Novel Pathways of Endocrine Disruption Through Pesticides Interference With Human Mineralocorticoid Receptors

Novel Pathways of Endocrine Disruption Through Pesticides Interference With Human... Abstract Mineralocorticoid receptor (MR) is one member of the steroid receptor family. In addition to its important role in Na+/K+ homeostasis, MR is reported as a tumor-suppressor in carcinogenesis. So far, little was known about the ability of pesticides to interfere with MR. In this study, a total of 43 pesticides and/or metabolites were investigated for their potential effects on human MR. None of the tested pesticides exhibited MR agonistic potency, whereas 16 compounds showed antagonistic activities. Further investigations indicated that these 16 chemicals individually antagonized aldosterone-induced alkaline phosphatase expression in vascular smooth muscle cells and aldosterone-inhibited hepatocellular carcinoma cell proliferation at higher concentrations, and the mixture of these 16 pesticides at environmentally relevant concentrations significantly disrupted MR activity. The additional quantitative mixture experiments indicated a good agreement between the combined anti-mineralocorticoidic activities of 16 pesticides and the responses predicted by concentration addition model instead of independent action model. The interruption of nuclear translocation of MR was clarified as a main mechanism for the anti-mineralocorticoidic activities by these pesticides. These data suggest that the health risk may increase when multiple MR antagonists cooperate following concentration addition model and exhibit a combined effect. Our findings emphasize that comprehensive risk assessment of adverse effects of environmental MR ligands on human health should be considered. endocrine-disrupting chemicals, pesticides, steroid hormone receptors, mineralocorticoid receptor, anti-mineralocorticoid activity Most prior research on environmental endocrine-disrupting chemicals (EDCs) focused on their actions on estrogen receptor (ER) and androgen receptor (AR). Estrogen receptor and AR belong to the steroid receptor subfamily of nuclear transcription factors (Jaffe and Mendelsohn, 2005). Other members of the steroid receptor family, including progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR), are also involved in steroid-regulated biological processes that may similarly be disrupted if agonists or antagonists are present in the environment. Recently, there has been increasing evidence for the effects of EDCs on other members of this steroid receptor class such as PR and GR (Rehan et al., 2015; Zhang et al., 2016). Very few studies also showed the potential impact of EDCs on MR. Bodwell et al. (2006) reported a biphasic response of MR activity to arsenic (As) exposure, with stimulation of activity at low doses of As and inhibition at higher doses. A fungicide, vinclozolin, and its primary metabolite were identified as MR antagonists using in vitro assay (Molina-Molina et al., 2006). However, data regarding the possible environmental ligands for MR are still extremely sparse in EDCs research. Mineralocorticoid receptor mediates the effects of mineralocorticoids, a class of essential physiological corticosteroids, on a variety of target tissues such as kidney, colon, adipose tissue, cardiovascular and central nervous systems (Berger et al., 1998; Pippal and Fuller, 2008; Rogerson and Fuller, 2000; Zennaro et al., 1995). As a nuclear transcription factor, MR translocates to the nucleus upon ligation by cognate ligand and binds to specific DNA sequence and regulates the corresponding responsive gene expression, critically involved in Na+/K+ homeostasis, blood pressure regulation, and cell proliferation (Gaeggeler et al., 2005; Grossmann et al., 2010; Sekizawa et al., 2011). Loss of MR function in deficient mice and human resulted in neonate mortality because of severe dehydration by renal sodium and water loss (Berger et al., 1998; Fuller and Rogerson, 2002; Geller et al., 1998). Blockade of MR also reported to impair stress-related learning and lead to anxiety behavior in animal models (Douma et al., 1998; Yau et al., 1999). Recently, the tumor-suppressive role of MR has been appreciated in cancer development and progression, such as colorectal cancer, lung cancer, and hepatocellular carcinoma (HCC) (Jeong et al., 2010; Nie et al., 2015; Tiberio et al., 2013). Due to the indispensable physiological functions of MR, it is urgent to recognize potential MR agonists or antagonists among environmental chemicals and potential interferences with MR should be considered for the safety assessment of EDCs. The ubiquitous usage of pesticides with an annual global amount of 1–2.5 million tons has resulted in a type of the most widespread and significant environmental pollution (Fenner et al., 2013). Pesticides are now suspected of being a kind of important EDCs. Numerous pesticides have been reported to have estrogenic or anti-androgenic activity via interfering with ER or AR (Kojima et al., 2004). Our recent study found that more than one-third of tested pesticides were potential GR antagonists, suggesting that many pesticides could affect steroid-regulated biological processes via binding to other members of steroid receptor class such as GR (Zhang et al., 2016). Thus, to identify common pesticides for MR agonists or antagonists and to further explore the possible mechanism are imperative to expand the understanding of latent risks of pesticides. In this study, 43 pesticides (listed in Supplementary Table 1) were screened for MR activities using luciferase reporter gene assay. The results characterized a total of 16 pesticides as potential MR antagonists. The effects of these potential MR ligands on mineralocorticoid signaling were further confirmed by the inhibition of the expression of mineralocorticoid-responsive gene alkaline phosphatase (Alk) in human vascular smooth muscle cells (VSMCs). It was also demonstrated that these pesticides reversed MR-mediated suppression of HCC proliferation. Furthermore, we uncovered a mechanism for anti-mineralocorticoidic activities by these pesticides that mainly involved the inhibition of mineralocorticoid-induced nuclear translocation of MR. More importantly, our findings indicated that the mixture of these 16 pesticides at environmentally relevant concentrations could disrupt MR transactivity and inhibit MR-mediated function. The observed responses of the mixture composed of these 16 chemicals agreed very well with the predicted regression curves of concentration addition (CA) model. MATERIALS AND METHODS Chemicals Aldosterone (>97% pure) was purchased from J&K Scientific (Beijing, China). Spironolactone (>99% pure) was obtained from Selleck Chemicals (Boston, Massachusetts). The 43 pesticides and pesticide metabolites listed in Supplementary Table 1 were obtained from Sigma-Aldrich (St Louis, Missouri). Stock solutions of chemicals were prepared using dimethylsulfoxide (DMSO) as a solvent and stored at −20 °C, except for paraquat that was dissolved in deionized water. Plasmid constructs The human mineralocorticoid receptor (hMR) expression plasmid EGFP-C1-hMR was kindly provided by Dr Claudia Großmann (Martin Luther University, Germany) (Grossmann et al., 2005; Ouvrard-Pascaud et al., 2004). The mineralocorticoid response element containing reporter plasmid pMMTV-luc was kindly provided by Dr Evangelia Charmandari (Biomedical Research Foundation of the Academy of Athens, Greece) (Nicolaides et al., 2014). In dual-luciferase reporter assays, pRL-TK (Promega, Madison) was used as an internal control as previously described (Zhang et al., 2016). Cell cultures Chinese hamster ovary K1 cell line (CHO-K1) and human HCC cell line (SMMC-7721) were maintained at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, Logan, Utah) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 100 U/ml streptomycin-penicillin (Hyclone) under saturating humidity. Primary cultured human VSMCs were kindly provided by Dr Luyang Yu (College of Life Science, Zhejiang University). VSMCs were maintained in M199 media (Gibco, Grand Island, New York) with 20% FBS (Gibco), 1% glutamine (Gibco), and 100 U/ml streptomycin-penicillin (Yu et al., 2011). Low-passage VSMCs primary cultures were used for real-time quantitative PCR. For all exposure experiments, the cells were cultured with phenol red–free DMEM or M199 supplemented with charcoal/dextran-treated FBS. Cell proliferation assay As previously described, cell proliferation was assessed after exposure to tested chemicals at the concentration of 10−5 or 10−6 M using CellTiter 96 AQueous One Solution Cell Proliferation (Promega, Madison, Wisconsin) (Liu et al., 2012; Zhang et al., 2014). The exposure periods were 24 h for CHO-K1 cells and 48 h for SMMC-7721 cells. The absorbance at 490 nm was detected using microplate reader (Infinite M200 PRO, Tecan, Switzerland). Reporter gene assay The reporter gene assay was performed as previously described (Zhang et al., 2016). Briefly, after transient transfection with pEGFP-C1-hMR, pMMTV-luc, and pRL-TK plasmids, CHO-K1 cells were exposed to tested chemicals or 0.1% DMSO (vehicle control) to measure the agonistic activity of hMR. For antagonistic activity measurement, transfected cells were treated with 10−9.5 M aldosterone in combination with tested chemicals after 30 min pretreatment with the tested compound alone. Firefly luciferase and Renilla luciferase activities were measured using the Dual-luciferase Reporter Assay Kit (Promega) after 24 h exposure and the ratio of firefly to Renilla luciferase activity was used to present the relative transcriptional activity. The transfected cells were exposed to the mixture of all 16 potential antagonists with an equal concentration of 10−10 to10−6 M for a plain combined antagonistic effect evaluation. Relative inhibition rate (RIR) is obtained at the highest tested concentration of chemicals as percent decrease of aldosterone response. The Weibull regression model was applied for the individual concentration-response analyses. The concentrations of tested chemicals that inhibited 20% (IC20) or 50% (IC50) of the luciferase activity induced by aldosterone were calculated.   E=1-exp⁡(-exp⁡ (α+ β log10  c )), (1) E, effect, the fraction of antagonistic effect (0 ≤ E ≤ 1); α and β, model parameters that varied depending on the individual concentration-response curve; c, the concentration of tested chemicals. Calculation of mixture-effect predictions To further predict the combined effects of 16 potential MR antagonists, the mixtures were designed as fixed-ratio equipotent mixtures that were calculated based on the effect concentrations (EC) of the individual components that led to an inhibition of aldosterone effects by 10% (here termed as inhibitory concentrations 10% [IC10]) based on Weibull regression model (Faust et al., 2001). The mathematical and statistical procedures used to calculate predictions of the effect concentrations (ECxmix) of the mixture according to CA and IA models were well-described in previous studies (Faust et al., 2001; Orton et al., 2012; Xing et al., 2012). The equations that predicted the effect mixture concentrations under the hypothesis of CA (equation 1) and IA (equation 2) were listed below: CA:   ECxmix=∑i=1npiECxi-1, (2)pi, the relative proportions of the individual component in the whole mixture; ECxi, the equivalent effect concentrations of the individual component. IA:   x%=1-∏i=1n1-Fipi×ECxmix. (3) In this equation, Fi are calculated from the concentration-response functions and the x% represents the total effect. Real-time quantitative PCR VSMCs were treated with the tested chemicals for 24 h and then cells were lysed for total RNA isolation and reverse transcription using SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo, Tokyo, Japan) according to the manufacturer’s instruction. SYBR Green PCR Master Mix (Toyobo, Japan) was used for real-time quantitative PCR on Mx3000P (Agilent Technologies, Palo Alto) as previously described (Zhang et al., 2016). The primer sequences are listed in Supplementary Material. Relative gene expression level was calculated using the ΔΔ threshold cycle (Ct) method and normalized to the endogenous reference gene gapdh. Immunofluorescence and confocal microscopy Immunofluorescence of MR was performed as previously described (Grossmann et al., 2010). Briefly, CHO-K1 cells were seeded on glass coverslips, transiently transfected with EGFP-C1-hMR, and then treated with 10−9.5 M aldosterone in combination with tested chemicals for 1 h after 30 min pretreatment with tested chemicals. Cells were fixed and then incubated with anti-MR antibody (dilution 1:200, Santa Cruz Biotechnology, Dallas) overnight at 4 °C. After further incubation with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, ThermoFisher Scientific, Eugene) and 4ʹ,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame), the cells were analyzed by confocal microscopy (LSM780, Zeiss, Gottingen, Germany). Western blotting After transfection with pEGFP-C1-hMR plasmids, CHO-K1 cells were exposed to 10−9.5 M aldosterone in combination with tested chemicals for 1 h after 30 min pretreatment with the tested compound alone. Nuclear protein was isolated using the CelLytic NuCLEAR extraction kit (Sigma-Aldrich). Western blotting was performed as previously described (Liu et al., 2012), and details are shown in Supplementary Material. Statistical analysis All experimentations were repeated at least 3 times. The data are presented as mean ± SD of at least 3 independent assays with triplicates. Statistical analysis was carried out using SPSS version 16.0 (SPSS, Chicago) and Origin 8.0 (OriginLab, Northampton). The significance of difference was evaluated by one-way ANOVA followed by Dunnett’s post hoc test, and differences were considered significant if p < .05. RESULTS Agonistic Effects of Tested Chemicals in MR Assays The results of MTS assay showed that exposure to resmethrin, carbendazim, or tolylfluanid at a concentration of 10−6 M and other chemicals at a concentration of 10−5 M for 24 h did not affect cell viability (Supplementary Figure 1). The endogenous mineralocorticoid aldosterone stimulated the MR transcriptional activity in a concentration-response manner and the maximal activity reached at 10−9.5 M aldosterone or higher (Supplementary Figure 2A). Therefore, the relative induction rate compared with the MR activity obtained by 10−9.5 M aldosterone was used to evaluate the agonistic effects of 43 pesticides and metabolites at non-cytotoxic concentrations. There was no significant induction observed, suggesting that none of the tested chemicals has MR agonistic activity (Supplementary Figure 3). Antagonistic Effects of Tested Chemicals in MR Assays Spironolactone, an MR antagonist that suppressed the aldosterone-induced MR transactivation in a concentration-dependent manner, was used as a positive control to evaluate MR antagonistic activity (Supplementary Figure 2B). A total of 16 pesticides among the 43 tested chemicals, including 4 pyrethroids (bifenthrin, cypermethrin, fenvalerate, and permethrin), 4 organochlorines (o,p′-dichlorodiphenyltrichloroethane [DDT], p,p′-dichlorodiphenyldichloroethylene [DDE], p,p′-DDE, and methoxychlor), 2 organophosphates (acephate and dimethoate), triazine herbicide terbuthylazine, and metabolites of atrazine (atrazine-desethyl [DEA] and atrazine-desisopropyl [DIA]), as well as alachlor, zineb, and fipronil, significantly attenuated aldosterone (10−9.5 M) induced MR transcriptional activity at the highest tested non-cytotoxic concentration (Figure 1), suggesting that these 16 chemicals potentially exhibited MR antagonistic properties. And the inhibition of transcriptional activity was reversed by a higher concentration of aldosterone (10−6 M), indicating the antagonism would be due to competitive binding to the receptor (Figure 1E). Subsequently, the concentration-dependent MR antagonistic activities of these 16 potential antagonists were determined at doses of 10−9 to 10−5 M (Figs. 2A–D). The concentrations of the tested chemicals reducing 20% of 10−9.5 M aldosterone-induced MR activity (20% inhibitory concentration, IC20), 50% inhibitory concentration (IC50), and the RIR of these 16 chemicals at the highest tested concentrations that represented as percent decrease of aldosterone response were calculated from the concentration-response curves (Table 1). These 16 chemicals exhibited an antagonistic activity against MR with IC20 between 10−7 M and 10−6 M, indicating that these pesticides were weak MR antagonists when compared with the pharmaceutical antagonist spironolactone (Table 1). Table 1. Inhibitory Effects of 16 Pesticides and Metabolites on MR Transcriptional Activity Induced by Aldosterone Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Aldosterone      100  4. Organophosphates        Spironolactone  4.17 × 10−8  1.71 × 10−7  83.3  Acephate  4.07 × 10−7  8.52 × 10−6  52.6  1. Pyrethroids        Dimethoate  2.87 × 10−7  NA  45.4  Bifenthrin  3.76 × 10−7  7.06 × 10−6  54.3          Cypermethrin  2.43 × 10−7  4.96 × 10−6  56.0  5. Carbamates        Fenvalerate  1.03 × 10−6  7.40 × 10−6  52.1  Zineb  1.93 × 10−7  8.21 × 10−6  46.4  Permethrin  1.60 × 10−7  7.27 × 10−6  54.0                  6. Triazines        2. Organochlorines        DEA  8.35 × 10−7  NA  41.3  o,pʹ-DDT  1.26 × 10−6  4.04 × 10−6  81.2  DIA  1.02 × 10−7  4.35 × 10−6  48.1  p,pʹ-DDE  2.41 × 10−6  6.01 × 10−6  72.4  Terbuthylazine  3.86 × 10−7  NA  43.5  p,pʹ-DDT  1.00 × 10−6  3.29 × 10−6  80.5          Methoxychlor  2.19 × 10−6  8.13 × 10−6  69.2  8. Others                Fipronil  1.30 × 10−6  NA  41.6  3. Amids                Alachlor  1.04 × 10−7  5.13 × 10−6  58.2          Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Aldosterone      100  4. Organophosphates        Spironolactone  4.17 × 10−8  1.71 × 10−7  83.3  Acephate  4.07 × 10−7  8.52 × 10−6  52.6  1. Pyrethroids        Dimethoate  2.87 × 10−7  NA  45.4  Bifenthrin  3.76 × 10−7  7.06 × 10−6  54.3          Cypermethrin  2.43 × 10−7  4.96 × 10−6  56.0  5. Carbamates        Fenvalerate  1.03 × 10−6  7.40 × 10−6  52.1  Zineb  1.93 × 10−7  8.21 × 10−6  46.4  Permethrin  1.60 × 10−7  7.27 × 10−6  54.0                  6. Triazines        2. Organochlorines        DEA  8.35 × 10−7  NA  41.3  o,pʹ-DDT  1.26 × 10−6  4.04 × 10−6  81.2  DIA  1.02 × 10−7  4.35 × 10−6  48.1  p,pʹ-DDE  2.41 × 10−6  6.01 × 10−6  72.4  Terbuthylazine  3.86 × 10−7  NA  43.5  p,pʹ-DDT  1.00 × 10−6  3.29 × 10−6  80.5          Methoxychlor  2.19 × 10−6  8.13 × 10−6  69.2  8. Others                Fipronil  1.30 × 10−6  NA  41.6  3. Amids                Alachlor  1.04 × 10−7  5.13 × 10−6  58.2          Eight chemicals groups are listed in Supplementary Table 1. NA, not available because that the calculated concentration is out of the range of concentration tested in this study. IC20 and IC50: concentration of tested chemicals that inhibited 20% or 50% of the luciferase activity induced by aldosterone. RIR obtained at highest tested concentration of chemicals as percent decrease of aldosterone response. Figure 1. View largeDownload slide Antagonistic effects of pesticides and their metabolites in the mineralocorticoid receptor (MR) transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with 0.1% dimethylsulfoxide (DMSO) (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with pesticides at non-cytotoxic concentrations. A, Antagonistic effects of 10−5 M of bifenthrin, λ-cyhalothrin, cypermethrin, fenvalerate, permethrin, 3-PBald, 3-PBA, 3-PBalc, and 10−6 M of resmethrin. B, Antagonistic effects of 10−5 M of α-BHC, β-BHC, δ-BHC, γ-BHC, o,p′-DDT, p,p′-DDT, p,p′-DDE, methoxychlor, alachlor, dimethenamid, metolachlor, and S-metolachlor. C, Antagonistic effects of 10−5 M of acephate, chlorpyrifos, diazinon, dimethoate, omethoate, parathion-ethyl, carbaryl, ethiofencarb, fenobucarb, pirimicarb, and zineb. D, Antagonistic effects of 10−5 M of atrazine, DEA, DIA, metribuzin, terbuthylazine, benomyl, fipronil, fomesafen, paraquat, and 10−6 M of carbendazim and tolylfluanid. E, The antagonistic effect of chemicals at 10−5 M in the presence of 10−9.5 M or 10−6 M aldosterone. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (Ald) (= 100%). #p < .05, ##p < .01, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Figure 1. View largeDownload slide Antagonistic effects of pesticides and their metabolites in the mineralocorticoid receptor (MR) transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with 0.1% dimethylsulfoxide (DMSO) (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with pesticides at non-cytotoxic concentrations. A, Antagonistic effects of 10−5 M of bifenthrin, λ-cyhalothrin, cypermethrin, fenvalerate, permethrin, 3-PBald, 3-PBA, 3-PBalc, and 10−6 M of resmethrin. B, Antagonistic effects of 10−5 M of α-BHC, β-BHC, δ-BHC, γ-BHC, o,p′-DDT, p,p′-DDT, p,p′-DDE, methoxychlor, alachlor, dimethenamid, metolachlor, and S-metolachlor. C, Antagonistic effects of 10−5 M of acephate, chlorpyrifos, diazinon, dimethoate, omethoate, parathion-ethyl, carbaryl, ethiofencarb, fenobucarb, pirimicarb, and zineb. D, Antagonistic effects of 10−5 M of atrazine, DEA, DIA, metribuzin, terbuthylazine, benomyl, fipronil, fomesafen, paraquat, and 10−6 M of carbendazim and tolylfluanid. E, The antagonistic effect of chemicals at 10−5 M in the presence of 10−9.5 M or 10−6 M aldosterone. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (Ald) (= 100%). #p < .05, ##p < .01, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Figure 2. View largeDownload slide The concentration-response effects of antagonistic pesticides in the MR transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with pesticides at the concentrations range from 10−9 M to 10−5 M in the presence of 10−9.5 M aldosterone. A, The concentration-response antagonistic effects of bifenthrin, cypermethrin, fenvalerate, and permethrin. B, The concentration-response antagonistic effects of o,p′-DDT, p,p′-DDE, p,p′-DDT, and methoxychlor. C, The concentration-response antagonistic effects of acephate, dimethoate, DEA, DIA, and terbuthylazine. D, The concentration-response antagonistic effects of alachlor, zineb, and fipronil. E, The concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−10 M to10−6 M. F, The predicted and observed concentration-response antagonistic effects of fixed-ratio equipotent mixtures composed in the ratio of their IC10. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (= 100%). Figure 2. View largeDownload slide The concentration-response effects of antagonistic pesticides in the MR transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with pesticides at the concentrations range from 10−9 M to 10−5 M in the presence of 10−9.5 M aldosterone. A, The concentration-response antagonistic effects of bifenthrin, cypermethrin, fenvalerate, and permethrin. B, The concentration-response antagonistic effects of o,p′-DDT, p,p′-DDE, p,p′-DDT, and methoxychlor. C, The concentration-response antagonistic effects of acephate, dimethoate, DEA, DIA, and terbuthylazine. D, The concentration-response antagonistic effects of alachlor, zineb, and fipronil. E, The concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−10 M to10−6 M. F, The predicted and observed concentration-response antagonistic effects of fixed-ratio equipotent mixtures composed in the ratio of their IC10. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (= 100%). Because of coexistence of multiple chemicals in the realistic environment, transfected cells were exposed to the mixture of these 16 potential antagonists at their individual concentrations of 10−10 to 10−6 M to evaluate their combined effects. As shown in Figure 2E, exposure to the mixture caused a dose-dependent reduction in aldosterone-induced MR transcriptional activity. Notably, a significant inhibition was observed at a dose as low as 10−9 M (Figure 2E). The mixture modeling was further explored by comparing the observed responses and the combined effects predicted by CA and IA models based on fixed-ratio concentration-response analyses. The regression model parameters and proportion of the individual components were listed in Supplementary Table 2. There was a good agreement of the responses of mixtures comprised by IC10 ratio aliquots with predictions by CA over the concentration range from 10−8 M to 10−5 M (Figure 2F). Disruption of Mineralocorticoid-Responsive Gene Expression To further assess the anti-mineralocorticoid potency, the expression of mineralocorticoid-induced gene Alk that plays an important role in vascular calcification was evaluated in the primary cultured human VSMCs that express endogenous MR (Jaffe and Mendelsohn, 2005). As shown in Figure 3, all tested compounds at a concentration of 10−6 or 10−5 M significantly antagonized aldosterone-induced Alk expression in VSMCs (Figs. 3A and 3B). The concentrations of pesticides used for treatment did not cause obvious cytotoxic effects on VSMCs (Supplementary Figure 4A). The expression of housekeeping gene gapdh had high stability and its relative Ct values did not change in VSMCs after pesticide treatment, suggesting that these chemicals at non-cytotoxic concentrations had no effects on this non-MR target gene (Supplementary Figure 4B). A total of 12 chemicals among these 16 candidate pesticides, including 4 pyrethroids (bifenthrin, cypermethrin, fenvalerate and permethrin), 2 organochlorines (o,pʹ-DDT and methoxychlor), herbicide alachlor, organophosphate insecticide dimethoate, carbamate fungicide zineb, atrazine metabolite DIA, triazine herbicide terbuthylazine, and fipronil, remarkably antagonized aldosterone (10−9.5 M) upregulated Alk gene expression by more than 50% when compared with the aldosterone-treated positive control (Figure 3). The repression of Alk expression was significantly reversed when cells were exposed to aldosterone at higher concentration of 10−6 M (Figs. 3A and 3B). By exposing VSMCs to the mixture of these 16 pesticides at their individual concentrations in the range of 10−9 to 10−5 M, a concentration-dependent decrease in aldosterone-induced Alk expression was observed and the significant inhibition occurred at a dose as low as 10−8 M of the mixture (Figure 3C). Figure 3. View largeDownload slide The inhibitory effects of pesticides on aldosterone-induced expression of mineralocorticoid-responsive gene Alk. VSMCs were treated with 0.1% DMSO (vehicle control), 10−9.5 M or 10−6 M aldosterone (Ald, positive control), 10−5 M spironolactone (antagonist control), or the combination of 10−9.5 M or 10−6 M aldosterone with pesticides at non-cytotoxic concentrations. A, Inhibitory effects of 10−5 M bifenthrin, cypermethrin, fenvalerate, permethrin, and 10−6 M o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor. B, Inhibitory effects of 10−5 M alachlor, acephate, dimethoate, zineb, DEA, DIA, terbuthylazine, and fipronil. C, Concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−9 M to10−6 M, in the presence of 10−9.5 M aldosterone. The relative mRNA levels of Alk in VSMCs are present as percent induction with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (10−9.5 M Ald) (= 100%). #p < .05, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Figure 3. View largeDownload slide The inhibitory effects of pesticides on aldosterone-induced expression of mineralocorticoid-responsive gene Alk. VSMCs were treated with 0.1% DMSO (vehicle control), 10−9.5 M or 10−6 M aldosterone (Ald, positive control), 10−5 M spironolactone (antagonist control), or the combination of 10−9.5 M or 10−6 M aldosterone with pesticides at non-cytotoxic concentrations. A, Inhibitory effects of 10−5 M bifenthrin, cypermethrin, fenvalerate, permethrin, and 10−6 M o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor. B, Inhibitory effects of 10−5 M alachlor, acephate, dimethoate, zineb, DEA, DIA, terbuthylazine, and fipronil. C, Concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−9 M to10−6 M, in the presence of 10−9.5 M aldosterone. The relative mRNA levels of Alk in VSMCs are present as percent induction with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (10−9.5 M Ald) (= 100%). #p < .05, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Exposure of Tested Chemicals Alters the Aldosterone-Induced HCC Growth Suppression It has been reported that MR exerted a suppressive role in HCC progression (Nie et al., 2015). In this study, we further examined the effects of these 16 potential MR antagonists on the MR-mediated suppression of HCC proliferation. SMMC-7721 cells were exposed to aldosterone in combination with the tested chemicals at a non-toxic concentration (10−5 or 10−6 M) (Supplementary Figs. 5A and 5B). As expected, the native ligand aldosterone significantly suppressed the proliferation of HCC cell line SMMC-7721 in a concentration-dependent manner (Supplementary Figure 5C), whereas aldosterone-induced suppression was reversed by MR antagonist spironolactone (Figure 4). The result indicated that 14 of these 16 chemicals remarkably attenuated aldosterone-induced HCC growth suppression (Figs. 4A and 4B). Furthermore, the blockage of aldosterone-induced suppression of SMMC-7721 cells was also observed after a treatment with the mixture of these 16 pesticides at concentrations in the range of 10−9 to 10−7 M (Figure 4C). Figure 4. View largeDownload slide Effects of pesticides on the aldosterone-induced HCC growth suppression. SMMC-7721 cells were treated with 0.1% DMSO (vehicle control), 10−6 M aldosterone (Ald, positive control), spironolactone (antagonist control) in the presence of 10−6 M aldosterone, or the combination of 10−6 M aldosterone with pesticides at non-cytotoxic concentrations (10−6 M for bifenthrin, o,p′-DDT, p,p′-DDT, and methoxychlor, or 10−5 M for other compounds). *p < .05, **p < .01, compared with vehicle control (DMSO) (= 100%). Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction. Figure 4. View largeDownload slide Effects of pesticides on the aldosterone-induced HCC growth suppression. SMMC-7721 cells were treated with 0.1% DMSO (vehicle control), 10−6 M aldosterone (Ald, positive control), spironolactone (antagonist control) in the presence of 10−6 M aldosterone, or the combination of 10−6 M aldosterone with pesticides at non-cytotoxic concentrations (10−6 M for bifenthrin, o,p′-DDT, p,p′-DDT, and methoxychlor, or 10−5 M for other compounds). *p < .05, **p < .01, compared with vehicle control (DMSO) (= 100%). Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction. Effects of Tested Chemicals on Aldosterone-Induced Nuclear Translocation of MR To explore the mechanistic basis for these antagonistic effects, we examined whether coexposure of tested chemicals alters aldosterone-induced nuclear translocation of MR. Mineralocorticoid receptor immunolabeling is located in both cytoplasm and nucleus in the majority of transfected cells in the absence of ligand aldosterone, but not nucleoli (Figure 5A). After treatment with aldosterone, MR predominately translocated to the nucleus and detectable MR almost totally disappeared from the cytosolic pool (Figure 5B), which was consistent with previous observations (Fejes-Toth et al., 1998). Coexposure of most tested chemicals and aldosterone resulted in an obvious appearance of MR in the cytosol (Figure 5). A total of 14 among 16 tested antagonists significantly suppressed the aldosterone-induced nuclear translocation of MR (Figs. 5C–P). Moreover, the results of Western blotting also showed that coexposure to tested antagonists remarkably decreased the amount of MR in nucleus when compared with the aldosterone-treated control (Figs. 5Q and 5R). However, among these 16 pesticides, fipronil and fenvalerate did not appreciably alter the aldosterone-stimulated nuclear accumulation of MR (Supplementary Figure 6). Figure 5. View largeDownload slide Effects of tested pesticides on nuclear translocation of MR. CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to A, vehicle control (0.1% DMSO); B, positive control (10−9.5 M aldosterone), or the combination of 10−9.5 M aldosterone with C, bifenthrin; D, cypermethrin; E, permethrin; F, o,p′-DDT; G, p,p′-DDE; H, p,p′-DDT; I, methoxychlor; J, alachlor; K, acephate; L, dimethoate; M, zineb; N, DEA; O, DIA; P, terbuthylazine; Q, CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to 0.1% DMSO (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with bifenthrin (BF), cypermethrin (CP), permethrin (PM), o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor (MXC), alachlor (ALA), acephate (AP), dimethoate (DIM), zineb, DEA, DIA, and terbuthylazine (TBA). Lamin A/C was present as an internal control for the total amount of nuclear protein. R, Grayscale quantization of LaminA/C-normalized nuclear MR from at least 3 independent experiments. *p < .05, **p < .01, compared with positive control (Ald) (= 100%). Figure 5. View largeDownload slide Effects of tested pesticides on nuclear translocation of MR. CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to A, vehicle control (0.1% DMSO); B, positive control (10−9.5 M aldosterone), or the combination of 10−9.5 M aldosterone with C, bifenthrin; D, cypermethrin; E, permethrin; F, o,p′-DDT; G, p,p′-DDE; H, p,p′-DDT; I, methoxychlor; J, alachlor; K, acephate; L, dimethoate; M, zineb; N, DEA; O, DIA; P, terbuthylazine; Q, CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to 0.1% DMSO (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with bifenthrin (BF), cypermethrin (CP), permethrin (PM), o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor (MXC), alachlor (ALA), acephate (AP), dimethoate (DIM), zineb, DEA, DIA, and terbuthylazine (TBA). Lamin A/C was present as an internal control for the total amount of nuclear protein. R, Grayscale quantization of LaminA/C-normalized nuclear MR from at least 3 independent experiments. *p < .05, **p < .01, compared with positive control (Ald) (= 100%). DISCUSSION Although the estrogenic and/or anti-androgenic potency of a number of pesticides has been observed, little was known about the disrupting effects of pesticides via nonsexual steroid hormone receptors, especially MR. In this study, 43 pesticides from both legacy and current-use types were evaluated for MR-mediated mineralocorticoid activity. Similar to what was found in our previous GR study (Zhang et al., 2016), none of the test pesticides exhibited mineralocorticoidic activity, but 16 of 43 chemicals showed MR antagonistic effects. To the best of our knowledge, this is the first study to recognize these 16 pesticides as MR antagonists. We found that 4 DDT analogs (o,p′-DDT, p,p′-DDT, p,p′-DDE, and methoxychlor) and 2 pyrethroids (bifenthrin and cypermethrin) exhibited both GR and MR antagonistic activities (Zhang et al., 2016). The overlap of antagonism of these pesticides toward GR and MR could be explained by the high homology in structure and function between GR and MR. In fact, the native MR ligand aldosterone is able to bind to the GR but the affinity between aldosterone and GR is several orders of magnitude lower than MR, whereas native GR ligand cortisol can also bind to MR (Fuller et al., 2000). However, the identity of amino acid sequences between the ligand binding domains of GR and MR was only 57%, which results in the difference in protein spatial structure and subsequently the diversity of xenobiotics with antagonistic effects (Arriza et al., 1987). For example, in our studies, atrazine showed antagonistic potency for GR rather than MR, whereas 2 metabolites of atrazine, DEA and DIA, exhibited MR antagonistic effects. It is likely that chemical structures had an important impact on MR antagonistic activity. For example, pyrethroids are all esters and are hydrolyzed into the acid and alcohol metabolites (Kaneko, 2011). In our study, most of the parent compounds of pyrethroids rather than their common acid and alcohol metabolites exhibited MR antagonistic activity, suggesting that the structure of an ester in this group of chemicals is required for their MR antagonistic activity. In addition, among the organochlorine group, all 4 DDT analogues tested in this study showed MR antagonistic activity, but none of the 4 benzene hexachloride isoforms. However, among the different groups, these 16 chemicals have a variety of different structures and MR exhibits promiscuity to these potential environmental ligands. Actually, other members of the family of steroid receptors, such as ER, AR, and GR, also can be activated or antagonized by a large set of synthetic drugs and pollutants in different chemical classes with highly variant structures (Kojima et al., 2004, 2009, 2013; Takeuchi et al., 2005, 2011; Zhang et al., 2016). The mechanism(s) of the promiscuous sensitivity of steroid receptors to exogenous substances remains unclear and is worth to further investigate. In this study, 16 compounds were considered as relative weak MR antagonists with the IC20 ranging from 10−7 M to 10−6 M (approximately equivalent to 44.5–419 ppb), which likely exceeds the realistic residual levels of these pesticides in the environment. However, a significant MR antagonistic effect was observed when these 16 chemicals were combined at an individual concentration as low as 10−9 M (approximately equal to 0.17–0.44 ppb). Consistently, a mixture of candidate pesticides at individual concentrations of 10−8 M produced combined MR antagonist effects on the expression of mineralocorticoid-responsive gene Alk in human VSMCs. The cumulative anti-mineralocorticoidic effects of a mixture of pesticides at low concentrations were also observed in the MR-mediated suppression of HCC proliferation. Noticeably, the realistic environmental levels of some chemicals among these candidate MR antagonists approach or exceed their corresponding concentrations in the mixture exposure. For instance, as the most ubiquitous metabolite of DDT, p,pʹ-DDE was reported to mean level of 1.64–5.18 ppb in the population of developed countries (Carreno et al., 2007; Zubero et al., 2015), whereas the effective level of p,pʹ-DDE in the mixture of this study was 0.32 ppb. As the most widely used pyrethroids, permethrin and cypermethrin were detected in human breast milk in South Africa with a mean concentration of 10.38 and 1.71 ppb, respectively (Bouwman et al., 2006), which is higher than their effective concentrations in the mixture (0.39 ppb for permethrin and 0.42 ppb for cypermethrin). Moreover, the dealkylated triazine metabolites (DEA, DIA, and 2-chlorodiminoatrazine) were detected in urine samples from pregnant women in France with a mean values approximately 1.29–1.66 ppb (Chevrier et al., 2014), which is higher than the total effective levels of DEA and DIA (0.36 ppb) observed in the mixture exposure group. Furthermore, the fixed-ratio mixture predictions showed that the mixture effects of 16 compounds are quietly predictable by CA model, suggesting that a mixture of widely used pesticides at environmentally relevant levels could act additively as MR antagonists by competitive antagonism of mineralocorticoids binding to the ligand-binding domain of the receptor. In this study, we further demonstrated the mechanism by which 14 of 16 candidate pesticides inhibited MR-dependent transcription appeared to involve modifications in mineralocorticoid-induced nuclear translocation. Upon ligand binding, the alteration in conformation of MR results in exposure of receptor nuclear localization signaling motif and translocation from cytoplasm to nucleus (Nakatani et al., 2013). Like other steroids, the native MR ligand aldosterone is a small molecular lipophilic chemical (Sutanto and Dekloet, 1991). Most of the MR-antagonistic pesticides identified in this study are also lipophilic chemicals. It is highly possible that these 14 pesticides are able to enter the ligand binding pocket of MR, competitively binding to the receptor but unable to induce the correct conformation change and remained in the cytoplasm. However, other 2 pesticides, fenvalerate and fipronil, are unable to prevent the translocation of MR. Similar effects on other members of the nonsexual steroid receptor were observed in the As-inhibited GR activation that was independent of steroid-induced GR nuclear translocation (Kaltreider et al., 2001). It suggests a different mechanism that may act by disrupting downstream progress such as DNA binding or cofactor recruiting receptor function after nuclear translocation. So far, little was known about the adverse effects of a continuous MR function impairment by environmental contaminants. In this study, 16 pesticides and/or their metabolites were reported first time to exhibit anti-mineralocorticoidic activity and 14 of them interrupted MR transactivation through disrupting nuclear translocation process. The additively combined effects of multiple pesticides were observed in a quite predictable manner, indicating that a mixture composed of a larger number of MR antagonists would have produced additive adverse health effects at correspondingly lower concentrations, approaching realistic environmental levels. Indeed, the compounds tested in this study were only a small portion of the great number of xenobiotics. Other man-made chemicals should be recruited for MR antagonist/agonist screening, and it will provide important knowledge in evaluating the overall role of environmental MR ligands-induced health effects. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING National Natural Science Foundation of China (21377113 and 21320102007); Zhejiang Provincial Natural Science Foundation of China (LR15B070001). REFERENCES Arriza J. L., Weinberger C., Cerelli G., Glaser T. M., Handelin B. L., Housman D. E., Evans R. M. ( 1987). Cloning of human mineralocorticoid receptor complementary-DNA—Structural and functional kinship with the glucocorticoid receptor. Science  237, 268– 275. Google Scholar CrossRef Search ADS PubMed  Barraza-Vazquez A., Borja-Aburto V. H., Bassol-Mayagoitia S., Monrroy A., Recio-Vega R. ( 2008). Dichlorodiphenyldichloroethylene concentrations in umbilical cord of newborns and determinant maternal factors. J. Appl. Toxicol . 28, 27– 34. Google Scholar CrossRef Search ADS PubMed  Berger S., Bleich M., Schmid W., Cole T. J., Peters J., Watanabe H., Kriz W., Warth R., Greger R., Schutz G. ( 1998). Mineralocorticoid receptor knockout mice: Pathophysiology of Na+ metabolism. Proc. Natl. Acad. Sci. U.S.A . 95, 9424– 9429. Google Scholar CrossRef Search ADS PubMed  Bodwell J. E., Gosse J. A., Nomikos A. P., Hamilton J. W. ( 2006). Arsenic disruption of steroid receptor gene activation: Complex dose–response effects are shared by several steroid receptors. Chem. Res. Toxicol . 19, 1619– 1629. Google Scholar CrossRef Search ADS PubMed  Bouwman H., Sereda B., Meinhardt H. M. ( 2006). Simultaneous presence of DDT and pyrethroid residues in human breast milk from a malaria endemic area in South Africa. Environ. Pollut . 144, 902– 917. http://dx.doi.org/10.1016/j.envpol.2006.02.002 Google Scholar CrossRef Search ADS PubMed  Carreno J., Rivas A., Granada A., Lopez-Espinosa M. J., Mariscal M., Olea N., Olea-Serrano F. ( 2007). Exposure of young men to organochlorine pesticides in Southern Spain. Environ. Res . 103, 55– 61. Google Scholar CrossRef Search ADS PubMed  Chevrier C., Serrano T., Lecerf R., Limon G., Petit C., Monfort C., Hubert-Moy L., Durand G., Cordier S. ( 2014). Environmental determinants of the urinary concentrations of herbicides during pregnancy: The PELAGIE mother-child cohort (France). Environ. Int . 63, 11– 18. Google Scholar CrossRef Search ADS PubMed  Douma B. R. K., Korte S. M., Buwalda B., la Fleur S. E., Bohus B., Luiten P. G. M. ( 1998) Repeated blockade of mineralocorticoid receptors, but not of glucocorticoid receptors impairs food rewarded spatial learning. Psychoneuroendocrinology  23, 33– 44. Google Scholar CrossRef Search ADS PubMed  Faust M., Altenburger R., Backhaus T., Blanck H., Boedeker W., Gramatica P., Hamer V., Scholze M., Vighi M., Grimme L. H. ( 2001). Predicting the joint algal toxicity of multi-components—Triazine mixtures at low-effect concentrations of individual toxicants. Aquat. Toxicol . 56, 13– 32. Google Scholar CrossRef Search ADS PubMed  Fejes-Toth G., Pearce D., Naray-Fejes-Toth A. ( 1998). Subcellular localization of mineralocorticoid receptors in living cells: Effects of receptor agonists and antagonists. Proc. Natl. Acad. Sci. U.S.A . 95, 2973– 2978. Google Scholar CrossRef Search ADS PubMed  Fenner K., Canonica S., Wackett L. P., Elsner M. ( 2013). Evaluating pesticide degradation in the environment: Blind spots and emerging opportunities. Science  341, 752– 758. http://dx.doi.org/10.1126/science.1236281 Google Scholar CrossRef Search ADS PubMed  Fuller P. J., Lim-Tio S. S., Brennan F. E. ( 2000). Specificity in mineralocorticoid versus glucocorticoid action. Kidney Int . 57, 1256– 1264. http://dx.doi.org/10.1046/j.1523-1755.2000.00959.x Google Scholar CrossRef Search ADS PubMed  Fuller P. J., Rogerson F. M. ( 2002). Pseudohypoaldosteronism: Kidney, lungs and colon. Clin. Endocrinol . 56, 571– 572. http://dx.doi.org/10.1046/j.1365-2265.2002.01512.x Google Scholar CrossRef Search ADS   Gaeggeler H. P., Gonzalez-Rodriguez E., Jaeger N. F., Loffing-Cueni D., Norregaard R., Loffing J., Horisberger J. D., Rossier B. C. ( 2005) Mineralocorticoid versus glucocorticoid receptor occupancy mediating aldosterone-stimulated sodium transport in a novel renal cell line. J. Am. Soc. Nephrol . 16, 878– 891. Google Scholar CrossRef Search ADS PubMed  Geller D. S., Rodriguez-Soriano J., Boado A. V., Schifter S., Bayer M., Chang S. S., Lifton R. P. ( 1998). Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I. Nat. Genet . 19, 279– 281. Google Scholar CrossRef Search ADS PubMed  Grossmann C., Benesic A., Krug A. W., Freudinger R., Mildenberger S., Gassner B., Gekle M. ( 2005). Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions. Mol. Endocrinol . 19, 1697– 1710. Google Scholar CrossRef Search ADS PubMed  Grossmann C., Husse B., Mildenberger S., Schreier B., Schuman K., Gekle M. ( 2010). Colocalization of mineralocorticoid and EGF receptor at the plasma membrane. Mol. Cell. Res . 1803, 584– 590. Jaffe I. Z., Mendelsohn M. E. ( 2005). Angiotensin II and aldosterone regulate gene transcription via functional mineralocortocoid receptors in human coronary artery smooth muscle cells. Circ. Res . 96, 643– 650. http://dx.doi.org/10.1161/01.RES.0000159937.05502.d1 Google Scholar CrossRef Search ADS PubMed  Jeong Y., Xie Y., Xiao G. H., Behrens C., Girard L., Wistuba I. I., Minna J. D., Mangelsdorf D. J. ( 2010). Nuclear receptor expression defines a set of prognostic biomarkers for lung cancer. PLoS Med . 7, e1000378. Kaltreider R. C., Davis A. M., Lariviere J. P., Hamilton J. W. ( 2001). Arsenic alters the function of the glucocorticoid receptor as a transcription factor. Environ. Health Perspect . 109, 245– 251. Google Scholar CrossRef Search ADS PubMed  Kaneko H. ( 2011). Pyrethroids: Mammalian metabolism and toxicity. J. Agric. Food Chem . 59, 2786– 2791. http://dx.doi.org/10.1021/jf102567z Google Scholar CrossRef Search ADS PubMed  Kojima H., Katsura E., Takeuchi S., Niiyama K., Kobayashi K. ( 2004). Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect . 112, 524– 531. Google Scholar CrossRef Search ADS PubMed  Kojima H., Takeuchi S., Itoh T., Iida M., Kobayashi S., Yoshida T. ( 2013). In vitro endocrine disruption potential of organophosphate flame retardants via human nuclear receptors. Toxicology  314, 76– 83. Google Scholar CrossRef Search ADS PubMed  Kojima H., Takeuchi S., Uramaru N., Sugihara K., Yoshida T., Kitamura S. ( 2009). Nuclear hormone receptor activity of polybrominated diphenyl ethers and their hydroxylated and methoxylated metabolites in transactivation assays using Chinese hamster ovary cells. Environ. Health Perspect . 117, 1210– 1218. Google Scholar CrossRef Search ADS PubMed  Liu J., Zhao M. R., Zhuang S. L., Yang Y., Yang Y., Liu W. P. ( 2012). Low concentrations of o,p′-DDT inhibit gene expression and prostaglandin synthesis by estrogen receptor-independent mechanism in rat ovarian cells. PLoS One  7, e49916. Molina-Molina J. M., Hillenweck A., Jouanin I., Zalko D., Cravedi J. P., Fernandez M. F., Pillon A., Nicolas J. C., Olea N., Balaguer P. ( 2006). Steroid receptor profiling of vinclozolin and its primary metabolites. Toxicol. Appl. Pharmacol . 216, 44– 54. Google Scholar CrossRef Search ADS PubMed  Nakatani Y., Amano T., Takeda H. ( 2013). Corticosterone suppresses the proliferation of RAW264.7 macrophage cells via glucocorticoid, but not mineralocorticoid, receptor. Biol. Pharm. Bull . 36, 592– 601. http://dx.doi.org/10.1248/bpb.b12-00968 Google Scholar CrossRef Search ADS PubMed  Nicolaides N. C., Roberts M. L., Kino T., Braatvedt G., Hurt D.E., Katsantoni E., Sertedaki A., Chrousos G. P., Charmandari E. ( 2014). A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: Dissociation of the transactivating and transreppressive activities. J. Clin. Endocrinol. Metab . 99, E902– E907. Google Scholar CrossRef Search ADS PubMed  Nie H. Z., Li J., Yang X. M., Cao Q. Z., Feng M. X., Xue F., Wei L., Qin W. X., Gu J. R., Xia Q., et al.   ( 2015). Mineralocorticoid receptor suppresses cancer progression and the Warburg effect by modulating the miR-338-3p-PKLR axis in hepatocellular carcinoma. Hepatology  62, 1145– 1159. Google Scholar CrossRef Search ADS PubMed  Niu J. J., Lin Y., Guo Z. N., Niu M., Su C. H. ( 2016). The epidemiological investigation on the risk factors of hepatocellular carcinoma: A case-control study in Southeast China. Medicine  95, e2758. Orton F., Rosivatz E., Scholze M., Kortenkamp A. ( 2012). Competitive androgen receptor antagonism as a factor determining the predictability of cumulative antiandrogenic effects of widely used pesticides. Environ. Health Perspect . 120, 1578– 1584. Google Scholar CrossRef Search ADS PubMed  Ouvrard-Pascaud A., Puttini S., Saint-Marie Y., Athman R., Fontaine V., Cluzeaud F., Farman N., Rafestin-Oblin M. E., Blot-Chabaud M., Jaisser F. ( 2004). Conditional gene expression in renal collecting duct epithelial cells: Use of the inducible Cre-lox system. Am. J. Physiol. Renal Physiol . 286, F180– F187. Google Scholar CrossRef Search ADS PubMed  Pippal J. B., Fuller P. J. ( 2008). Structure-function relationships in the mineralocorticoid receptor. J. Mol. Endocrinol . 41, 405– 413. http://dx.doi.org/10.1677/JME-08-0093 Google Scholar CrossRef Search ADS PubMed  Rehan M., Ahmad E., Sheikh I. A., Abuzenadah A. M., Damanhouri G. A., Bajouh O. S., AlBasri S. F., Assiri M. M., Beg M. A. ( 2015). Androgen and progesterone receptors are targets for bisphenol A (BPA), 4-methyl-2,4-bis-(p-hydroxyphenyl)pent-1-ene-A potent metabolite of BPA, and 4-tert-octylphenol: A computational insight. PLoS One  10, e0138438. Rogerson F. M., Fuller P. J. ( 2000). Mineralocorticoid action. Steroids  65, 61– 73. http://dx.doi.org/10.1016/S0039-128X(99)00087-2 Google Scholar CrossRef Search ADS PubMed  Rossi L., Barbieri O., Sanguineti M., Cabral J. R. P., Bruzzi P., Santi L. ( 1983). Carcinogenicity study with technical-grade dichlorodiphenyltrichloroethane and 1,1-dichloro-2,2-bis(para-chlorophenyl)ethylene in hamsters. Cancer Res . 43, 776– 781. Google Scholar PubMed  Sekizawa N., Yoshimoto T., Hayakawa E., Suzuki N., Sugiyama T., Hirata Y. ( 2011). Transcriptome analysis of aldosterone-regulated genes in human vascular endothelial cell lines stably expressing mineralocorticoid receptor. Mol. Cell Endocrinol . 341, 78– 88. Google Scholar CrossRef Search ADS PubMed  Sifaki-Pistolla D., Karageorgos S. A., Koulentaki M., Samonakis D., Stratakou S., Digenakis E., Kouroumalis E. ( 2016). Geoepidemiology of hepatocellular carcinoma in the island of Crete, Greece. A possible role of pesticides. Liver Int . 36, 588– 594. Google Scholar CrossRef Search ADS PubMed  Sutanto W., Dekloet E. R. ( 1991). Mineralocorticoid receptor ligands—Biochemical, pharmacological, and clinical aspects. Med. Res. Rev . 11, 617– 639. Google Scholar CrossRef Search ADS PubMed  Takeuchi S., Iida M., Kobayashi S., Jin K., Matsuda T., Kojima H. ( 2005). Differential effects of phthalate esters on transcriptional activities via human estrogen receptors alpha and beta, and androgen receptor. Toxicology  210, 223– 233. Google Scholar CrossRef Search ADS PubMed  Takeuchi S., Shiraishi F., Kitamura S., Kuroki H., Jin K., Kojima H. ( 2011). Characterization of steroid hormone receptor activities in 100 hydroxylated polychlorinated biphenyls, including congeners identified in humans. Toxicology  289, 112– 121. Google Scholar CrossRef Search ADS PubMed  Tiberio L., Nascimbeni R., Villanacci V., Casella C., Fra A., Vezzoli V., Furlan L., Meyer G., Parrinello G., Baroni M. D., et al.   ( 2013). The decrease of mineralocorticoid receptor drives angiogenic pathways in colorectal cancer. PLoS One  8, e59410. VoPham T., Brooks M. M., Yuan J. M., Talbott E. O., Ruddell D., Hart J. E., Chang C. C. H., Weissfeld J. L. ( 2015). Pesticide exposure and hepatocellular carcinoma risk: A case-control study using a geographic information system (GIS) to link SEER-Medicare and California pesticide data. Environ. Res . 143, 68– 82. Google Scholar CrossRef Search ADS PubMed  Xing L., Sun J., Liu H., Yu H. ( 2012). Combined toxicity of three chlorophenols 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol to Daphnia magna. J. Environ. Monit . 14, 1677. http://dx.doi.org/10.1039/c2em30185g Google Scholar CrossRef Search ADS PubMed  Yau J. L. W., Noble J., Seckl J. R. ( 1999). Continuous blockade of brain mineralocorticoid receptors impairs spatial learning in rats. Neurosci. Lett . 277, 45– 48. http://dx.doi.org/10.1016/S0304-3940(99)00858-7 Google Scholar CrossRef Search ADS PubMed  Yu L. Y., Qin L. F., Zhang H. F., He Y., Chen H., Pober J. S., Tellides G., Min W. ( 2011). AIP1 prevents graft arteriosclerosis by inhibiting interferon-gamma-dependent smooth muscle cell proliferation and intimal expansion. Circ. Res . 109, 418– 427. Google Scholar CrossRef Search ADS PubMed  Zennaro M. C., Keightley M. C., Kotelevtsev Y., Conway G. S., Soubrier F., Fuller P. J. ( 1995). Human mineralocorticoid receptor genomic structure and identification of expressed isoforms. J. Biol. Chem . 270, 21016– 21020. Google Scholar CrossRef Search ADS PubMed  Zhang J. Y., Zhang J., Liu R., Gan J., Liu J., Liu W. P. ( 2016). Endocrine-disrupting effects of pesticides through interference with human glucocorticoid receptor. Environ. Sci. Technol . 50, 435– 443. Google Scholar CrossRef Search ADS PubMed  Zhang Q., Ye J. J., Chen J. Y., Xu H. J., Wang C., Zhao M. R. ( 2014). Risk assessment of polychlorinated biphenyls and heavy metals in soils of an abandoned e-waste site in China. Environ. Pollut . 185, 258– 265. Google Scholar CrossRef Search ADS PubMed  Zubero M. B., Aurrekoetxea J. J., Murcia M., Ibarluzea J. M., Goni F., Jimenez C., Ballester F. ( 2015). Time trends in serum organochlorine pesticides and polychlorinated biphenyls in the general population of Biscay, Spain. Arch. Environ. Contam. Toxicol . 68, 476– 488. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. 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Novel Pathways of Endocrine Disruption Through Pesticides Interference With Human Mineralocorticoid Receptors

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Abstract

Abstract Mineralocorticoid receptor (MR) is one member of the steroid receptor family. In addition to its important role in Na+/K+ homeostasis, MR is reported as a tumor-suppressor in carcinogenesis. So far, little was known about the ability of pesticides to interfere with MR. In this study, a total of 43 pesticides and/or metabolites were investigated for their potential effects on human MR. None of the tested pesticides exhibited MR agonistic potency, whereas 16 compounds showed antagonistic activities. Further investigations indicated that these 16 chemicals individually antagonized aldosterone-induced alkaline phosphatase expression in vascular smooth muscle cells and aldosterone-inhibited hepatocellular carcinoma cell proliferation at higher concentrations, and the mixture of these 16 pesticides at environmentally relevant concentrations significantly disrupted MR activity. The additional quantitative mixture experiments indicated a good agreement between the combined anti-mineralocorticoidic activities of 16 pesticides and the responses predicted by concentration addition model instead of independent action model. The interruption of nuclear translocation of MR was clarified as a main mechanism for the anti-mineralocorticoidic activities by these pesticides. These data suggest that the health risk may increase when multiple MR antagonists cooperate following concentration addition model and exhibit a combined effect. Our findings emphasize that comprehensive risk assessment of adverse effects of environmental MR ligands on human health should be considered. endocrine-disrupting chemicals, pesticides, steroid hormone receptors, mineralocorticoid receptor, anti-mineralocorticoid activity Most prior research on environmental endocrine-disrupting chemicals (EDCs) focused on their actions on estrogen receptor (ER) and androgen receptor (AR). Estrogen receptor and AR belong to the steroid receptor subfamily of nuclear transcription factors (Jaffe and Mendelsohn, 2005). Other members of the steroid receptor family, including progesterone receptor (PR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR), are also involved in steroid-regulated biological processes that may similarly be disrupted if agonists or antagonists are present in the environment. Recently, there has been increasing evidence for the effects of EDCs on other members of this steroid receptor class such as PR and GR (Rehan et al., 2015; Zhang et al., 2016). Very few studies also showed the potential impact of EDCs on MR. Bodwell et al. (2006) reported a biphasic response of MR activity to arsenic (As) exposure, with stimulation of activity at low doses of As and inhibition at higher doses. A fungicide, vinclozolin, and its primary metabolite were identified as MR antagonists using in vitro assay (Molina-Molina et al., 2006). However, data regarding the possible environmental ligands for MR are still extremely sparse in EDCs research. Mineralocorticoid receptor mediates the effects of mineralocorticoids, a class of essential physiological corticosteroids, on a variety of target tissues such as kidney, colon, adipose tissue, cardiovascular and central nervous systems (Berger et al., 1998; Pippal and Fuller, 2008; Rogerson and Fuller, 2000; Zennaro et al., 1995). As a nuclear transcription factor, MR translocates to the nucleus upon ligation by cognate ligand and binds to specific DNA sequence and regulates the corresponding responsive gene expression, critically involved in Na+/K+ homeostasis, blood pressure regulation, and cell proliferation (Gaeggeler et al., 2005; Grossmann et al., 2010; Sekizawa et al., 2011). Loss of MR function in deficient mice and human resulted in neonate mortality because of severe dehydration by renal sodium and water loss (Berger et al., 1998; Fuller and Rogerson, 2002; Geller et al., 1998). Blockade of MR also reported to impair stress-related learning and lead to anxiety behavior in animal models (Douma et al., 1998; Yau et al., 1999). Recently, the tumor-suppressive role of MR has been appreciated in cancer development and progression, such as colorectal cancer, lung cancer, and hepatocellular carcinoma (HCC) (Jeong et al., 2010; Nie et al., 2015; Tiberio et al., 2013). Due to the indispensable physiological functions of MR, it is urgent to recognize potential MR agonists or antagonists among environmental chemicals and potential interferences with MR should be considered for the safety assessment of EDCs. The ubiquitous usage of pesticides with an annual global amount of 1–2.5 million tons has resulted in a type of the most widespread and significant environmental pollution (Fenner et al., 2013). Pesticides are now suspected of being a kind of important EDCs. Numerous pesticides have been reported to have estrogenic or anti-androgenic activity via interfering with ER or AR (Kojima et al., 2004). Our recent study found that more than one-third of tested pesticides were potential GR antagonists, suggesting that many pesticides could affect steroid-regulated biological processes via binding to other members of steroid receptor class such as GR (Zhang et al., 2016). Thus, to identify common pesticides for MR agonists or antagonists and to further explore the possible mechanism are imperative to expand the understanding of latent risks of pesticides. In this study, 43 pesticides (listed in Supplementary Table 1) were screened for MR activities using luciferase reporter gene assay. The results characterized a total of 16 pesticides as potential MR antagonists. The effects of these potential MR ligands on mineralocorticoid signaling were further confirmed by the inhibition of the expression of mineralocorticoid-responsive gene alkaline phosphatase (Alk) in human vascular smooth muscle cells (VSMCs). It was also demonstrated that these pesticides reversed MR-mediated suppression of HCC proliferation. Furthermore, we uncovered a mechanism for anti-mineralocorticoidic activities by these pesticides that mainly involved the inhibition of mineralocorticoid-induced nuclear translocation of MR. More importantly, our findings indicated that the mixture of these 16 pesticides at environmentally relevant concentrations could disrupt MR transactivity and inhibit MR-mediated function. The observed responses of the mixture composed of these 16 chemicals agreed very well with the predicted regression curves of concentration addition (CA) model. MATERIALS AND METHODS Chemicals Aldosterone (>97% pure) was purchased from J&K Scientific (Beijing, China). Spironolactone (>99% pure) was obtained from Selleck Chemicals (Boston, Massachusetts). The 43 pesticides and pesticide metabolites listed in Supplementary Table 1 were obtained from Sigma-Aldrich (St Louis, Missouri). Stock solutions of chemicals were prepared using dimethylsulfoxide (DMSO) as a solvent and stored at −20 °C, except for paraquat that was dissolved in deionized water. Plasmid constructs The human mineralocorticoid receptor (hMR) expression plasmid EGFP-C1-hMR was kindly provided by Dr Claudia Großmann (Martin Luther University, Germany) (Grossmann et al., 2005; Ouvrard-Pascaud et al., 2004). The mineralocorticoid response element containing reporter plasmid pMMTV-luc was kindly provided by Dr Evangelia Charmandari (Biomedical Research Foundation of the Academy of Athens, Greece) (Nicolaides et al., 2014). In dual-luciferase reporter assays, pRL-TK (Promega, Madison) was used as an internal control as previously described (Zhang et al., 2016). Cell cultures Chinese hamster ovary K1 cell line (CHO-K1) and human HCC cell line (SMMC-7721) were maintained at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, Logan, Utah) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 100 U/ml streptomycin-penicillin (Hyclone) under saturating humidity. Primary cultured human VSMCs were kindly provided by Dr Luyang Yu (College of Life Science, Zhejiang University). VSMCs were maintained in M199 media (Gibco, Grand Island, New York) with 20% FBS (Gibco), 1% glutamine (Gibco), and 100 U/ml streptomycin-penicillin (Yu et al., 2011). Low-passage VSMCs primary cultures were used for real-time quantitative PCR. For all exposure experiments, the cells were cultured with phenol red–free DMEM or M199 supplemented with charcoal/dextran-treated FBS. Cell proliferation assay As previously described, cell proliferation was assessed after exposure to tested chemicals at the concentration of 10−5 or 10−6 M using CellTiter 96 AQueous One Solution Cell Proliferation (Promega, Madison, Wisconsin) (Liu et al., 2012; Zhang et al., 2014). The exposure periods were 24 h for CHO-K1 cells and 48 h for SMMC-7721 cells. The absorbance at 490 nm was detected using microplate reader (Infinite M200 PRO, Tecan, Switzerland). Reporter gene assay The reporter gene assay was performed as previously described (Zhang et al., 2016). Briefly, after transient transfection with pEGFP-C1-hMR, pMMTV-luc, and pRL-TK plasmids, CHO-K1 cells were exposed to tested chemicals or 0.1% DMSO (vehicle control) to measure the agonistic activity of hMR. For antagonistic activity measurement, transfected cells were treated with 10−9.5 M aldosterone in combination with tested chemicals after 30 min pretreatment with the tested compound alone. Firefly luciferase and Renilla luciferase activities were measured using the Dual-luciferase Reporter Assay Kit (Promega) after 24 h exposure and the ratio of firefly to Renilla luciferase activity was used to present the relative transcriptional activity. The transfected cells were exposed to the mixture of all 16 potential antagonists with an equal concentration of 10−10 to10−6 M for a plain combined antagonistic effect evaluation. Relative inhibition rate (RIR) is obtained at the highest tested concentration of chemicals as percent decrease of aldosterone response. The Weibull regression model was applied for the individual concentration-response analyses. The concentrations of tested chemicals that inhibited 20% (IC20) or 50% (IC50) of the luciferase activity induced by aldosterone were calculated.   E=1-exp⁡(-exp⁡ (α+ β log10  c )), (1) E, effect, the fraction of antagonistic effect (0 ≤ E ≤ 1); α and β, model parameters that varied depending on the individual concentration-response curve; c, the concentration of tested chemicals. Calculation of mixture-effect predictions To further predict the combined effects of 16 potential MR antagonists, the mixtures were designed as fixed-ratio equipotent mixtures that were calculated based on the effect concentrations (EC) of the individual components that led to an inhibition of aldosterone effects by 10% (here termed as inhibitory concentrations 10% [IC10]) based on Weibull regression model (Faust et al., 2001). The mathematical and statistical procedures used to calculate predictions of the effect concentrations (ECxmix) of the mixture according to CA and IA models were well-described in previous studies (Faust et al., 2001; Orton et al., 2012; Xing et al., 2012). The equations that predicted the effect mixture concentrations under the hypothesis of CA (equation 1) and IA (equation 2) were listed below: CA:   ECxmix=∑i=1npiECxi-1, (2)pi, the relative proportions of the individual component in the whole mixture; ECxi, the equivalent effect concentrations of the individual component. IA:   x%=1-∏i=1n1-Fipi×ECxmix. (3) In this equation, Fi are calculated from the concentration-response functions and the x% represents the total effect. Real-time quantitative PCR VSMCs were treated with the tested chemicals for 24 h and then cells were lysed for total RNA isolation and reverse transcription using SuperPrep Cell Lysis & RT Kit for qPCR (Toyobo, Tokyo, Japan) according to the manufacturer’s instruction. SYBR Green PCR Master Mix (Toyobo, Japan) was used for real-time quantitative PCR on Mx3000P (Agilent Technologies, Palo Alto) as previously described (Zhang et al., 2016). The primer sequences are listed in Supplementary Material. Relative gene expression level was calculated using the ΔΔ threshold cycle (Ct) method and normalized to the endogenous reference gene gapdh. Immunofluorescence and confocal microscopy Immunofluorescence of MR was performed as previously described (Grossmann et al., 2010). Briefly, CHO-K1 cells were seeded on glass coverslips, transiently transfected with EGFP-C1-hMR, and then treated with 10−9.5 M aldosterone in combination with tested chemicals for 1 h after 30 min pretreatment with tested chemicals. Cells were fixed and then incubated with anti-MR antibody (dilution 1:200, Santa Cruz Biotechnology, Dallas) overnight at 4 °C. After further incubation with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, ThermoFisher Scientific, Eugene) and 4ʹ,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame), the cells were analyzed by confocal microscopy (LSM780, Zeiss, Gottingen, Germany). Western blotting After transfection with pEGFP-C1-hMR plasmids, CHO-K1 cells were exposed to 10−9.5 M aldosterone in combination with tested chemicals for 1 h after 30 min pretreatment with the tested compound alone. Nuclear protein was isolated using the CelLytic NuCLEAR extraction kit (Sigma-Aldrich). Western blotting was performed as previously described (Liu et al., 2012), and details are shown in Supplementary Material. Statistical analysis All experimentations were repeated at least 3 times. The data are presented as mean ± SD of at least 3 independent assays with triplicates. Statistical analysis was carried out using SPSS version 16.0 (SPSS, Chicago) and Origin 8.0 (OriginLab, Northampton). The significance of difference was evaluated by one-way ANOVA followed by Dunnett’s post hoc test, and differences were considered significant if p < .05. RESULTS Agonistic Effects of Tested Chemicals in MR Assays The results of MTS assay showed that exposure to resmethrin, carbendazim, or tolylfluanid at a concentration of 10−6 M and other chemicals at a concentration of 10−5 M for 24 h did not affect cell viability (Supplementary Figure 1). The endogenous mineralocorticoid aldosterone stimulated the MR transcriptional activity in a concentration-response manner and the maximal activity reached at 10−9.5 M aldosterone or higher (Supplementary Figure 2A). Therefore, the relative induction rate compared with the MR activity obtained by 10−9.5 M aldosterone was used to evaluate the agonistic effects of 43 pesticides and metabolites at non-cytotoxic concentrations. There was no significant induction observed, suggesting that none of the tested chemicals has MR agonistic activity (Supplementary Figure 3). Antagonistic Effects of Tested Chemicals in MR Assays Spironolactone, an MR antagonist that suppressed the aldosterone-induced MR transactivation in a concentration-dependent manner, was used as a positive control to evaluate MR antagonistic activity (Supplementary Figure 2B). A total of 16 pesticides among the 43 tested chemicals, including 4 pyrethroids (bifenthrin, cypermethrin, fenvalerate, and permethrin), 4 organochlorines (o,p′-dichlorodiphenyltrichloroethane [DDT], p,p′-dichlorodiphenyldichloroethylene [DDE], p,p′-DDE, and methoxychlor), 2 organophosphates (acephate and dimethoate), triazine herbicide terbuthylazine, and metabolites of atrazine (atrazine-desethyl [DEA] and atrazine-desisopropyl [DIA]), as well as alachlor, zineb, and fipronil, significantly attenuated aldosterone (10−9.5 M) induced MR transcriptional activity at the highest tested non-cytotoxic concentration (Figure 1), suggesting that these 16 chemicals potentially exhibited MR antagonistic properties. And the inhibition of transcriptional activity was reversed by a higher concentration of aldosterone (10−6 M), indicating the antagonism would be due to competitive binding to the receptor (Figure 1E). Subsequently, the concentration-dependent MR antagonistic activities of these 16 potential antagonists were determined at doses of 10−9 to 10−5 M (Figs. 2A–D). The concentrations of the tested chemicals reducing 20% of 10−9.5 M aldosterone-induced MR activity (20% inhibitory concentration, IC20), 50% inhibitory concentration (IC50), and the RIR of these 16 chemicals at the highest tested concentrations that represented as percent decrease of aldosterone response were calculated from the concentration-response curves (Table 1). These 16 chemicals exhibited an antagonistic activity against MR with IC20 between 10−7 M and 10−6 M, indicating that these pesticides were weak MR antagonists when compared with the pharmaceutical antagonist spironolactone (Table 1). Table 1. Inhibitory Effects of 16 Pesticides and Metabolites on MR Transcriptional Activity Induced by Aldosterone Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Aldosterone      100  4. Organophosphates        Spironolactone  4.17 × 10−8  1.71 × 10−7  83.3  Acephate  4.07 × 10−7  8.52 × 10−6  52.6  1. Pyrethroids        Dimethoate  2.87 × 10−7  NA  45.4  Bifenthrin  3.76 × 10−7  7.06 × 10−6  54.3          Cypermethrin  2.43 × 10−7  4.96 × 10−6  56.0  5. Carbamates        Fenvalerate  1.03 × 10−6  7.40 × 10−6  52.1  Zineb  1.93 × 10−7  8.21 × 10−6  46.4  Permethrin  1.60 × 10−7  7.27 × 10−6  54.0                  6. Triazines        2. Organochlorines        DEA  8.35 × 10−7  NA  41.3  o,pʹ-DDT  1.26 × 10−6  4.04 × 10−6  81.2  DIA  1.02 × 10−7  4.35 × 10−6  48.1  p,pʹ-DDE  2.41 × 10−6  6.01 × 10−6  72.4  Terbuthylazine  3.86 × 10−7  NA  43.5  p,pʹ-DDT  1.00 × 10−6  3.29 × 10−6  80.5          Methoxychlor  2.19 × 10−6  8.13 × 10−6  69.2  8. Others                Fipronil  1.30 × 10−6  NA  41.6  3. Amids                Alachlor  1.04 × 10−7  5.13 × 10−6  58.2          Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Group, Compound  IC20 (M)  IC50 (M)  RIR (%)  Aldosterone      100  4. Organophosphates        Spironolactone  4.17 × 10−8  1.71 × 10−7  83.3  Acephate  4.07 × 10−7  8.52 × 10−6  52.6  1. Pyrethroids        Dimethoate  2.87 × 10−7  NA  45.4  Bifenthrin  3.76 × 10−7  7.06 × 10−6  54.3          Cypermethrin  2.43 × 10−7  4.96 × 10−6  56.0  5. Carbamates        Fenvalerate  1.03 × 10−6  7.40 × 10−6  52.1  Zineb  1.93 × 10−7  8.21 × 10−6  46.4  Permethrin  1.60 × 10−7  7.27 × 10−6  54.0                  6. Triazines        2. Organochlorines        DEA  8.35 × 10−7  NA  41.3  o,pʹ-DDT  1.26 × 10−6  4.04 × 10−6  81.2  DIA  1.02 × 10−7  4.35 × 10−6  48.1  p,pʹ-DDE  2.41 × 10−6  6.01 × 10−6  72.4  Terbuthylazine  3.86 × 10−7  NA  43.5  p,pʹ-DDT  1.00 × 10−6  3.29 × 10−6  80.5          Methoxychlor  2.19 × 10−6  8.13 × 10−6  69.2  8. Others                Fipronil  1.30 × 10−6  NA  41.6  3. Amids                Alachlor  1.04 × 10−7  5.13 × 10−6  58.2          Eight chemicals groups are listed in Supplementary Table 1. NA, not available because that the calculated concentration is out of the range of concentration tested in this study. IC20 and IC50: concentration of tested chemicals that inhibited 20% or 50% of the luciferase activity induced by aldosterone. RIR obtained at highest tested concentration of chemicals as percent decrease of aldosterone response. Figure 1. View largeDownload slide Antagonistic effects of pesticides and their metabolites in the mineralocorticoid receptor (MR) transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with 0.1% dimethylsulfoxide (DMSO) (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with pesticides at non-cytotoxic concentrations. A, Antagonistic effects of 10−5 M of bifenthrin, λ-cyhalothrin, cypermethrin, fenvalerate, permethrin, 3-PBald, 3-PBA, 3-PBalc, and 10−6 M of resmethrin. B, Antagonistic effects of 10−5 M of α-BHC, β-BHC, δ-BHC, γ-BHC, o,p′-DDT, p,p′-DDT, p,p′-DDE, methoxychlor, alachlor, dimethenamid, metolachlor, and S-metolachlor. C, Antagonistic effects of 10−5 M of acephate, chlorpyrifos, diazinon, dimethoate, omethoate, parathion-ethyl, carbaryl, ethiofencarb, fenobucarb, pirimicarb, and zineb. D, Antagonistic effects of 10−5 M of atrazine, DEA, DIA, metribuzin, terbuthylazine, benomyl, fipronil, fomesafen, paraquat, and 10−6 M of carbendazim and tolylfluanid. E, The antagonistic effect of chemicals at 10−5 M in the presence of 10−9.5 M or 10−6 M aldosterone. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (Ald) (= 100%). #p < .05, ##p < .01, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Figure 1. View largeDownload slide Antagonistic effects of pesticides and their metabolites in the mineralocorticoid receptor (MR) transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with 0.1% dimethylsulfoxide (DMSO) (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with pesticides at non-cytotoxic concentrations. A, Antagonistic effects of 10−5 M of bifenthrin, λ-cyhalothrin, cypermethrin, fenvalerate, permethrin, 3-PBald, 3-PBA, 3-PBalc, and 10−6 M of resmethrin. B, Antagonistic effects of 10−5 M of α-BHC, β-BHC, δ-BHC, γ-BHC, o,p′-DDT, p,p′-DDT, p,p′-DDE, methoxychlor, alachlor, dimethenamid, metolachlor, and S-metolachlor. C, Antagonistic effects of 10−5 M of acephate, chlorpyrifos, diazinon, dimethoate, omethoate, parathion-ethyl, carbaryl, ethiofencarb, fenobucarb, pirimicarb, and zineb. D, Antagonistic effects of 10−5 M of atrazine, DEA, DIA, metribuzin, terbuthylazine, benomyl, fipronil, fomesafen, paraquat, and 10−6 M of carbendazim and tolylfluanid. E, The antagonistic effect of chemicals at 10−5 M in the presence of 10−9.5 M or 10−6 M aldosterone. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (Ald) (= 100%). #p < .05, ##p < .01, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Figure 2. View largeDownload slide The concentration-response effects of antagonistic pesticides in the MR transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with pesticides at the concentrations range from 10−9 M to 10−5 M in the presence of 10−9.5 M aldosterone. A, The concentration-response antagonistic effects of bifenthrin, cypermethrin, fenvalerate, and permethrin. B, The concentration-response antagonistic effects of o,p′-DDT, p,p′-DDE, p,p′-DDT, and methoxychlor. C, The concentration-response antagonistic effects of acephate, dimethoate, DEA, DIA, and terbuthylazine. D, The concentration-response antagonistic effects of alachlor, zineb, and fipronil. E, The concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−10 M to10−6 M. F, The predicted and observed concentration-response antagonistic effects of fixed-ratio equipotent mixtures composed in the ratio of their IC10. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (= 100%). Figure 2. View largeDownload slide The concentration-response effects of antagonistic pesticides in the MR transactivation assays. EGFP-C1-hMR transfected CHO-K1 cells were treated with pesticides at the concentrations range from 10−9 M to 10−5 M in the presence of 10−9.5 M aldosterone. A, The concentration-response antagonistic effects of bifenthrin, cypermethrin, fenvalerate, and permethrin. B, The concentration-response antagonistic effects of o,p′-DDT, p,p′-DDE, p,p′-DDT, and methoxychlor. C, The concentration-response antagonistic effects of acephate, dimethoate, DEA, DIA, and terbuthylazine. D, The concentration-response antagonistic effects of alachlor, zineb, and fipronil. E, The concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−10 M to10−6 M. F, The predicted and observed concentration-response antagonistic effects of fixed-ratio equipotent mixtures composed in the ratio of their IC10. Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction, with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (= 100%). Because of coexistence of multiple chemicals in the realistic environment, transfected cells were exposed to the mixture of these 16 potential antagonists at their individual concentrations of 10−10 to 10−6 M to evaluate their combined effects. As shown in Figure 2E, exposure to the mixture caused a dose-dependent reduction in aldosterone-induced MR transcriptional activity. Notably, a significant inhibition was observed at a dose as low as 10−9 M (Figure 2E). The mixture modeling was further explored by comparing the observed responses and the combined effects predicted by CA and IA models based on fixed-ratio concentration-response analyses. The regression model parameters and proportion of the individual components were listed in Supplementary Table 2. There was a good agreement of the responses of mixtures comprised by IC10 ratio aliquots with predictions by CA over the concentration range from 10−8 M to 10−5 M (Figure 2F). Disruption of Mineralocorticoid-Responsive Gene Expression To further assess the anti-mineralocorticoid potency, the expression of mineralocorticoid-induced gene Alk that plays an important role in vascular calcification was evaluated in the primary cultured human VSMCs that express endogenous MR (Jaffe and Mendelsohn, 2005). As shown in Figure 3, all tested compounds at a concentration of 10−6 or 10−5 M significantly antagonized aldosterone-induced Alk expression in VSMCs (Figs. 3A and 3B). The concentrations of pesticides used for treatment did not cause obvious cytotoxic effects on VSMCs (Supplementary Figure 4A). The expression of housekeeping gene gapdh had high stability and its relative Ct values did not change in VSMCs after pesticide treatment, suggesting that these chemicals at non-cytotoxic concentrations had no effects on this non-MR target gene (Supplementary Figure 4B). A total of 12 chemicals among these 16 candidate pesticides, including 4 pyrethroids (bifenthrin, cypermethrin, fenvalerate and permethrin), 2 organochlorines (o,pʹ-DDT and methoxychlor), herbicide alachlor, organophosphate insecticide dimethoate, carbamate fungicide zineb, atrazine metabolite DIA, triazine herbicide terbuthylazine, and fipronil, remarkably antagonized aldosterone (10−9.5 M) upregulated Alk gene expression by more than 50% when compared with the aldosterone-treated positive control (Figure 3). The repression of Alk expression was significantly reversed when cells were exposed to aldosterone at higher concentration of 10−6 M (Figs. 3A and 3B). By exposing VSMCs to the mixture of these 16 pesticides at their individual concentrations in the range of 10−9 to 10−5 M, a concentration-dependent decrease in aldosterone-induced Alk expression was observed and the significant inhibition occurred at a dose as low as 10−8 M of the mixture (Figure 3C). Figure 3. View largeDownload slide The inhibitory effects of pesticides on aldosterone-induced expression of mineralocorticoid-responsive gene Alk. VSMCs were treated with 0.1% DMSO (vehicle control), 10−9.5 M or 10−6 M aldosterone (Ald, positive control), 10−5 M spironolactone (antagonist control), or the combination of 10−9.5 M or 10−6 M aldosterone with pesticides at non-cytotoxic concentrations. A, Inhibitory effects of 10−5 M bifenthrin, cypermethrin, fenvalerate, permethrin, and 10−6 M o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor. B, Inhibitory effects of 10−5 M alachlor, acephate, dimethoate, zineb, DEA, DIA, terbuthylazine, and fipronil. C, Concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−9 M to10−6 M, in the presence of 10−9.5 M aldosterone. The relative mRNA levels of Alk in VSMCs are present as percent induction with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (10−9.5 M Ald) (= 100%). #p < .05, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Figure 3. View largeDownload slide The inhibitory effects of pesticides on aldosterone-induced expression of mineralocorticoid-responsive gene Alk. VSMCs were treated with 0.1% DMSO (vehicle control), 10−9.5 M or 10−6 M aldosterone (Ald, positive control), 10−5 M spironolactone (antagonist control), or the combination of 10−9.5 M or 10−6 M aldosterone with pesticides at non-cytotoxic concentrations. A, Inhibitory effects of 10−5 M bifenthrin, cypermethrin, fenvalerate, permethrin, and 10−6 M o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor. B, Inhibitory effects of 10−5 M alachlor, acephate, dimethoate, zineb, DEA, DIA, terbuthylazine, and fipronil. C, Concentration-response antagonistic effects of mixture of all 16 potential antagonists with an equal individual component concentration of 10−9 M to10−6 M, in the presence of 10−9.5 M aldosterone. The relative mRNA levels of Alk in VSMCs are present as percent induction with 100% activity defined as the activity obtained with positive control (10−9.5 M aldosterone). *p < .05, **p < .01, compared with positive control (10−9.5 M Ald) (= 100%). #p < .05, difference between the combined treatment of chemical with 10−9.5 M aldosterone and that of 10−6 M aldosterone. Exposure of Tested Chemicals Alters the Aldosterone-Induced HCC Growth Suppression It has been reported that MR exerted a suppressive role in HCC progression (Nie et al., 2015). In this study, we further examined the effects of these 16 potential MR antagonists on the MR-mediated suppression of HCC proliferation. SMMC-7721 cells were exposed to aldosterone in combination with the tested chemicals at a non-toxic concentration (10−5 or 10−6 M) (Supplementary Figs. 5A and 5B). As expected, the native ligand aldosterone significantly suppressed the proliferation of HCC cell line SMMC-7721 in a concentration-dependent manner (Supplementary Figure 5C), whereas aldosterone-induced suppression was reversed by MR antagonist spironolactone (Figure 4). The result indicated that 14 of these 16 chemicals remarkably attenuated aldosterone-induced HCC growth suppression (Figs. 4A and 4B). Furthermore, the blockage of aldosterone-induced suppression of SMMC-7721 cells was also observed after a treatment with the mixture of these 16 pesticides at concentrations in the range of 10−9 to 10−7 M (Figure 4C). Figure 4. View largeDownload slide Effects of pesticides on the aldosterone-induced HCC growth suppression. SMMC-7721 cells were treated with 0.1% DMSO (vehicle control), 10−6 M aldosterone (Ald, positive control), spironolactone (antagonist control) in the presence of 10−6 M aldosterone, or the combination of 10−6 M aldosterone with pesticides at non-cytotoxic concentrations (10−6 M for bifenthrin, o,p′-DDT, p,p′-DDT, and methoxychlor, or 10−5 M for other compounds). *p < .05, **p < .01, compared with vehicle control (DMSO) (= 100%). Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction. Figure 4. View largeDownload slide Effects of pesticides on the aldosterone-induced HCC growth suppression. SMMC-7721 cells were treated with 0.1% DMSO (vehicle control), 10−6 M aldosterone (Ald, positive control), spironolactone (antagonist control) in the presence of 10−6 M aldosterone, or the combination of 10−6 M aldosterone with pesticides at non-cytotoxic concentrations (10−6 M for bifenthrin, o,p′-DDT, p,p′-DDT, and methoxychlor, or 10−5 M for other compounds). *p < .05, **p < .01, compared with vehicle control (DMSO) (= 100%). Data represent the mean ± SD of triplicate measurements in 3 independent experiments as percent induction. Effects of Tested Chemicals on Aldosterone-Induced Nuclear Translocation of MR To explore the mechanistic basis for these antagonistic effects, we examined whether coexposure of tested chemicals alters aldosterone-induced nuclear translocation of MR. Mineralocorticoid receptor immunolabeling is located in both cytoplasm and nucleus in the majority of transfected cells in the absence of ligand aldosterone, but not nucleoli (Figure 5A). After treatment with aldosterone, MR predominately translocated to the nucleus and detectable MR almost totally disappeared from the cytosolic pool (Figure 5B), which was consistent with previous observations (Fejes-Toth et al., 1998). Coexposure of most tested chemicals and aldosterone resulted in an obvious appearance of MR in the cytosol (Figure 5). A total of 14 among 16 tested antagonists significantly suppressed the aldosterone-induced nuclear translocation of MR (Figs. 5C–P). Moreover, the results of Western blotting also showed that coexposure to tested antagonists remarkably decreased the amount of MR in nucleus when compared with the aldosterone-treated control (Figs. 5Q and 5R). However, among these 16 pesticides, fipronil and fenvalerate did not appreciably alter the aldosterone-stimulated nuclear accumulation of MR (Supplementary Figure 6). Figure 5. View largeDownload slide Effects of tested pesticides on nuclear translocation of MR. CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to A, vehicle control (0.1% DMSO); B, positive control (10−9.5 M aldosterone), or the combination of 10−9.5 M aldosterone with C, bifenthrin; D, cypermethrin; E, permethrin; F, o,p′-DDT; G, p,p′-DDE; H, p,p′-DDT; I, methoxychlor; J, alachlor; K, acephate; L, dimethoate; M, zineb; N, DEA; O, DIA; P, terbuthylazine; Q, CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to 0.1% DMSO (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with bifenthrin (BF), cypermethrin (CP), permethrin (PM), o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor (MXC), alachlor (ALA), acephate (AP), dimethoate (DIM), zineb, DEA, DIA, and terbuthylazine (TBA). Lamin A/C was present as an internal control for the total amount of nuclear protein. R, Grayscale quantization of LaminA/C-normalized nuclear MR from at least 3 independent experiments. *p < .05, **p < .01, compared with positive control (Ald) (= 100%). Figure 5. View largeDownload slide Effects of tested pesticides on nuclear translocation of MR. CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to A, vehicle control (0.1% DMSO); B, positive control (10−9.5 M aldosterone), or the combination of 10−9.5 M aldosterone with C, bifenthrin; D, cypermethrin; E, permethrin; F, o,p′-DDT; G, p,p′-DDE; H, p,p′-DDT; I, methoxychlor; J, alachlor; K, acephate; L, dimethoate; M, zineb; N, DEA; O, DIA; P, terbuthylazine; Q, CHO-K1 cells were transiently transfected with EGFP-C1-hMR plasmid and exposed to 0.1% DMSO (vehicle control), 10−9.5 M aldosterone (Ald, positive control), or the combination of 10−9.5 M aldosterone with bifenthrin (BF), cypermethrin (CP), permethrin (PM), o,p′-DDT, p,p′-DDE, p,p′-DDT, methoxychlor (MXC), alachlor (ALA), acephate (AP), dimethoate (DIM), zineb, DEA, DIA, and terbuthylazine (TBA). Lamin A/C was present as an internal control for the total amount of nuclear protein. R, Grayscale quantization of LaminA/C-normalized nuclear MR from at least 3 independent experiments. *p < .05, **p < .01, compared with positive control (Ald) (= 100%). DISCUSSION Although the estrogenic and/or anti-androgenic potency of a number of pesticides has been observed, little was known about the disrupting effects of pesticides via nonsexual steroid hormone receptors, especially MR. In this study, 43 pesticides from both legacy and current-use types were evaluated for MR-mediated mineralocorticoid activity. Similar to what was found in our previous GR study (Zhang et al., 2016), none of the test pesticides exhibited mineralocorticoidic activity, but 16 of 43 chemicals showed MR antagonistic effects. To the best of our knowledge, this is the first study to recognize these 16 pesticides as MR antagonists. We found that 4 DDT analogs (o,p′-DDT, p,p′-DDT, p,p′-DDE, and methoxychlor) and 2 pyrethroids (bifenthrin and cypermethrin) exhibited both GR and MR antagonistic activities (Zhang et al., 2016). The overlap of antagonism of these pesticides toward GR and MR could be explained by the high homology in structure and function between GR and MR. In fact, the native MR ligand aldosterone is able to bind to the GR but the affinity between aldosterone and GR is several orders of magnitude lower than MR, whereas native GR ligand cortisol can also bind to MR (Fuller et al., 2000). However, the identity of amino acid sequences between the ligand binding domains of GR and MR was only 57%, which results in the difference in protein spatial structure and subsequently the diversity of xenobiotics with antagonistic effects (Arriza et al., 1987). For example, in our studies, atrazine showed antagonistic potency for GR rather than MR, whereas 2 metabolites of atrazine, DEA and DIA, exhibited MR antagonistic effects. It is likely that chemical structures had an important impact on MR antagonistic activity. For example, pyrethroids are all esters and are hydrolyzed into the acid and alcohol metabolites (Kaneko, 2011). In our study, most of the parent compounds of pyrethroids rather than their common acid and alcohol metabolites exhibited MR antagonistic activity, suggesting that the structure of an ester in this group of chemicals is required for their MR antagonistic activity. In addition, among the organochlorine group, all 4 DDT analogues tested in this study showed MR antagonistic activity, but none of the 4 benzene hexachloride isoforms. However, among the different groups, these 16 chemicals have a variety of different structures and MR exhibits promiscuity to these potential environmental ligands. Actually, other members of the family of steroid receptors, such as ER, AR, and GR, also can be activated or antagonized by a large set of synthetic drugs and pollutants in different chemical classes with highly variant structures (Kojima et al., 2004, 2009, 2013; Takeuchi et al., 2005, 2011; Zhang et al., 2016). The mechanism(s) of the promiscuous sensitivity of steroid receptors to exogenous substances remains unclear and is worth to further investigate. In this study, 16 compounds were considered as relative weak MR antagonists with the IC20 ranging from 10−7 M to 10−6 M (approximately equivalent to 44.5–419 ppb), which likely exceeds the realistic residual levels of these pesticides in the environment. However, a significant MR antagonistic effect was observed when these 16 chemicals were combined at an individual concentration as low as 10−9 M (approximately equal to 0.17–0.44 ppb). Consistently, a mixture of candidate pesticides at individual concentrations of 10−8 M produced combined MR antagonist effects on the expression of mineralocorticoid-responsive gene Alk in human VSMCs. The cumulative anti-mineralocorticoidic effects of a mixture of pesticides at low concentrations were also observed in the MR-mediated suppression of HCC proliferation. Noticeably, the realistic environmental levels of some chemicals among these candidate MR antagonists approach or exceed their corresponding concentrations in the mixture exposure. For instance, as the most ubiquitous metabolite of DDT, p,pʹ-DDE was reported to mean level of 1.64–5.18 ppb in the population of developed countries (Carreno et al., 2007; Zubero et al., 2015), whereas the effective level of p,pʹ-DDE in the mixture of this study was 0.32 ppb. As the most widely used pyrethroids, permethrin and cypermethrin were detected in human breast milk in South Africa with a mean concentration of 10.38 and 1.71 ppb, respectively (Bouwman et al., 2006), which is higher than their effective concentrations in the mixture (0.39 ppb for permethrin and 0.42 ppb for cypermethrin). Moreover, the dealkylated triazine metabolites (DEA, DIA, and 2-chlorodiminoatrazine) were detected in urine samples from pregnant women in France with a mean values approximately 1.29–1.66 ppb (Chevrier et al., 2014), which is higher than the total effective levels of DEA and DIA (0.36 ppb) observed in the mixture exposure group. Furthermore, the fixed-ratio mixture predictions showed that the mixture effects of 16 compounds are quietly predictable by CA model, suggesting that a mixture of widely used pesticides at environmentally relevant levels could act additively as MR antagonists by competitive antagonism of mineralocorticoids binding to the ligand-binding domain of the receptor. In this study, we further demonstrated the mechanism by which 14 of 16 candidate pesticides inhibited MR-dependent transcription appeared to involve modifications in mineralocorticoid-induced nuclear translocation. Upon ligand binding, the alteration in conformation of MR results in exposure of receptor nuclear localization signaling motif and translocation from cytoplasm to nucleus (Nakatani et al., 2013). Like other steroids, the native MR ligand aldosterone is a small molecular lipophilic chemical (Sutanto and Dekloet, 1991). Most of the MR-antagonistic pesticides identified in this study are also lipophilic chemicals. It is highly possible that these 14 pesticides are able to enter the ligand binding pocket of MR, competitively binding to the receptor but unable to induce the correct conformation change and remained in the cytoplasm. However, other 2 pesticides, fenvalerate and fipronil, are unable to prevent the translocation of MR. Similar effects on other members of the nonsexual steroid receptor were observed in the As-inhibited GR activation that was independent of steroid-induced GR nuclear translocation (Kaltreider et al., 2001). It suggests a different mechanism that may act by disrupting downstream progress such as DNA binding or cofactor recruiting receptor function after nuclear translocation. So far, little was known about the adverse effects of a continuous MR function impairment by environmental contaminants. In this study, 16 pesticides and/or their metabolites were reported first time to exhibit anti-mineralocorticoidic activity and 14 of them interrupted MR transactivation through disrupting nuclear translocation process. The additively combined effects of multiple pesticides were observed in a quite predictable manner, indicating that a mixture composed of a larger number of MR antagonists would have produced additive adverse health effects at correspondingly lower concentrations, approaching realistic environmental levels. Indeed, the compounds tested in this study were only a small portion of the great number of xenobiotics. Other man-made chemicals should be recruited for MR antagonist/agonist screening, and it will provide important knowledge in evaluating the overall role of environmental MR ligands-induced health effects. SUPPLEMENTARY DATA Supplementary data are available at Toxicological Sciences online. FUNDING National Natural Science Foundation of China (21377113 and 21320102007); Zhejiang Provincial Natural Science Foundation of China (LR15B070001). REFERENCES Arriza J. L., Weinberger C., Cerelli G., Glaser T. M., Handelin B. L., Housman D. E., Evans R. M. ( 1987). Cloning of human mineralocorticoid receptor complementary-DNA—Structural and functional kinship with the glucocorticoid receptor. Science  237, 268– 275. Google Scholar CrossRef Search ADS PubMed  Barraza-Vazquez A., Borja-Aburto V. H., Bassol-Mayagoitia S., Monrroy A., Recio-Vega R. ( 2008). Dichlorodiphenyldichloroethylene concentrations in umbilical cord of newborns and determinant maternal factors. J. Appl. Toxicol . 28, 27– 34. Google Scholar CrossRef Search ADS PubMed  Berger S., Bleich M., Schmid W., Cole T. J., Peters J., Watanabe H., Kriz W., Warth R., Greger R., Schutz G. ( 1998). Mineralocorticoid receptor knockout mice: Pathophysiology of Na+ metabolism. Proc. Natl. Acad. Sci. U.S.A . 95, 9424– 9429. Google Scholar CrossRef Search ADS PubMed  Bodwell J. E., Gosse J. A., Nomikos A. P., Hamilton J. W. ( 2006). Arsenic disruption of steroid receptor gene activation: Complex dose–response effects are shared by several steroid receptors. Chem. Res. Toxicol . 19, 1619– 1629. Google Scholar CrossRef Search ADS PubMed  Bouwman H., Sereda B., Meinhardt H. M. ( 2006). Simultaneous presence of DDT and pyrethroid residues in human breast milk from a malaria endemic area in South Africa. Environ. Pollut . 144, 902– 917. http://dx.doi.org/10.1016/j.envpol.2006.02.002 Google Scholar CrossRef Search ADS PubMed  Carreno J., Rivas A., Granada A., Lopez-Espinosa M. J., Mariscal M., Olea N., Olea-Serrano F. ( 2007). Exposure of young men to organochlorine pesticides in Southern Spain. Environ. Res . 103, 55– 61. Google Scholar CrossRef Search ADS PubMed  Chevrier C., Serrano T., Lecerf R., Limon G., Petit C., Monfort C., Hubert-Moy L., Durand G., Cordier S. ( 2014). Environmental determinants of the urinary concentrations of herbicides during pregnancy: The PELAGIE mother-child cohort (France). Environ. Int . 63, 11– 18. Google Scholar CrossRef Search ADS PubMed  Douma B. R. K., Korte S. M., Buwalda B., la Fleur S. E., Bohus B., Luiten P. G. M. ( 1998) Repeated blockade of mineralocorticoid receptors, but not of glucocorticoid receptors impairs food rewarded spatial learning. Psychoneuroendocrinology  23, 33– 44. Google Scholar CrossRef Search ADS PubMed  Faust M., Altenburger R., Backhaus T., Blanck H., Boedeker W., Gramatica P., Hamer V., Scholze M., Vighi M., Grimme L. H. ( 2001). Predicting the joint algal toxicity of multi-components—Triazine mixtures at low-effect concentrations of individual toxicants. Aquat. Toxicol . 56, 13– 32. Google Scholar CrossRef Search ADS PubMed  Fejes-Toth G., Pearce D., Naray-Fejes-Toth A. ( 1998). Subcellular localization of mineralocorticoid receptors in living cells: Effects of receptor agonists and antagonists. Proc. Natl. Acad. Sci. U.S.A . 95, 2973– 2978. Google Scholar CrossRef Search ADS PubMed  Fenner K., Canonica S., Wackett L. P., Elsner M. ( 2013). Evaluating pesticide degradation in the environment: Blind spots and emerging opportunities. Science  341, 752– 758. http://dx.doi.org/10.1126/science.1236281 Google Scholar CrossRef Search ADS PubMed  Fuller P. J., Lim-Tio S. S., Brennan F. E. ( 2000). Specificity in mineralocorticoid versus glucocorticoid action. Kidney Int . 57, 1256– 1264. http://dx.doi.org/10.1046/j.1523-1755.2000.00959.x Google Scholar CrossRef Search ADS PubMed  Fuller P. J., Rogerson F. M. ( 2002). Pseudohypoaldosteronism: Kidney, lungs and colon. Clin. Endocrinol . 56, 571– 572. http://dx.doi.org/10.1046/j.1365-2265.2002.01512.x Google Scholar CrossRef Search ADS   Gaeggeler H. P., Gonzalez-Rodriguez E., Jaeger N. F., Loffing-Cueni D., Norregaard R., Loffing J., Horisberger J. D., Rossier B. C. ( 2005) Mineralocorticoid versus glucocorticoid receptor occupancy mediating aldosterone-stimulated sodium transport in a novel renal cell line. J. Am. Soc. Nephrol . 16, 878– 891. Google Scholar CrossRef Search ADS PubMed  Geller D. S., Rodriguez-Soriano J., Boado A. V., Schifter S., Bayer M., Chang S. S., Lifton R. P. ( 1998). Mutations in the mineralocorticoid receptor gene cause autosomal dominant pseudohypoaldosteronism type I. Nat. Genet . 19, 279– 281. Google Scholar CrossRef Search ADS PubMed  Grossmann C., Benesic A., Krug A. W., Freudinger R., Mildenberger S., Gassner B., Gekle M. ( 2005). Human mineralocorticoid receptor expression renders cells responsive for nongenotropic aldosterone actions. Mol. Endocrinol . 19, 1697– 1710. Google Scholar CrossRef Search ADS PubMed  Grossmann C., Husse B., Mildenberger S., Schreier B., Schuman K., Gekle M. ( 2010). Colocalization of mineralocorticoid and EGF receptor at the plasma membrane. Mol. Cell. Res . 1803, 584– 590. Jaffe I. Z., Mendelsohn M. E. ( 2005). Angiotensin II and aldosterone regulate gene transcription via functional mineralocortocoid receptors in human coronary artery smooth muscle cells. Circ. Res . 96, 643– 650. http://dx.doi.org/10.1161/01.RES.0000159937.05502.d1 Google Scholar CrossRef Search ADS PubMed  Jeong Y., Xie Y., Xiao G. H., Behrens C., Girard L., Wistuba I. I., Minna J. D., Mangelsdorf D. J. ( 2010). Nuclear receptor expression defines a set of prognostic biomarkers for lung cancer. PLoS Med . 7, e1000378. Kaltreider R. C., Davis A. M., Lariviere J. P., Hamilton J. W. ( 2001). Arsenic alters the function of the glucocorticoid receptor as a transcription factor. Environ. Health Perspect . 109, 245– 251. Google Scholar CrossRef Search ADS PubMed  Kaneko H. ( 2011). Pyrethroids: Mammalian metabolism and toxicity. J. Agric. Food Chem . 59, 2786– 2791. http://dx.doi.org/10.1021/jf102567z Google Scholar CrossRef Search ADS PubMed  Kojima H., Katsura E., Takeuchi S., Niiyama K., Kobayashi K. ( 2004). Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells. Environ. Health Perspect . 112, 524– 531. Google Scholar CrossRef Search ADS PubMed  Kojima H., Takeuchi S., Itoh T., Iida M., Kobayashi S., Yoshida T. ( 2013). In vitro endocrine disruption potential of organophosphate flame retardants via human nuclear receptors. Toxicology  314, 76– 83. Google Scholar CrossRef Search ADS PubMed  Kojima H., Takeuchi S., Uramaru N., Sugihara K., Yoshida T., Kitamura S. ( 2009). Nuclear hormone receptor activity of polybrominated diphenyl ethers and their hydroxylated and methoxylated metabolites in transactivation assays using Chinese hamster ovary cells. Environ. Health Perspect . 117, 1210– 1218. Google Scholar CrossRef Search ADS PubMed  Liu J., Zhao M. R., Zhuang S. L., Yang Y., Yang Y., Liu W. P. ( 2012). Low concentrations of o,p′-DDT inhibit gene expression and prostaglandin synthesis by estrogen receptor-independent mechanism in rat ovarian cells. PLoS One  7, e49916. Molina-Molina J. M., Hillenweck A., Jouanin I., Zalko D., Cravedi J. P., Fernandez M. F., Pillon A., Nicolas J. C., Olea N., Balaguer P. ( 2006). Steroid receptor profiling of vinclozolin and its primary metabolites. Toxicol. Appl. Pharmacol . 216, 44– 54. Google Scholar CrossRef Search ADS PubMed  Nakatani Y., Amano T., Takeda H. ( 2013). Corticosterone suppresses the proliferation of RAW264.7 macrophage cells via glucocorticoid, but not mineralocorticoid, receptor. Biol. Pharm. Bull . 36, 592– 601. http://dx.doi.org/10.1248/bpb.b12-00968 Google Scholar CrossRef Search ADS PubMed  Nicolaides N. C., Roberts M. L., Kino T., Braatvedt G., Hurt D.E., Katsantoni E., Sertedaki A., Chrousos G. P., Charmandari E. ( 2014). A novel point mutation of the human glucocorticoid receptor gene causes primary generalized glucocorticoid resistance through impaired interaction with the LXXLL motif of the p160 coactivators: Dissociation of the transactivating and transreppressive activities. J. Clin. Endocrinol. Metab . 99, E902– E907. Google Scholar CrossRef Search ADS PubMed  Nie H. Z., Li J., Yang X. M., Cao Q. Z., Feng M. X., Xue F., Wei L., Qin W. X., Gu J. R., Xia Q., et al.   ( 2015). Mineralocorticoid receptor suppresses cancer progression and the Warburg effect by modulating the miR-338-3p-PKLR axis in hepatocellular carcinoma. Hepatology  62, 1145– 1159. Google Scholar CrossRef Search ADS PubMed  Niu J. J., Lin Y., Guo Z. N., Niu M., Su C. H. ( 2016). The epidemiological investigation on the risk factors of hepatocellular carcinoma: A case-control study in Southeast China. Medicine  95, e2758. Orton F., Rosivatz E., Scholze M., Kortenkamp A. ( 2012). Competitive androgen receptor antagonism as a factor determining the predictability of cumulative antiandrogenic effects of widely used pesticides. Environ. Health Perspect . 120, 1578– 1584. Google Scholar CrossRef Search ADS PubMed  Ouvrard-Pascaud A., Puttini S., Saint-Marie Y., Athman R., Fontaine V., Cluzeaud F., Farman N., Rafestin-Oblin M. E., Blot-Chabaud M., Jaisser F. ( 2004). Conditional gene expression in renal collecting duct epithelial cells: Use of the inducible Cre-lox system. Am. J. Physiol. Renal Physiol . 286, F180– F187. Google Scholar CrossRef Search ADS PubMed  Pippal J. B., Fuller P. J. ( 2008). Structure-function relationships in the mineralocorticoid receptor. J. Mol. Endocrinol . 41, 405– 413. http://dx.doi.org/10.1677/JME-08-0093 Google Scholar CrossRef Search ADS PubMed  Rehan M., Ahmad E., Sheikh I. A., Abuzenadah A. M., Damanhouri G. A., Bajouh O. S., AlBasri S. F., Assiri M. M., Beg M. A. ( 2015). Androgen and progesterone receptors are targets for bisphenol A (BPA), 4-methyl-2,4-bis-(p-hydroxyphenyl)pent-1-ene-A potent metabolite of BPA, and 4-tert-octylphenol: A computational insight. PLoS One  10, e0138438. Rogerson F. M., Fuller P. J. ( 2000). Mineralocorticoid action. Steroids  65, 61– 73. http://dx.doi.org/10.1016/S0039-128X(99)00087-2 Google Scholar CrossRef Search ADS PubMed  Rossi L., Barbieri O., Sanguineti M., Cabral J. R. P., Bruzzi P., Santi L. ( 1983). Carcinogenicity study with technical-grade dichlorodiphenyltrichloroethane and 1,1-dichloro-2,2-bis(para-chlorophenyl)ethylene in hamsters. Cancer Res . 43, 776– 781. Google Scholar PubMed  Sekizawa N., Yoshimoto T., Hayakawa E., Suzuki N., Sugiyama T., Hirata Y. ( 2011). Transcriptome analysis of aldosterone-regulated genes in human vascular endothelial cell lines stably expressing mineralocorticoid receptor. Mol. Cell Endocrinol . 341, 78– 88. Google Scholar CrossRef Search ADS PubMed  Sifaki-Pistolla D., Karageorgos S. A., Koulentaki M., Samonakis D., Stratakou S., Digenakis E., Kouroumalis E. ( 2016). Geoepidemiology of hepatocellular carcinoma in the island of Crete, Greece. A possible role of pesticides. Liver Int . 36, 588– 594. Google Scholar CrossRef Search ADS PubMed  Sutanto W., Dekloet E. R. ( 1991). Mineralocorticoid receptor ligands—Biochemical, pharmacological, and clinical aspects. Med. Res. Rev . 11, 617– 639. Google Scholar CrossRef Search ADS PubMed  Takeuchi S., Iida M., Kobayashi S., Jin K., Matsuda T., Kojima H. ( 2005). Differential effects of phthalate esters on transcriptional activities via human estrogen receptors alpha and beta, and androgen receptor. Toxicology  210, 223– 233. Google Scholar CrossRef Search ADS PubMed  Takeuchi S., Shiraishi F., Kitamura S., Kuroki H., Jin K., Kojima H. ( 2011). Characterization of steroid hormone receptor activities in 100 hydroxylated polychlorinated biphenyls, including congeners identified in humans. Toxicology  289, 112– 121. Google Scholar CrossRef Search ADS PubMed  Tiberio L., Nascimbeni R., Villanacci V., Casella C., Fra A., Vezzoli V., Furlan L., Meyer G., Parrinello G., Baroni M. D., et al.   ( 2013). The decrease of mineralocorticoid receptor drives angiogenic pathways in colorectal cancer. PLoS One  8, e59410. VoPham T., Brooks M. M., Yuan J. M., Talbott E. O., Ruddell D., Hart J. E., Chang C. C. H., Weissfeld J. L. ( 2015). Pesticide exposure and hepatocellular carcinoma risk: A case-control study using a geographic information system (GIS) to link SEER-Medicare and California pesticide data. Environ. Res . 143, 68– 82. Google Scholar CrossRef Search ADS PubMed  Xing L., Sun J., Liu H., Yu H. ( 2012). Combined toxicity of three chlorophenols 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol to Daphnia magna. J. Environ. Monit . 14, 1677. http://dx.doi.org/10.1039/c2em30185g Google Scholar CrossRef Search ADS PubMed  Yau J. L. W., Noble J., Seckl J. R. ( 1999). Continuous blockade of brain mineralocorticoid receptors impairs spatial learning in rats. Neurosci. Lett . 277, 45– 48. http://dx.doi.org/10.1016/S0304-3940(99)00858-7 Google Scholar CrossRef Search ADS PubMed  Yu L. Y., Qin L. F., Zhang H. F., He Y., Chen H., Pober J. S., Tellides G., Min W. ( 2011). AIP1 prevents graft arteriosclerosis by inhibiting interferon-gamma-dependent smooth muscle cell proliferation and intimal expansion. Circ. Res . 109, 418– 427. Google Scholar CrossRef Search ADS PubMed  Zennaro M. C., Keightley M. C., Kotelevtsev Y., Conway G. S., Soubrier F., Fuller P. J. ( 1995). Human mineralocorticoid receptor genomic structure and identification of expressed isoforms. J. Biol. Chem . 270, 21016– 21020. Google Scholar CrossRef Search ADS PubMed  Zhang J. Y., Zhang J., Liu R., Gan J., Liu J., Liu W. P. ( 2016). Endocrine-disrupting effects of pesticides through interference with human glucocorticoid receptor. Environ. Sci. Technol . 50, 435– 443. Google Scholar CrossRef Search ADS PubMed  Zhang Q., Ye J. J., Chen J. Y., Xu H. J., Wang C., Zhao M. R. ( 2014). Risk assessment of polychlorinated biphenyls and heavy metals in soils of an abandoned e-waste site in China. Environ. Pollut . 185, 258– 265. Google Scholar CrossRef Search ADS PubMed  Zubero M. B., Aurrekoetxea J. J., Murcia M., Ibarluzea J. M., Goni F., Jimenez C., Ballester F. ( 2015). Time trends in serum organochlorine pesticides and polychlorinated biphenyls in the general population of Biscay, Spain. Arch. Environ. Contam. Toxicol . 68, 476– 488. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

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Toxicological SciencesOxford University Press

Published: Mar 1, 2018

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