Under the modern lifestyle, humans are exposed to various chemicals such as pesticide residuals in fruits and vegetables, antibiotics in meat and milk, preservatives in cosmetics and personal care products [1
]. These chemicals are usually in low doses and may not have a short term significant impact on the human body, but they can cause long term damages to health [3
]. The effects of low-dose compounds on human health are mainly related to the endocrine system [4
]. These compounds can in fact mimic or influence the action of endogenous hormones through various mechanisms, being therefore referred to as endocrine disrupting chemicals (EDCs) [7
The modern industrial and agricultural system relies heavily on pesticides. The compelling need of high food crop increasingly demands the use of chemicals. This results in the extensive applications of millions of tons of pesticides every year [9
]. Among the many pesticides available on the market, glyphosate is one of the most widely used herbicides in the world. In plants, it affects the synthesis of essential aromatic amino acids by inhibiting the activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway [11
]. As a result, glyphosate is considered harmless to mammals since they do not contain the EPSPS enzyme [12
]. Conversely, neonicotinoids are currently the most widely used agricultural insecticides [13
]. These selective insecticides specifically bind to the α -subunit of nicotinic acetylcholine receptors (nAChR), which is common in all insects [14
]. Due to their difficulty to penetrate the blood-brain barrier, they should exert low toxicity in vertebrates. However, different direct, indirect, and suspect toxic effects of these compounds on vertebrate wildlife and human health have been reported [16
]. Neonicotinoids were introduced in the market in the 1990s, therefore their sales volume has enormously proliferated in recent decades, exceeding 25% of the market in 2010 [19
]. Imidacloprid has become the world’s best-selling insecticide next to glyphosate [20
]. Although the European Union banned the outdoor application of imidacloprid, clothianidin, and thiamethoxam in 2018 due to a high risk for bees that are responsible for pollinating most crops worldwide [21
], neonicotinoids remain the most extensively applied insecticides in the world [22
The intensive use of glyphosate-based herbicides and neonicotinoid insecticides has caused the contamination of soil, water, air, and agricultural products [23
]. The half-life of glyphosate in the field is usually 47 days and it is primarily degraded into aminomethylphosphonic acid (AMPA) and glyoxylic acid by soil microorganisms [29
]. Neonicotinoids can exist for a long time in soil and water, and their half-life being as long as three years [22
]. Given these observations and the increased use of pesticides, concerns are raising about their potential impact on human health and the environment. In particular, mounting evidence discloses that exposure to pesticides can affect the endocrine system. Toxicological and epidemiological studies indicate possible genotoxic and cytotoxic effects as well as birth defects and neurotoxicity in different cell lines and animal models [18
Aiming at establishing the interference of pollutants with human health, we monitored the effect of glyphosate and two neonicotinoids on two critical targets of the endocrine system: Aromatase, the enzyme responsible for estrogen biosynthesis, and estrogen receptor (ER) alpha, the main protein promoting estrogen signaling.
Aromatase catalyzes the transformation of androgens to estrogens [31
]. In vitro experiments showed that glyphosate causes changes of aromatase mRNA levels and activity in placental JEG3 cells and human HEK293 cells, thus interfering with steroid conversion to estradiol [32
]. In addition, the expression of androgen and ERα was inhibited in glyphosate treated HepG2 cells [34
] where also the transcription and activity of aromatase were altered [35
]. Insecticides such as thiacloprid, thiamethoxam, and imidacloprid affect aromatase expression and activity in a co-culture model of fetoplacental steroidogenesis, increasing estrone and estradiol production, while estriol production is decreased [36
]. Thiacloprid, imidacloprid, and thiamethoxam have also been demonstrated to increase aromatase expression and activity in H295R and Hs578t cells [37
Here, a recombinant form of human aromatase is used to test the possible effect of the three pesticides on estrogen biosynthesis, gaining new information at molecular level [39
]. Moreover, all-atom molecular dynamics simulations provided structural insights on the ability of these molecules to target the aromatase enzyme.
At a cellular level, the estrogen-regulated signaling is mainly due to estrogen receptors (ERs). The main ERs are the ERα and ERβ nuclear receptors and the G-protein-coupled estrogen receptor 1, which is a membrane receptor [42
]. Estrogen receptor α (ERα) is a nuclear hormone receptor and a ligand-regulated transcription factor, which mediates the activity of estrogens in vital processes (i.e., reproduction, cardiovascular maintenance, bone density/remodeling). ERα is composed of five functional domains, among which the ligand-binding domain, activated upon estrogen binding, stimulates cell growth and proliferation. After the menopause, increased estrogen levels due to a deregulated activity of aromatase bind as an agonist to ERα, exerting a pro-oncogenic effect by either decreasing apoptosis or promoting cell proliferation [43
]. Therefore, estrogen selective modulators have been developed and one of them, tamoxifen, is extensively used in adjuvant therapy of breast cancer for its ability to act as an ERα antagonist.
Previous studies have investigated the ability of glyphosate to induce an estrogenic activity mediated by ERs. The results of the study of Thongprakaisang et al., 2013 [44
] demonstrated that glyphosate induces an ER-mediated estrogenic activity, mediated by ER activation, similar to 17β-estradiol (E2) on T47D-KBluc cells. However, recently another study, using the same cells, showed that this pesticide induces an estrogenic activity, mediated by ER activation, lower than E2 and that this activation is probably induced by a ligand-independent mechanism [45
]. Moreover, additional studies on different transfected cells showed that glyphosate did not induce any ER-mediated estrogenic activity and did not produce any anti-estrogenic effect when tested in combination with E2 [35
]. Therefore, whether glyphosate can trigger an ER-mediated estrogenic activity remains controversial. To our knowledge, only three studies have been performed on the estrogenic activity of imidacloprid and thiacloprid. In particular, in the study of Mesnage et al., 2018 [47
] the proliferative effect of both pesticides was investigated on estrogen-sensitive cells, while Kojima et al., 2004 [46
] and Westlund and Yargeau, 2017 [48
] assessed the ER-mediated estrogenic activity of imidacloprid and thiacloprid on mammalian or yeast cells, respectively. The results of the three studies demonstrated that the two pesticides induce no proliferative effect and no estrogenic activity was mediated by ERs, while an anti-estrogenic activity was detected testing imidacloprid in combination with E2 on yeast cells.
In this work, in order to increase the knowledge on the estrogenic activity of glyphosate, imidacloprid, and thiacloprid, the gene reporter assay on estrogen-sensitive human breast cancer MCF-7 cells transfected with the ERE-βGlob-Luc-SVNeo plasmid (MELN cells) is applied to test the three pesticides for their possible ER-mediated estrogenic activity.
Possible additive and/or antagonist effects are also investigated. Moreover, docking simulations provide atomic level insights on the potential binding mode of these molecules to the primary ligand (estrogen) binding site as well as to a peripheral allosteric site which may be responsible for the experimental observed additive effect of the pollutant with the endogenous ligand.
2. Materials and Methods
All reagents are analytically pure by purchase from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of chemical compounds were prepared in absolute ethanol or dimethyl sulfoxide (DMSO). Before each experiment, the test sample was diluted into a fresh buffer solution, and the final organic solvent concentration was less than 0.1%. The recombinant human aromatase (Aro) and the human recombinant cytochrome P450 reductase (hCPR) were expressed and purified as previously described [39
2.2. ELISA Assay
An estrone direct competitive ELISA kit (BioVendor, Brno, Czech Republic) was used to evaluate the effect of pesticides on aromatase activity. Different reaction mixtures were set up by mixing 5 nM Aro, 5 nM hCPR, 0.5 mM NADPH, 50 nM androstenedione, and three concentrations of pesticides (500, 1000, and 1500 nM) in a 100 mM potassium buffer (KPi) containing 20% glycerol, 1 mM β-mercaptoethanol at pH 7.0. Reactions were carried out for 10 min at 30 °C, heat-inactivated for 10 min at 90 °C, and centrifuged for 10 min at 11,000 rpm. After centrifugation, the supernatant was diluted 1:8 in the Calibrator A provided by the ELISA kit and the product estrone quantified performing ELISA according to the manufacturer’s instructions. Reactions in the presence of anastrozole or without hCPR were used as negative controls. The concentration of estrone was extrapolated from a calibration curve with known concentrations of estrone.
For the experiment where the catalytic parameters were derived, four substrate concentrations were applied in the reaction mixture (ranging from 25 to 250 nM) in the absence and presence of 1000 and 5000 nM of glyphosate.
2.3. Computational Studies
In order to explain the molecular terms for the action of glyphosate on the aromatase enzyme we docked it into the two possible allosteric sites previously identified [50
]. Docking has been performed with the GLIDE software, release 2020-1 (Schrödinger, LLC, New York, NY, USA) using the single-precision protocol [51
]. The two neonicotinoids, thiacloprid and imidacloprid, were instead docked into the ERαs active site, using as a starting structure the crystal structure of 17-β-estradiol (EST)-bound ERα dimer (PDB id: 1qku) [52
]. In this structure, we have searched for putative allosteric pockets using the SiteMap algorithm [53
In order to account for its flexibility of the receptor and since is flexibility resulted to be of paramount importance for the identification of novel allosteric inhibitors [54
], we performed classical Molecular Dynamics (MD) simulations on the complex with the aromatase enzyme. We employed as a starting structure of our simulation the equilibrated enzyme model which was embedded in a mimic of a membrane bilayer by using the CHARMMGUI webserver [55
]. This consisted of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and 6 wt% of cholesterol (CHL) in order to mimic the endoplasmic reticulum membrane. Physiological protonation states were calculated with the webserver H++ [56
]. Asp309 was considered in its neutral form consistently with other literature studies [57
]. The glyphosate molecule was considered in the most likely protonation state at physiological pH. According to literature data, the first protonation of the molecule occurs on its phosphate group [58
The Parm99SB AMBER force field (FF) [59
] and lipid14 FF [61
] were used for the protein and the lipids, respectively.
The Shahrokh et al. parameters were used for the heme moiety and Cys437 [62
]. Simulations were done in the presence of the substrate androstenedione (ASD) in the active site and of glyphosate in the allosteric pockets for which the general Amber FF (GAFF) was employed [63
]. For the organic ligands the electrostatic potential (ESP) charges [64
] were calculated by performing geometry optimization of the substrates at the Hartree-Fock level of theory using a 6-31G* basis set with the Gaussian 09 software (Gaussian Inc., Wallingford, CT, USA) [65
]. These were later transformed in RESP charges by using the Antechamber tool [66
The system was then explicitly solvated using the TIP3P water model, leading to a total of 131,454 atoms. Topology, built with AmberTools 18, was later converted in a GROMACS format using the acpype algorithm [67
]. MD simulations were performed with GROMACS 5.0.4 [68
]. An integration time step of 2 fs was used and all covalent bonds involving hydrogen atoms were constrained with the LINCS algorithm. The Particle Mesh Ewald algorithm [69
] was used in order to account for electrostatic interactions. Simulations were done in the isothermal-isobaric NPT ensemble, at a temperature of 300 K, using a velocity-rescaling thermostat [70
]. Preliminary energy minimization was done with the steepest descend algorithm.
An initial equilibration of the membrane was performed for 100 ns with the protein atoms harmonically restrained with a force constant of 1000 kJ mol−1 nm−2, reaching a constant value (92 × 92 × 151 Å3) of the simulation box size. Constraints were then slowly released, and the system was thermalized to the target temperature of 300 K in about 10 ns. Then, the aromatase in complex with glyphosate was relaxed by performing a 100 ns MD simulation rescaling the motion of the center of mass of aromatase and the ligand, followed by an unbiased 100 ns MD simulation.
2.4. MELN cell Culture
MELN cells were provided by Dr. P. Balaguer (INSERM, Montpellier, France). They are estrogen-sensitive human breast cancer cells (MCF-7) transfected with the ERE-βGlob-Luc-SVNeo plasmid (ERE-β
]. The integrated plasmid contains a luciferase reporter gene, the estrogen-responsive elements (ERE) and an antibiotic resistance selection gene (SVNeo). MELN cells were cultured at 37 °C and 5% CO2
in Dulbecco’s Modified Eagle’s Medium Nutrient Mixture F12-Ham (DMEM-F12), supplemented with phenol red, fetal bovine serum (FBS) (5% v/v), l
-glutamine (4 mM), penicillin-streptomycin (100 U/mL–100 µg/mL), and G418 (1 mg/mL).
2.5. MELN Gene Reporter Assay
The assay was carried out as described by Balaguer et al., 1999 [73
] with slight modifications [74
]. For three days the cells were adapted to a test medium: DMEM-F12 without phenol red and supplemented with dextran-coated charcoal-treated FBS (5% v/v), l
-glutamine (4 mM), and penicillin-streptomycin (100 U/mL-100 µg/mL). Then, the cells were seeded at a density of 40,000 cells/well, in 96-well plates (100 μL/well). After 24 h, the test medium of each well was replaced with a test medium containing pesticides (100 μL/wells), and the cells were incubated for 16 h. After the incubation, the luciferase activity was assessed adding 100 μL/well of the One Glo Reagent (One-Glo Luciferase Assay System, Promega, Madison, USA), mixing (5 min) and measuring the luminescence of each well by a luminometer (Infinite Reader M200 Pro, Tecan, Männedorf, Switzerland).
The stock solutions of thiacloprid and imidacloprid were prepared in DMSO, while the stock solution of glyphosate was prepared in a test medium. The stock solutions were stored at −20 °C and, shortly before exposure, different concentrations of pesticides were prepared in a test medium (glyphosate and imidacloprid: From 10−8 to 10−3 M; thiacloprid from 10−8 to 5 × 10−4 M, due to lower solubility). The final DMSO concentration was less than 0.1%. Cells exposed to the test medium were used as a negative control and five concentrations of E2 (from 10−12 to 10−8 M) were tested to obtain a standard positive curve of the reference compound (E2).
The estrogenic activity was calculated as the ratio of the activity induced by the treatment over the activity induced by the positive control with 17-βestradiol (E2). It was expressed in percentage considering the relative luciferase activity of E2 (10−8
M) as 100%. Since all experiments were performed in quadruplicate (four wells for each experimental condition), the estrogenic activity was expressed as the mean and standard deviation of four values. The estrogenic activity of pesticides was also evaluated by the determination of the relative potency of each pesticide in comparison with the reference compound (E2) and it was expressed as the E2 equivalency factor (EEF) [75
]. The EEF was calculated using the concentrations of E2 and pesticides at which 50% of biological effect is achieved (EC50) through the formula: EEF = E2 EC50/pesticide EC50.
Three concentrations of pesticides (10−5, 2.5 × 10−4, and 5 × 10−4 M) were also tested: In combination with an ER-antagonist (tamoxifen 10−6 M), in order to confirm whether the observed effects were due to the ER activation, and in combination with E2 (10−10 M), in order to investigate the interaction between pesticides and E2 in MELN cells. The estrogenic activity of these treatments was expressed as relative luciferase activity and it was calculated as percentage of activity induced by the treatment with respect to the activity induced by the E2 10−10 M (relative luciferase activity of E2 10−10 M = 100%). The stock solutions of E2 and tamoxifen were prepared in ethanol and stored at −20 °C.
2.6. Data Analysis
Statistical analysis was performed using IBM SPSS Statistics 25.0 (IBM, Armonk, USA). The EC50 of E2 and pesticides was calculated by dose–response curves, which were estimated through a probit regression between the relative luciferase activity and Log transformed-concentrations of E2 or pesticides.
Data collected with the MELN gene reporter assay were not normally distributed, so the non-parametric Kruskal-Wallis test followed by the post-hoc Dunnett test was used to assess significant differences among the different experimental conditions. The differences were considered significant with p-value < 0.05.
In this study, the effect of three pesticides on two key targets of the endocrine system was evaluated using a combination of experimental and in silico methods that allowed an investigation at the molecular level.
The first target considered was aromatase, the key enzyme for estrogen production that resulted in being partially inhibited by glyphosate. The increase obtained in the aromatase activity in the presence of imidacloprid and thiacloprid, even if not statistically significant, can be interesting to be further investigated. Indeed, these two compounds could directly act on aromatase as allosteric activators or they could exert their action indirectly on CPR that has an essential role in catalysis and acts as an effector on human aromatase conformation [80
Glyphosate is the most widely used active compound among herbicides and was already reported to affect aromatase expression and activity in cells [33
]. The impact for human health has already been demonstrated: Indeed, an alteration in androgens/estrogens balance due to a lower aromatase expression as a consequence of the glyphosate presence changed the sperm nuclear quality impacting mammalian reproduction [82
However, the direct interaction between glyphosate and the enzyme together with the inhibition mechanism was not reported yet. In this study, the ELISA assay revealed that the inhibition of aromatase by glyphosate is partial and weak and strongly depends on the substrate concentration. Moreover, at the lower concentration of glyphosate tested (1 μM), the inhibition was found to be non-competitive, while at the higher concentration used (5 μM), the inhibition turned into a mixed inhibition mode. These data suggest that glyphosate binds to an allosteric site both when the enzyme is free and in complex with the substrate. Classical MD simulations supply a structural model for glyphosate binding to aromatase, explaining at the atomic-level that this pollutant may exert its inhibitory activity at a low concentration by binding to the allosteric Site 1 previously identified by Magistrato et al. [50
] and demonstrated to bind small-molecules inhibitors [54
]. When the concentration increases, a mixed inhibition is observed again compatible with the presence of an allosteric site. The binding of glyphosate probably becomes stronger or another allosteric site is occupied by the inhibitor. However, MD simulations showed that glyphosate did not stably bind to the second allosteric pocket (Site 2) identified in previous studies.
The second important target studied was the estrogen receptor that is responsible for estrogen binding and signal transduction in cells. MCF-7 cells, stably transfected with an estrogen-regulated luciferase gene (MELN cells), were used to assess the estrogenic activity of the pesticides. Glyphosate induced no estrogenic activity on MELN cells, moreover glyphosate did not change the effect induced by tamoxifen or E2. These results are in accordance with the results of the studies of Kojima et al., 2004 [46
] and Gasnier et al., 2009 [35
], who did not find any agonistic or antagonistic effect of glyphosate on ER. On the contrary, our results are different than the results of Thongprakaisang et al., 2013 [44
] and Mesnage et al., 2017 [45
] who found a significant increase of ER-induced estrogenic activity. A possible explanation of this discrepancy could be that in our study the estrogenic activity was evaluated on MELN cells while the other two studies applied T47D-KBluc cells, which might be more sensitive to the glyphosate activity than MELN cells. Moreover, our results are consistent with the results of the Endocrine Disruptor Screening Program (EDSP) conducted by the United States Environmental Protection Agency (US EPA, 2015) which concluded that “there is no convincing evidence of a potential interaction with the estrogen pathway for glyphosate”.
In the present study, both imidacloprid and thiacloprid induced a dose-response estrogenic activity mediated by the ER activation, in particular starting from 10−4
M. A consistent result was found in the study of Kojima et al., 2004 [46
] in which no estrogenic activity was detected exposing transfected-cells to imidacloprid at concentrations lower than 10−5
M; a contradicting result was found by Westlund and Yargeau, 2017 [48
], who did not find any estrogenic activity testing thiacloprid at concentrations comparable to ours. The conflicting results of thiacloprid should be interpreted considering the different cell models applied in our study with respect to the study of Westlund and Yargeau, 2017 (i.e., the present study was performed on mammalian cells while the previous one on yeast cells) [48
]. Indeed, yeast cells are characterized by a different membrane permeability, transport proteins, and signal transduction pathways with respect to mammalian cells which may have influenced the results [42
], as reported before also for other nuclear receptors [83
The estrogenic activity of imidacloprid and thiacloprid was observed at concentrations higher than the pesticide levels measured in human biological samples [76
]. Although such high levels of pesticides are not found in human biological samples, low doses of these pesticides should not be considered harmless. Indeed, in biological fluids these pesticides may be present in combination with other EDCs with a similar action mode and, thus, these pesticides together with other EDCs might cause an overall estrogenic effect. Moreover, these high concentrations of pesticides are similar to levels in some environmental matrices, where they could cause adverse health effects on wildlife animals. For example, in guttation water or morning dew of plants, imidacloprid was found to be present at a concentration up to 346 mg/L [85
Interestingly, the results of the present study showed that, when the ER-antagonist tamoxifen was added to these pesticides, the estrogenic activity was still higher than the negative control. This result can be explained by the competition between each pesticide and tamoxifen for ER binding. Moreover, in the presence of high concentrations of imidacloprid and thiacloprid, an additive effect with E2 was also observed. The docking calculation suggested that these two neonicotinoids may bind to both the orthosteric and allosteric pockets of ERα, suggesting a putative mechanism to rationalize their observed estrogenic activity.