Glyphosate modifies the gene expression and migration of trophoblastic cells without altering the process of angiogenesis or the implantation of blastocysts in vitro.

Adverse pregnancy outcomes have been associated with the presence of glyphosate (G) in umbilical cord, serum, and urine samples from pregnant women. Our aim was to study the effect of G on blastocyst implantation using an in vitro mouse model, and the migration and acquisition of endothelial phenotype of the human trophoblastic HTR8/SVneo (H8) cells. In mouse blastocysts, no differences in attachment time and implantation outgrowth area were observed after G exposure. H8 cell migration was stimulated by 0.625 µM G without cytotoxicity. After 6 h, the mRNA expression of vascular endothelial growth factor (VEGF) and C-C motif chemokine ligand 2 (CCL2) was upregulated in H8 cells exposed to 1.25 µM G when compared vehicle-treated cells (p ≤ 0.05). No differences were observed in interleukin 11, VEGF receptor 1, and coagulation factor II thrombin receptor in H8 cells exposed to different concentrations of G for 6 h compared to the vehicle. Interestingly, exposure to G did not alter angiogenesis as measured by a tube formation assay. Taken all together, these results suggest that G exposure may contribute as a risk factor during pregnancy, due to its ability to alter trophoblast migration and gene expression.

Branching morphogenesis of the mouse mammary gland after exposure to benzophenone-3.

Pubertal mammary branching morphogenesis is a hormone-regulated process susceptible to exposure to chemicals with endocrine disruptive capacity, such as the UV-filter benzophenone-3 (BP3). Our aim was to assess whether intrauterine or in vitro exposure to BP3 modified the branching morphogenesis of the female mouse mammary gland. For this, pregnant mice were dermally exposed to BP3 (0.15 or 50 mg/kg/day) from gestation day (GD) 8.5 to GD18.5. Sesame oil treatment served as control. Changes of the mammary glands of the offspring were studied on postnatal day 45. Further, mammary organoids from untreated mice were cultured under branching induction conditions and exposed for 9 days to BP3 (1 × 10-6 M, 1 × 10-9 M, or 1 × 10-12 M with 0.01% ethanol as control) to evaluate the branching progression. Mice that were exposed to BP3 in utero showed decreased mRNA levels of progesterone receptor (PR) and WNT4. However, estradiol and progesterone serum levels, mammary histomorphology, proliferation, and protein expression of estrogen receptor alpha (ESR1) and PR were not significantly altered. Interestingly, direct exposure to BP3 in vitro also decreased the mRNA levels of PR, RANKL, and amphiregulin without affecting the branching progression. Most effects were found after exposure to 50 mg/kg/day or 1 × 10-6 M of BP3, both related to sunscreen application in humans. In conclusion, exposure to BP3 does not impair mammary branching morphogenesis in our models. However, BP3 affects PR transcriptional expression and its downstream mediators, suggesting that exposure to BP3 might affect other developmental stages of the mammary gland.

Mammary gland development in male rats perinatally exposed to propiconazole, glyphosate, or their mixture.

This study aimed to assess whether perinatal exposure to propiconazole (PRO), glyphosate (GLY) or their mixture (PROGLY) alters key endocrine pathways and the development of the male rat mammary gland. To this end, pregnant rats were orally exposed to vehicle, PRO, GLY, or a mixture of PRO and GLY from gestation day 9 until weaning. Male offspring were euthanized on postnatal day (PND) 21 and PND60. On PND21, GLY-exposed rats showed reduced mammary epithelial cell proliferation, whereas PRO-exposed ones showed increased ductal p-Erk1/2 expression without histomorphological alterations. On PND60, GLY-exposed rats showed reduced mammary gland area and estrogen receptor alpha expression and increased aromatase expression, whereas PRO-exposed ones showed enhanced lobuloalveolar development and increased lobular hyperplasia. However, PROGLY did not modify any of the endpoints evaluated. In summary, PRO and GLY modified the expression of key molecules and the development of the male mammary gland individually but not together.

Postnatal exposure to a glyphosate-based herbicide interferes with the development and growth of the mammary gland of pre-pubertal Ewe lambs.

The aim of the present study was to evaluate whether early postnatal exposure to a glyphosate-based herbicide (GBH) alters pre-pubertal mammary development in Friesian lambs. To this end, from postnatal day 1-14, ewe lambs were exposed subcutaneously or orally to GBH (2 mg/kg bw/day) or vehicle (control) and mammary gland biopsies were obtained at 45 days of age. GBH-exposed lambs exhibited larger mammary ducts and less area occupied by terminal duct lobular units than controls, accompanied by an increase in the area of adipocytes in the mammary stroma. Lambs subcutaneously exposed to GBH showed increased protein expression of estrogen receptor alpha; however, both GBH-exposed groups had decreased mRNA expression of this receptor. Control lambs showed nuclear progesterone receptor (PR) protein expression, whereas GBH-exposed animals showed cytoplasmic PR expression; both GBH-exposed groups exhibited decreased mRNA expression of PR. GBH-exposed lambs also had decreased epithelial cell proliferation. Regarding insulin-like growth factors, both groups showed similar IGF-1 mRNA and protein expression but decreased expression of its receptor, and increased IGFBP5 expression. In addition, phosphorylated AKT was only observed in the mammary gland of control lambs. Our results show that early postnatal exposure to GBH, regardless of the exposure route, affects the IGF-1 system and the AKT/protein kinase B pathway, interfering with steroid hormone receptor expression and cell proliferation. This consequently modifies the growth and development of the pre-pubertal mammary gland of Frisian lambs.

Prenatal exposure to a mixture of different phthalates increases the risk of mammary carcinogenesis in F1 female offspring.

Phthalates metabolites have been detected in the urine of pregnant and breastfeeding women. Thus, this study evaluated the adverse effects of maternal exposure to a mixture of six phthalates (Pth mix) on the mammary gland development and carcinogenesis in F1 female offspring. Pregnant female Sprague-Dawley rats were exposed daily to vehicle or Pth mix (35.22% diethyl-phthalate, 21.03% di-(2-ethylhexyl)-phthalate, 14.91% dibutyl-phthalate, 15.10% diisononyl-phthalate, 8.61% diisobutyl-phthalate, and 5.13% benzylbutyl-phthalate) by gavage at 20 µg/kg, 200 µg/kg or 200 mg/kg during gestational day 10 (GD 10) to postnatal day 21 (PND 21). After weaning (PND 22), some female offspring were euthanized for mammary gland analyses while other females received a single dose of N-methyl-N-nitrosourea (MNU, 50 mg/kg) or vehicle and then tumor incidence and multiplicity were recorded until PND 180. Maternal Pth mix exposure increased the number of Ki-67 and progesterone receptor-positive epithelial cells in the mammary gland from Pth mix 200 at µg/kg and 200 mg/kg groups. In addition, tumor incidence and mean number were higher only in Pth mix at 200 mg/kg when compared to the vehicle-treated group, and percentage of tumor-free animals was lower in Pth mix at 200 µg/kg and 200 mg/kg groups. The findings indicate that perinatal Pth mixture exposure increased susceptibility to MNU-induced mammary carcinogenesis in adult F1 female offspring.

Maternal Low-Protein Diet Deregulates DNA Repair and DNA Replication Pathways in Female Offspring Mammary Gland Leading to Increased Chemically Induced Rat Carcinogenesis in Adulthood.

Studies have shown that maternal malnutrition, especially a low-protein diet (LPD), plays a key role in the developmental mechanisms underlying mammary cancer programming in female offspring. However, the molecular pathways associated with this higher susceptibility are still poorly understood. Thus, this study investigated the adverse effects of gestational and lactational low protein intake on gene expression of key pathways involved in mammary tumor initiation after a single dose of N-methyl-N-nitrosourea (MNU) in female offspring rats. Pregnant Sprague-Dawley rats were fed a normal-protein diet (NPD) (17% protein) or LPD (6% protein) from gestational day 1 to postnatal day (PND) 21. After weaning (PND 21), female offspring (n = 5, each diet) were euthanized for histological analysis or received NPD (n = 56 each diet). At PND 28 or 35, female offspring received a single dose of MNU (25 mg/kg body weight) (n = 28 each diet/timepoint). After 24 h, some females (n = 10 each diet/timepoint) were euthanized for histological, immunohistochemical, and molecular analyses at PDN 29 or 36. The remaining animals (n = 18 each diet/timepoint) were euthanized when tumors reached ≥2 cm or at PND 250. Besides the mammary gland development delay observed in LPD 21 and 28 groups, the gene expression profile demonstrated that maternal LPD deregulated 21 genes related to DNA repair and DNA replication pathways in the mammary gland of LPD 35 group after MNU. We further confirmed an increased γ-H2AX (DNA damage biomarker) and in ER-α immunoreactivity in mammary epithelial cells in the LPD group at PND 36. Furthermore, these early postnatal events were followed by significantly higher mammary carcinogenesis susceptibility in offspring at adulthood. Thus, the results indicate that maternal LPD influenced the programming of chemically induced mammary carcinogenesis in female offspring through increase in DNA damage and deregulation of DNA repair and DNA replication pathways. Also, Cidea upregulation gene in the LPD 35 group may suggest that maternal LPD could deregulate genes possibly leading to increased risk of mammary cancer development and/or poor prognosis. These findings increase the body of evidence of early-transcriptional mammary gland changes influenced by maternal LPD, resulting in differential response to breast tumor initiation and susceptibility and may raise discussions about lifelong prevention of breast cancer risk.

Bisphenol A and benzophenone-3 exposure alters milk protein expression and its transcriptional regulation during functional differentiation of the mammary gland in vitro.

The plastic monomer and plasticizer bisphenol A (BPA), and the UV-filter benzophenone-3 (BP3) have been shown to have estrogenic activities that could alter mammary gland development. Our aim was to analyze whether BPA or BP3 direct exposure affects the functional differentiation of the mammary gland using an in vitro model. Mammary organoids were obtained and isolated from 8 week-old virgin female C57BL/6 mice and were differentiated on Matrigel with medium containing lactogenic hormones and exposed to: a) vehicle (0.01% ethanol); b) 1 × 10-9 M or 1 × 10-6 M BPA; or c) 1 × 10-12 M, 1 × 10-9 M or 1 × 10-6 M BP3 for 72 h. The mRNA and protein expression of estrogen receptor alpha (ESR1) and progesterone receptor (PR) were assessed. In addition, mRNA levels of PR-B isoform, glucocorticoid receptor (GR), prolactin receptor (PRLR) and Stat5a, and protein expression of pStat5a/b were evaluated at 72 h. The mRNA and protein expression of milk proteins and their DNA methylation status were also analyzed. Although mRNA level of PRLR and GR was similar between treatments, mRNA expression of ESR1, total PR, PR-B and Stat5a was increased in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. Total PR expression was also increased with 1 × 10-6 M BPA. Nuclear ESR1 and PR expression was observed in all treated organoids; whereas nuclear pStat5a/b alveolar cells was observed only in organoids exposed to 1 × 10-9 M BPA and 1 × 10-12 M BP3. The beta-casein mRNA level was increased in both BPA concentrations and 1 × 10-12 M BP3, which was associated with hypomethylation of its promoter. The beta-casein protein expression was only increased with 1 × 10-9 M BPA or 1 × 10-12 M BP3. In contrast, BPA exposure decreased alpha-lactalbumin mRNA expression and increased DNA methylation level in different methylation-sensitive sites of the gene. Also, 1 × 10-9 M BPA decreased alpha-lactalbumin protein expression. Our results demonstrate that BPA or BP3 exposure alters milk protein synthesis and its transcriptional regulation during mammary gland differentiation in vitro.

Perinatal exposure to Bisphenol A disturbs the early differentiation of male germ cells.

Understanding the effects of Bisphenol A (BPA) on early germ cell differentiation and their consequences in adult life is an area of growing interest in the field of endocrine disruption. Herein, we investigate whether perinatal exposure to BPA affects the differentiation of male germ cells in early life using a transgenic mouse expressing the GFP reporter protein under the Oct4 promoter. In this model, the expression of GFP reflects the expression of the Oct4 gene. This pluripotency gene is required to maintain the spermatogonial stem cells in an undifferentiated stage. Thus, GFP expression was used as a parameter to evaluate the effect of BPA on early germ cell development. Female pregnant transgenic mice were exposed to BPA by oral gavage, from embryonic day 5.5 to postnatal day 7 (PND7). The effects of BPA on male germ cell differentiation were evaluated at PND7, while sperm quality, testicular morphology, and protein expression of androgen receptor and proliferating cell nuclear antigen were studied at PND130. We found that perinatal/lactational exposure to BPA up-regulates the expression of Oct4-driven GFP in testicular cells at PND7. This finding suggests a higher proportion of undifferentiated spermatogonia in BPA-treated animals compared with non-exposed mice. Moreover, in adulthood, the number of spermatozoa per epididymis was reduced in those animals perinatally exposed to BPA. This work shows that developmental exposure to BPA disturbed the normal differentiation of male germ cells early in life, mainly by altering the expression of Oct4 and exerted long-lasting sequelae at the adult stage, affecting sperm count and testis.

Exposure to a Glyphosate-based Herbicide Alters the Expression of Key Regulators of Mammary Gland Development on Pre-pubertal Male Rats.

We previously reported that exposure during gestation and lactation to a low dose of glyphosate-based herbicide (GBH) reduced the area and perimeter of male offspring mammary gland at postnatal day 60 (PND60), whereas a higher dose increased the longitudinal growth of the gland. Here, our aim was to assess whether perinatal exposure to GBH exhibits endocrine disruptive action in male mammary gland at an early time point (pre-puberty), which could be related to the changes observed after puberty. We also wanted to explore whether an early evaluation of the male rat mammary gland is appropriate to assess exposure to potential endocrine disrupting chemicals (EDCs). Pregnant rats were orally exposed, through the diet, to vehicle (saline solution), 3.5 or 350 mg/kg/day of GBH from gestational day 9 until weaning. At PND21, the male offspring were euthanized, and mammary gland samples were collected. The histology and proliferation index of the mammary glands were evaluated, and the mRNA expression of estrogen (ESR1) and androgen (AR) receptors, cyclin D1 (Ccnd1), amphiregulin (Areg), insulin-like growth factor 1 (IGF1), epidermal growth factor receptor (EGFR) and IGF1 receptor (IGF1R) were assessed. Moreover, the phosphorylated-Erk1/2 (p-ERK1/2) protein expression was determined. No differences were observed in mammary epithelial structures and AR expression between experimental groups; however, the proliferation index was reduced in GBH3.5-exposed males. This result was associated with decreased ESR1, Ccnd1, Areg, IGF1, EGFR and IGF1R mRNA expressions, as well as reduced p-Erk1/2 protein expression in these animals. ESR1, Ccnd1, IGF1R and EGFR expressions were also reduced in GBH350-exposed males. In conclusion, the mammary gland development of pre-pubertal male rats is affected by perinatal exposure to GBH. Although further studies are still needed to understand the molecular mechanisms involved in GBH350 exposure, the present results may explain the alterations observed in mammary gland growth of post-pubertal males exposed to low doses of GBH. Our results also suggest that early evaluation of the male rat mammary gland is useful in assessing exposure to potential EDCs. However, analysis of EDCs effects at later time points should not be excluded.

AhR ligands reactivate LINE-1 retrotransposon in triple-negative breast cancer cells MDA-MB-231 and non-tumorigenic mammary epithelial cells NMuMG.

Breast cancer is the most common cancer type in females worldwide. Environmental exposure to pesticides affecting hormonal homeostasis does not necessarily induce DNA mutations but may influence gene expression by disturbances in epigenetic regulation. Expression of long interspersed nuclear element-1 (LINE-1) has been associated with tumorigenesis in several cancers. In nearly all somatic cells, LINE-1 is silenced by DNA methylation in the 5́'UTR and reactivated during disease initiation and/or progression. Strong ligands of aryl hydrocarbon receptor (AhR) activate LINE-1 through the transforming growth factor-ß1 (TGF-ß1)/Smad pathway. Hexachlorobenzene (HCB) and chlorpyrifos (CPF), both weak AhR ligands, promote cell proliferation and migration in breast cancer cells, as well as tumor growth in rat models. In this context, our aim was to examine the effect of these pesticides on LINE-1 expression and ORF1p localization in the triple-negative breast cancer cell line MDA-MB-231 and the non-tumorigenic epithelial breast cell line NMuMG, and to evaluate the role of TGF-ß1 and AhR pathways. Results show that 0.5 µM CPF and 0.005 µM HCB increased LINE-1 mRNA expression through Smad and AhR signaling in MDA-MB-231. In addition, the methylation of the first sites in 5́'UTR of LINE-1 was reduced by pesticide exposure, although the farther sites remained unaffected. Pesticides modulated ORF1p localization in MDA-MB-231: 0.005 µM HCB and 50 µM CPF increased nuclear translocation, while both induced cytoplasmic retention at 0.5 and 5 µM. Moreover, both stimulated double-strand breaks, enhancing H2AX phosphorylation, coincidentally with ORF1p nuclear localization. In NMuMG similar results were observed, since they heighten LINE-1 mRNA levels. CPF effect was through AhR and TGF-ß1 signaling, whereas HCB action depends only of AhR. In addition, both pesticides increase ORF1p expression and nuclear localization. Our results provide experimental evidence that HCB and CPF exposure modify LINE-1 methylation levels and induce LINE-1 reactivation, suggesting that epigenetic mechanisms could contribute to pesticide-induced breast cancer progression.

Culturing Rat Whole Ovary for UV Filter Benzophenone-3 Treatment.

We describe a detailed protocol to establish a newborn rat whole ovary culture, which enables the study of direct effects (independent of hypothalamic-pituitary-gonadal axis) of endocrine disrupting chemicals (EDCs), such as benzophenone-3 (BP-3). This method is useful to understand changes in follicle formation, primordial to primary transition, and expression of regulatory molecules linked to these processes and also provides an alternative to animal models. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Rat ovarian surgery Basic Protocol 2: Whole organ/ovarian culture Basic Protocol 3: RNA isolation and quantitative real-time PCR Basic Protocol 4: Histological processing and staining.

Impact of gestational low protein diet and postnatal bisphenol A exposure on chemically induced mammary carcinogenesis in female offspring rats.

This study evaluated the effect of gestational low protein diet (LPD) and/or postnatal bisphenol A (BPA) exposure on mammary gland development and carcinogenesis in female offspring. Pregnant Sprague-Dawley rats were fed a normal protein diet (NPD, 17% protein) or LPD (6% protein). At weaning, female offspring were distributed in four groups (NPD, LPD, NPD + BPA, and LPD + BPA) and received vehicle or BPA in drinking water (0.1%), during postnatal day (PND) 21 to 51. On PND 51, some female offspring were euthanized or received a single dose of 7,12-dimethylbenzoanthracene (DMBA, 30 mg/kg, i.g.) and were euthanized on PND 250. On PND 51, neither gestational LPD nor postnatal BPA exposure, individually or in combination, significantly altered the development of mammary gland tree, mean number of terminal structures or estrogen receptor beta (ER-ß), proliferating cell nuclear antigen (PCNA) or caspase-3 protein expression in the mammary tissue. A significant reduction in mammary epithelial area (%) was observed in both LPD groups and a significant increase in ER-α protein expression was detected only in LPD group. In LPD + BPA group was observed a significant increase in both fat pad area (%) and in mean number of mammary epithelial cells positive for progesterone receptor (PR). On PND 250, the groups that received BPA presented lower latency and higher tumor incidence and tumor multiplicity and LPD + BPA group more aggressive tumors. These findings suggest that postnatal BPA exposure associated with gestational LPD is able to induce morphological changes in the mammary gland and increase susceptibility to mammary carcinogenesis.

Hyperandrogenism Induces Histo-Architectural Changes in the Rat Uterus.

The effects of androgens on the uterus have been poorly studied and they need to be clarified to understand why androgen excess, such as observed in women with polycystic ovary syndrome (PCOS), is a risk factor for the development of endometrial hyperplasia, cancer, and infertility. Thus, uterine histomorphology in a PCOS experimental model was evaluated. Beginning at weaning, female rats were injected daily with dehydroepiandrosterone (DHEA, 6 mg/100 g body weight) or vehicle (sesame oil) for 20 consecutive days. On postnatal day 41 (PND41), DHEA-treated animals showed high serum testosterone levels. In addition, uterine histological analysis showed a significant increase in luminal epithelial height and glandular density without changes in cell proliferation. The thickness of the subepithelial stroma and myometrium also increased in these animals. The effect of DHEA on uterine thickness was accompanied by a significant reduction in cell density in both tissue compartments (subepithelial stroma and myometrium). Cell proliferation was not altered in the myometrium, whereas a decrease in the proliferative activity was seen at PND41 in the subepithelial stroma of DHEA animals. The analysis of the extracellular space showed that the changes in the thickness of the subepithelial stroma and myometrium were related to an increase in the organization of collagen fibers and water imbibition. The latter was associated with higher aquaporin 3 and 8 expression. This study provides evidence to further the understanding of PCOS-associated hyperandrogenism effects on uterine architecture. This could have implications for the regulation of uterine function and the development of uterine lesions.

Male mammary gland development and methylation status of estrogen receptor alpha in Wistar rats are modified by the developmental exposure to a glyphosate-based herbicide.

Postnatal treatment with glyphosate-based herbicides (GBHs) induces endocrine-disrupting effects on the male rat mammary gland. In this study, the effects of developmental exposure to GBH on mammary gland growth and development, and the possible molecular mechanisms involved, were evaluated in pre- and post-pubertal male rats. To this end, pregnant rats were orally exposed through the food to 0, 3.5 or 350 mg GBH/kg bw/day from gestational day 9 until weaning. Mammary gland development and estradiol (E2) and testosterone (T) serum levels of male offspring were evaluated on postnatal day (PND)21 and PND60. Besides, prolactin (PRL) serum levels, proliferation index, androgen (AR) and estrogen receptor alpha (ESR1) expression, ESR1 alternative transcript mRNA levels, and DNA methylation status of ESR1 promoters were assessed on PND60. No differences between groups were observed in mammary gland development at PND21 or in E2 and T levels on both PNDs studied. On PND60, GBH3.5-exposed animals presented similar mammary gland histology but higher AR protein expression than controls, whereas GBH350-exposed males presented a less developed mammary gland, accompanied by a lower proliferation index, similar AR levels, and slightly increased PRL serum levels than controls. In both exposed groups, ESR1 expression was lower than in control rats, being lower in GBH350-exposed rats. GBH also altered the abundance of ESR1 transcript variants by hypermethylation of ESR1 promoters. GHB3.5 decreased only ESR1-OS expression, whereas GBH350 affected ESR1-O, OT and E1 expression. Our results show that developmental exposure to GBH induces epigenetic changes in ESR1, which could be responsible for the altered male mammary gland development observed in GBH350-exposed animals.

Postnatal exposure to a glyphosate-based herbicide modifies mammary gland growth and development in Wistar male rats.

Our aim was to evaluate whether postnatal exposure to a glyphosate-based herbicide (GBH) modifies mammary gland development in pre- and post-pubertal male rats. From postnatal day 1 (PND1) to PND7, male rats were injected subcutaneously every 48 h with either saline solution (vehicle) or 2 mg GBH/kg·bw. On PND21 and PND60, mammary gland and blood samples were collected. Estradiol (E2) and testosterone (T) serum levels, mammary gland histology, collagen fiber organization, mast cell infiltration, proliferation index, and estrogen (ESR1) and androgen receptor (AR) expression levels were evaluated. At PND21, GBH-exposed male rats exhibited greater development of the mammary gland with increased stromal collagen organization and terminal end buds (TEBs) compared to control rats. At PND60, the number of TEBs remained high and was accompanied by an increase in mast cell infiltration, proliferation and ESR1 expression in GBH-exposed male rats. In contrast, no effects were observed in E2 and T serum levels and AR expression in both days studied. Our results showed that a postnatal subacute treatment with GBH induces endocrine-disrupting effects in the male mammary gland in vivo, altering its normal development.

Early postnatal exposure to endosulfan interferes with the normal development of the male rat mammary gland.

Our aim was to evaluate whether postnatal exposure to endosulfan (ENDO) modifies mammary gland (MG) development in pre- and post-pubertal male rats. From postnatal day 1 (PND1) to PND7, male rats were injected subcutaneously every 48h with either corn oil (vehicle) or 600µg ENDO/kg.bw. On PND21 and PND60, MG and blood samples were collected. Estradiol (E2) and testosterone (T) serum levels, MG histology, collagen fiber organization, proliferation index, and estrogen (ESR1) and androgen receptor (AR) expressions were evaluated. On PND21, E2 and T levels were similar between groups, whereas MG area, perimeter, number of terminal end buds and ESR1 expression were increased in ENDO-exposed rats. These changes were associated with alveolar development and increased organized collagen in the stroma. On PND60, a higher proliferation index in ENDO-exposed rats was correlated with a more developed lobuloalveolar structure. Hyperplastic alveoli and, hyperplastic ducts surrounded by a dense stroma were also observed in this group. T levels and ESR1 expression were similar between groups, whereas E2 levels and AR expression were decreased in ENDO-exposed rats. The exposure to ENDO in the first week of life interferes with the normal development of the MG and induces pre-malignant lesions in post-pubertal male rats.

Bisphenol A disrupts the temporal pattern of histofunctional changes in the female reproductive tract of Caiman latirostris.

Recently, we have described the ontogeny of histofunctional differentiation changes in the oviduct of Caiman latirostris. The expression of estrogen receptor alpha and progesterone receptor shows that the caiman oviduct could be a target of the action of xenoestrogens such as the widely environmentally present Bisphenol A (BPA), early in life. The aims of this study were: to complement oviduct characterization by establishing the ontogenetic changes in androgen receptor (AR) expression and assessing the effects of early postnatal exposure to 17-ß-estradiol (E2) or BPA on the histofunctional features of the oviduct. AR was expressed in all the stages studied. The spatial pattern of AR immunostaining changed from neonatal to juvenile caimans. In the luminal epithelium, changes were at the subcellular level, from cytoplasmic to nuclear. In the subepithelium, although both cytoplasmic and nuclear AR expression was observed, changes were mainly at tissue level, from the subepithelial compartment to the outer muscular layer. The oviduct was highly sensitive to E2 and BPA at the early postnatal developmental stage. E2- and BPA-exposed caimans showed increased luminal epithelial height and higher proliferative activity. Changes in histomorphological features (measured by a scoring system), steroid hormone receptors, collagen remodeling and muscle-associated proteins suggest a precocious oviduct histofunctional differentiation in E2- and BPA-exposed caimans. The modification of the temporal pattern of oviductal biomarkers suggests that organizational changes could impair C. latirostris reproductive health later in life. The alterations in the caiman female reproductive tract exposed to BPA highlight the importance of preserving aquatic environments from plastic pollution.

Perinatal Exposure to Bisphenol A or Diethylstilbestrol Increases the Susceptibility to Develop Mammary Gland Lesions After Estrogen Replacement Therapy in Middle-Aged Rats.

The development of the mammary gland is a hormone-regulated event. Several factors can dysregulate its growth and make the gland more susceptible to cellular transformation. Among these factors, perinatal exposure to xenoestrogens and hormone replacement therapy has been associated with increased risk of developing breast cancer. Here, we assessed the effects induced by estrogen replacement therapy (ERT) in ovariectomized (OVX) middle-aged rats and whether perinatal exposure to diethylstilbestrol (DES) or bisphenol A (BPA) modified these effects in the mammary gland. Pregnant rats were orally exposed to vehicle, 5 µg DES/kg/day, or 0.5 or 50 µg BPA/kg/day from gestational day 9 until weaning. Then, 12-month-old offspring were OVX and treated with 17ß-estradiol for 3 months. Morphological changes and the percentage of epithelial cells that proliferated or expressed estrogen receptor alpha (ESR1) and progesterone receptor (PR) were analyzed in mammary gland samples of 15-month-old animals. ERT induced lobuloalveolar hyperplasia and ductal cysts in the mammary gland of middle-aged rats, associated with a higher proliferation index of epithelial cells. Perinatal exposure to DES followed by ERT increased the number of cysts and induced the formation of fibroadenoma and ductal carcinoma in situ, without modifying the expression of ESR1 or PR. Also, after 3 months of ERT, BPA-exposed rats had a higher incidence of ductal hyperplasia and atypical lobular hyperplasia than animals under ERT alone. In conclusion, perinatal exposure to xenoestrogens increases the susceptibility of the mammary gland to develop cysts and hyperplastic lesions when confronted with ERT later in life.

Perinatal exposure to bisphenol A modifies the transcriptional regulation of the ß-Casein gene during secretory activation of the rat mammary gland.

With the aim to analyze whether bisphenol A (BPA) modifies ß-Casein (ß-Cas) synthesis and transcriptional regulation in perinatally exposed animals, here, pregnant F0 rats were orally exposed to 0, 0.6 or 52 µg BPA/kg/day from gestation day 9 until weaning. Then, F1 females were bred and mammary glands were obtained on lactation day 2. Perinatal BPA exposure decreased ß-Cas expression without modifying the activation of prolactin receptor. It also decreased the expression of glucocorticoid receptor in BPA52-exposed dams and ß1 and α6 integrins as well as dystroglycan in both BPA groups. In addition, BPA exposure altered the expression of histone-modifying enzymes and induced histone modifications and DNA methylation in the promoter, enhancer and exon VII of the ß-Cas gene. An impaired crosstalk between the extracellular matrix and lactogenic hormone signaling pathways and epigenetic modifications of the ß-Cas gene could be the molecular mechanisms by which BPA decreased ß-Cas expression.