Male mammary gland development and methylation status of estrogen receptor alpha in Wistar rats are modified by the developmental exposure to a glyphosate-based herbicide

https://doi.org/10.1016/j.mce.2018.11.005Get rights and content

Highlights

  • Developmental exposure to GBH modifies mammary gland development in male rats.

  • GBH decreases ESR1 protein and mRNA expression in the male mammary gland.

  • GBH induces epigenetic changes in ESR1 promoters in post-pubertal males.

Abstract

Postnatal treatment with glyphosate-based herbicides (GBHs) induces endocrine-disrupting effects on the male rat mammary gland. In this study, the effects of developmental exposure to GBH on mammary gland growth and development, and the possible molecular mechanisms involved, were evaluated in pre- and post-pubertal male rats. To this end, pregnant rats were orally exposed through the food to 0, 3.5 or 350 mg GBH/kg bw/day from gestational day 9 until weaning. Mammary gland development and estradiol (E2) and testosterone (T) serum levels of male offspring were evaluated on postnatal day (PND)21 and PND60. Besides, prolactin (PRL) serum levels, proliferation index, androgen (AR) and estrogen receptor alpha (ESR1) expression, ESR1 alternative transcript mRNA levels, and DNA methylation status of ESR1 promoters were assessed on PND60. No differences between groups were observed in mammary gland development at PND21 or in E2 and T levels on both PNDs studied. On PND60, GBH3.5-exposed animals presented similar mammary gland histology but higher AR protein expression than controls, whereas GBH350-exposed males presented a less developed mammary gland, accompanied by a lower proliferation index, similar AR levels, and slightly increased PRL serum levels than controls. In both exposed groups, ESR1 expression was lower than in control rats, being lower in GBH350-exposed rats. GBH also altered the abundance of ESR1 transcript variants by hypermethylation of ESR1 promoters. GHB3.5 decreased only ESR1-OS expression, whereas GBH350 affected ESR1-O, OT and E1 expression. Our results show that developmental exposure to GBH induces epigenetic changes in ESR1, which could be responsible for the altered male mammary gland development observed in GBH350-exposed animals.

Introduction

Glyphosate (N-phosphonomethyl glycine) is an active ingredient of broad-spectrum herbicide formulations, whose primary mechanism of action is the inhibition of 5-enolpyruvylshikimate 3-phosphate synthase, an enzyme essential for the formation of aromatic amino acids in plants (Steinrucken and Amrhein, 1980). The use of glyphosate-based herbicides (GBHs) has rapidly increased over the last two decades and is expected to continue to increase due to the emergence of glyphosate-resistant weeds and increased applications of GBHs, such as for pre-harvest crop desiccation (Benbrook, 2016; Vandenberg et al., 2017). In Argentina, more than 200 million liters of GBHs are applied every year (Aparicio et al., 2013) and their extensive use has been accompanied by the constant increase in transgenic glyphosate-resistant soybean production (CASAFE, 2012). Recent monitoring studies have evidenced the presence of glyphosate residues and its metabolite aminomethylphosphonic acid (AMPA) in different regions of Argentina, such as Buenos Aires, Corrientes, Córdoba, Entre Ríos, La Pampa and Santa Fe. These compounds have been detected in surface water, sediments, and soil (Aparicio et al., 2013; Bonansea et al., 2017; Mac Loughlin et al., 2017; Primost et al., 2017; Ronco et al., 2016), as well as in the respirable dust emitted by agricultural soil (Mendez et al., 2017). Furthermore, these compounds have been detected in a variety of crops at harvest and in processed food (EFSA, 2017a; Myers et al., 2016), as well as in human urine samples (Connolly et al., 2017; Mills et al., 2017; Niemann et al., 2015; Parvez et al., 2018) and maternal and umbilical cord serum (Kongtip et al., 2017). This widespread presence of these compounds shows that there is a risk of environmental exposure and concern about their possible effects on the environment and human health. Several studies have reported adverse effects of GBH exposure on both the female and male reproductive systems at both low and environmentally relevant doses (Mesnage et al., 2015; Székács and Darvas, 2018). In adult female rats, it has been shown that GBH affects the expression of estrogen-sensitive genes in the uterus (Varayoud et al., 2017), whereas early postnatal exposure alters uterine development (Guerrero Schimpf et al., 2017), the reproductive performance and uterine signaling (Ingaramo et al., 2016, 2017) and enhances the sensitivity of the rat uterus to estradiol (E2) (Guerrero Schimpf et al., 2018). Also, perinatal exposure to GBH impairs the female reproductive performance and induces structural congenital anomalies in F2 offspring (Milesi et al., 2018). In male rodents, a systematic review has shown that glyphosate exposure decreases sperm concentration (Cai et al., 2017). In addition in rats, adult and perinatal exposure to GBH decreases testosterone (T) serum concentration, increases aromatase expression levels in the testis, and increases abnormal sperm morphology (Cassault-Meyer et al., 2014; Dallegrave et al., 2007; Owagboriaye et al., 2017). However, Romano et al. (2010) and Romano et al. (2012) showed that, depending on the window of exposure, the effects on steroid hormone serum levels are different. Gestational and early postnatal exposure to GBH increases T and E2 concentration (Romano et al., 2012), whereas peri-pubertal exposure to GBH decreases T but does not affect E2 levels (Romano et al., 2010). Also, it has been reported that glyphosate may induce human breast cancer cell proliferation by directly activating estrogen receptor alpha (ESR1) in vitro (Thongprakaisang et al., 2013) or by activating ESR1 through a ligand-independent mechanism (Mesnage et al., 2017). Despite all the above-mentioned experimental data, there is still controversy about the endocrine disrupting potential of glyphosate, considering that no evidence of the potential interaction of glyphosate with endocrine pathways has been detected by the United States Environmental Protection Agency (US EPA) (EPA, 2015) or the European Food Safety Authority (EFSA) (EFSA, 2017b).

Exposure to certain endocrine-disrupting chemicals has been shown to alter the development of the male rat mammary gland (Filgo et al., 2016; Kass et al., 2015; Mandrup et al., 2016). Some of them induce developmental changes only in the male mammary gland (You et al., 2002), whereas others induce alterations in both the male and female mammary gland, affecting males earlier than females (Mandrup et al., 2012). Based on these results, the mammary gland of male rats constitutes a useful model to study the effects of potential endocrine disruptors. Recently, we have shown that postnatal exposure to the endocrine disruptor endosulfan induces premalignant lesions in the mammary gland of post-pubertal male rats (Altamirano et al., 2017a), and that an early postnatal subcutaneous treatment with a GBH increases mammary gland development of pre-pubertal male rats and increases mast cell infiltration, proliferation index, and ESR1 expression in post-pubertal ones (Altamirano et al., 2018). Considering that both the susceptibility window and the administration route to which animals are exposed may have different impacts on the endocrine system (Bergman et al., 2012; Yang et al., 2015), in the present study, we decided to assess the effects of an oral exposure to a GBH during development (in utero + lactation). Gestational and neonatal stages are critical periods of mammary gland development, which could be altered by exposure to endocrine disruptors. Since the primary ducts are formed and begin their extension and branching during these stages, any interference of these events could lead to altered mammary development, leaving lasting effects on the gland (Fenton, 2006). Given that, as mentioned above, glyphosate residues have been detected in water courses (Ronco et al., 2016), crops at harvest and food (Myers et al., 2016), as well as maternal and umbilical cord serum (Kongtip et al., 2017), the risk of oral and in utero exposure exists. Thus, the aims of the present study were to determine whether oral developmental exposure to a GBH affects mammary gland growth and development in pre- and post-pubertal male rats, and to evaluate the possible mechanisms involved.

Section snippets

Animals

All the experimental protocols were approved by the Ethical Committee of the Facultad de Bioquímica y Ciencias Biológicas of the Universidad Nacional del Litoral (FBCB-UNL), Santa Fe, Argentina. Animals were treated humanely and with regard for alleviation of suffering. Sexually mature female and male rats (90 days old) of a Wistar-derived strain bred at the Department of Human Physiology (FBCB-UNL) were used. The animals were maintained in a controlled environment (22 ± 2 °C; 14 h of light)

Results

As previously described by Milesi et al. (2018), there were no changes in the average body weight or food consumption of F0 dams, and glyphosate serum concentrations were 0.039 ± 0.006 mg/l in GBH3.5-exposed dams and 3.80 ± 1.20 mg/l in GBH350 ones, whereas glyphosate was not detectable in control F0 dams. In addition, no AMPA was detected in the F0 dams of any of the groups studied. Also, the treatment with GBH from GD9 until weaning, administered through the diet, produced no signs of embryo

Discussion

Currently, due to the increasing and widespread use of glyphosate formulations (Mesnage et al., 2015; Vandenberg et al., 2017) and their potential endocrine-disrupting properties (Cai et al., 2017; Guerrero Schimpf et al., 2017; Ingaramo et al., 2017; Varayoud et al., 2017), their possible consequences on human health are an issue of concern. In the present study, the effect of developmental exposure to a GBH was evaluated on the male rat mammary gland, given the high sensitivity of this organ

Conflicts of interest

The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by grants from Universidad Nacional del Litoral [CAI+D 2016 50420150100088LI] and the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) [PICT2014 N°1348], Argentina. These funding sources were not involved in the study design, the collection, analysis or interpretation of the data, the writing of the report, or the decision to submit the article for publication.

Acknowledgments

We thank Melisa Delconte and Laura Bergero from the Instituto de Salud y Ambiente del Litoral (UNL-CONICET), and Juan Grant and Eduardo Masat from the Facultad de Bioquímica y Ciencias Biológicas (UNL) for technical assistance and animal care. G.A.A. and A.L.G. are fellows and L.K. and V.L.B. are Career Investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

References (79)

  • T. Doshi et al.

    Hypermethylation of estrogen receptor promoter region in adult testis of rats exposed neonatally to bisphenol A

    Toxicology

    (2011)
  • M. Guerrero Schimpf et al.

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

    Toxicology

    (2017)
  • P.I. Ingaramo et al.

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

    Reprod. Toxicol.

    (2017)
  • L. Kass et al.

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

    Reprod. Toxicol.

    (2015)
  • J. Kato et al.

    The multiple untranslated first exons and promoters system of the oestrogen receptor gene in the brain and peripheral tissues of the rat and monkey and the developing rat cerebral cortex

    J. Steroid Biochem. Mol. Biol.

    (1998)
  • T.M. Mac Loughlin et al.

    Pesticide impact study in the peri-urban horticultural area of Gran La Plata, Argentina

    Sci. Total Environ.

    (2017)
  • K.R. Mandrup et al.

    Mixtures of environmentally relevant endocrine disrupting chemicals affect mammary gland development in female and male rats

    Reprod. Toxicol.

    (2015)
  • M.J. Mendez et al.

    Glyphosate and Aminomethylphosphonic acid (AMPA) contents in the respirable dust emitted by an agricultural soil of the central semiarid region of Argentina

    Aeolian Research

    (2017)
  • R. Mesnage et al.

    Potential toxic effects of glyphosate and its commercial formulations below regulatory limits

    Food Chem. Toxicol.

    (2015)
  • R. Mesnage et al.

    Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents

    Food Chem. Toxicol.

    (2017)
  • M.M. Milesi et al.

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

    Mol. Cell. Endocrinol.

    (2017)
  • F.O. Owagboriaye et al.

    Reproductive toxicity of Roundup herbicide exposure in male albino rat

    Exp. Toxicol. Pathol.

    (2017)
  • S.D. Parlee et al.

    Quantifying size and number of adipocytes in adipose tissue

    Methods Enzymol.

    (2014)
  • D.A. Parodi et al.

    Alteration of mammary gland development and gene expression by in utero exposure to arsenic

    Reprod. Toxicol.

    (2015)
  • J.E. Primost et al.

    Glyphosate and AMPA, "pseudo-persistent" pollutants under real-world agricultural management practices in the Mesopotamic Pampas agroecosystem, Argentina

    Environ. Pollut.

    (2017)
  • T.T. Schug et al.

    Endocrine disrupting chemicals and disease susceptibility

    J. Steroid Biochem. Mol. Biol.

    (2011)
  • J.P. Stanko et al.

    Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts

    Reprod. Toxicol.

    (2015)
  • H.C. Steinrucken et al.

    The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase

    Biochem. Biophys. Res. Commun.

    (1980)
  • S. Thongprakaisang et al.

    Glyphosate induces human breast cancer cells growth via estrogen receptors

    Food Chem. Toxicol.

    (2013)
  • G.M. Williams et al.

    Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans

    Regul. Toxicol. Pharmacol.

    (2000)
  • P. Andrews et al.

    Sensitive detection of the endocrine effects of the estrogen analogue ethinylestradiol using a modified enhanced subacute rat study protocol (OECD Test Guideline no. 407)

    Arch. Toxicol.

    (2002)
  • C.M. Benbrook

    Trends in glyphosate herbicide use in the United States and globally

    Environ. Sci. Eur.

    (2016)
  • A. Bergman et al.

    State of the Science of Endocrine Disrupting Chemicals 2012

    (2012)
  • R.I. Bonansea et al.

    The fate of glyphosate and AMPA in a freshwater endorheic basin: an ecotoxicological risk assessment

    Toxics

    (2017)
  • K.L. Burnstein

    Regulation of androgen receptor levels: implications for prostate cancer progression and therapy

    J. Cell. Biochem.

    (2005)
  • R.H. Cardy

    Sexual dimorphism of the normal rat mammary gland

    Vet. Pathol.

    (1991)
  • CASAFE

    Mercado Argentino 2012 de productos fitosanitarios. Camara Argentina de Sanidad Agropecuaria y Fertilizantes

    (2012)
  • S. Cikos et al.

    Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis

    BMC Mol. Biol.

    (2007)
  • R. Czerniak

    Gender-based differences in pharmacokinetics in laboratory animal models

    Int. J. Toxicol.

    (2001)
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