Evaluation of Estrogen Receptor Alpha Activation by Glyphosate-Based Herbicide Constituents

Evaluation of Estrogen Receptor Alpha Activation by Glyphosate-Based Herbicide Constituents

King’s Research Portal DOI: 10.1016/j.fct.2017.07.025 Document Version Publisher's PDF, also known as Version of record Link to publication record in King's Research Portal Citation for published version (APA): Mesnage, R., Phedonos, A., Biserni, M., Arno, M., Balu, S., Corton, J. C., Ugarte, R., & Antoniou, M. N. (2017). Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents. Food and Chemical Toxicology, 108, 30-42. https://doi.org/10.1016/j.fct.2017.07.025 Citing this paper Please note that where the full-text provided on King's Research Portal is the Author Accepted Manuscript or Post-Print version this may differ from the final Published version. If citing, it is advised that you check and use the publisher's definitive version for pagination, volume/issue, and date of publication details. 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Download date: 26. Sep. 2021 Food and Chemical Toxicology 108 (2017) 30e42 Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox Evaluation of estrogen receptor alpha activation by glyphosate-based herbicide constituents Robin Mesnage a, Alexia Phedonos a, Martina Biserni a, Matthew Arno b, Sucharitha Balu b, * J. Christopher Corton c, Ricardo Ugarte d, Michael N. Antoniou a, a Gene Expression and Therapy Group, King's College London, Faculty of Life Sciences & Medicine, Department of Medical and Molecular Genetics, 8th Floor, Tower Wing, Guy's Hospital, Great Maze Pond, London SE1 9RT, United Kingdom b Genomics Centre, King's College London, Waterloo Campus, 150 Stamford Street, London SE1 9NH, United Kingdom c Integrated Systems Toxicology Division, US Environmental Protection Agency, 109 T.W. Alexander Dr MD-B143-06, Research Triangle Park, NC 27711, United States d Instituto de Ciencias Químicas, Facultad de Ciencias, Universidad Austral de Chile, Independencia 641, Valdivia, Chile article info abstract Article history: The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a Received 20 March 2017 matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and Received in revised form polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (10,000 mg/L or 20 June 2017 59 mM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also Accepted 11 July 2017 increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D- Available online 12 July 2017 KBluc cells, which was blocked by the estrogen antagonist ICI 182,780. Commercial GBH formulations or their adjuvants alone did not exhibit estrogenic effects in either assay. Transcriptomics analysis of Keywords: MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced Endocrine disrupting effects a Pesticides cell proliferation but did not overlap with an ER gene expression biomarker. Calculation of glyphosate À1 Glyphosate binding energy to ERa predicts a weak and unstable interaction (À4.10 kcal mol ) compared to estradiol À Breast cancer (À25.79 kcal mol 1), which suggests that activation of this receptor by glyphosate is via a ligand- Estrogen receptor independent mechanism. Induction of ERE-luc expression by the PKA signalling activator IBMX shows that ERE-luc is responsive to ligand-independent activation, suggesting a possible mechanism of glyphosate-mediated activation. Our study reveals that glyphosate, but not other components present in GBHs, can activate ERa in vitro, albeit at relatively high concentrations. © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction surfactants. They are generally included in commercial GBH for- mulations, but are also sold and used separately as adjuvants and Glyphosate-based herbicides (GBHs) are the most widely used added during the preparation of the agriculturally applied pesticide pesticides worldwide. Glyphosate acts as a herbicide by inhibiting mixture. In 2014, the amount of GBH sprayed by farmers was the enzyme 5-enolpyruvylshikimate 3-phosphate synthase equivalent to glyphosate being applied at 0.53 kg/hectare on all (EPSPS), causing a shortage of aromatic amino acids (Boocock and cropland worldwide (Benbrook, 2016). Glyphosate is routinely Coggins, 1983). Glyphosate occupies the binding site of the sec- detected in foodstuffs (EFSA, 2014), air and rain (Majewski et al., ond substrate of EPSPS (phosphoenol pyruvate), mimicking an in- 2014). The half-life of glyphosate is variable depending on envi- termediate state of the ternary enzyme substrate complex ronmental conditions. For example, it ranged from 47 to 315 days (Schonbrunn et al., 2001). GBH are commercialized in the form of depending on light and temperature in a study using coastal mixtures consisting of glyphosate and various co-formulants, seawater from the Great Barrier Reef (Mercurio et al., 2014). which are required to stabilise and allow penetration of glypho- Epidemiological data on the human body burden of GBH residues is sate into plants. The major class of co-formulants is represented by very limited but evidence suggests that glyphosate and its metab- olites are wide-spread (Niemann et al., 2015). Although the contamination of human biological fluids by compounds used as * Corresponding author. co-formulants in commercial pesticides is poorly investigated, their E-mail address: michael.antoniou@kcl.ac.uk (M.N. Antoniou). http://dx.doi.org/10.1016/j.fct.2017.07.025 0278-6915/© 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). R. Mesnage et al. / Food and Chemical Toxicology 108 (2017) 30e42 31 presence in food (Ferrer et al., 2011) and in the environment (Berge Segars, 2002). These different modes of action can be revealed by et al., 2012; Tush and Meyer, 2016) is well documented. transcriptomics analysis as they have distinct gene expression A number of in vivo toxicity studies have suggested effects of signatures (Dudek and Picard, 2008). The quantum mechanical glyphosate and its commercial formulations on reproductive or- behaviour of glyphosate ions within the ligand binding domain gans (Mesnage et al., 2015a). This includes studies on the uterus (LBD) of ERa was assessed using molecular dynamic simulation. (Guerrero Schimpf et al., 2017), the hypothalamice Own N-layered Integrated Molecular Orbital and Molecular me- pituitaryegonadal axis (Romano et al., 2012), testis (Cassault- chanics (ONIOM) calculations were undertaken from the lowest Meyer et al., 2014) and ovaries (Armiliato et al., 2014). However, energy structures of molecular dynamic simulations in order to it is not clear whether toxic effects observed in these studies are evaluate the binding energy of complexes. due to endocrine disrupting mechanisms or result from a more Our results reveal that glyphosate (>10,000 mg/L) but none of general cytotoxicity mechanism. Glyphosate was suggested to have the other compounds tested activate ERa. The predicted weak endocrine interference properties by inhibiting aromatase enzyme interaction between ERa and glyphosate, suggests that gene acti- activity (Richard et al., 2005) and activating estrogen receptors (ER) vation by this compound is through other mechanisms (for (Thongprakaisang et al., 2013). In contrast, no evidence of potential example, a non-ligand binding mechanism) than direct binding to interaction of glyphosate with the estrogen pathway has been the receptor. detected in the Endocrine Disruptor Screening Program (EDSP) conducted by the US Environmental Protection Agency (EPA) (US 2. Material and methods EPA, 2015). Thus the endocrine disruptive capability of glyphosate remains uncertain. 2.1. Reagents Other modes of action can also be postulated for glyphosate- induced toxicity in a number of diverse species. Glyphosate inhi- All reagents and chemicals, unless otherwise specified, were of bition of EPSPS in plants is by competitive inhibition of its substrate analytical grade and were purchased from Sigma-Aldrich (Gilling- phosphoenolpyruvate (PEP) binding at the enzyme active site ham, Dorset, UK). Glyphosate (CAS Number: 1071-83-6) used was ® (Schonbrunn et al., 2001). Enzymes binding PEP are regulators of the PESTANAL analytical standard (98.0%) obtained from Sigma- energy metabolism, in particular through the TCA

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