Glyphosate-based herbicide worsens alterations induced by cafeteria diet on rat uterus.

Exposure to glyphosate-based herbicides (GBH) and consumption of cafeteria (CAF) diet, which are widespread in Western society, seem to be associated with endometrial hyperplasia (EH). Here, we aimed to evaluate the effects of a subchronic low dose of GBH added to the CAF diet on the rat uterus. Female Wistar rats were fed from postnatal day (PND)21 until PND240 with chow (control) or CAF diet. Since PND140, rats also received GBH (2 mg of glyphosate/kg/day) or water through food, yielding four experimental groups: control, CAF, GBH, and CAF+GBH. On PND240, CAF and CAF+GBH animals showed an increased adiposity index. With respect to the control group, no changes in the serum levels of 17ß-estradiol and progesterone were found. However, progesterone levels were higher in the CAF+GBH group than in the CAF and GBH groups. In the uterus, both studied factors alone and in combination induced morphological and molecular changes associated with EH. Furthermore, the addition of GBH provoked an increased thickness of subepithelial stroma in rats fed with the CAF diet. As a consequence of GBH exposure, CAF+GBH rats exhibited an increased density of abnormal gland area, considered preneoplastic lesions, as well as a reduced PTEN and p27 expression, both tumor suppressor molecules that inhibit cell proliferation, with respect to control rats. These results indicate that the addition of GBH exacerbates the CAF effects on uterine lesions and that the PTEN/p27 signaling pathway seems to be involved. Further studies focusing on the interaction between unhealthy diets and environmental chemicals should be encouraged to better understand uterine pathologies.

A Reliable Method for Quantifying Plasma Cell-Free DNA Using an Internal Standard Strategy: Evaluation in a Cohort of Non-Pregnant and Pregnant Women.

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-µl volume, actin-ß (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Epigenetic disruption of placental genes by chronic maternal cafeteria diet in rats.

Maternal diet has impact on reproduction, fetal development and offspring behavior, although molecular mechanisms remained unknown. Our aims were to assess (1) the effects of a cafeteria (CAF) diet (western diet habits) on female reproductive performance, fetal and placental parameters on gestational day 21 and litter size and pup weight at birth; and (2) placental messenger RNA (mRNA) expression and epigenetic regulation of Insulin-Like Growth Factor (Igf) and Vascular Endothelial Growth Factor (Vegf) and their receptors. Female Wistar rats were fed with control or CAF diet from weaning until parturition. At week 14 after diets started, females were mated and half of the animals were euthanized on gestational day 21 to evaluate reproductive parameters including the pregnancy rate, number of corpora lutea, implantation sites and resorption sites. Moreover, fetal weight and length, placental weight, and placental index were recorded. Placentas were collected for mRNA quantification and DNA methylation analysis. The remaining animals were allowed to give birth and the number and weight of the pups were evaluated. CAF diet did not affect reproductive performance or fetal weight and length. However, CAF-fed animals showed a decrease in placental weight and index and the pups exhibited a low birth weight. Additionally, we found an upregulation of Igf2 and a down regulation of Vegf placental mRNA expression in CAF dams, associated with methylation status changes of their promoters. We conclude that female chronic CAF diet consumption impairs feto-placental development and could be explained by an epigenetic disruption of Igf and Vegf systems.

Steroidogenic enzymes in the hippocampus: Transcriptional regulation aspects.

Neurosteroids are steroids synthesized de novo from cholesterol in brain regions, and regulate processes associated with the development and functioning of the nervous system. Enzymes and proteins involved in the synthesis of these steroids have been detected in several brain regions, including hippocampus, hypothalamus, and cerebral cortex. Hippocampus has long been associated with learning and memory functions, while the loss of its functionality has been linked to neurodegenerative pathologies. In this sense, neurosteroids are critical for the maintenance of hippocampal functions and neuroprotective effects. Moreover, several factors have been shown to deregulate expression of steroidogenic enzymes in the rodent brain, including aging, enrichment experiences, diet habits, drug/alcohol consumption, hormone fluctuations, neurodegenerative processes and other diseases. These transcriptional deregulations are mediated mainly by transcription factors and epigenetic mechanisms. An epigenetic modification of chromatin involves changes in bases and associated proteins in the absence of changes in the DNA sequence. One of the most well-studied mechanisms related to gene silencing is DNA methylation, which involves a reversible addition of methyl groups in a cytosine base. Importantly, these epigenetic marks could be maintained over time and could be transmitted transgenerationally. The aim of this chapter is to present the most relevant steroidogenic enzymes described in rodent hippocampus; to discuss about their transcriptional regulation under different conditions; to show the main gene control regions and to propose DNA methylation as an epigenetic mechanism through which the expression of these enzymes could be controlled.

Aberrant Hoxa10 gene methylation as a mechanism for endosulfan-induced implantation failures in rats.

DNA methylation is a well-established epigenetic mechanism controlling gene expression. Environmental chemicals, such as pesticides have been shown to alter DNA methylation. We have previously shown that the insecticide endosulfan impairs female fertility in rats by increasing the rate of preimplantation embryo losses. In this study, we evaluated whether early postnatal exposure to endosulfan affects long-term transcriptional regulation of Homeobox A10 (Hoxa10) gene, which is a key marker of endometrial receptivity. Female rats were neonatally exposed to 6 or 600 µg/kg/day (ENDO6 and ENDO600, respectively) of endosulfan and uterine samples collected on gestational day (GD) 5. Hoxa10 protein and mRNA levels were assessed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), respectively. In silico analysis of enzyme-specific restriction sites and predicted transcription factors were performed to investigate the methylation status of the regulatory regions of Hoxa10 gene by methylation-sensitive restriction enzymes-PCR technique. The expression of the DNA methyltransferases (Dnmts) was also evaluated. ENDO600 showed a decreased uterine Hoxa10 expression at protein and transcript level, while ENDO6 decreased only the level of transcripts, during the receptive stage. In addition, endosulfan increased levels of Dnmt3a and Dnmt3b. Dysregulation of DNA methylation patterns of Hoxa10 regulatory regions was detected in ENDO6- and ENDO600-treated rats. All these results suggest that aberrant DNA methylation in Hoxa10 gene could be an underlining mechanism contributing to explain endosulfan-induced preimplantation losses.

AHR agonistic effects of 6-PN contribute to potential beneficial effects of Hops extract.

Hops (Humulus lupulus) is used as an alternative to hormone replacement therapy due to the phytoestrogen, 8-prenylnaringenin (8-PN). To examine the potential risks/benefits of hops extract and its compounds (8-PN and 6-prenylnaringenin, 6-PN), we aimed to evaluate the estrogen receptor α (ERα) and aryl hydrocarbon receptor (AHR) signaling pathways in human endometrial cancer cells. Hops extract, 8-PN and 6-PN showed estrogenic activity. Hops extract and 6-PN activated both ERα and AHR pathways. 6-PN increased the expression of the tumor suppressor gene (AHRR), and that of genes involved in the estrogen metabolism (CYP1A1, CYP1B1). Although 6-PN might activate the detoxification and genotoxic pathways of estrogen metabolism, hops extract as a whole only modulated the genotoxic pathway by an up-regulation of CYP1B1 mRNA expression. These data demonstrate the relevant role of 6-PN contained in the hops extract as potential modulator of estrogen metabolism due to its ERα and AHR agonist activity.

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.

Altered uterine angiogenesis in rats treated with a glyphosate-based herbicide.

Glyphosate-based herbicides (GBHs) are the agrochemicals most used around the globe. However, they might have adverse effects on human and animal health. Previously, we showed that female rats neonatally exposed to GBHs exhibit altered expression of morphogenetic molecules and biomarkers of uterine development. We also observed a reduction in the size of implantation sites, altered expression of decidualization-related molecules, and increased post-implantation losses. Since decidualization comprises morphogenetic, biochemical and vascular changes, here we investigated the effects of neonatal GBH exposure on uterine angiogenesis in neonatal and pregnant rats. To achieve this, Wistar female rats were exposed to saline solution or GBH (2 mg glyphosate/kg-bw/day) on post-natal days (PND) 1, 3, 5 and 7. On PND8, uterine samples were collected for developmental studies. On PND90, the remaining females were mated and in the morning of gestational day (GD) 9, the implantation sites were collected. Angiogenesis-related molecules and cells involved in this process were identified and/or measured by immunohistochemistry or RT-PCR. On PND8, GBH-treated rats showed increased vascular endothelial growth factor (VEGF) expression and decreased Notch1, inducible nitric oxide synthase (iNOS) and Angiopoietin-2 (Ang2) mRNA levels. Vascular area, vessel diameter, endothelial cell proliferation, VEGF and Nestin protein expression, and VEGF, Notch1, iNOS and cyclooxygenase-2 (Cox-2) genes were downregulated in implantation sites of exposed females, while Ang2, VEGF receptor 1 and interleukin-10 (IL-10) were increased. Mast cells and macrophages were increased on PND8 and GD9 of treated rats. The increased Transforming growth factor-beta expression in the antimesometrial zone and IL-10 mRNA expression suggest that the M2 type is the predominant population of macrophages on implantation sites. In conclusion, neonatal GBH exposure alters the expression of angiogenesis-related molecules at neonatal uterine development and decidual reaction, suggesting altered vascular support. These alterations might contribute to the increased post-implantation losses observed in GBH-treated rats.

Glyphosate Herbicide: Reproductive Outcomes and Multigenerational Effects.

Glyphosate base herbicides (GBHs) are the most widely applied pesticides in the world and are mainly used in association with GBH-tolerant crop varieties. Indiscriminate and negligent use of GBHs has promoted the emergence of glyphosate resistant weeds, and consequently the rise in the use of these herbicides. Glyphosate, the active ingredient of all GBHs, is combined with other chemicals known as co-formulants that enhance the herbicide action. Nowadays, the safety of glyphosate and its formulations remain to be a controversial issue, as evidence is not conclusive whether the adverse effects are caused by GBH or glyphosate, and little is known about the contribution of co-formulants to the toxicity of herbicides. Currently, alarmingly increased levels of glyphosate have been detected in different environmental matrixes and in foodstuff, becoming an issue of social concern. Some in vitro and in vivo studies have shown that glyphosate and its formulations exhibit estrogen-like properties, and growing evidence has indicated they may disrupt normal endocrine function, with adverse consequences for reproductive health. Moreover, multigenerational effects have been reported and epigenetic mechanisms have been proved to be involved in the alterations induced by the herbicide. In this review, we provide an overview of: i) the routes and levels of human exposure to GBHs, ii) the potential estrogenic effects of glyphosate and GBHs in cell culture and animal models, iii) their long-term effects on female fertility and mechanisms of action, and iv) the consequences on health of successive generations.

Epigenetic Changes Associated With Exposure to Glyphosate-Based Herbicides in Mammals.

Glyphosate is a phosphonomethyl amino acid derivative present in a number of non-selective and systemic herbicides. During the last years the use of glyphosate-based herbicide (GBH) has been increasing exponentially around the world, including Argentina. This fact added to the detection of glyphosate, and its main metabolite, amino methylphosphonic acid (AMPA), in environmental matrices such as soil, sediments, and food, has generated great concern about its risks for humans, animals, and environment. During the last years, there were controversy and intense debate regarding the toxicological effects of these compounds associated with the endocrine system, cancer, reproduction, and development. The mechanisms of action of GBH and their metabolites are still under investigation, although recent findings have shown that they could comprise epigenetic modifications. These are reversible mechanisms linked to tissue-specific silencing of gene expression, genomic imprinting, and tumor growth. Particularly, glyphosate, GBH, and AMPA have been reported to produce changes in global DNA methylation, methylation of specific genes, histone modification, and differential expression of non-coding RNAs in human cells and rodents. Importantly, the epigenome could be heritable and could lead to disease long after the exposure has ended. This mini-review summarizes the epigenetic changes produced by glyphosate, GBHs, and AMPA in humans and rodents and proposes it as a potential mechanism of action through which these chemical compounds could alter body functions.

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.

The impact of sensory and motor enrichment on the epigenetic control of steroidogenic-related genes in rat hippocampus.

In the present study, we analyzed the effects of a short-term environmental enrichment on the mRNA expression and DNA methylation of steroidogenic enzymes in the hippocampus. Thus, young adult (80-day-old) and middle-aged (350-day-old) Wistar female rats were exposed to sensory (SE) or motor (ME) enrichment during 10 days and compared to animals housed under standard conditions. SE was provided by an assortment of objects that included plastic tubes and toys; for ME, rodent wheels were provided. In young adult animals, SE and ME increased the mRNA expression of cytochrome P450 17α-hydroxylase/c17,20-lyase, steroid 5α-reductase type 1 (5αR-1) and 3α-hydroxysteroid dehydrogenase and decreased the methylation levels of 5αR-1 gene. In middle-aged rats, ME and SE upregulated the gene expression of aldosterone synthase and decreased the methylation state of its promoter. These results propose that SE and ME differentially regulate the transcription of neurosteroidogenic enzymes through epigenetic mechanisms in young and aged rats.

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.