPA-exposed females, estrogen receptor alpha (ER alpha) expression was down-regulated in both the medial preoptic (MPN) and ventromedial nucleus (VMHvl), while repressor of estrogen receptor activity (REA) expression was up-regulated in the VMHvl. Interestingly, BPA-exposed females displayed significantly lower levels of proceptive behavior. Our results show that BPA permanently alters the hypothalamic estrogen-dependent mechanisms that govern sexual behavior in the adult female rat.

Endosulfan modulates estrogen-dependent genes like a non-uterotrophic dose of 17β-estradiol

The estrogenic activity of environmentally relevant doses of endosulfan was investigated using an animal model. Ovariectomized adult rats were injected once a day for 3 days with sesame oil (control), 0.02mg/kg/day 17beta-estradiol (an uterotrophic dose; UE(2)), 0.0002mg/kg/day 17beta-estradiol (a non-uterotrophic dose; NUE(2)), or 0.006, 0.06, 0.6 or 6mg/kg/day endosulfan. After 24h of treatment, the uteri were weighed (uterotrophic assay) and the luminal epithelial cell height (LECH) and progesterone receptor (PR), and estrogen receptor alpha (ERalpha) protein levels were measured. PR, ERalpha, and complement factor-3 (C3) mRNAs were evaluated using real-time PCR. Uterine weight and LECH were only increased in UE(2)-treated rats. PR, ERalpha and C3 expression levels were modified in most of the endosulfan-treated groups, showing an identical pattern of expression to the NUE(2)-group. Our results show that the pesticide endosulfan mimics non-uterotrophic E(2) actions, strengthening the hypothesis that endosulfan is a widespread xenoestrogen.