Risk assessment of the endocrine-disrupting effects of nine chiral pesticides

Accepted Manuscript Title: Risk assessment of the endocrine-disrupting effects of nine chiral pesticides Authors: Qin Song, Yi Zhang, Lu Yan, Jinghua Wang, Chensheng Lu, Quan Zhang, Meirong Zhao PII: DOI: Reference:

S0304-3894(17)30358-8 http://dx.doi.org/doi:10.1016/j.jhazmat.2017.05.015 HAZMAT 18574

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Journal of Hazardous Materials

Received date: Revised date: Accepted date:

10-11-2016 6-5-2017 10-5-2017

Please cite this article as: Qin Song, Yi Zhang, Lu Yan, Jinghua Wang, Chensheng Lu, Quan Zhang, Meirong Zhao, Risk assessment of the endocrine-disrupting effects of nine chiral pesticides, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Risk assessment of the endocrine-disrupting effects of nine chiral pesticides

Qin Song1, Yi Zhang1, Lu Yan1, Jinghua Wang1, Chensheng Lu2, Quan Zhang1,2* and Meirong Zhao1,2

1. Key Laboratory of Microbial Technology for Industrial Pollution Control of Zheji ang Province, College of Environment, Zhejiang University of Technology, Hangzhou, Zhejiang, 310032, China 2. Department of Environmental Health, Harvard T.H. Chan School of Public Health, Landmark Center West, Boston, MA, 02215, USA

*

To whom correspondence should be addressed. Phone: +86 571 88871579;

Fax: +86-571-88320265.Email: [email protected] (Q Zhang).

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Table of Content Highlights: 1. o,p'-DDT and its metabolite o,p'-DDD had the same enantioselectivity direction. 2. Five chiral pesticides showed significant anti-estrogenic effects. 3. 1R,3R-cis-αS-λ-cyhalothrin and R-(-)-myclobutanil showed anti-thyroid effects. 4. Dramatic differences between enantiomers were observed in EDEs.

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Abstract The increased release of chiral pesticides into the environment has generated interest in the role of enantioselectivity in the environmental fate and ecotoxicological effects of these compounds. However, the information on the endocrine disrupting effects (EDEs) of chiral pesticides is still limited and discrepancies are also usually observed among different assays. In this study, we investigated the enantioselectivity of EDEs via estrogen and thyroid hormone receptors for nine chiral pesticides using in vitro and in silico approaches. The results of the luciferase reporter gene assays showed 7 chiral pesticides possessed enantioselective estrogenic activities and 2 chiral pesticides exerted thyroid hormone antagonistic effects. Proliferation assays in MCF-7 and GH3 cells were also used to verify the results of the dual-luciferase reporter gene assays. At last, the molecular docking results indicated that the enantioselective EDEs of chiral pesticides were partially due to enantiospecific binding affinities with receptors. Our data not only show enantioselective EDEs of nine chiral pesticides, but also would be helpful to better understanding the molecular biological mechanisms of enantioselectivity in EDEs of chiral pesticides. Keywords: Risk assessment; Enantioselectivity; Chiral chemicals; Endocrine-disrupting effects.

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1. Introduction Over time, more and more pesticides have been extensively used in agricultural and non-agricultural (e.g., urban landscaping, industry protection and public health protection sites) production. A well-known fact is that many pesticides contain chiral structures and thus consist of enantiomers. Among the widely used pesticides, over 25% are chiral pesticides, and up to 40% of pesticides in China are chiral chemicals [1, 2]. It is expected that more chiral pesticides will be introduced into the market [3].Many studies have revealed that enantiomers selectively interact with biological systems and may behave as completely different chemicals. Dramatic differences between enantiomers had been observed in their acute toxicity, cytotoxicity, developmental toxicity and immune toxicity to non-target organisms; in addition, their microbial degradation rates in soil and food were different [4-9]. However, the role of enantioselectivity in the EDEs of chiral pesticides is poorly understood, and the gap is mainly due to the complexity of the endocrine system and the chemical structure of chiral chemicals. EDEs are an important index of the ecological and health risks of environmental pollutants. Although the investigation into the environmental safety of chiral pesticides has been ongoing for 30 years, few reports have indicated that the estrogen-like activity of chiral pesticides is significantly enantioselective [7, 10-12]. Reports from the US Environmental Protection Agency (EPA) and other accumulated evidence have shown that a large number of racemic pesticides (e.g., organochlorines, diphenyl ethers, organophosphorus, carbamates, pyrethroids and acid amides) possess

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estrogen, androgen, thyroid and glucocorticoid EDEs [13-15]. Previous reports revealed that chiral persistent organic pollutants (POPs) (o,p´-DDT) and currently used pyrethroids pesticides (bifenthrin and permethrin) have enantioselective EDEs [7, 10, 11]. The EDEs mediated by nuclear receptors are diverse because these receptors include steroid receptors (e.g., the estrogen receptor (ER), androgen receptor (AR), and glucocorticoid receptor (GR)) and non-steroid hormone receptors (e.g., the thyroid hormone receptor (TR) and pregnane X receptor (PXR)). Thus, studies that only focus on individual EDEs induced by single chiral pesticides are incomprehensive. Evaluation of the multiple EDEs induced by the many types of chiral pesticides (i.e., insecticides, herbicides and fungicides) is necessary to properly assess environmental risk and understand the common enantioselective mechanisms of chiral pollutants in EDEs. In vertebrates, the reproductive and thyroid endocrine systems are primarily controlled by the hypothalamic-pituitary-gonad (HPG) axis and hypothalamic-pituitary-thyroid (HPT) axis, respectively, and these systems can be disrupted by exogenous chemicals [16].Estrogen and thyroid hormones are the main agents that regulate protein synthesis, secretion, transport, and metabolism, and these hormones support the reproductive system, immune system and energy metabolism. Unfortunately, there is no available information on enantioselectivity in the ER-mediated antiestrogenic effects of chiral pesticides. Antiestrogenic effects are indispensable for the proper functioning of a myriad of biochemical reactions. Furthermore, little is known about enantioselectivity in thyroid hormone-disrupting

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effects and the mechanism of action for a large number of chiral pesticides. The enantioselectivity of chiral metabolites is rarely reported in studies of environmental fate and ecotoxicological effects. Importantly, misleading conclusions may be deduced when the toxicity of a chiral chemical is assessed only with its parent chemical. Zhang Q et al [17] reported that diclofop-methyl and its metabolite diclofop acid had an enantioselective effect on non-target plants. Wang C et al [18] also reported that o,p'-DDT and its daughter metabolite o,p'-DDD produced enantioselective toxicity in PC12 cells, and the R-enantiomer was the more toxic stereo structure for both o,p´-DDT and o,p'-DDD. Therefore, these results remind us the enantioselectivity of chiral metabolites also should aroused great concerns. In the present study, we used three in vitro methods (Luciferase reporter gene assay, E-screen and T-screen assay) to rapidly screen the estrogen and thyroid EDEs of 9 chiral pesticides and their metabolites that had a high frequency of use and a high detection rate in China and other countries [19, 20]. Furthermore, molecular docking was used to explain the relationship between the chemical structure and hormonal activity of enantiomers on receptors. We are the first to document enantioselectivity in thyroid EDEs and provide information on the impact of the order of the magnitude of the concentration and enantioselective direction on the EDEs of chiral pesticides and their metabolites. 2. Materials and Methods 2.1. Chemicals 17β-estradiol (E2, >97% pure) and dimethyl sulfoxide (DMSO) were purchased

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from Sigma-Aldrich (St. Louis, MO, U.S.A.). Liothyronine (T3, 95% pure) was purchased from J&K (Beijing, China). The 9 chiral pesticides tested in this study are listed in Table S1. The racemic mixtures of these pesticides were purchased from Dr. Ehrenstorfer in Germany and were 97.5-99.5% pure. The enantiomers of 9 chiral pesticides were separated using previously described methods [18, 21-30]. Stock solutions were prepared in DMSO at concentrations of 10-1 M for E2 and 10-3 M for T3. The 9 pesticides were dissolved in DMSO at the initial concentration of 10-2 M. All of the solutions were stored at −20 °C. Based on previous studies, all pesticides were directly diluted to the experimental concentrations using the appropriate medium [31]. The final DMSO concentration in the culture medium did not exceed 0.1%, and this concentration did not affect cell yield. 2.2. Cells culture and plasmids Chinese hamster ovary cell (CHO) cells, human breast adenocarcinoma (MCF-7) cells and rat pituitary adenoma (GH3) cells were obtained from the cell bank at the Chinese Academy of Sciences (Shanghai, China; original sources from ATCC, Manassas, USA). CHO ,MCF-7 and GH3 cells were routinely cultured in a 25-cm2 flask (Corning, NY, USA) with 5 mL of complete medium containing 90% RPMI-1640 medium (Hyclone, Logan, UT) and 10% FBS (Hyclone). All cells are living in the incubation conditions of 37°C, 5% CO2 with saturating humidity. pERE-AUG-Luc+, rat 2u globulin promoter and rER/pCI plasmids were a gift from Dr. M. Takeyoshi (Chemicals Assessment Center, Chemicals Evaluation and Research Institute, Oita, Japan) and were used in the ER luciferase reporter gene assay.

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PGal4-L-hTRβ and pUAS-tk-Luc plasmids were kindly provided by Dr. Ronald M. Evans (Gene Expression Laboratory, Howard Hughes Medical Institute, San Diego, CA, USA) and were used in the TR reporter gene assay. The plasmid phRL-tk purchased from Promega (Madison, WI, U.S.A.) was used as an internal control for transfection effciency. The detail cell culture conditions were described in our previous publications [32, 33]. 2.3. MTS assay CHO cells were treated with test chemicals at the concentration ranging from 10−10 to 10−5 M for 24 h. Cell viability was measured using Cell Titer 96® Aqueous One Solution Cell Proliferation Assay (Promega, USA) as the manufacturer's instructions and previously described [33]. The absorbance was measured at 490 nm using the 96-well plate reader (Infinite M200, Tecan, Switzerland) to determine the formazan concentration, which is proportional to the number of live cells. The exposure concentrations of no cell cytotoxicity were used for the dual-luciferase reporter gene assay, E-screen and T-screen assay. 2.4. Dual-luciferase assay for ER and TRβ The procedure for the dual-luciferase reporter gene assay was based on our previous study [34]. The CHO cells were plated in 96-well microtiter plates at a density of 15,000 cells/well for 24 h. For the detection of ER activity, cells were transfected with 40 ng of rERα/pCI, 135 ng of pERE-AUG-Luc, 15 ng of phRL-tk and 0.5 μL of lipofectamine 2000 per well. For the detection of TRβ activity, cells were transfected with 50 ng of pGal4-L-hTRβ, 131 ng of pUAS-tk-luc, 22 ng of

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phRL-tk and 0.5 μL of lipofectamine 2000 per well. The culture medium was changed after the 4 h transfection. After culturing overnight, cells were dosed with various concentrations of the selected chiral pesticides, 10-10 M E2 or 2×10–7 M T3. After 24 h, we measured luciferase and Renilla luciferase activity with a fluorescence spectrophotometer (Infinite M200, Tecan, Switzerland).The relative transcriptional activity was presented as the ratio of firefly to renilla luciferase activity. 2.5. E-screen and T-screen assays To verify the reliability of the results from the dual-luciferase reporter gene assay, E-screen and T-screen assays were used to characterize estrogen- and thyroid hormone-disrupting effects, respectively. E-screen had been previously described [33].GH3 cells were plated in 96-well plates with experimental medium (phenol red-free RPMI-1640 with 5% CD-FBS) for 24 h to allow the attachment to the bottom. To measure the thyroid hormone agonistic activity of tested chemicals, cells were treated at the concentration ranging from 10−8 to 10−6 M of tested chemicals or 0.1% DMSO (vehicle control), while for the measurement of antagonistic activity, the cells were exposed to 2 × 10-8 M T3 in combination with tested chemicals at doses from 10−8 to 10−6 M. After 5 days of incubation, cell proliferation was detected by the MTS kit (Cell Titer 96 Aqueous One Solution) following manufacturer's instructions [33]. 2.6. Molecular docking Docking studies were completed using CDOCKER module of Discovery Studio 2.5 (Accelrys, Inc., San Diego, CA, USA). The crystal structure of human ERα (PDB entry code: 1ERE and 1ERR) and human TRβ (PDB code: 2PIN) used in the docking

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process were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/). The reference (ligands) of 1ERE, 1ERR and 2PIN are E2 (C18H24O2), raloxifene (C28H27NO4S) and 1-(4-hexylphenyl) prop-2-en-1-one (C15H20O) respectively. Before docking, the ligands were optimized by minimization with the CHARMM force field using the Simulation module of Discovery Studio 2.5. The default values for CDOCKER parameters were used, and 100 poses for each ligand were saved. The -CDOCKER energy between the ligand and receptor was calculated, and the conformation with the highest -CDOCKER energy for each ligand was selected as the most likely bioactive conformation. The CDOCKER molecular docking analysis provided information on the interactions between ligands and receptors that can aid in the explanation of the in vitro assay results. 2.7. Statistical analysis The statistical analysis was conducted using Origin 8.0 (OriginLab, Northampton, MA). All data are presented as the mean ± SD (standard deviation). The data were obtained from three separate experiments each consisting of duplicate wells. A significant level of p< 0.05 was selected for the dual-luciferase reporter gene and MTS assays. The mean differences between the experimental and control group were evaluated using a one-way analysis of variance (ANOVA) with a Dennett’s post-hoc test. 3. Results 3.1. Cytotoxicity of chiral pesticides The cytotoxicity of 9 chiral pesticides was determined using a MTS assay prior to

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performing dual-luciferase reporter gene assays. S-metolachlor (MC) showed weak cytotoxic effects at 10-5 M on CHO cell proliferation. No cytotoxic effects were observed for the other chiral pesticides at the tested concentrations (see Figure S1 in Supporting Information (SI)). Therefore, we performed dual-luciferase reporter gene assays at no cytotoxic concentrations. 3.2. Enantioselectivity in estrogenic activity of 9 chiral pesticides Before dual-luciferase reporter gene assays, we fitted the dose response curve of E2 at various concentrations using logistic model. Then maximal effect concentration (10-10 M E2) and approximate EC80 (5 × 10−11 M E2) value (approximately 80% of the maximal effect) was selected for the positive control in agonistic and antagonistic activities assays, respectively [14]. As shown in Figure 1A and Table S2, R-(-)-o,p'-DDT, rac-o,p'-DDT, R-(+)-o,p'-DDD and rac-o,p'-DDD showed significant estrogenic activity with the REC20 values of 6.30 × 10-7 M, 8.11 × 10-7 M, 5.58 × 10-7M and 7.52 × 10-7M respectively. o,p'-DDT and o,p'-DDD, heptachlor (HCH), fipronil (FR) and β-cypermethrin (β-CP) exhibited enantioselective antiestrogenic effects (Figure 2A). The RIC20 values of S-(+)-o,p'-DDT, rac-o,p'-DDT, S-(-)-o,p'-DDD, rac-o,p'-DDD, (+)-HCH, rac-HCH, R-(-)-FR, rac- FR, 1R-cis-αS-β-CP and rac-β-CP were 6.35 × 10-7 M, 8.13 × 10-7 M, 4.82 × 10-7 M, 7.79 × 10-7 M, 2.82 × 10-9 M, 1.32 × 10-7 M, 2.17 × 10-7 M, 5.22 × 10-6 M, 6.41 × 10-7 M and 8.75 × 10-7 M respectively (Table S2). All tested compounds with positive results were dose-dependent in ERα mediated estrogenic activity. The lowest observed effect level (LOEL) for R-(+)-o,p'-DDD, (+)-HCH, rac-HCH, R-(+)-FR and rac-FR were

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10-7 M, 10-7 M, 5 × 10-7 M, 5 × 10-7 M and 5 × 10-6 M in reporter gene respectively. All other chemicals’ LOEL were 10-6 M. No estrogenic/anti-estrogenic activities were observed for λ-cyhalothrin (λ-CT), metalaxyl (ML), MC, and myclobutanil (MT) within the concentration range tested (Figure S2 and Figure S3). E-screen assay was used to confirm the results obtained from the dual-luciferase reporter gene assay. The dose-dependent proliferation of MCF-7cells was induced by E2. The half-maximal effective concentration (EC50) was 1.2 × 10–13 M. o,p'-DDT, o,p'-DDD and MT displayed significant estrogenic activity and induced the proliferation of MCF-7 cells(Figure 1B). The order of relative proliferative effects for MCF-7 cells at a concentration of 1 × 10−6 M was R-(-)-o,p'-DDT > rac-o,p'-DDT > S-(+)-o,p'-DDT, R-(+)-o,p'-DDD > rac-o,p'-DDD > S-(-)-o,p'-DDD, R-(-)-MT > rac-MT > S-(+)-MT. No estrogenic activities were observed for the other 6 chiral pesticides (Figure S6). Figure 2B shows the antagonistic effects of 9 chiral pesticides measured via the E-screen assay. The order of anti-estrogenic activity was S-(+)-o,p'-DDT > rac-o,p'-DDT > R-(-)-o,p'-DDT, S-(-)-o,p'-DDD > rac-o,p'-DDD > R-(+)-o,p'-DDD, (+)-HCH > rac-HCH > (-)-HCH, 1R-cis-S-β-CP > rac-β-CP and its’ other enantiomers and R-(-)-FR > rac-FR > S-(+)-FR. The other 4 chiral pesticides showed no antagonistic activity (Figure S7). Overall, the results from the dual-luciferase reporter gene and E-screen assays were nearly identical. 3.3. Enantioselectivity in thyroid hormone activities of 9 chiral pesticides Before dual-luciferase reporter gene assays, the dose response curve of T3 was fitted at various concentrations using logistic model. Then maxima approximate EC80

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(2 × 10−7 M T3) value was selected for the positive control in antagonistic activities assays [14].In the dual-luciferase reporter gene assay for TRβ, none of the 9 chiral pesticides showed TRβ agonistic activity (Figure S4) . However, when co-administered with 2 × 10–7 M of T3, 1R,3R-cis-αS-λ-CT and R-(-)-MT showed increased antagonistic activity with RIC20 values of 2.01 × 10-8 M and 4.98 × 10-7 M respectively (Table S3). As shown in Figure 3A, λ-CT and MT significantly inhibited the response induced by 2 × 10–7 M T3. The LOEL of 1R,3R-cis-αS-λ-CT and R-(-)-MT were 5 × 10-7 M and 10-6 M respectively. No anti-thyroid hormone effects were observed for the other 7 chiral chemicals (Figure S5). To verify the results from the dual-luciferase reporter gene assay. T-screen assay was used to determine the agonistic or antagonistic activity of 9 chiral pesticides in GH3 cells. The dose-dependent proliferation of GH3 cells was induced by T3. The half-maximal effective concentration (EC50) value was 2.0 × 10–8M. In the T-screen assay, none of the 9 chiral pesticides exerted agonistic activity (Figure S8). The pesticides λ-CT, MT and HCH showed significant enantioselective antagonistic activity and inhibited the proliferation induced by T3 in GH3 cells. The order of anti-estrogenic activity was 1R,3R-cis-αS-λ-CT > rac-λ-CT >1S, 3S-cis-αR-λ-CT, R-(-)-MT > rac-MT > S-(+)-MT, (+)-HCH > rac-HCH > (-)-HCH (Figure 3B). The other 6 chiral pesticides did not show anti-estrogenic activity (Figure S9). The results from dual-luciferase reporter gene assay and T-screen assays were nearly identical. 3.4. Molecular Docking Molecular docking was used to analyze the interactions between chiral pesticide

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enantiomers and ER/TR. As shown in Figure 4, the 6 chiral pesticides were docked into the active pocket of hERα/hTRβ. The active sites of the receptors mainly consisted of the following residues: HIS524, ILE424, THR347, GLU353, MET388, MET343, MET421, LEU391, LEU428, LEU349, LEU442, LEU525, PHE425, GLY521, ARG394 (1ERE); HIS524, ASP351, ALA350, TYR526, THR347, GLY521, GLU353, MET528, MET343, MET522, MET543, MET421, LEU525, LEU391, LEU428, LEU387, LEU354, LEU538, LEU539, PRO535, PHE404 (1ERR); and PHE272, PHE269, PHE455, PHE439, ALA279, ILE353, ILE275, ILE276, GLY521, GLY345, GLY332, GLY344, MET310, MET442, MET313, LEU346, LEU341, LEU330, ARG320, ARG316, ASN331, VAL283 (2PIN). There were three hydrogen bonds observed between E2 and the ER (1ERE), and four hydrogen bonds and one π-σ bonds between raloxifene and the ER (1ERR). Meanwhile, we found that S-(+)o,p'-DDT had π-σ bond with LEU525 and 1R-cis-αS-β-CP had hydrogen bond with ASP351. We also observed that R-(-)-MT had π-π bond with PHE272 and hydrogen bond with ASN331 and 1R, 3R-cis-αS-λ-CT had π-cation bond with ARG316. However, for 1-(4-hexylphenyl) prop-2-en-1-one, there was no bonds with the amino acid residues in TRβ. The -CDOCKER energy between each ligand and hERα/hTRβ is shown in Table S4. 4. Discussion 4.1. EDEs of the chiral metabolite o,p´-DDD To date, few studies have examined the EDEs of chiral POP metabolites. Dechlorination is the major metabolic pathway for the transformation of o,p'-DDT to

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o,p'-DDD in the natural environment. Through dechlorination, R-(-)-o,p'-DDT is metabolized to R-(+)-o,p'-DDD, while S-(+)-o,p'-DDT is metabolized to S-(-)-o,p'-DDD [34]. After decades of degradation in the natural environment following o,p'-DDT application, its metabolite o,p'-DDD is the predominant residue in the environment. Therefore, when the EDEs and enantioselectivity of chiral daughter chemicals are different from the parent chemical, conclusions from risk assessments that only study the parent chemical may be misleading. Our results showed that the R-enantiomer for both o,p'-DDD and o,p'-DDT were stronger estrogen analogs at concentrations of the same order of magnitude (10-6 M). Thus, the order of magnitude of the concentration and enantioselectivity direction are consistent between chiral parent (o,p'-DDT) and metabolite chemicals (o,p'-DDD) with regards to EDEs. This phenomenon may be due to dechlorination that failed to change the spatial structure of the parental chemical and thus failed to alter its function. The data suggest that the risk assessment for EDEs caused by o,p'-DDD may be predicted from the chiral parent chemical (o,p'-DDT). However, chiral POP metabolites have other toxicological effects during natural degradation processes, and it is unknown whether the order of magnitude of the concentration and enantioselective direction are consistent between chiral parent and daughter metabolite. Further studies on the enantiospecific effects of chiral metabolites will enable a more accurate evaluation of risks. 4.2. Molecular Docking According to the docking scores of 100 poses [35], effective enantiomers had

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higher energy scores than non-effective enantiomers (Table S4). It was suggested that the enantioselective EDEs of chiral pesticides may be partially due to enantiospecific binding affinities with ER and TR. Compared with reference (E2 and raloxifene), effective enantiomers had higher energy scores, which hinted these effective enantiomers had a good binding abilities with ER. For docking results of TR model, effective enantiomers (R-(-)-MT and1R, 3R-cis-αS-λ-CT) have lower scores than 1-(4-hexylphenyl) prop-2-en-1-one. It may be possible that MT and λ-CT have weak thyroid hormone antagonist activity. In addition, model’s factors (solvents and pH) and biological process (uptake and transportation) may lead to different results between CDOCKER model and reporter gene assay [61, 62]. Furthermore, we analyzed the interaction modes of the test chemicals in the ligand-binding pocket of hERα and hTRβ. R-(-)-o,p'-DDT and R-(+)-o,p'-DDD were located deep in the hydrophobic cavity (Figure 4A and B). Both of them share the same residues with E2 [36]. And these residues were crucial for the interaction of E2 with ERα, which indicated that the binding modes of test chemicals in this study were similar to that of E2 with ERα. Similarly, our docking results showed that S-(+)-o,p'-DDT, S-(-)-o,p'-DDD, R-(-)-FR and1R-cis-αS-β-CP also occupied the hydrophobic cavity and had similar binding modes with raloxifene with ERα (Figure 4C, D, E and F). Meanwhile S-(+)-o,p'-DDT and 1R-cis-αS-β-CP had π-σ and hydrogen bonds with the amino acids around respectively [36]. Thus, different chemicals may bind to ER through different interactions. For TRβ, R-(-)-MT formed hydrogen bond with ASN331and π-π bond with PHE272. 1R,3R-cis-αS-λ-CT had π-cation bond with

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ARG316. These acting forces may provide strong evidence for them as an antagonist for TRβ (Figure 4G and H). 4.3. Ecotoxicological Implications We compared the LOEL with the residue levels in previous reports, assuming negligible differences between species, bioavailability, and metabolism. The LOEL can be calculated as follows: o,p´-DDT and its´ enantiomers (10−6 M = 10−6 M/L × molar mass (354 g/M) = 354 μg/L = 354ppb), o,p´-DDD (10−6 M = 320 ppb) and R-(+)- o,p´-DDD (10−7 M = 32 ppb), HCH (5×10−7 M = 186.5 ppb) and (+)-HCH (10−7 M = 37.3 ppb), FR (5×10−6 M = 2185 ppb) and R-(-)-FR (5×10−7 M = 218.5 ppb), β-CT and 1R-cis-S (10−6 M = 416 ppb), λ-CT (10−6 M= 446 ppb) and 1R, 3R-cis-S -λ-CT (5×10−7 M= 223 ppb), MT and R-(-)-MT (10−6 M= 288 ppb). According to the previous study, o,p´-DDT in maize consumed by infants from southwest Ethiopia was up to 1770 ppb [37]. o,p´-DDD was the metabolite of o,p´-DDT, both of o,p´-DDD and o,p´-DDT were detected in breast milk in at maximal levels of 69 and 2120 ppb, respectively[38]. λ-CT was up to 828 ppb in cabbage samples collected from Niaga [39]. HCH was up to 110 ppb in grey partridge eggs in French cereal ecosystems [19]. MT was up to 981 ppb in pollen samples collected from North American honey bee [40]. We can clearly concluded that the residue levels of o,p´-DDT, o,p´-DDD, λ-CT, HCH and MT are higher than their corresponding LOELs, the residue levels of their enantiomers may be higher than LOELs. It suggested that the existence of four chiral pesticides could potentially induce adverse effects and possess ecological risks. β-CT (max of 30 ppb in tea) are

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lower than their LOELs in our study, but risks would not be receded due to lipophilic, stable, additive and synergistic effects[41]. For an accurate risk assessment of chiral pesticides, enantioselectivity in toxicity and degradation should be simultaneously considered. For instance, the S-(+)-enantiomer of the insecticide FR is more efficient at killing insects than the R-(-)-enantiomer [27]; however, the R-(-)-enantiomer showed greater toxicity in non-target organisms and was preferentially enriched in sulfidogenic sediment and the absorption phase of Eichhornia crassipes [42, 43]. Therefore, the use of a racemic mixture (FR) will result in an underestimate of ecotoxicity. In this study, 7 chiral pesticides (o,p'-DDT, o,p'-DDD, HCH, FR, β-CP, MT and λ-CT) displayed enantioselective EDEs. Previous reports stated that 6 of these chemicals (o,p'-DDT, o,p'-DDD, FR, β-CP, MT and λ-CT) showed enantioselective degradation [28, 44-60]. Therefore, if specific enantiomers possess significant toxicity, then the adverse ecotoxicological effects will closely rely on the behavior of the active enantiomer rather than the racemate. As shown in Table S5, the toxicologically active enantiomers [1R-cis-αS-β-CP, (-)-enantiomer of o,p'-DDT, FR, and MT] were preferentially enriched in air, soil, water and biotic components. Thus, the use of a racemate mixture of these chemicals (o,p'-DDT, FR, MT and β-CP) will result in an underestimate of ecotoxicity [63]. Conversely, the toxicologically active enantiomer [(+)-enantiomer] of o,p'-DDD was preferentially degraded in air, soil, water and food. In this case, the use of a racemate could result in an overestimate or underestimate of ecotoxicity. In addition, the proportion of enantiomers in different trophic levels of

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the food chain varies [64]. As shown in Table S5, there is a general trend of deviation from EF (enantiomer fraction) = 0.5 for chiral o,p'-DDT based on the EF model as follows [63]:air < soil < water < grass < mollusks or whale < maternal colostrum (i.e., lower trophic biotic < higher trophic biotic). The deviation of the racemic mixture mainly depends on microbial community abundance in the abiotic environment. Air, water and soil are home to various microorganisms and can be assumed to be the decomposition of food chain. Biotic components showed a higher deviation with the racemic mixture, which was attributed to stereospecific metabolization and enzymatic transport processes [63]. Based on this deviation, ignoring chirality will result in a more serious overestimate or underestimate of ecological risks for higher trophic biotics than lower trophic biotics. Thus, it is important to establish the enantioselective toxicity and bioaccumulation of chiral chemicals for higher trophic biotics, especially humans. We compared the enantioselectivity of endocrine disruption, target toxicity, acute toxicity, cytotoxicity, developmental toxicity, and immune toxicity for six chiral pesticides (o,p'-DDT, o,p'-DDD, HCH, FR, β-CP, MT and λ-CT). R-(-)-o,p'-DDT, R-(+)-o,p'-DDD, (+)-HCH, and 1R-cis-αS-β-CP were toxicologically active enantiomers that showed endocrine disrupting toxicity [65]. Furthermore, these toxicologically active enantiomers displayed cytotoxicity, target toxicity, acute toxicity, and developmental toxicity. However, it is unclear whether these chiral pesticides will possess the same toxicologically active enantiomers in other types of toxicity because chiral pesticides have different mechanisms of action that depend on

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the target organism. (+)-FR was more acutely toxic to C. dubia than the (-)-enantiomer based on the 48h LC50 and number of offspring. In contrast, (-)-FR was the toxicologically active enantiomer that caused acute toxicity and endocrine disruption in Eiseniafeotida; thus, the enantioselectivity of toxicological effects for FR is diverse [66]. The enantioselective toxicity of chiral pesticides, such as endocrine disruption, developmental toxicity, neurotoxicity and immune toxicity, is closely related to human health. However, the available information is very limited. Research into the risk assessment of chiral chemicals, including their environmental behavior and toxic effects, is greatly needed. Our study show the enantioselectivity of multiple EDEs induced by multiple types of chiral pesticides. More than 70% of the selected chiral pesticides exhibited enantioselective EDEs. However, there is no legislation for the use and management of chiral pesticides. In addition, an ecotoxicological implications analysis showed that a chiral chemical with enantioselective toxicity and degradation for one specific species, cell type or tissue, did not guarantee that it had enantioselective effects in other species. To avoid inaccurate risk assessments of chiral chemicals, more attention should be paid to both enantioselective toxicity and environmental fate. On the other hand, many new pesticides were launched as the substitution of high toxicity pesticides. They were considered low toxicity to non-target organism and cannot be easily accumulated in the environment media. However, with the ban of some pesticides, the usage of them was increased year by year. Now they were more frequently detected in environment than before, but the joint effects of new launched pesticides were still unknown. Moreover, our precious study revealed the toxicity of binary mixtures of enantiomers in chiral organophosphorus insecticides [67].

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Although the new chiral pesticides have been determined with the low concentrations compared with the banned insecticides such as DDT, the joint effects of their enantiomers have not been well studied especially at environmental relevant concentrations. It needs future investigations.

Acknowledgements This study was funded by the National Natural Science Foundation of China (21337005, 21377119 and 21307109) and Zhejiang Provincial Xinmiao Talent Plan (2016R403070).

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References [1] A.W. Garrison, Probing the enantioselectivity of chiral pesticides, Environ.Sci &Technol, 40 (2006) 16-23. [2] J. Ye, J. Wu, W.P. Liu, Enantioselective separation and analysis of chiral pesticides by high-performance liquid chromatography, Trac-Trend Anal. Chem, 28 (2009) 1148-1163. [3] b.S. kankan Zhang, yang Song, research overreview on analysis of chiral pesticides, The 3rd.Sym.Mol.Chirality on china, (2011) 61-68. [4] W. Liu, J. Gan, D. Schlenk, W.A. Jury, Enantioselectivity in environmental safety of current chiral insecticides, Proc. Natl. Acad. Sci. U S A, 102 (2005) 701-706. [5] Z.H. Yang, G.D. Ji, Stereoselective degradation and molecular ecological mechanism of chiral pesticides beta-cypermethrin in soils with different ph values,Environ.Sci &Technol, 49 (2015) 14166-14175. [6] Q. Zhang, X.D. Hua, H.Y. Shi, J.S. Liu, M.M. Tian, M.H. Wang, Enantioselective bioactivity, acute toxicity and dissipation in vegetables of the chiral triazole fungicide flutriafol, J. Hazard Mater, 284 (2015) 65-72. [7] M. Zhao, Y. Zhang, S. Zhuang, Q. Zhang, C. Lu, W. Liu, Disruption of the hormonal network and the enantioselectivity of bifenthrin in trophoblast: maternal-fetal health risk of chiral pesticides, Environ.Sci &Technol, 48 (2014) 8109-8116. [8] C. Xu, W. Tu, C. Lou, Y. Hong, M. Zhao, Enantioselective separation and zebrafish embryo toxicity of insecticide beta-cypermethrin, J. of Environ.Sci, 22 (2010) 738-743. [9] M. Zhao, F. Chen, C. Wang, Q. Zhang, J. Gan, W. Liu, Integrative assessment of enantioselectivity in endocrine disruption and immunotoxicity of synthetic pyrethroids, Environ. Pollut, 158 (2010) 1968-1973. [10] P.F. Hoekstra, B.K. Burnison, T. Neheli, D.C. Muir, Enantiomer-specific activity of o,p'-DDT with the human estrogen receptor, Toxicol. Lett, 125 (2001) 75-81. [11] L. Wang, S. Zhou, K. Lin, M. Zhao, W. Liu, J. Gan, Enantioselective estrogenicity of o,p'-dichlorodiphenyltrichloroethane in the MCF-7 human breast carcinoma cell line, Environ. Toxicol. Chem, 28 (2009) 1-8. [12] C. Wang, Q. Zhang, X.F. Zhang, J. Liu, W.P. Liu, Understanding the endocrine disruption of chiral pesticides: The enantioselectivity in estrogenic activity of synthetic pyrethroids, Sci. China Chem, 53 (2010) 1003-1009. [13] J. Zhang, J. Zhang, R. Liu, J. Gan, J. Liu, W. Liu, Endocrine-Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor, Environ.Sci &Technol, 50 (2016) 435-443. [14] H. Kojima, E. Katsura, S. Takeuchi, K. Niiyama, K. Kobayashi, Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using chinese hamster ovary cells, Environ, Health Persp, 112 (2004) 524-531. [15] M. Lacasana, I. Lopez-Flores, M. Rodriguez-Barranco, C. Aguilar-Garduno, J. Blanco-Munoz, O. Perez-Mendez, R. Gamboa, S. Bassol, M.E. Cebrian, Association between organophosphate pesticides exposure and thyroid hormones in floriculture workers, Toxicol. Appl. Pharmacol, 243 (2010) 19-26. [16] C.S. Liu, X.W. Zhang, J. Deng, M. Hecker, A. Al-Khedhairy, J.P. Giesy, B.S. Zhou, Effects of prochloraz or propylthiouracil on the cross-talk between the hpg, hpa, and hpt axes in zebrafish, Environ.Sci &Technol, 45 (2011) 769-775. [17] Q. Zhang, M.R. Zhao, H.F. Qian, T. Lu, Q. Zhang, W.P. Liu, Enantioselective damage of diclofop acid mediated by oxidative stress and acetyl-coa carboxylase in nontarget plant arabidopsis

22

thaliana,Environ.Sci &Technol, 46 (2012) 8405-8412. [18] C. Wang, Z. Li, Q. Zhang, M. Zhao, W. Liu, Enantioselective induction of cytotoxicity by o,p'-DDD in PC12 cells: implications of chirality in risk assessment of POPs metabolites, Environ.Sci &Technol, 47 (2013) 3909-3917. [19] E. Bro, J. Devillers, F. Millot, A. Decors, Residues of plant protection products in grey partridge eggs in French cereal ecosystems,Environ.Sci. Pollut.R, 23(2016)9559-9573. [20] O. Akoto, S. Gavor, M.K. Appah, J. Apau, Estimation of human health risk associated with the consumption of pesticide-contaminated vegetables from Kumasi, Ghana, Environ. Monit. Assess, 187 (2015) 244. [21] X. Chao, T. Wenqing, L. Chun, H. Yingying, Z. M eirong, Enantioselective separation and zebrafish embryo toxicity of insecticide beta-cypermethrin, J. Environ. Sci, 22 (2010) 738-743. [22] V. Perez-Fernandez, M.A. Garcia, M.L. Marina, Chiral separation of agricultural fungicides, J. Chromatogr A, 1218 (2011) 6561-6582. [23] W.L. Champion, J. Lee, A.W. Garrison, J.C. DiMarco, A. Matabe, K.B. Prickett, Liquid chromatographic separation of the enantiomers of trans-chlordane, cis-chlordane, heptachlor, heptachlor epoxide and α-hexachlorocyclohexane with application to small-scale preparative separation, J. Chromatogr A, 1024 (2004) 55-62. [24] C. Xu, J. Wang, W. Liu, G. Daniel Sheng, Y. Tu, Y. Ma, Separation and aquatic toxicity of enantiomers of the pyrethroid insecticide lambda-cyhalothrin, Environ. Toxicol. Chem, 27 (2008) 174-181. [25] F. Chen, Q. Zhang, C. Wang, Y.C. Lu, M.R. Zhao, Enantioselectivity in estrogenicity of the organochlorine insecticide acetofenate in human trophoblast and MCF-7 cells, Reprod. Toxicol, 33 (2012) 53-59. [26] S. Chen, W. Liu, Enantioselective degradation of metalaxyl in anaerobic activated sewage sludge, Bull. Environ. Contam. Toxicol, 82 (2009) 327-331. [27] M.G. Nillos, K. Lin, J. Gan, S. Bondarenko, D. Schlenk, Enantioselectivity in fipronil aquatic toxicity and degradation, Environ. Toxicol. Chem, 28 (2009) 1825-1833. [28] C. Bosch, J.O. Grimalt, P. Fernandez, Enantiomeric fraction and isomeric composition to assess sources of DDT residues in soils, Chemosphere, 138 (2015) 40-46. [29] J. You, M.J. Lydy, A solution for isomerization of pyrethroid insecticides in gas chromatography, J. Chromatogr A, 1166 (2007) 181-190. [30] W.P. Liu, J.J. Gan, Separation and analysis of diastereomers and enantiomers of cypermethrin and cyfluthrin by gas chromatography, J. Agr. Food Chem, 52 (2004) 755-761. [31] G. Du, O. Shen, H. Sun, J. Fei, C. Lu, L. Song, Y. Xia, S. Wang, X. Wang, Assessing hormone receptor activities of pyrethroid insecticides and their metabolites in reporter gene assays, Toxicol. Sci, 116 (2010) 58-66. [32] Q. Zhang, M. Lu, X. Dong, C. Wang, C. Zhang, W. Liu, M. Zhao, Potential estrogenic effects of phosphorus-containing flame retardants, Environ.Sci &Technol, 48 (2014) 6995-7001. [33] Q. Zhang, C. Ji, X. Yin, L. Yan, M. Lu, M. Zhao, Thyroid hormone-disrupting activity and ecological risk assessment of phosphorus-containing flame retardants by in vitro, in vivo and in silico approaches, Environ. Pollut, 210 (2016) 27-33. [34] A.W. Garrison, V.A. Nzengung, J.K. Avants, J.J. Ellington, W.J. Jones, D. Rennels, N.L. Wolfe, Phytodegradation of p,p '-DDT and the enantiomers of o,p '-DDT, Environ.Sci &Technol, 34 (2000) 1663-1670.

23

[35] M.I. Ahmad, A. Usman, M. Ahmad, Computational study involving identification of endocrine disrupting potential of herbicides: Its implication in TDS and cancer progression in CRPC patients, Chemosphere, 173 (2017) 395-403. [36] A.M. Brzozowski, A.C. Pike, Z. Dauter, R.E. Hubbard, T. Bonn, O. Engstrom, L. Ohman, G.L. Greene, J.A. Gustafsson, M. Carlquist, Molecular basis of agonism and antagonism in the oestrogen receptor, Nature, 389 (1997) 753-758. [37] S. Mekonen, C. Lachat, A. Ambelu, W. Steurbaut, P. Kolsteren, L. Jacxsens, M. Wondafrash, M. Houbraken, P. Spanoghe, Risk of DDT residue in maize consumed by infants as complementary diet in southwest Ethiopia, Sci. Total Environ, 511 (2015) 454-460. [38] H. Bouwman, B. Sereda, H.M. Meinhardt, Simultaneous presence of DDT and pyrethroid residues in human breast milk from a malaria endemic area in South Africa, Environ. Pollut, 144 (2006) 902-917. [39] A. Diop, Y.M. Diop, D.D. Thiare, F. Cazier, S.O. Sarr, A. Kasprowiak, D. Landy, F. Delattre, Monitoring survey of the use patterns and pesticide residues on vegetables in the Niayes zone, Senegal, Chemosphere, 144 (2016) 1715-1721. [40] C.A. Mullin, M. Frazier, J.L. Frazier, S. Ashcraft, R. Simonds, D. vanEngelsdorp, J.S. Pettis, High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health, Plos One, 5 (2010)9754. [41] C.P. Zhang, H. Zhao, M. Wu, X.Q. Hu, X.M. Cai, L.F. Ping, Z. Li, Simultaneous determination of procymidone, pyridaben and beta-cypermethrin residues in tea solution by gc-ecd, J. Chromatogr. Sci, 50 (2012) 940-944. [42] T. Liu, P. Wang, Y. Lu, G. Zhou, J. Diao, Z. Zhou, Enantioselective bioaccumulation of soil-associated fipronil enantiomers in Tubifex tubifex, J. Hazard Mater, 50 (2012) 219-220. [43] Q. Zhang, C. Wang, X. Zhang, D. Jin, C. Huang, M. Zhao, Enantioselective aquatic toxicity of current chiral pesticides, Journal of environmental monitoring : J.Environ.Monitor, 14 (2012) 465-472. [44] H. Tan, Y. Cao, T. Tang, K. Qian, W.L. Chen, J. Li, Biodegradation and chiral stability of fipronil in aerobic and flooded paddy soils, Sci. Total. Environ, 407 (2008) 428-437. [45] M. Venier, R.A. Hites, Chiral organochlorine pesticides in the atmosphere, Atmos. Environ, 41 (2007) 768-775. [46] G.L. Yuan, Y. Sun, J.X. Qin, J. Li, G.H. Wang, Chiral signature of alpha-HCH and o,p'-DDT in the soil and grass of the Central Tibetan Plateau, China, Sci. Total. Environ, 500-501 (2014) 147-154. [47] C.S. Wong, P.F. Hoekstra, H. Karlsson, S.M. Backus, S.A. Mabury, D.C. Muir, Enantiomer fractions of chiral organochlorine pesticides and polychlorinated biphenyls in standard and certified reference materials, Chemosphere, 49 (2002) 1339-1347. [48] X.Z. Meng, Y. Guo, B.X. Mai, E.Y. Zeng, Enantiomeric signatures of chiral organochlorine pesticides in consumer fish from South China, J. Agric. Food Chem, 57 (2009) 4299-4304. [49] F. Dong, L. Cheng, X. Liu, J. Xu, J. Li, Y. Li, Z. Kong, Q. Jian, Y. Zheng, Enantioselective analysis of triazole fungicide myclobutanil in cucumber and soil under different application modes by chiral liquid chromatography/tandem mass spectrometry, J Agric Food Chem, 60 (2012) 1929-1936. [50] D. Liu, P. Wang, W. Zhu, X. Gu, W. Zhou, Z. Zhou, Enantioselective degradation of fipronil in Chinese cabbage (Brassica pekinensis), Food Chem, 110 (2008) 399-405. [51] W. Jackjines, C. Stophers, M. Jonhnf, K. Neke, A. Waynegarrison, Enantioselective microbial transformation of the phenylpyrazole insecticide fipronil in anoxic sediments,Environ.Sci &Technol, 41 (2007) 8301–8307.

24

[52] C. Corcellas, E. Eljarrat, D. Barcelo, First report of pyrethroid bioaccumulation in wild river fish: a case study in Iberian river basins (Spain), Environ. Int, 75 (2015) 110-116. [53] C.Y. Xu, M.L. Tang, H.H. Zhang, C.L. Zhang, W.P. Liu, Levels and patterns of DDTs in maternal colostrum from an island population and exposure of neonates, Environ. Pollut, 209 (2016) 132-139. [54] A.W. Garrison, M. Cyterski, K.D. Roberts, D. Burdette, J. Williamson, J.K. Avants, Occurrences and fate of DDT principal isomers/metabolites, DDA, and o,p'-DDD enantiomers in fish, sediment and water at a DDT-impacted Superfund site, Environ. Pollut, 194 (2014) 224-234. [55] Y. Huang, Y. Xu, J. Li, W. Xu, G. Zhang, Z. Cheng, J. Liu, Y. Wang, C. Tian, Organochlorine pesticides in the atmosphere and surface water from the equatorial Indian Ocean: enantiomeric signatures, sources, and fate, Environ.Sci &Technol, 47 (2013) 13395-13403. [56] H. Zhang, X. Wang, M. Qian, X. Wang, H. Xu, M. Xu, Q. Wang, Residue analysis and degradation studies of fenbuconazole and myclobutanil in strawberry by chiral high-performance liquid chromatography-tandem mass spectrometry, J. Agric. Food Chem, 59 (2011) 12012-12017. [57] S. Zhou, Q. Tang, M. Jin, W. Liu, L. Niu, H. Ye, Residues and chiral signatures of organochlorine pesticides in mollusks from the coastal regions of the Yangtze River Delta: source and health risk implication, Chemosphere, 114 (2014) 40-50. [58] J. Yan, P. Zhang, X.R. Wang, Y. Wang, Z.Q. Zhou, W.T. Zhu, Stereoselective Degradation of Chiral Fungicide Myclobutanil in Rat Liver Microsomes, Chirality, 26 (2014) 51-55. [59] D. Lu, D. Liu, X. Gu, J. Diao, Z. Zhou, Stereoselective metabolism of fipronil in water hyacinth (Eichhornia crassipes), Pestic. Biochem. Phys, 97 (2010) 289-293. [60] Y. Naude, E.R. Rohwer, Two multidimensional chromatographic methods for enantiomeric analysis of o,p'-DDT and o,p'-DDD in contaminated soil and air in a malaria area of South Africa, Anal. Chim. Acta, 730 (2012) 120-126. [61] F. Li, Q. Xie, X. Li, P. Chi, J. W. Chen, Z. J.Wang, C. Hao, Hormone activity of hydroxylated polybrominated diphenyl ethers on human thyroid receptor-β: in vitro and in silico investigations. Environ. Health Persp, 118 (2009), 602-606. [62] L. Zhe, S. W. Charles, Factors affecting phase I stereoselective biotransformation of chiral polychlorinated biphenyls by rat cytochrome P-450 2B1 isozyme. Environ.Sci &Technol, 45 (2011), 8298-8305. [63] W.J. Hegeman, R.W. Laane, Enantiomeric enrichment of chiral pesticides in the environment, Rev. Environ.Contam, 173 (2002) 85-116. [64] M. Zhao, C. Wang, C. Zhang, Y. Wen, W. Liu, Enantioselective cytotoxicity profile of o,p'-DDT in PC 12 cells, Plos One, 7 (2012) e43823. [65] F. Qin, Y. Gao, P. Xu, B. Guo, J. Li, H. Wang, Enantioselective bioaccumulation and toxic effects of fipronil in the earthworm Eisenia foetida following soil exposure, Pest Manag. Sci, 71 (2015) 553-561. [66] W.A. Wilson, B.J. Konwick, A.W. Garrison, J.K. Avants, M.C. Black, Enantioselective chronic toxicity of fipronil to ceriodaphnia dubia, arc.h environ. Contam. Toxicol, 54 (2008) 36-43. [67] Q. Zhang, C. Wang, Toxicity of binary mixtures of enantiomers in chiral organophosphorus insecticides: the significance of joint effects between enantiomers, Chirality, 25 (2013) 787-792.

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Figure 1. (A) The estrogen agonist activities of 9 chiral pesticides were measured using ER-mediated reporter gene assays. CHO cells were transiently transfected with rERα/pCI, pERE-AUG-Luc and phRL-tk. Then, CHO cells were administered with various concentrations of the test chemicals. Luciferase activity was normalized based on the Renilla luciferase activity of the co-transfected phRL-tk plasmid. The agonist activities are presented as percent induction, with 100% activity defined as the activity achieved with 10-10 M E2. (B) The agonistic effects of 9 chiral pesticides were measured using the E-screen assay. All data were presented as the mean ± SD (standard deviation) of at least three independent assays with 3 to 5 repetition (DMSO did not exceed 0.1%). *Significantly different from the control (DMSO) (p<0.05).

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Figure 2. (A) The estrogen antagonism effects of 9 chiral pesticides were measured using ER-mediated reporter gene assays. CHO cells were transiently transfected with rERα/Pci, pERE-AUG-Luc and phRL-tk. Then CHO cells were co-exposure with various concentrations of the test chemicals and 5 × 10-11 M of E2. Luciferase activity was normalized based on the Renilla luciferase activity of the co-transfected phRL-tk plasmid. The antagonist activities are presented as percent induction, with 100% activity defined as the activity achieved with 5 × 10-11 M of E2. (B) The antagonistic effects of 9 chiral pesticides were measured using the E-screen assay. All data were presented as the mean ± SD (standard deviation) of at least three independent assays with 3 to 5 repetition (DMSO did not exceed 0.1%). *Significantly different from the control (E2) (p<0.05). 27

Figure 3. (A) The thyroid hormone antagonist activities of 9 chiral pesticides were measured using TRβ-mediated reporter gene assays. CHO cells were transiently transfected with pGal4-L-hTRβ, pUAS-tk-luc and phRL-tk. Then CHO cells were co-exposure with various concentrations of the test chemicals and 2 × 10-7 M of T3. Luciferase activity was normalized based on the Renilla luciferase activity of the co-transfected phRL-tk plasmid. The antagonist activities are presented as percent induction, with 100% activity defined as the activity achieved with 2 × 10-7 M of T3. (B) The antagonistic effects of 9 chiral pesticides were measured using the T-screen assay. *Significantly different from the control (T3) (p<0.05).

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Figure 4. Docking of ligands in the binding pocket of hERα and hTRβ. Yellow molecules: references, blue molecules: test chemicals. Estrogen agonist activity: (A) R-(-)-o,p'-DDT and E2, (B) R-(+)-o,p'-DDD and E2. Estrogen antagonist activity: (C) S-(+)-o,p'-DDT and raloxifene, (D) S-(-)-o,p'-DDD and raloxifene, (E) 1R-cis-S-β-CP and raloxifene, (F) R-(-)-FR and raloxifene. Thyroid hormone antagonist activity: (G) R-(-)-MT and 1-(4-hexylphenyl) prop-2-en-1-one, (H) 1R, 3R-cis-αS-λ-CT and 1-(4-hexylphenyl) prop-2-en-1-one.

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