Milena Durando

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.

Prenatal exposure to pesticides disrupts testicular histoarchitecture and alters testosterone levels in male Caiman latirostris

The increased use of agrochemical pesticides, such as atrazine (ATZ) and endosulfan (END), may have a significant impact on ecosystem health and biodiversity. The aim of this study was to investigate the consequences of in ovum exposure to ATZ and END on Caiman latirostris gonadal histo-functional features. Caiman eggs were collected from environmentally pristine areas and incubated in controlled conditions at male producing temperature (33 degrees C). At stage 20 of embryonic development, the sensitive stage for gonadal sex determination, eggs were exposed to one dose of either END or ATZ. Gonadal histo-morphology was examined in caiman hatchlings and serum levels of testosterone were measured. Regardless of treatment condition, all eggs incubated at 33 degrees C resulted in male hatchlings. Tortuous seminiferous tubules with increased perimeter, disrupted distribution of peritubular myoid cells (desmin positive), and emptied tubular lumens characterized the testes of pesticide-exposed caiman. An imbalance between proliferative activity and cell death was observed in the testes of caiman exposed to the higher doses of END, mainly due to a high frequency of apoptosis in intratubular cells. This altered cell turnover was associated with decreased testosterone levels. Prenatal exposure to only one dose of END and ATZ disrupted neonatal male gonadal histo-functional features. Alterations described here could have detrimental effects on the sexual maturation of the caiman and, ultimately, on the success of male caiman reproduction.

Association of increased estrogen receptor β2 expression with parity-induced alterations in the rat mammary gland

In this study, we investigated the cellular and molecular events involved in parity-related alterations in mammary gland (MG) proliferation and differentiation. Rat MGs were removed on day 9 of either first (nulliparous), second (primiparous) or third (multiparous) pregnancy. Expression of steroid hormone receptors along with cellular biomarkers of proliferation and differentiation were quantified in all MG tissue compartments by immunohistochemistry. Wnt-4 (a Wingless-like morphogenic gene involved in MG development), ERbeta and ERbeta2 mRNA were evaluated by RT-PCR analysis. Serum levels of mammotrophic hormones were measured. In comparison to nulliparous and primiparous rats, multiparous animals exhibited decreased luminal cell proliferation and PR levels, whereas alpha-lactalbumin, ERalpha, ERbeta and ERbeta2 expression were increased. In myoepithelial cells, while parity induced a decrease in proliferative activity, subsequent pregnancies and lactations lead to an increased state of differentiation. Our results showed that at least two periods of pregnancy and lactation were necessary to modify the studied parameters. The lower proliferative activity and higher differentiation state of the multiparous MG are associated with both a decreased PR expression and increased ERalpha and ERbeta expression. Since ERbeta and/or ERbeta2 isoform expression was related to parity history, results suggest that the decreased proliferative activity and PR expression observed in the MG of multiparous animals may be associated with overexpression of ERbeta and/or the ERbeta2 isoform, thereby antagonizing the proliferative effects associated with ERalpha.

Neonatal expression of amh, sox9 and sf-1 mRNA in Caiman latirostris and effects of in ovo exposure to endocrine disrupting chemicals

Caiman latirostris is a reptilian species that exhibits temperature-dependent sex determination (TSD). Male-to-female sex reversal can be achieved after in ovo estrogen/xenoestrogen exposure. This is known as hormone-dependent sex determination (HSD). The amh, sox9 and sf-1 genes are involved in sex determination, sex differentiation, and steroidogenesis. The aims of this study were: (a) to establish the expression patterns of amh, sox9 and sf-1 mRNA in the gonad-adrenal-mesonephros (GAM) complexes of neonatal TSD-male and TSD-female caimans, (b) to compare the expression of these genes between TSD-females and HSD-females (born from E2-exposed eggs incubated at the male-producing temperature) and (c) to evaluate whether in ovo exposure to a low dose of E2 or bisphenol A (BPA) or to a high dose of endosulfan (END) modifies amh, sox9 or sf-1 mRNA expressions in neonatal males. The mRNA expressions of amh, sox9 and sf-1 in GAM complexes from TSD-males and TSD-females and from HSD-females were quantitatively compared by RT-PCR. A sexually dimorphic pattern of amh and sox9 mRNA expression was found, with a higher expression in TSD-males than in TSD-females. sf-1 mRNA did not differ between TSD-males and TSD-females. HSD-females exhibited a higher expression of sox9 than TSD-females. In males, increased mRNA expression of sex-determining genes was observed after in ovo exposure to END. E2 decreased sox9 but increased sf-1 mRNA expression. Changes induced by BPA were evident although not significant. These results provide new insights into the potential mechanisms that lead to the gonadal histo-functional alterations observed in caimans exposed to contaminated environments.

Prenatal Bisphenol A exposure delays the development of the male rat mammary gland

Our aims were to evaluate whether exposure to Bisphenol A (BPA) modifies the development of the male rat mammary gland (MG) and to evaluate whether this modification is gender specific. From gestational day 9, pregnant rats were exposed either subcutaneously to 0, 25 or 250μg BPA/kgbw/day until parturition or orally to 0 and 64μg BPA/kgbw/day until weaning. MG development was analyzed on postnatal days (PND) 5, 15 and 30. On PND30, steroid hormone receptor expression and mammary growth were also evaluated. On PND30, the exposure to 64BPA and 250BPA induced a delay in male MG development, evidenced by reduced ductal growth, decreased number of terminal structures and lower expression of androgen receptor (AR). In contrast, female mammary ductal growth was altered only by 250BPA. Regardless of the administration route and length of the exposure period, BPA induced a delay in MG development and modified AR expression in prepubertal male rats.

Ovarian dysfunctions in adult female rat offspring born to mothers perinatally exposed to low doses of bisphenol A.

The study of oral exposure to the environmental estrogen bisphenol A (BPA) during the perinatal period and its effects on ovarian functionality in adulthood has generated special interest. Thus, our objective was to investigate ovarian folliculogenesis and steroidogenesis in adult female rat offspring born to mothers exposed to low doses of BPA (BPA50: 50μg/kgday; BPA0.5: 0.5μg/kgday) by the oral route during gestation and breastfeeding. Ovaries from both BPA-treated groups showed reduced primordial follicle recruitment and a greater number of corpora lutea, indicating an increased number of ovulated oocytes, coupled with higher levels of mRNA expression of 3β-hydroxysteroid dehydrogenase and serum progesterone. BPA50-treated animals had lower expression of androgen receptor (AR) at different stages of the growing follicle population. BPA0.5-treated rats evidenced an imbalance of AR expression between primordial/primary follicles, with higher mRNA-follicle-stimulating hormone receptor expression. These results add to the growing evidence that folliculogenesis and steroidogenesis are targets of BPA within the ovary.

Histomorphological changes in testes of broad-snouted caimans (Caiman latirostris) associated with in ovo exposure to endocrine-disrupting chemicals

Studies regarding the effects of endocrine-disrupting chemicals (EDCs) on the reproductive functions of wild animals have raised increasing concern. Thus, here we evaluated the consequences of in ovo exposure to endosulfan (END) and bisphenol A (BPA) in testes from neonatal to juvenile (Juv) caimans (Caiman latirostris). Caiman eggs were collected from areas with low to moderate anthropogenic intervention and incubated at male-producing temperature. At stage 20 of embryonic development (previous to gonad sex determination), eggs were exposed to either END (20 ppm) or BPA (1.4 ppm) and male gonad histomorphology examined in 10-day-old, 90-day-old, and Juv caimans. The relative seminiferous tubular area (RTA) was measured in testes and the proliferation index and the expression of estrogen receptor alpha (ERα) were quantified in intratubular cells. Regardless of the treatment, all eggs resulted in male hatchlings. The testes of EDC-exposed caimans presented tortuous seminiferous tubules with empty tubular lumens. The RTA of 10-day-old caimans exposed to BPA was decreased. The percentage of cells expressing ERα was not different after in ovo treatment with EDCs (compared to the Control group), although caimans exposed to END showed a different ERα distribution pattern. The proliferation index was lower in 90-day-old caimans exposed to END, and higher in Juv caimans exposed to BPA. In ovo exposure to END or BPA modified sensitive parameters of C. latirostris male gonads. The alterations described here might compromise not only the sexual maturation but also the reproductive performance of adult caimans. J. Exp. Zool. 325A:84-96, 2016.

Effects of a glyphosate-based herbicide on the uterus of adult ovariectomized rats.

Glyphosate is the active ingredient of several herbicide formulations. Different reports suggest that glyphosate‐based herbicides (GBHs) may act as endocrine disruptors. We evaluated the potential estrogenic effects of a GBH formulation using the uterotrophic assay. Adult ovariectomized rats were sc injected for 3 consecutive days with: saline solution (vehicle control), 2.10−5 g E2/kg/day (uterotrophic dose; UE2), 2.10−7 g E2/kg/day (nonuterotrophic dose; NUE2), or 0.5, 5, or 50 mg GBH/kg/day of the. Twenty‐four hours after the last injection, the uterus was removed and weighed and processed for histopathology and mRNA extraction. Epithelial cell proliferation and height and expression of estrogen‐responsive genes were evaluated (estrogen receptors, ERα and ERβ; progesterone receptor, PR; complement 3, C3). Uterine weight and epithelial proliferation were not affected by GBH. However, the luminal epithelial cell height increased at GBH0.5. ERα mRNA was downregulated by all GBH doses and E2 groups, whereas PR and C3 mRNA were diminished by GBH0.5. GBH5‐, GBH50‐, and UE2‐treated rats showed downregulated ERα protein expression in luminal epithelial cells, while the receptor was upregulated in the stroma. GBH upregulated ERβ (GBH0.5–50) and PR (GBH5) expressions in glandular epithelial cells, similar effect to that of NUE2 group. These results indicate that, although the uterine weight was not affected, GBH modulates the expression of estrogen‐sensitive genes.

Postnatal development and histofunctional differentiation of the oviduct in the broad-snouted caiman (Caiman latirostris).

Caiman latirostris is a South American crocodilian species characterized as a sentinel of the presence of endocrine-disrupting compounds (EDCs). Evaluating developmental events in hormone-dependent organs, such as the oviduct, is crucial to understand physiological postnatal development, to identify putative periods of exposure sensitive to EDCs, and/or to identify biomarkers useful to evaluate the effects of EDC exposure. In this study, we describe the histomorphological features of C. latirostris oviducts by establishing the ontogeny of changes at cellular, tissue and molecular levels from the neonatal to the pre-pubertal juvenile stages. Since the histological diagnosis of the adenogenic oviduct lies on a group of features, here we defined a histofunctional score system and a cut-off value to distinguish between preadenogenic and adenogenic oviducts. Our results showed that the maturation of the C. latirostris oviduct is completed postnatally and characterized by changes that mimic the pattern of histological modifications described for the mammalian uterus. Ontogenic changes in the oviductal epithelium parallel changes at subepithelial level, and include collagen remodeling and characteristic spatial-temporal patterns of α-actin and desmin. The expression pattern of estrogen receptor alpha and progesterone receptor evidenced that, even at early postnatal developmental stages, the oviduct of C. latirostris is a target organ of endogenous and environmental hormones. Besides, oviductal adenogenesis seems to be an estrogen-dependent process. Results presented here provide not only insights into the histophysiological aspect of caiman female reproductive ducts but also new tools to better characterize caimans as sentinels of endocrine disruption.

Induction of uterine hyperplasia after cafeteria diet exposure

Our aim was to evaluate whether chronic administration of CAF affects the uterus and induces the morphological and molecular changes associated with endometrial hyperplasia. Female Wistar rats exposed to CAF from weaning for 20 weeks displayed increased energy intake, body weight and fat depots, but did not develop metabolic syndrome. The adult uteri showed an increase in glandular volume fraction and stromal area. The epithelial proliferation rate and protein expression of oestrogen receptor alpha (ERα) were also increased. The CAF diet enhanced leptin serum levels and the long form of leptin receptor (Ob-Rb) mRNA expression in the uterus. No changes were detected in either insulin serum levels or those of insulin growth factor I (IGF-I) mRNA expression. However the levels of IGF-I receptor (IGF-IR) mRNA were lower in CAF-fed animals. Overall, the results indicate that our rat model of the CAF diet produces morphological and molecular changes associated with uterine hyperplasia and could predispose to endometrial carcinogenesis.