Elsevier

Toxicology

Volume 376, 1 February 2017, Pages 2-14
Toxicology

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

https://doi.org/10.1016/j.tox.2016.06.004Get rights and content

Highlights

  • Neonatal exposure to GBH lead to endometrial hyperplasia and increase proliferation.

  • GBH disrupts proteins involved in uterine organogenetic differentiation.

  • GBH exposure induced persistent increase of PR and Hoxa10 proteins.

Abstract

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, 2 mg/kg) by sc injection every 48 h 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.

Introduction

Glyphosate (N-phosphonomethyl glycine) is the active ingredient of a number of broad-spectrum herbicide formulations, widely used all over the world to control weeds on both cropland and non-cropland areas (Baylis, 2000, Woodburn, 2000, Cerdeira et al., 2007, Duke and Powles, 2008). Commercial formulations of glyphosate include other chemical compounds that act as solvents, adjuvants, preservatives or surfactants. Although these substances are classified as inert compounds, it has been demonstrated that the formulations of glyphosate are more toxic than the compound in its technical grade (Richard et al., 2005, Benachour and Seralini, 2009, Mesnage et al., 2014). In Argentina, the areas of lands in transgenic glyphosate-resistant soybean production have extensively increased, and that has been accompanied by an increase in the herbicide use (Cerdeira et al., 2011). To date, more than 200 million litters of GBHs are applied every year in our country (Aparicio et al., 2013).

Although glyphosate has been considered to have low persistency, the magnitude of environmental impact depends on the rate and frequency of glyphosate application (Mamy et al., 2010). In Argentina, a monitoring study carried out within the main area of soybean production, revealed levels of glyphosate range from 0.1 to 0.7 mg/l in surface waters and 0.5–5 mg/kg in sediments and soil (Peruzzo et al., 2008, Aparicio et al., 2013). Other studies reported the presence of glyphosate residues in pre-harvest soybean (Arregui et al., 2004; Test Biotech, 2013) and in crops at harvest (Agricultural Marketing Service − U.S. Department of Agriculture, 2013). In addition, Curwin et al., 2007a, Curwin et al., 2007b reported glyphosate detection in the urine of families living in farms and nonfarm households, although the estimated exposure levels to glyphosate were several orders of magnitude below thereference dose (RfD) proposed by the U.S. Environmental Protection Agency (U.S. EPA, 1993).

In a recent report, a consensus statement analyzeddifferent results related to GBHs (Myers et al., 2016).Some studies indicate that GBHs disrupt endocrine-signalling systems in vitro (Richard et al., 2005, Gasnier et al., 2009, Thongprakaisang et al., 2013, Defarge et al., 2016). Few in vivo studies have dealt with the effects of GBHs, and no reports are available regarding the consequence of GBHs exposure during critical periods of developmentonthe female reproductive tract.

The female reproductive tract and particularly the uterus are highly sensitive to developmentally disruptive effects of hormonal steroids and natural or synthetic endocrine-disrupting chemicals (EDCs) (Spencer et al., 2012, Varayoud et al., 2014). Transient disruption of the normal developmental program has long-term adverse consequence for uterine function and reproductive health (Varayoud et al., 2008, Varayoud et al., 2011, Milesi et al., 2012, Milesi et al., 2015). In the present work we hypothesized that early postnatal exposure to a GBH might interfere with normal uterine development and differentiation. We evaluated the effects of neonatal exposure to a low dose of a GBH on the uterine morphology, the cell proliferation and the expression of proteins involved in uterine organogenetic differentiation, such as, ERα, PR, Hoxa10 (a member of the Hox gene family) and Wnt7a (a member of the Wnt gene family). The effects were determined at two time points: i) shortly after the end of the exposure period (PND8, neonatal period) to evaluate the acute response to GBH exposure, and ii) two weeks after the end of the exposure period (PND21, prepubertal period), to investigate whether the effects persisted and/or were manifested in a stage distant from the GBH exposure. The selection of proteins to be evaluated was based on their role in uterine organogenetic differentiation. Hoxa10 and Wnt7a, regulate several developmental pathways that guide uterine growth and differentiation during embryogenesis and postnatal development (Benson et al., 1996, Miller and Sassoon, 1998, Spencer et al., 2012). These molecules are also dynamically expressed in adult endometrium, where they play a pivotal role on embryo implantation (Bagot et al., 2000, Dunlap et al., 2011). Because of many EDCs exert their actions through the interaction with sex steroid hormone receptors (Roy et al., 2009), we postulate that uterine ERα and PR proteins could be affected by a GBH developmental exposure.

Section snippets

Animals

All procedures used in this study were approved by the Institutional Ethic Committee of the School of Biochemistry and Biological Sciences (Universidad Nacional del Litoral, Santa Fe, Argentina), and were performed in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals issued by the U.S. National Academy of Sciences. Inbred Wistar strain rats were bred at the Department of Human Physiology (Santa Fe, Argentina) and housed under a

Results

No alterations in maternal care and nursing were detected between the experimental groups. No signs of acute or chronic toxicity were observed in the litters, and no significant differences in weight gain between treated and control pups were recorded during the experiment. At the end of both the neonatal (PND8) and the prepubertal period (PND21) puṕsbody weights were similar between the GBH-exposed animals (PND8: 14.28 ± 0.20 g and PND21: 35.56 ± 0.59 g) and control animals (PND8: 14.22 ± 0.30 g and

Discussion

To our knowledge, this is the first study showing that postnatal exposure to a GBH affects the uterine morphology and the expression of proteins that regulate uterine organogenetic differentiation in neonatal and prepubertal rats. The most relevant effects were incidence of LEH (75% of animals), increase in stromal and circular myometrium thickness, increase in epithelial and stromal proliferation, and induction of Hoxa10, PR and ERα on PND8. Two weeks after the end of the GBH-exposure, some

Conclusion

In summary, our results show that early postnatal exposure to a GBH, at dose similar to the RfD, alters uterine morphology and the expression of proteins involved in uterine development and differentiation. We considered that more studied should be conducted to fully understand the effects of GBH on the female reproductive health, as well as, its molecular mechanism of action. Currently, further experiments are underway to investigate whether the deregulation of steroid receptors, Hoxa10 and

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

Financial support from the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT 2011-1491, PICT 2014-1522;PICT 2014-1522), the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 2011, 11220110100494) and the Universidad Nacional del Litoral (CAI+D 2011, 501 20110100423 LI).

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