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.

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.

Long-term effects of in ovo exposure to an environmentally relevant dose of atrazine on the thyroid gland of Caiman latirostris.

The increased incidence of human thyroid disorders, particularly in women, suggests that the exposure to endocrine-disrupting compounds (EDCs) together with sex-related factors could play a role in thyroid dysregulation. Since the herbicide atrazine (ATZ) is an environmental EDC suspected to behave as a thyroid disruptor, and Caiman latirostris is a crocodilian species highly sensitive to endocrine disruption that can be exposed to ATZ, this study aimed to describe the histoarchitecture and sexually dimorphic features of the thyroid gland of C. latirostris, and to determine the long-term effects of in ovo exposure to an environmentally relevant dose of ATZ (0.2 ppm) on its thyroid gland and growth. Control caimans showed no sexual dimorphisms. In contrast, ATZ-exposed caimans showed altered embryo growth but an unaltered temporal pattern of development and a sexually dimorphic response in the body condition index growth curves postnatally, which suggests a female-related increase in fat storage. Besides, both male and female exposed caimans showed increases in the size of the thyroid stromal compartment, content of interstitial collagen, and follicular hyperplasia, and decreases in the expression of androgen receptor in the follicular epithelium. ATZ-exposed females, but not males, also showed evidences of thyroid enlargement, colloid depletion, increased follicular epithelial height and increased presence of microfollicular structures. Our results demonstrate that prenatal exposure of caimans to ATZ causes thyroid disruption and that females were more vulnerable to ATZ than males. The effects were organizational and observed long after exposure ended. These findings alert on ATZ side-effects on the growth, metabolism, reproduction and development of non-target exposed organisms, particularly females.