Endocrine disruption is an environmental and public health problem (Yilmaz et al., 2020). Several natural or synthetic compounds, among them pesticides, have been recognized as “endocrine disruptor chemicals” (EDCs) due to their molecular structure capable of mimicking or antagonizing the activity of natural hormones (Uslu et al., 2013; McKinlay et al., 2008; Yaşar et al., 2017; Diamanti-Kandarakis et al., 2009). Robust evidence demonstrates that human exposure to EDCs is associated with adverse health outcomes, including reduced fertility, birth defects of reproductive organs, changes in the onset of puberty, and endocrine-related neoplasm such as uterine and breast cancer (Rachoń, 2015; Morgan et al., 2017; Jacobsen et al., 2010).
Glyphosate, or N-(phosphonomethyl)glycine, is a derivative amino acid used as an active ingredient in a set of formulations known as Glyphosate-Based Herbicides (GBHs), Roundup® being the most well-known brand name (Muñoz et al., 2021). Its main mechanism of action consists of the inhibition of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), involved in the biosynthesis of aromatic amino acids in plants and microorganisms (Muñoz et al., 2021; Ingaramo et al., 2020). Besides competitive inhibition of EPSP, glyphosate acts as a chelating agent that binds macro and micronutrients, such as iron, manganese, copper, and zinc, decreasing their uptake and availability in plants (Mertens et al., 2018).
GBHs usage has been successfully diversified and exponentially increased worldwide due to its high effectiveness, particularly after introducing genetically modified glyphosate-tolerant weeds in the mid-'90s (Benbrook, 2016a). Only in the last decade, 6.1 billion kgs of glyphosate have been applied (Benbrook, 2016b). Therefore, trace levels of glyphosate and aminomethylphosphonic acid (AMPA), its main metabolite, can now easily be detected in soil, air, water, food (Demonte et al., 2018; Mercurio et al., 2014; Simonetti et al., 2019) and in various human fluids (Philipp Schledorn, 2014; Steinborn et al., 2016; Gillezeau et al., 2019), raising concerns about possible side effects. In this context, the European Food Safety Authority (EFSA) asserted there is no convincing evidence for supporting the potential endocrine-disrupting effects of glyphosate (EFSA, 2015). However, a large number of recent in vitro and in vivo toxicological studies have questioned its safety, showing that GBHs lead to a plethora of adverse effects typical of endocrine disruption, mainly by affecting physiological processes regulated by sex steroid hormones, such as reproduction, pregnancy, gestation, gonadal development and offspring (Muñoz et al., 2021; Serra et al., 2021; Dai et al., 2016; Guerrero Schimpf et al., 2017; Varayoud et al., 2017; Ingaramo et al., 2016; Perego et al., 2017; Zhao et al., 2021; Zhang et al., 2020; Manservisi et al., 2019; Pham et al., 2019; Romano et al., 2010, 2012; Clair et al., 2012; Cassault-Meyer et al., 2014).
The most predominant circulating estrogen is the 17β-estradiol (E2), a pivotal steroidal hormone involved in a wide variety of effects on diverse cell and organ systems (Yaşar et al., 2017). The effects of E2 are exerted mainly by estrogen receptor alpha (ERα), a member of the nuclear receptor superfamily of transcription factors (Yaşar et al., 2017). Upon E2 binding, ERα undergoes a conformational change that results in its dimerization and nuclear translocation. There, it can directly bind to estrogen response elements (EREs), in the promoter region of estrogen response genes, eliciting transcriptional responses (direct genomic effects) (McDonnell and Norris, 2002; Tsai and O'Malley, 1994). ERα is also the target of several posttranslational modifications (PTMs), such as phosphorylation, glycosylation, and acetylation. Of these, phosphorylation at its N-terminal domain is considered the most important PTM since alters ERα activity through distinct mechanisms (Likhite et al., 2006). The ERα phosphorylation can be stimulated by ligand-dependent mechanism, through cyclin-dependent kinase 7 (Cdk7) among others (Chen et al., 2000), and by ligand-independent mechanisms through kinases such as ERK, S6K1, PKA, PI3K/Akt, GSK3, Cdk2/cyclin A, in response to extracellular signals such as peptide growth factors and cytokines (Thomas et al., 2008; Medunjanin et al., 2005; Chen et al., 2002; Cheng et al., 2007; Lannigan, 2003; Anbalagan and Rowan, 2015; Vrtačnik et al., 2014). On the other hand, E2 can trigger rapid cytoplasmic signaling cascades that do not involve ERα binding directly to DNA, but rather a binding to transcription factors already bound to the promoter (non-genomic effects) (Levin, 2009).
The US Environmental Protection Agency (USEPA) through its Endocrine Disruptor Screening Programs (EDSP) stated that glyphosate has not the potential to interact with the estrogen signaling pathway (USEPA and EDSP, 2015), which has been supported by results of independent researchers (Kojima et al., 2004; Tóth et al., 2020). However, recent findings suggest that glyphosate display estrogenic activity in animal models and hormone-sensitive cell lines (Thongprakaisang et al., 2013; Verderame and Scudiero, 2019; De Almeida et al., 2018). The first evidence was shown by Hokanson et al. who reported that glyphosate disrupted estrogen-regulated genes in breast cancer cells (Hokanson et al., 2007). Further, Thongprakaisang et al. demonstrated that glyphosate treatment induces proliferation in human breast cancer cells via ERα (Thongprakaisang et al., 2013), which was later supported by Mesnage et al. whose results showed that glyphosate increases ERα transcriptional activity (Mesnage et al., 2017). Since the molecular architecture of glyphosate does not display the structural features of classical ERα agonists, the authors of this article assumed that it is unlikely that glyphosate can bind directly to ERα. Therefore, based on their results, they hypothesized that glyphosate possibly exerts its effects in a ligand-independent pathway through cAMP-dependent protein kinase A (Mesnage et al., 2017). This hypothesis was endorse by recent results that demonstrate that glyphosate can induce ERα activity stimulating mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathway in a cholangiocarcinoma cell model (Sritana et al., 2018). However, a new study suggests that a direct binding between glyphosate and ERα ligand binding site to promote estrogenic activity is chemically feasible (Liu et al., 2022).
Therefore, since the estrogenic activity of glyphosate and the eventual mechanism by which it is exerted is still a matter of debate, here we evaluated the molecular pathways related to ERα activity and the functional consequences associated with exposure to glyphosate in breast cancer cell lines positives for ERα, as MCF7 and T47D.