Disruption of the Hormonal Network and the Enantioselectivity of

Jun 17, 2014 - Endocrine-disrupting chemicals (EDCs) can interfere with normal hormone signaling to increase health risks to the maternal–fetal syst...
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Disruption of the Hormonal Network and the Enantioselectivity of Bifenthrin in Trophoblast: Maternal−Fetal Health Risk of Chiral Pesticides Meirong Zhao,†,‡,∥ Ying Zhang,§,⊥,∥ Shulin Zhuang,⊥ Quan Zhang,† Chengsheng Lu,‡ and Weiping Liu*,⊥ †

College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, China Department of Environmental Health, Harvard School of Public Health, Landmark Center West, Boston, Massachusetts 02115, United States § School of Ecological and Environmental Science, East China Normal University, Shanghai 200241, China ⊥ College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China ‡

S Supporting Information *

ABSTRACT: Endocrine-disrupting chemicals (EDCs) can interfere with normal hormone signaling to increase health risks to the maternal−fetal system, yet few studies have been conducted on the currently used chiral EDCs. This work tested the hypothesis that pyrethroids could enantioselectively interfere with trophoblast cells. Cell viability, hormone secretion, and steroidogenesis gene expression of a widely used pyrethroid, bifenthrin (BF), were evaluated in vitro, and the interactions of BF enantiomers with estrogen receptor (ER) were predicted. At low or noncytotoxic concentrations, both progesterone and human chorionic gonadotropin secretion were induced. The expression levels of progesterone receptor and human leukocyte antigen G genes were significantly stimulated. The key regulators of the hormonal cascade, GnRH type-I and its receptor, were both upregulated. The expression levels of selected steroidogenic genes were also significantly altered. Moreover, a consistent enantioselective interference of hormone signaling was observed, and S-BF had greater effects than R-BF. Using molecular docking, the enantioselective endocrine disruption of BF was predicted to be partially due to enantiospecific ER binding affinity. Thus, BF could act through ER to enantioselectively disturb the hormonal network in trophoblast cells. These converging results suggest that the currently used chiral pesticides are of significant concern with respect to maternal−fetal health.

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widespread human exposures.5 Although the half-lives of pyrethroids in the body are short, pyrethroids have been detected in blood samples of pregnant women.6 In addition, 25- to 30-year-old females have a higher average daily intake than males.7 Although no data are currently available regarding the characteristics of residual pyrethroids in the female reproductive organs and placental tissues, a combined analysis of prenatal and neonatal matrices revealed a significant exposure to environmental pyrethroids.8 Collectively, the data imply that cumulative pyrethroid exposures are potentially hazardous to pregnant women and their fetuses. As a large family of chiral pesticides, pyrethroid enantiomers have nonsuperposable structures sharing identical physical− chemical properties, but have different physiological and biochemical properties.9 Due to the enantioselective interactions with chiral biomacromolecules, dramatic differences may appear

INTRODUCTION The networks of endocrine and nervous systems are essential for reproductive function via the framework of hormone signaling. However, endocrine-disrupting chemicals (EDCs) can act through different hormone nuclear receptors, including the estrogen receptor (ER), to disrupt the normal hormone networks of the endocrine and reproductive systems.1 Exposure to exogenous compounds during the critical prenatal period may result in abortion, adverse birth outcomes, and even subsequent developmental defects and adult complications.2 A group of EDCs, most of which are well-known persistent organic pollutants and restricted pesticides, have showed adverse reproductive effects in mammals and humans.3,4 Because of the rapidly increasing application and human exposure to currently used compounds, comprehensive risk assessments must be performed and serious public concerns must be raised to such chemicals regarding the potential adverse effects on both mothers and fetuses. Among the currently used EDCs, chiral pyrethroids are of particular concern. Because of their high efficacy against insects and relatively low mammalian toxicity, pyrethroids are increasingly used throughout the world, which results in © 2014 American Chemical Society

Received: Revised: Accepted: Published: 8109

October 3, 2013 June 15, 2014 June 17, 2014 June 17, 2014 dx.doi.org/10.1021/es501903b | Environ. Sci. Technol. 2014, 48, 8109−8116

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(for cell viability assay), 2 days (for ELISA assay), or 1 day (for qRT-PCR). Ethanol was used as the negative control, and all test solutions were prepared in ethanol with the final concentrations at 0.1% by volume. Assessment of Cell Viability. The cell viability of JEG-3 cells after exposure to 10−7 to 10−5 mol/L rac-BF or 5 × 10−6 mol/L BF enantiomers was measured using thiazolyl blue assay. The results were expressed as the relative viability. The details of the experiment are shown in the Supporting Information (SI). At least 4 independent replicates were performed for each treatment. Analysis of Hormones Secretion. Cells were cultured with 10−7 to 10−5 mol/L rac-BF or 10−6 mol/L BF enantiomers, and the culture supernatants were collected. To evaluate the influence of the pure antiestrogen, cells were also cotreated with 10−6 mol/L rac-BF and 10−9 mol/L ICI 182,780. The concentrations of progesterone and human chorionic gonadotropin (hCG) were measured by ELISA kits (BioSource International Inc., Camarillo, CA, USA). All assays were performed according to the manufacturer’s instructions. At least 3 independent replicates were performed for each treatment. Real-Time Quantitative PCR. JEG-3 cells were treated with 10−7 to 10−6 mol/L rac-BF or 5 × 10−7 mol/L BF enantiomers. ICI 182,780 (10−9 mol/L) was used as the antagonist. Total RNA was extracted and the expression levels of progesterone receptor (PR), human leukocyte antigen G (HLA-G), gonadotropin-releasing hormone type-I (GnRHI), gonadotropin-releasing hormone receptor type-I (GnRHRI), cytochrome P450c17 (CYP17), cytochrome P450c19 (CYP19), 3 beta-hydroxysteroid dehydrogenase (3β-HSD), and 17 betahydroxysteroid dehydrogenase (17β-HSD) were analyzed. Experiment details were shown in the SI. At least 3 independent replicates were performed for each treatment. Molecular Docking. To explore the binding modes of R-BF and S-BF to ER α and ER β, molegro virtual docker (MVD) 4.2 program was used.17 The atomic coordinates of the ER α and ER β ligand binding domain (LBD) were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/home/ home.do; PDB ID of ER α LBD: 1XPC, PDB ID of ER β LBD: 3OLS). Explicit hydrogen atoms were created and the heteroatoms such as waters, ions, and cofactors were removed. The binding pocket was defined as a sphere with a radius of 15 Å around the center of the bounding box spanning the receptors. Grid resolution was set to 0.30 Å. The heuristic search algorithm MolDock SE was used as the search algorithm and the number of runs was set to 10. Energetic evaluations were calculated using MOLDOCK score algorithm. The best pose with the highest docking score was finally selected. Statistical Analysis. The results were presented as mean ± standard error of mean (SEM) and tested for statistical significance by analysis of variance (ANOVA) followed by post hoc Dunnett’s test using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant when the p values were less than 0.05 or 0.01.

between the enantiomers. Studies so far show that enantioselectivity commonly occurs in the environmental fate, bioaccumulation, and toxicological effects of different chiral pesticides,10 but most data come from ecotoxicological research. Even though the role of enantioselectivity in human health risks is not yet fully understood, the knowledge gap is very important for the application of these chiral compounds. Previous studies have showed the enantioselective estrogenic potential of pyrethroids via the estrogen response pathway acting though ER.11 There is speculation as to whether prenatal exposure to such chiral chemicals could also cause enantioselective adverse effects in the maternal−fetal system. The placenta is the primary demarcation in the maternal− fetal unit, and performs crucial endocrine functions in response to hormonal signals.12 The complex activities of the placenta are sensitive to direct toxicity by chemicals and indirect effects through endocrine disrupters, as shown by the high occurrence of abortion and pregnancy diseases.13,14 Trophoblast cells are the only cells of the fetal−placental unit in direct and extensive contact with the maternal tissue, which can provide a vital barrier to prevent chemicals from entering the growing embryo.15 However, various organic compounds such as organochlorine pesticides and their metabolites have been shown to pass through the barrier and reach the placenta during pregnancy.16 Moreover, the presence of ER α and ER β in trophoblast cells allow the placenta to be a potential target of EDCs. Bifenthrin (BF) is among the most widely used pyrethroids in orchards, nurseries, and homes, and is widespread in diverse environmental media.5 We selected BF as a representative of pyrethroids to test the hypothesis that estrogen-like pyrethroids could enantioselectively interfere with the maternal−fetal system. JEG-3 choriocarcinoma cells, which retain characteristics of normal pregnancy trophoblast cells and express both ERs, were used as an in vitro model. The possible actions of BF on cell viability, hormone secretion, and steroidogenesis gene expression levels were evaluated, and the interaction modes of BF enantiomers with ER were further predicted by molecular docking. The approaches developed in the present study will be helpful for better assessing the health risks of the currently used chiral EDCs exposure to pregnant women and their fetuses.

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MATERIALS AND METHODS Chemicals and Preparation of the Enantiomers. An analytical standard of racemic BF (rac-BF; 2-methylbiphenyl-3ylmethyl-(Z) -(1RS)-cis-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)2,2-dimethylcyclopropane carboxylate, 99.5%) was purchased from Sigma Chemical (St. Louis, MO, USA). The enantiomers of BF were separated as previously described and redissolved in ethanol for subsequent experiments.10 The ER antagonist ICI 182,780 (> 99%) was obtained from Tocris (Avonmouth, Bristol, UK). Other chemicals or solvents used were of cell culture, HPLC, or analytical grade. Cell Culture and Treatments. The JEG-3 cell line (cell bank of the Chinese Academy of Science, Shanghai, China) was cultured in Dulbecco’s Modified Eagle Medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% of charcoal-dextran treated fetal bovine serum (CDFBS; HyClone, Logan, UT, USA) at 37 °C in a 5% CO2 humidified incubator. The culture media was replaced with the experimental medium (phenol-red-free DMEM containing 5% CDFBS) for 1 day before treatment to reduce the effect of serum and phenol. The cells were then treated with the dosing medium (the experimental medium along with test compounds) for 1−4 days

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RESULTS Induction of Cytotoxicity. The potential response of JEG-3 cells to BF was initially assessed by measuring cell viability. As shown in Figure 1A and B, rac-BF inhibited cell viability in both concentration- and time-dependent manners. The proliferation was significantly inhibited upon treatment with 10−6 to 10−5 mol/L BF (p < 0.01), with the most significant inhibition of 36.3% at 10−5 mol/L. Significant cytotoxicity was induced after a 8110

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Figure 1. Effects of bifenthrin (BF) on the viability of JEG-3 cells. JEG-3 cells were exposed to 10−7 to 10−5 mol/L rac-BF for 96 h (A) or 5 × 10−6 mol/L racemate or two enantiomers of BF for 24 to 96 h (B) followed by the MTT assay. The results are presented as the mean ± SEM. ** Indicates p < 0.01 compared to the negative control. Different letters indicate a significant difference (p < 0.05) between individual enantiomers or between an enantiomer and racemate, while the same letter indicates no significant difference.

Figure 2. Stimulation of hormone secretion by bifenthrin (BF). JEG-3 cells were treated with 10−7 to 10−5 mol/L rac-BF or cotreated with 10−6 mol/L rac-BF and 10−9 mol/L ICI 182,780 (A), as well as 5 × 10−6 mol/L racemate and enantiomers of BF (B). The secretions of progesterone and hCG were measured by ELISA. The results are presented as the mean ± SEM. ** Indicates p < 0.01. Different letters indicate a significant difference (p < 0.05) between different treatments, while the same letter indicates no significant difference.

3-day exposure to R-BF and S-BF (p < 0.01) (Figure 1B). A consistent enantioselectivity was shown in inhibitory effects on JEG-3 cell viability induced by the two enantiomers. Compared to the 4-day control, S-BF inhibited cell growth to 53.8% and 69.8% after 3 and 4 days of exposure, while R-BF decreased cell viability to 62.5% and 85.2%, respectively. The results suggested that S-BF could induce stronger cytotoxic effects than R-BF (p < 0.05). Based on these results, the low or nontoxic concentrations of 10−7 to 10−6 mol/L were chosen for further investigations. Stimulation of Hormones Secretion. Progesterone is essential for the establishment and maintenance of pregnancy,18 and the promotion of progesterone production requires hCG. Both progesterone and hCG could be induced by rac-BF in

dose-dependent relationships (Figure 2A). Progesterone secretion increased significantly upon treatment with 5 × 10−7 to 10−5 mol/L rac-BF (p < 0.01) with the highest secretion level occurring at 5 × 10−6 mol/L. The stimulation of progesterone production by rac-BF could not be blocked by ICI 182,780. The secretion of hCG was continuously induced from 254.5 to 472.3 mIU/mL upon treatment with rac-BF concentrations ranging from 10−7 to 10−5 mol/L (p < 0.01) compared to the control at 158.4 mIU/mL. The secretion was blocked by 10−9 mol/L ICI 182,780 (p < 0.05). The two enantiomers also significantly stimulated progesterone and hCG secretion (p < 0.01) (Figure 2B). Cells treated with S-BF produced 12.5 ng/mL progesterone while cells exposure to R-BF produced 8111

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function.20 In the present study, the first identified form, GnRHI, and its receptor GnRHRI were analyzed. As shown in Figure 3C, the GnRHI gene expression level was upregulated by 2.27 and 2.59 times when treated with 5 × 10−7 and 10−6 mol/L rac-BF (p < 0.01), while the GnRHRI expression level was induced by 2.82 and 5.65 times, respectively (p < 0.01) (Figure 3D). Following cotreatment with ICI 182,780, upregulation was significantly decreased. There was no difference in GnRHI compared to the negative control, and the induction of GnRHRI was reduced to 3.46-times (p < 0.01). For the two enantiomers, S-BF significantly altered the expression level, with 4.24 times for GnRHI and 6.54 times for GnRHRI (p < 0.01), while R-BF at the same concentration had no effect (Figure 4C and D). The inductions of GnRHI and GnRHRI genes by S-BF were approximately 2.58 and 4.24 times greater than those by R-BF, respectively. Alteration of Steroidogenic Genes Expression. CYPs and HSDs are the main steroidogenic enzymes involved in progesterone biosynthesis and metabolism. At all tested concentrations, rac-BF significantly altered the expression level of CYP17 with the maximal induction by 2.69 times occurring at the concentration of 10−6 mol/L (p < 0.01) (Figure 3E). The mRNA levels of CYP19 were significantly upregulated to 2.12 and 3.44 times by 5 × 10−7 and 10−6 mol/L rac-BF, respectively (p < 0.01) (Figure 3F). Moreover, the 3β-HSD expression level was upregulated to 3.23 and 5.17 times by the two higher concentrations of rac-BF (p < 0.01) (Figure 3G), while the mRNA level of 17β-HSD was significantly downregulated to 40.6 and 19.8% (p < 0.01) (Figure 3H). In addition, S-BF had no effect on the CYP17 expression level, while R-BF could significantly induce the CYP17 mRNA level to 3.34 times (p < 0.01) (Figure 4E). The mRNA level of CYP19 was upregulated to 2.73 times by R-BF and 4.91 times by S-BF (p < 0.01) (Figure 4F). Furthermore, S-BF could significantly alter the mRNA expression of 3β-HSD and 17β-HSD (p < 0.01), with the maximal upregulation to 7.83 times for 3β-HSD and the maximal downregulation to 17.2% for 17β-HSD (Figure 4G and H). Overall, the steroidogenic gene expression results suggested a consistent enantioselectivity with S-BF showing a greater effect than R-BF. Interaction between BF and ER. Molecular docking was used to gain insight into the interactions of the two BF enantiomers with ER α and ER β. Even though the expression of ER in JEG-3 cells is not high, our results showed that the endocrine disruption of BF in trophoblast mediated partly by ER. As shown in Figure 5A, both of the two enantiomers fit into the ER α LBD binding pocket but showed different orientations. R-BF could not form hydrogen bonds with ER α LBD residues, comparatively, while S-BF forms hydrogen bond with residue Thr347 of ER α LBD. To quantitatively evaluate the binding affinity of the two isomers, the interactions were scored using MOLDOCK score algorithm. The obtained binding scores for R-BF and S-BF with ER α LBD were −100.84 and −116.53 kJ/mol, respectively, indicating higher binding affinity for S-BF. For the interactions with ER β LBD, the two enantiomers also showed distinct binding modes (Figure 5B). R-BF formed two hydrogen bonds with residue Arg346 of the ER β LBD, whereas S-BF formed one hydrogen bond with residue Leu343 in addition to two hydrogen bonds formed with residue Arg346. The binding scores of R-BF and S-BF were −108.88 and −117.07 kJ/mol, respectively. The binding score analysis indicated that the binding affinity of S-BF was comparatively stronger than that of R-BF. Molecular docking

9.2 ng/mL progesterone. The production of hCG was 1.7 times greater after S-BF treatment compared to R-BF treatment. The two BF enantiomers showed significant enantioselective effects on hormones secretion (p < 0.05). Regulation of PR and HLA-G Gene Expression. HLA-G plays a critical role in the maternal−fetal interface, and progesterone can regulate placental HLA-G gene expression via the binding to PR.19 Rac-BF significantly induced PR and HLA-G gene expression in a dose-dependent manner. The expression levels of PR and HLA-G were increased by 3.46 and 5.33 times, respectively, upon treatment with 10−6 mol/L rac-BF (p < 0.01). The upregulation of both genes could be partially blocked by 10−9 mol/L ICI 182,780 (p < 0.05) (Figure 3A and B). While 5 × 10−7 mol/L R-BF did not

Figure 3. Alteration of gene expression by rac-BF. PR (A), HLA-G (B), GnRHI (C), GnRHRI (D), CYP17 (E), CYP19 (F), 3β-HSD (G), and 17β-HSD (H) mRNA expression levels of JEG-3 cells exposed to 10−7 to 10−6 mol/L racemate BF and 10−9 mol/L ICI 182,780 were calculated by real-time quantitative PCR. The results are presented as the mean ± SEM. * Indicates p < 0.05 and ** indicates p < 0.01 compared to solvent control. Different letters indicate significant differences between different treatments (p < 0.05), while the same letter indicates no significant difference.

significantly stimulate PR or HLA-G gene expression, the mRNA expression levels of the two genes after S-BF exposure reached 3.09 and 8.50 times, respectively (p < 0.01) (Figure 3A and B). The results revealed a significant difference in the upregulation of PR and HLA-G gene expression between the two enantiomers. Induction of GnRHI and GnRHRI Gene Expression. Synthesis and release of the decapeptidic hormone GnRH is precisely regulated to achieve the appropriate reproductive 8112

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Figure 4. Gene expressions induced by the racemate and enantiomers of BF. JEG-3 cells were exposed to 5 × 10−7 mol/L rac-BF and two BF enantiomers, and PR (A), HLA-G (B), GnRHI (C), GnRHRI (D), CYP17 (E), CYP19 (F), 3β-HSD (G), and 17β-HSD (H) mRNA expression were induced. ** Indicates a significant difference from the solvent control (p < 0.01). Different letters indicate significant differences between different treatments of BF (p < 0.05).

Figure 5. Binding mode of BF enantiomers to ER α LBD (A) and ER β LBD (B). The ligands are represented in sticks, and proteins are presented in cartoons. 8113

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while the upregulation of GnRHRI was only partially blocked, showing a requirement for ER. Based on the data and the previous discovery of the estrogen response element (ERE) in the GnRHI gene,27 we hypothesize that GnRH may be induced by BF as an estrogen mimic through ERs directly binding to EREs located in GnRHI or as a subsequent feedback of hormone interactions. During embryo implantation, GnRH precisely regulates the production and release of hCG in trophoblast,28 which plays a regulatory role in the proliferation, differentiation, and apoptosis of different cell types involved in placental trophoblast formation.29 The secretion of hCG was continuously induced by BF at increasing concentrations, which may account for the inhibition of JEG-3 cell proliferation in response to BF treatment. Stimulation of hCG governs progesterone production in the corpus luteum at the initiation of pregnancy from implantation to gestation.30 Progesterone is a dominant steroid hormone involved in the regulation of gonadotropin and the GnRHI gene through a feedback mechanism.31 In contrast to previous studies showing that some pyrethroids inhibit progesterone production,32,33 BF exposure was found to significantly promote progesterone production and PR gene expression. The increase in progesterone secretion could not be blocked by ICI 182,780, demonstrating that progesterone production in response to BF exposure was ER independent. Moreover, the expression of PR was partially blocked by ICI 182,780 cotreatment, suggesting that PR expression may be related to ER-mediated activities. Conflicting results obtained in other studies34,35 may be due to the use of different pyrethroid chemicals and diverse cell lines. Progesterone has been shown to increase HLA-G expression in JEG-3 cells,19 and the expression level of HLA-G was also significantly induced by BF. This disruption of the gonadal hormone implies that adverse effects on immune tolerance in pregnancy may occur. Our findings, coupled with the previous literatures, suggest that hormones in trophoblast cells are sensitive to xenobiotic pollutants, and the reproductive effects may be mediated by ER and altered at all of the hormonal levels by pyrethroids. Coupled with the results of previous studies, these results suggest that disruption of the maternal hormone state by EDCs may not only influence the process of pregnancy, but also increase the risks for adverse health outcomes in the offspring and mother.36 The subsequent effects of maternal pyrethroid exposure and the underlying mechanisms should be further studied. Placental steroidogenesis requires the catalytic reactions of aromatase enzymes and involves the CYP family and HSDs. CYPs catalyze the synthesis of pregnenolone from cholesterol, which is subsequently converted to progesterone via 3β-HSD.37 A previous study has reported that the endocrine disruption induced by fenvalerate involves steroidogenesis signaling cascades and steroidogenic enzyme activity.38 Thus, BF-induced hormone alterations in trophoblast cells were proposed to be mediated through the modification of the steroidogenic enzymes. Given the pivotal functional roles of aromatase enzymes in progesterone production, the upregulation of CYPs as well as 3β-HSD in response to BF resulted in higher progesterone production in JEG-3 cells. As an essential enzyme that catalyzes the final step in the interconversion of estrone and estradiol,39 significant suppression of 17β-HSD mRNA levels by BF may decrease estrogen production. Thus, the negative feedback of estrogen to GnRH may be primarily due to BF rather than endogenous estrogen. Based on the results, BF may be an estrogen mimic that can bind to ER and other related receptors

studies suggested an enantiomer-specific affinity of BF to ER, which might partially contribute to the enantioselective disruption of the hormonal network in JEG-3 cells by BF.

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DISCUSSION Health risk assessments of the maternal−fetal unit are critically important for both mothers and fetuses. There are growing concerns regarding the possible health threats posed by EDCs, yet the potential enantioselective maternal−fetal health risks of the currently used chiral EDCs are not often considered. As one of the most widely used pesticides, pyrethroids are ubiquitous in the environment and therefore pose a great risk for human exposures. It has been reported that the bioaccumulation of pyrethroids in human breast milk samples has reached up to 1200 ng/g lw.21 A study of areas without pyrethroid use for malaria control showed that BF was the most abundant in Brazilian breast milk samples with concentrations up to 7.48 ng/g lw.22 At low or noncytotoxic concentrations, results of the present study show that BF can act through ER to disturb the balance of hormone signaling in trophoblast cells. The data further indicated that the two enantiomers induce enantiospecific hormone disruption, with significantly greater effects induced by S-BF partially due to its comparatively stronger binding affinity to ER. To ensure successful reproduction, a number of hormones are involved and strictly regulated during pregnancy. However, there is less concern regarding the pesticide interference with hormonal functions such as hormone synthesis and release, hormone receptor recognition and binding, since many studies primarily focus on the disruption of the estrogen or androgen receptor.23 Similar to the hypothalamic-pituitary−gonadal axis (HPG axis), which integrates the information from the endocrine, nervous, and reproductive systems, the placenta retains the common abilities to produce brain, pituitary, and gonadal hormones. Thus, the intraplacental mechanism is comparable to the regulation of the HPG axis in many aspects.12 Given that hormones produced by the HPG axis such as GnRH, estrogen, and progesterone can respond to chemical signals, this axis may be a useful indicator for the interaction with environmental toxicants, particularly EDCs.24 Estrogenic pyrethroids would most likely interfere with placental endocrine functions. According to the present study, endocrine disruption via pyrethroids is complex and may involve all three levels of the HPG axis: the hypothalamus (to influence GnRH production), the pituitary (to alter gonadotropins release), and the gonads (to interfere with steroid hormone secretion). All the alterations may not only influence the reproduction process, but also alter the normal endocrine, nervous, and immune functions when these hormones act on their targets of the HPG axis. As a key regulator of the reproductive hormonal cascade, GnRH is expressed mostly in neurosecretory cells throughout the hypothalamus. GnRH stimulates gonadotropin synthesis and release through pulsatile transport to the pituitary to regulate gonadal steroidogenesis and gametogenesis.25 Researchers have suggested that GnRH has negative effects in the early pregnancy period and may result in abortion through the action of corpus luteum.26 Both the levels of GnRHI and GnRHRI were upregulated in response to BF exposure, suggesting that BF exposure alters an essential part in the reproductive process. On the other hand, the gonadal hormones subsequently act through a negative feedback in serum activins to regulate GnRH and gonadotropins release, and gonadotropins can also trigger the release of the two forms of GnRH.20 The enhancement of GnRHI was completely blocked by ICI 182,780 cotreatment 8114

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extrapolated to other chemicals. Given the widespread exposure to currently used compounds, particular attention should be paid to developing more comprehensive health risk assessments and health-based chemical regulations for sensitive populations. The convergence results implicate that chiral pesticides are of significant concern to maternal−fetal health. Further research is required to detect the residential levels in reproductive organs and placental tissues of pregnant women, determine the association between maternal pyrethroid exposure and subsequent adverse effects, and explore the underlying mechanism. Moreover, enantioselective toxic effects of currently used chiral EDCs should be considered in health risk assessments and policy making, especially for the sensitive population of pregnant women and their fetuses.

to form a complex and interfere with hormones and enzymes in the trophoblast cells, which may then mediate various effects on the maternal−fetal system. Numerous studies have assessed the endocrine disruption potential of pyrethroids. However, relatively few cases considered enantioselectivity. Researchers have paid attention to the enantioselectivity of chiral organochlorines, organophosphates, and pyrethroids pesticides on their potential endocrine disruption, neurotoxicity, immunotoxicity, and cytotoxicity.40 It has been reported that the estrogenic potential of S-BF was greater than that of R-BF with the relative proliferative effect ratios of 74.2% and 20.9%, respectively.11 Similarly, the results of the present study indicated an enantioselective endocrine disruption potential with a stronger effect induced by S-BF. Both of the enantiomers were cytotoxic to JEG-3 cells, and a greater enantioselectivity was displayed over time. A consistent enantioselectivity was observed in regard to trophoblast hormone production, with S-BF showing a greater effect than R-BF. Moreover, hormone signaling in trophoblast cells was stereospecifically disrupted, and the results demonstrated that S-BF is a much more potent EDC. In addition, S-BF obviously altered the levels of steroidogenesis-related genes in trophoblast cells. In contrast, R-BF did not change the mRNA levels of these genes with the exception of CYP19. It is notable that the S-BF enantiomer possesses a higher threat to maternal−fetal health than R-BF. The results revealed that the two enantiomers of BF interfere with hormone functions in the same way as the racemic form, which is an equal (1:1) mixture of the two, and the endocrine disruption activity of rac-BF is most likely attributable to S-BF, whereas R-BF is less toxic. Our results also showed that rac-BF displayed an endocrine disruption potential as a combination of the two enantiomers as measured by all of the indicators used in the present study. The endocrine disruption potential of the compounds used in the present study could be ranked in the following manner: S-BF > rac-BF > R-BF. Like other EDCs, BF possesses chemical components with similarity to estrogen metabolites, and ER may mediate the effects induced by the two enantiomers. Using molecular docking, S-BF was found to have a comparatively stronger binding affinity for ER α and ER β than R-BF, which may partially explain the enantioselectivity of BF. The results were consistent with the findings of the hormone secretion and gene expression. A previous study on organochlorine insecticide acetofenate showed that the enantiomers possess enantioselective estrogenic potential in progesterone secretion, as well as expression levels of PR, HLA-G, CYP19, and 3β-HSD in JEG-3 cells.41 As acetofenate could also interact with ER α, the combined results of acetofenate and BF imply that chiral environmental chemicals binding to ER have been linked to the risks to maternal−fetus health. Because a considerable portion of pesticides contain chiral structures and consist of enantiomers possessing ER-mediated estrogenic activities,42 the remarkable differential biological activity of the pyrethroid enantiomers suggests that stereospecificity should be considered in a more comprehensive health risk assessment of chiral EDCs. In conclusion, our study showed that both the racemic mixture and individual enantiomers of BF could interfere with the hormone signaling of the endocrine−nervous−reproductive system communication in trophoblast cells. In addition, the enantioselectivity in endocrine disruption of BF may be partially due to enantiospecific binding to ER. As a large number of pesticides and other EDCs similarly interfere through ER, the enantioselective disruption of hormonal signaling by BF may be

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ASSOCIATED CONTENT

S Supporting Information *

Assessment of cell viability; real-time quantitative PCR; primer sequences for real-time quantitative PCR; effects of bifenthrin (BF) on the viability of JEG-3 cells. This material is available free of charge via the Internet at http://pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-571-8898-2341; fax: 86-571-8898-2341; e-mail: [email protected]. Author Contributions ∥

Authors M.Z. and Y.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundations of China (21337005 and 21377119), the Program for Changjiang Scholars and Innovative Research Team in Chinese Universities (IRT 13096), and the Foundation of State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (KF2013-19).

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