Disruption of developmental programming with long-term consequences after exposure to a glyphosate-based herbicide in a rat model.

Glyphosate-based herbicides (GBHs) have been associated with endocrine disrupting effects on reproductive organs. We examined whether postnatal exposure to GBH affects developmental programming of the uterus with long-term consequences. Female Wistar pups received vehicle (control) or GBH (2 mg of glyphosate/kg/day) from postnatal day (PND) 1 to PND7, where the developing uterus is highly sensitive to endocrine disruption. Short-, mid- and long-term effects were evaluated on PND8, PND120 and PND600, respectively. GBH induced hyperplasia and epigenetic alterations in the uterus of neonatal females (PND8). DNA hypermethylation, enrichment of H3K9me3 and reductions of H3K27me3 at regulatory regions of the morphoregulatory gene Hoxa10 resulted in gene downregulation. In young adult females (PND120), GBH increased 17ß-estradiol (E2) and decreased progesterone (P4) serum levels, altering estrous cyclicity. Aged females (PND600) exposed to GBH developed leiomyoma and pre-neoplastic glandular lesions in the uterus. Vaginal rhabdomyosarcoma and intrahepatic bile duct adenoma were also observed. In conclusion, neonatal exposure to GBH altered the expression and induced hypermethylation of the Hoxa10 gene in uterine tissue at early life, and increased E2/P4 ratio serum level at middle-age. We propose that epigenetic reprogramming of Hoxa10 in association with hormonal imbalance could be among the possible mechanisms underlying the long-term adverse effects detected in GBH-exposed rats.

Evaluation of Development of the Rat Uterus as a Toxicity Biomarker.

The developing uterus is highly sensitive to a brief exposure to different substances, in particular those with endocrine-disrupting activity. Thus, exposure to environmental, nutritional, chemical, and other xenobiotic factors affecting signaling events during critical organizational periods can alter the normal course of uterine development with lasting consequences. In this chapter, we provide an experimental protocol to evaluate the development of the rat uterus as a toxicity biomarker at two different developmental time points: (1) the neonatal period, on postnatal day (PND) 8, and (2) the prepubertal period, on PND21. In this experimental approach, we propose to assess: (1) uterine morphology and cytodifferentiation, (2) uterine cell proliferation, and (3) the expression of proteins involved in uterine organogenetic differentiation. All these morphological and molecular markers are useful tools to determine the consequences of exposure to toxicants with the potential to disrupt the uterine development.

Perinatal exposure to glyphosate or a glyphosate-based formulation disrupts hormonal and uterine milieu during the receptive state in rats.

We investigated the effects of perinatal exposure to a glyphosate-based herbicide (GBH) or glyphosate alone (Gly) on female fertility and the hormonal and uterine milieu during the preimplantation period. F0 pregnant rats orally received a GBH or Gly in a dose of 2 mg of glyphosate/kg/day from gestational day (GD) 9 until weaning. F1 females were evaluated to determine the reproductive performance on GD19; and the sex steroid serum levels, the expression of estrogen receptor alpha (ERα), progesterone receptor (PR) and implantation-related genes on GD5 (preimplantation period). GBH and Gly induced preimplantation losses in F1 rats. GBH and Gly groups exhibited higher 17ß-estradiol serum levels, without changes in progesterone. Both compounds increased the uterine ERα protein expression, with no differences at transcript level; and only Gly decreased PR mRNA expression. Also, GBH and Gly downregulated Hoxa10 and Lif genes, with no difference in Muc1 and Areg expression. To conclude, perinatal exposure to a GBH or Gly disrupted critical hormonal and uterine molecular targets during the receptive state, possibly associated with the implantation failures. Overall, similar results were found in GBH- and Gly-exposed rats, suggesting that the active principle might be the main responsible for the deleterious effects.

Correction to: Response to comments on: Perinatal exposure to a glyphosate-based herbicide impairs female reproductive outcomes and induces second-generation adverse effects in Wistar rats.

We report a mistake in the tables published in Milesi et al. (2019).

Glyphosate-based herbicide induces hyperplastic ducts in the mammary gland of aging Wistar rats.

Glyphosate-based herbicide (GBH) exposure is known to have adverse effects on endocrine-related tissues. Here, we aimed to determine whether early postnatal exposure to a GBH induces long-term effects on the rat mammary gland. Thus, female Wistar pups were injected with saline solution (Control) or GBH (2 mg glyphosate/kg/day) on postnatal days (PND) 1, 3, 5 and 7. At 20 months of age, mammary gland samples were collected to determine histomorphological features, proliferation index and the expression of steroid hormone receptors expression, by immunohistochemistry, and serum samples were collected to assess 17ß-estradiol (E2) and progesterone (P4) levels. GBH exposure induced morphological changes evidenced by a higher percentage of hyperplastic ducts and a fibroblastic-like stroma in the mammary gland. GBH-treated rats also showed a high expression of steroid hormone receptors in hyperplastic ducts. The results indicate that early postnatal exposure to GBH induces long-term alterations in the mammary gland morphology of aging female rats.

Acute uterine effects and long-term reproductive alterations in postnatally exposed female rats to a mixture of commercial formulations of endosulfan and glyphosate.

Endosulfan and glyphosate are widely used pesticides and have been associated to reproductive disorders. We examine the acute and long-term effects of postnatal exposure to commercial formulations of endosulfan (EF), glyphosate (glyphosate-based herbicide, GBH) and a mixture of both pesticides (MIX). After birth, female pups of Wistar rats received saline solution (CONTROL), EF (600 µg/kg of b.w/day), GBH (2 mg/kg of b.w/day) or a mixture (at the same doses) from postnatal day (PND) 1 to PND7. The uterine histology and expression of Hoxa10, estrogen (ERα) and progesterone (PR) receptors were evaluated on PND8. Reproductive performance was evaluated on gestational day 19. GBH and MIX rats showed an increment of 1) the incidence of luminal epithelial hyperplasia, 2) PR and Hoxa10 expression. EF modified ERα and Hoxa10 expression. During adulthood, MIX and GBH rats showed higher post-implantation losses while EF alone produced an increase of pre-implantation losses. We showed that the co-administration of both pesticides produced acute uterine effects and long-term deleterious reproductive effects that were similar to those induced by GBH alone. We consider important to highlight the necessity to evaluate the commercial pesticide mixture as a more representative model of human exposure to a high number of pesticides.

Effect of neonatal exposure to endosulfan on myometrial adaptation during early pregnancy and labor in rats.

Proper myometrial adaptation during gestation is crucial for embryo implantation, pregnancy maintenance and parturition. Previously, we reported that neonatal exposure to endosulfan alters uterine development and induces implantation failures. The present work investigates the effects of endosulfan exposure on myometrial differentiation at the pre-implantation period, and myometrial activation during labor. Newborn female rats were s.c. injected with corn oil (vehicle) or 600 µg/kg/day of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate: i) the myometrial differentiation on gestational day 5 (GD5, pre-implantation period), by assessment myometrial histomorphology, smooth muscle cells (SMCs) proliferation, and expression of proteins involved in myometrial adaptation for embryo implantation (steroid receptors, Wnt7a and Hoxa10); ii) the timing of parturition and myometrial activation during labor by determining the uterine expression of contraction-associated genes (oxytocin receptor, OTXR; prostaglandin F2α receptor, PTGFR and connexin-43, Cx-43). Endosulfan decreased the thickness of both myometrial layers, with a concomitant decrease in the collagen remodeling. Blood vessels relative area in the interstitial connective tissue between muscle layers was also decreased. Endo600 group showed lower myometrial proliferation in association with a downregulation of Wnt7a and Hoxa10. Although in all females labor occurred on GD23, the exposure to endosulfan altered the timing of parturition, by inducing advancement in the initiation of labor. This alteration was associated with an increased uterine expression of OTXR, PTGFR and Cx-43. In conclusion, neonatal exposure to endosulfan produced long-term effects affecting myometrial adaptation during early pregnancy and labor. These alterations could be associated with the aberrant effects of endosulfan on the implantation process and the timing of parturition.

Epigenetic disruption of estrogen receptor alpha is induced by a glyphosate-based herbicide in the preimplantation uterus of rats.

Previously, we have shown that perinatal exposure to a glyphosate-based herbicide (GBH) induces implantation failures in rats. Estrogen receptor alpha (ERα) is critical for successful implantation. ERα transcription is under the control of five promoters (E1, OT, O, ON, and OS), which yield different transcripts. Here, we studied whether perinatal exposure to a GBH alters uterine ERα gene expression and prompts epigenetic modifications in its regulatory regions during the preimplantation period. Pregnant rats (F0) were orally treated with 350 mg glyphosate/kg bw/day through food from gestational day (GD) 9 until weaning. F1 females were bred, and uterine samples were collected on GD5 (preimplantation period). ERα mRNA levels and its transcript variants were evaluated by RT-qPCR. Enzyme-specific restriction sites and predicted transcription factors were searched in silico in the ERα promoter regions to assess the methylation status using the methylation-sensitive restriction enzymes-PCR technique. Post-translational modifications of histones were studied by the chromatin immunoprecipitation assay. GBH upregulated the expression of total ERα mRNA by increasing the abundance of the ERα-O transcript variant. In addition, different epigenetic changes were detected in the O promoter. A decrease in DNA methylation was observed in one of the three sites evaluated in the O promoter. Moreover, histone H4 acetylation and histone H3 lysine 9 trimethylation (H3K9me3) were enriched in the O promoter in GBH-exposed rats, whereas H3K27me3 was decreased. All these alterations could account for the increase in ERα gene expression. Our findings show that perinatal exposure to a GBH causes long-term epigenetic disruption of the uterine ERα gene, which could be associated with the GBH-induced implantation failures.

Glyphosate-based herbicide enhances the uterine sensitivity to estradiol in rats.

In a previous work, we detected that postnatal exposure to a glyphosate-based herbicide (GBH) alters uterine development in prepubertal rats causing endometrial hyperplasia and increasing cell proliferation. Our goal was to determine whether exposure to low-dose of a GBH during postnatal development might enhance the sensitivity of the uterus to an estrogenic treatment. Female Wistar pups were subcutaneously injected with saline solution (control) or GBH using the reference dose (2 mg/kg/day, EPA) on postnatal days (PND) 1, 3, 5, and 7. At weaning (PND21), female rats were bilaterally ovariectomized and treated with silastic capsules containing 17ß-estradiol (E2, 1mg/ml) until they were two months of age. On PND60, uterine samples were removed and processed for histology, immunohistochemistry and mRNA extraction to evaluate: i) uterine morphology, ii) uterine cell proliferation by the detection of Ki67, iii) the expression of the estrogen receptors alpha (ESR1) and beta (ESR2), and iv) the expression of WNT7A and ß-catenin. GBH-exposed animals showed increased luminal epithelial height and stromal nuclei density. The luminal and glandular epithelium were markedly hyperplastic in 43% of GBH-exposed animals. GBH exposure caused an increase in E2-induced cell proliferation in association with an induction of both ESR1 and ESR2. GBH treatment decreased membranous and cytoplasmic expression of ß-catenin in luminal and glandular epithelial cells and increased WNT7A expression in the luminal epithelium. These results suggest that early postnatal exposure to a GBH enhances the sensitivity of the rat uterus to estradiol, and induces histomorphological and molecular changes associated with uterine hyperplasia.

Perinatal exposure to a glyphosate-based herbicide impairs female reproductive outcomes and induces second-generation adverse effects in Wistar rats.

Glyphosate-based herbicides (GBHs) are the most globally used herbicides raising the risk of environmental exposition. Here, we investigated whether perinatal exposure to low doses of a GBH alters the female reproductive performance, and/or induced second-generation effects related to congenital anomalies or growth alterations. Pregnant rats (F0) received a GBH through food, in a dose of 2 mg (GBH-LD: GBH-low dose group) or 200 mg (GBH-HD: GBH-high dose group) of glyphosate/kg bw/day from gestational day (GD) 9 until weaning. Body weight gain and vaginal canal-opening of F1 females were recorded. Sexually mature F1 females were mated to evaluate their reproductive performance by assessing the pregnancy rate, and on GD19, the number of corpora lutea, the implantation sites (IS) and resorption sites. To analyze second-generation effects on F2 offspring, we analyzed the fetal morphology on GD19, and assessed the fetal length and weight, and the placental weight. GBH exposure neither altered the body weight gain of F1 females, nor vaginal opening onset. Although all GBH-exposed F1 rats became pregnant, a lower number of IS was detected. F2 offspring from both GBH groups showed delayed growth, evidenced by lower fetal weight and length, associated with a higher incidence of small for gestational age fetuses. In addition, higher placental weight and placental index were found in F2 offspring from GBH-HD dams. Surprisingly, structural congenital anomalies (conjoined fetuses and abnormally developed limbs) were detected in the F2 offspring from GBH-HD group. In conclusion, perinatal exposure to low doses of a GBH impaired female reproductive performance and induced fetal growth retardation and structural congenital anomalies in F2 offspring.

Neonatal exposure to a glyphosate-based herbicide alters uterine decidualization in rats.

We investigated whether defective modulation of uterine signaling may cause decidualization failure in rats neonatally exposed to a glyphosate-based herbicide (GBH). Female pups received vehicle or 2mg/kg of GBH from postnatal day (PND) 1 to PND7. On PND8 and PND21, Wnt5a and ß-catenin expression was evaluated in uterine samples. On gestational day (GD) 9, Wnt5a, Wnt7a and ß-catenin expression and Dkk1 and sFRP4 mRNA were evaluated on implantation sites. On PND8, GBH-exposed rats showed increased Wnt5a and ß-catenin expression in luminal epithelium (LE), whereas on PND21, they showed increased Wnt5a and ß-catenin expression in subepithelial stroma but decreased ß-catenin expression in glandular epithelium. On GD9, GBH-exposed rats showed decreased Wnt5a and Wnt7a expression in the antimesometrial zone and LE respectively, without changes in ß-catenin expression, while Dkk1 and sFRP4 were up- and down-regulated respectively. We concluded that neonatal GBH exposure may lead to embryo losses by disturbing uterine signaling.

Uterine ERα epigenetic modifications are induced by the endocrine disruptor endosulfan in female rats with impaired fertility.

High ERα activity may disrupt the window of uterine receptivity, causing defective implantation. We investigated whether implantation failures prompted by endosulfan are associated with aberrant ERα uterine expression and DNA methylation status during the pre-implantation period. ERα-dependent target genes that play a crucial role in the uterine receptivity for embryo attachment and implantation were also investigated. Newborn female rats received corn oil (vehicle, Control), 6 µg/kg/d of endosulfan (Endo6) or 600 µg/kg/d of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5, and 7. On PND90, females were made pregnant and on gestational day 5 (GD5, pre-implantation period) uterine samples were collected. ERα expression was assessed at protein and mRNA levels by immunohistochemistry and real time RT-PCR, respectively. ERα transcript variants mRNA containing alternative 5'-untranslated regions (5'UTRs) were also evaluated. We searched for predicted transcription factors binding sites in ERα regulatory regions and assessed their methylation status by Methylation-Sensitive Restriction Enzymes-PCR technique (MSRE-PCR). The expression of the ERα-dependent uterine target genes, i.e. mucin-1 (MUC-1), insulin-like growth factor-1 (IGF-1), and leukemia inhibitory factor (LIF), was assessed by real time RT-PCR. Both doses of endosulfan increased the expression of ERα and its transcript variants ERα-OS, ERα-O, ERα-OT and ERα-E1. Moreover, a decreased DNA methylation levels were detected in some ERα regulatory regions, suggesting an epigenetic up-regulation of it transcription. ERα overexpression was associated with an induction of its downstream genes, MUC-1 and IGF-1, suggesting that endosulfan might alter the uterine estrogenic pathway compromising uterine receptivity. These alterations could account, at least in part, for the endosulfan-induced implantation failures.

Neonatal exposure to a glyphosate based herbicide alters the development of the rat uterus.

Glyphosate-based herbicides (GBHs) are extensively used to control weeds on both cropland and non-cropland areas. No reports are available regarding the effects of GBHs exposure on uterine development. We evaluated if neonatal exposure to a GBH affects uterine morphology, proliferation and expression of proteins that regulate uterine organogenetic differentiation in rats. Female Wistar pups received saline solution (control, C) or a commercial formulation of glyphosate (GBH, 2mg/kg) by sc injection every 48h from postnatal day (PND) 1 to PND7. Rats were sacrificed on PND8 (neonatal period) and PND21 (prepubertal period) to evaluate acute and short-term effects, respectively. The uterine morphology was evaluated in hematoxylin and eosin stained sections. The epithelial and stromal immunophenotypes were established by assessing the expression of luminal epithelial protein (cytokeratin 8; CK8), basal epithelial proteins (p63 and pan cytokeratin CK1, 5, 10 and 14); and vimentin by immunohistochemistry (IHC). To investigate changes on proteins that regulate uterine organogenetic differentiation we evaluated the expression of estrogen receptor alpha (ERα), progesterone receptor (PR), Hoxa10 and Wnt7a by IHC. The GBH-exposed uteri showed morphological changes, characterized by an increase in the incidence of luminal epithelial hyperplasia (LEH) and an increase in the stromal and myometrial thickness. The epithelial cells showed a positive immunostaining for CK8, while the stromal cells for vimentin. GBH treatment increased cell proliferation in the luminal and stromal compartment on PND8, without changes on PND21. GBH treatment also altered the expression of proteins involved in uterine organogenetic differentiation. PR and Hoxa10 were deregulated both immediately and two weeks after the exposure. ERα was induced in the stromal compartment on PND8, and was downregulated in the luminal epithelial cells of gyphosate-exposed animals on PND21. GBH treatment also increased the expression of Wnt7a in the stromal and glandular epithelial cells on PND21. Neonatal exposure to GBH disrupts the postnatal uterine development at the neonatal and prepubertal period. All these changes may alter the functional differentiation of the uterus, affecting the female fertility and/or promoting the development of neoplasias.

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 ERß; 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 ERß (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. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1191-1201, 2017.

Effects of neonatal exposure to a glyphosate-based herbicide on female rat reproduction.

In this study, we investigated whether neonatal exposure to a glyphosate-based herbicide (GBH) alters the reproductive performance and the molecular mechanisms involved in the decidualization process in adult rats. Newborn female rats received vehicle or 2 mg/kg/day of a GBH on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate (i) the reproductive performance on gestational day (GD) 19 and (ii) the ovarian steroid levels, uterine morphology, endometrial cell proliferation, apoptosis and cell cycle regulators, and endocrine pathways that regulate uterine decidualization (steroid receptors/COUP-TFII/Bmp2/Hoxa10) at the implantation sites (IS) on GD9. The GBH-exposed group showed a significant increase in the number of resorption sites on GD19, associated with an altered decidualization response. In fact, on GD9, the GBH-treated rats showed morphological changes at the IS, associated with a decreased expression of estrogen and progesterone receptors, a downregulation of COUP-TFII (Nr2f2) and Bmp2 mRNA and an increased expression of HOXA10 and the proliferation marker Ki67(Mki67) at the IS. We concluded that alterations in endometrial decidualization might be the mechanism of GBH-induced post-implantation embryo loss.

Endosulfan affects uterine development and functional differentiation by disrupting Wnt7a and ß-catenin expression in rats.

Neonatal exposure to a low dose of endosulfan may disrupt the expression of Wnt7a and ß-catenin during uterine development leading to the failure of uterine functional differentiation during implantation. New-born female Wistar rats were treated with vehicle, endosulfan (600 µg/kg/d, E600) or diethylstilbestrol (0.2 µg/kg/d, DES) on postnatal days (PNDs) 1, 3, 5 and 7. Subsequently, uterine histomorphology and the protein expression of Wnt7a and ß-catenin were evaluated on PND8, PND21 and gestational day (GD) 5 (pre-implantation period). In the E600 rats, Wnt7a and ß-catenin protein expression was increased in the epithelium on PND8, and Wnt7a expression was decreased in the endometrial glands on PND21. On GD5, the number of uterine glands was decreased in the E600-and DES-treated rats. In addition, Wnt7a expression was decreased in all uterine compartments, and ß-catenin expression was increased in the luminal and glandular epithelia of the E600-and DES-treated rats. Disruption of Wnt7a and ß-catenin uterine expression in the prepubertal and adult females altered the uterine preparation for embryo implantation, which could be associated with the subfertility triggered by endosulfan.

Neonatal exposure to low doses of endosulfan induces implantation failure and disrupts uterine functional differentiation at the pre-implantation period in rats.

We investigated whether neonatal exposure to low doses of endosulfan affects fertility and uterine functional differentiation at pre-implantation in rats. Newborn female rats received the vehicle, 0.2 µg/kg/d of diethylstilbestrol (DES), 6 µg/kg/d of endosulfan (Endo6) or 600 µg/kg/d of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5, and 7. On PND90, the rats were mated to evaluate their reproductive performance on gestational day (GD) 19 and their ovarian steroid serum levels, endometrial proliferation and implantation-associated proteins on GD5. DES and endosulfan decreased the pregnancy rate and the number of implantation sites. On GD5, DES and endosulfan did not change the serum levels of 17ß-estradiol (E2) and progesterone (P); the endometrial proliferation decreased, which was associated with silencing of Hoxa10 in the Endo600-treated rats. Both doses of endosulfan increased the progesterone receptor (PR) expression, whereas the higher dose led additionally to an increase in estrogen receptor alpha (ERα). In the Endo600-treated rats, the down-regulation of Hoxa10 was associated with a deregulation of the steroid receptor coregulators. Alterations in endometrial proliferation and the endocrine pathway of Hoxa10/steroid receptors/coregulators might be the mechanism of endosulfan-induced implantation failure.

Neonatal exposure to low doses of endosulfan disrupts the expression of proteins regulating uterine development and differentiation.

This study investigates the effects of neonatal exposure to low doses of endosulfan on the expression of proteins regulating uterine development and differentiation. Female pups received vehicle, endosulfan (Endo6: 6 µg/kg, Endo600: 600 µg/kg) or diethylstilbestrol (DES: 0.2 µg/kg) from postnatal day 1 (PND1) to PND7. The uterine expression of estrogen receptor alpha (ERα), progesterone receptor (PR), Hoxa10 and alpha smooth muscle actin (α-SMA) was detected by immunohistochemistry on PND8 (neonatal period) and PND21 (prepubertal period), to evaluate acute and short-term responses. ERα, Hoxa10 and α-SMA were induced in the Endo600 group in both ages, while a striking decrease in PR expression was detected in the prepubertal rats following each dose of endosulfan. DES treatment deregulated ERα and Hoxa10 uterine expression at each age. Studies are currently underway to investigate whether the dysregulation of steroid receptors, Hoxa10 and α-SMA observed following neonatal exposure to endosulfan affect uterine functions in adulthood.