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

Abstract

The safety, including the endocrine disruptive capability, of glyphosate-based herbicides (GBHs) is a matter of intense debate. We evaluated the estrogenic potential of glyphosate, commercial GBHs and polyethoxylated tallowamine adjuvants present as co-formulants in GBHs. Glyphosate (≥10,000 μg/L or 59 μM) promoted proliferation of estrogen-dependent MCF-7 human breast cancer cells. Glyphosate also increased the expression of an estrogen response element-luciferase reporter gene (ERE-luc) in T47D-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 MCF-7 cells treated with glyphosate revealed changes in gene expression reflective of hormone-induced cell proliferation but did not overlap with an ERα gene expression biomarker. Calculation of glyphosate binding energy to ERα predicts a weak and unstable interaction (-4.10 kcal mol) compared to estradiol (-25.79 kcal mol), which suggests that activation of this receptor by glyphosate is via a ligand-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 ERα in vitro, albeit at relatively high concentrations.

Re-registration Challenges of Glyphosate in the European Union

“…It has not been reported to exert estrogen agonist effects in the estrogen receptor activation-reporter assay on JEG3 cells, but was proven to be anti-androgenic at subagricultural and non-cytotoxic dilutions (Gasnier et al, 2009). In contrast, it has been indicated to exert estrogen receptor activation on human transfected estrogen-dependent breast adenocarcinoma cells (T47D-KBluc) with an EC 50 value of 0.005 ng/ml (Thongprakaisang et al, 2013) or in a later study 0.002 mg/ml (Mesnage et al, 2017a), i.e., two orders of magnitude below concentrations causing cell mortality. Glyphosate-based herbicide preparations, containing polyethoxylated tallowamine (POEA) as formulating surfactant, showed a somewhat similar pattern at dilutions corresponding to two orders of magnitude lower glyphosate concentrations, (between 0.001 and 0.1 mg/ml, corresponding to ∼0.005-5% of the dilution used in agricultural applications), with notable outstanding sensitivities for cell lines NE-4C and JAr.…”

“…Although reviews of genotoxicity studies deemed DNA damage by glyphosate and glyphosate-based formulations secondary to cytotoxic effects (Kier and Kirkland, 2013;Kier, 2015), DNA-damaging effects and genotoxicity of glyphosate and particularly of its formulations (Roundup R , Glyfos R , Glyphogan R , Glyphosate-Biocarb R , etc.) on vertebrates (murine and human cells) (Bolognesi et al, 1997;Koller et al, 2012;Young et al, 2015;Townsend et al, 2017), cytotoxic effects of glyphosate-based herbicides on human embryonic and placental cells (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009Gasnier et al, , 2010Mesnage et al, 2013a,b), indication of endocrine disrupting effects by showing activity on estrogen receptors in human hormone-dependent breast cancer cells (Thongprakaisang et al, 2013;Mesnage et al, 2017a), inhibition of the biosynthesis of testosterone and estradiol (Romano et al, 2010) and progesterone (Young et al, 2015) or inhibitory effects on aromatase, a key enzyme in steroid hormone biosynthesis (Cassault-Meyer et al, 2014;Defarge et al, 2016), teratogenic effects on vertebrates by inhibiting the retinoic acid signaling pathway (Lajmanovich et al, 2003;Paganelli et al, 2010;Carrasco, 2013), birth defects in rats , and nephrotoxic and hepatotoxic effects of Roundup R have been demonstrated in rats in connection to RR GM maize (originally published in the journal Food and Chemical Toxicology in September 2012, but retracted by the journal in November 2013 following an alleged intervention from the industry stakeholder (Foucart, 2016), and subsequently republished in another journal a year later) . The analysis of kidney and liver tissues from the same rats by molecular profiling (transcriptomics, proteomics, metabolomics) confirmed pathology of these organs in the lowest dose Roundup R treatment group culminating in non-alcoholic fatty liver disease (Mesnage et al, 2015a(Mesnage et al, ,b, 2017a.…”

“…Roundup Transorb R exerted cytotoxicity on a zebrafish (Danio rerio) hepatocyte cell line ZF-L at concentrations as low as 0.068-0.27 µg/ml (corresponding to glyphosate concentrations of 0.033-0.13 µg/ml), due mostly to lysosomal instability and inhibition of mitochondrial function, and slightly to impaired cell membrane integrity. Synergistic detrimental effects were observed when Roundup Transorb R was applied with an insecticide formulation (Furadan 350 FIGURE 3 | In vitro cytotoxicity of glyphosate (light columns) and its formulated preparation Roundup ® (dark columns) on various cell lines Raji: human hematopoietic Raji (Epstein-Barr virus transformed human lymphocyte) cells (Townsend et al, 2017), DIMF, diploid fin cell line from the Oriental weather loach Misgurnus anguillicaudatus ; HaCaT, human epithelial keratinocyte cells (Elie-Caille et al, 2010); NE-4C, murine stem cell-like neuroectodermal cells ; Hep-2, human epithelial type 2 (HeLa contaminant) cells (Mañas et al, 2009); GM38, human fibroblast cells (Monroy et al, 2005); HT1080, human fibrosarcoma cells (Monroy et al, 2005); HUVEC, primary neonate human umbilical vein endothelial cells (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009); HEK293, embryonic kidney cells (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009;Mesnage et al, 2013a); MC3T3-E1, murine osteoblast precursor cells (Farkas et al, 2018); HepG2, human hepatoma cells (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009); JAr, human chorioplacental cells (Young et al, 2015); JEG3, human choriocarcinoma cells (Benachour et al, 2007;Benachour and Séralini, 2009;Gasnier et al, 2009;Mesnage et al, 2013aMesnage et al, , 2017a. Plain and grid column patterns indicate cytotoxicity detected by MTT test and mutagenicity tests, respectively.…”

A comprehensive analysis of the animal carcinogenicity data for glyphosate from chronic exposure rodent carcinogenicity studies

“…Seralini et al (2014) [36] saw an increase in mammary tumors in female SD rats exposed to the GBH GT Plus with associated hypertrophies and hyperplasia. Glyphosate and GBHs have also been shown to disrupt estrogen receptor alpha in rats [79] and to alter cellular replication and genotoxicity in estrogen-sensitive cell lines [80][81][82][83][84][85][86].…”

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

“…Similarly, different in vitro and in vivo studies have revealed possible endocrine-mediated effects of glyphosate and its commercial formulations on hormone-dependent tissues such as testis (Cassault-Meyer et al 2014), ovary (Perego et al 2017) and uterus (Guerrero Schimpf et al 2017. Glyphosate was suggested to have endocrine-disrupting properties by inhibiting aromatase activity (Richard et al 2005) and activating the estrogen receptor alpha (ESR1) and beta (ESR2) in breast cancer cells (Thongprakaisang et al 2013, Mesnage et al 2017. However, no evidence of potential interaction of glyphosate with endocrine pathways has been detected in the Endocrine Disruptor Screening Program (EDSP) conducted by the US Environmental Protection Agency (EPA) (US EPA 2015).…”