Endocrine-Disrupting Effects of Pesticides through Interference with

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Endocrine Disrupting Effects of Pesticides through Interference with Human Glucocorticoid Receptor Jianyun Zhang, Jing Zhang, Rui Liu, Jay J. Gan, Jing Liu, and Weiping Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03731 • Publication Date (Web): 08 Dec 2015 Downloaded from http://pubs.acs.org on December 13, 2015

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Environmental Science & Technology

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Endocrine Disrupting Effects of Pesticides through Interference with Human Glucocorticoid

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Receptor

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Jianyun Zhanga, Jing Zhangb, Rui Liua, Jay Ganc, Jing Liua,*, Weiping Liub,*

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a

MOE Key Laboratory of Environmental Remediation and Ecosystem Health, bResearch Center for Air

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Pollution and Health, College of Environmental and Resource Sciences, Zhejiang University,

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Hangzhou 310058, China

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c

Department of Environmental Sciences, University of California, Riverside, California 92521

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AUTHOR INFORMATION

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Corresponding Author

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*Fax: +86-571-88982342. E-mail: [email protected] (J.L.); [email protected] (W.L.)

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ABBREVIATIONS

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AR

androgen receptor

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Arg

arginase

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BPA

bisphenol A

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CD-FBS

charcoal/dextran-treated FBS

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CHO-K1

Chinese hamster ovary K1

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DCPH

dicyclohexyl phthalate

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DDT

dichlorodiphenyltrichloroethane

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DMEM

dulbecco's modified eagle medium

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DMSO

dimethylsulfoxide

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EDCs

endocrine-disrupting chemicals

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ER

estrogen receptor

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FBS

fetal bovine serum

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GILZ

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GR

glucocorticoid receptor

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GRE

glucocorticoid response element

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hGRα

human glucocorticoid receptor α

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MeSO2-PCBs

methylsulfone metabolites of polychlorinated biphenyls

glucocorticoid-induced leucine zipper

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ACS Paragon Plus Environment


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MMTV

mouse mammary tumor virus

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PEPCK

phosphoenol pyruvate carboxykinase

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POPs

persistent organic pollutants

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RIC20

20% relative inhibitory concentration

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RLA

relative luciferase activity

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TAT

tyrosine aminotransferase

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Running title: hGR activities of pesticides

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Conflict of interest statement: The authors declare that there is no conflict of interest that would

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prejudice the impartiality of this scientific work.

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ABSTRACT

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Many pesticides have been identified as endocrine-disrupting chemicals (EDCs) due to their ability to

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bind sex-steroid hormone receptors. However, little attention has been paid to the ability of pesticides

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to interfere with other steroid hormone receptors such as glucocorticoid receptor (GR) that plays a

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critical role in metabolic, endocrine, immune and nervous systems. In this study, the glucocorticoidic

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and anti-glucocorticoidic effects of 34 pesticides on human GR were investigated using luciferase

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reporter gene assay. Surprisingly, none of the test chemicals showed GR agonistic activity, but 12

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chemicals exhibited apparent antagonistic effects. Bifenthrin, λ-cyhalothrin, cypermethrin, resmethrin,

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o,p´-DDT, p,p´-DDT, methoxychlor, ethiofencarb and tolylfluanid showed remarkable GR antagonistic

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properties with RIC20 lower than 10-6 M. The disruption of glucocorticoid-responsive genes in H4IIE

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and J774A.1 cells was further evaluated on these 12 GR antagonists. In H4IIEcells, four

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organochlorine insecticides, bifenthrin and 3-PBA decreased cortisol-induced PEPCK gene expression,

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while o,p´-DDT and methoxychlor inhibited cortisol-stimulated Arg and TAT genes expression.

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Cypermethrin and tolyfluanid attenuated cortisol-induced TAT expression. In J774A.1 cells,

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λ-cyhalothrin, resmethrin, 3-PBA, o,p´-DDT, p,p´-DDT, p,p´-DDE, methoxychlor and tolylfluanid

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reduced cortisol-stimulated GILZ expression. Furthermore, molecular docking simulation indicated

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that hydrophobic interactions may stabilize the binding between molecules and GR. Our findings

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suggest that comprehensive screening and evaluation of GR antagonists/agonists should be considered

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to better understand the health and ecological risks of man-made chemicals such as pesticides.

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KEY WORDS: endocrine-disrupting chemicals, steroid hormone receptors, glucocorticoid receptor,

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pesticides, anti-glucocorticoid activity

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TABLE OF CONTENT

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INTRODUCTION An annual amount of 1 to 2.5 million tons of pesticides is applied to agricultural, residential and

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public health protection sites globally.1 These man-made chemicals are deliberately released into the

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environment and their parent compounds or metabolites are often found in the environment and biota.1

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Thus, exposure to pesticides constitutes a type of the most significant chemical pollution in the modern

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era.

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An increasingly number of studies have shown that pesticides and/or their metabolites may act as

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endocrine-disrupting chemicals (EDCs) by interfering with hormone receptors as hormone agonists or

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antagonists.2 Studies demonstrated that some pesticides were capable of binding estrogen receptor (ER)

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and androgen receptor (AR) as xenoestrogens or antiandrogens.3 For example, o,p’-DDT was reported

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to be an estrogen agonist as early as in 1960s,4 and shown to have potential to cause developmental

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feminization of male birds and reproductive tract abnormalities in female birds under field conditions.2

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Studies further revealed that other pesticides and/or their metabolites also exhibited estrogenic and/or

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anti-androgenic activity.3

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Recently, several studies demonstrated that glucocorticoid receptor (GR), one member of the

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steroid hormone receptors to which cortisol and other glucocorticoids bind, was a potential target for

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EDCs.5,6,7,8 GR also plays instrumental roles in metabolic, endocrine, immune and nervous systems.9

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Deletion of GR in transgenic mice resulted in 98% death of perinatal mutant offspring due to severe

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lung atelectasis.10 Loss of GR function in the nervous system of mice caused approximately 10-fold

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increase in glucocorticoid levels that led to symptoms reminiscent of those observed in human Cushing

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syndrome.11 About 30% of GR-impaired mice had depressive-like behaviors and neuroendocrinological

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abnormalities similar to depressed patients.12 On the other hand, transgenic mice over-activating GR

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developed hyperglycemia and decreased insulin secretion which resulted in diabetes.13 Given the vital

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functions of GR, it is valuable to identify potential GR agonists or antagonists among common

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pesticides to expand the understanding of their risks.

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Only one pesticide, tolylfluanid, has been evaluated for GR interfering effects to date. An earlier

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study showed that tolylfluanid competitively bound to GR and functionally acted as an antagonist for

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GR in rat hepatoma cells.7 However, subsequent studies suggested that tolylfluanid activated the GR as

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an agonist in mice adipocytes.6,8 Data regarding the actions of pesticides on GR are in general

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extremely sparse. In this study, we screened 34 pesticides (listed in Table 1) for their GR activities 5



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using luciferase reporter gene assay. The influence of these chemicals on glucocorticoid signaling was

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further validated by examining the expression of three glucocorticoid-responsive genes, e.g., arginase

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(Arg), phosphoenol pyruvate carboxykinase (PEPCK) and tyrosine aminotransferase (TAT), in the rat

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hepatoma cell line H4IIE, as well as the expression of glucocorticoid-induced leucine zipper (GILZ) in

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mouse macrophage cell line J774A.1. Molecular docking was further used to explore the possible

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interactions between these chemicals and the ligand binding pocket of GR. Our finding that many

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pesticides or their metabolites possess anti-glucocorticoidic activities via GR bears significant

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implications for assessing the overall roles of these EDCs-induced health effects, and points to a new

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direction for research.

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MATERIALS AND METHODS

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Chemicals. Cortisol (>98% pure) was purchased from Aladdin Industrial Inc. (ShangHai, China).

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RU486 (>98% pure) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Analytical standards

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of 34 pesticides and metabolites listed in Table 1 were purchased from Sigma-Aldrich (St. Louis, MO).

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All chemicals were dissolved in dimethylsulfoxide (DMSO), except for paraquat that was dissolved in

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deionized water because of its insolubility in DMSO.

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Plasmid constructs. The human glucocorticoid receptor α (hGRα) expression plasmid

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pF25GFP-hGRα and the glucocorticoid response element (GRE) containing reporter plasmid

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pMMTV-luc were kindly provided by Dr. Evangelia Charmandari (Biomedical Research Foundation of

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the Academy of Athens, Greece).14 pRL-TK (Promega, Madison, WI, USA) was used as an internal

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control in the dual-luciferase reporter assays.

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Cell culture. Chinese hamster ovary K1(CHO-K1) cells, rat hepatoma cells H4IIE and mouse

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macrophage cells J774A.1 were maintained in dulbecco's modified eagle medium (DMEM) (Hyclone,

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Logan, UT) supplemented with 10% fetal bovine serum (FBS) (Hyclone,) and 100 U/mL

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streptomycin-penicillin (Hyclone) in an atmosphere of 5% CO2 at 37°C under saturating humidity. For

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reporter gene assay and exposure experiments, the cells were incubated with fresh phenol red-free

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DMEM supplemented with charcoal/dextran-treated FBS (CD-FBS).

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MTS assay. Cells were treated with test chemicals at the concentration of 10-5 or 10-6 M for 24 h. Cell

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viability was measured using CellTiter 96 AQueous One Solution Cell Proliferation (MTS assay)

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(Promega, Madison, WI) as previously described.15,16 The absorbance was measured at 490 nm in the 6


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microplate reader (Infinite M200 PRO, Tecan, Switzerland) to determine the formazan concentration,

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which is proportional to the number of live cells.

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Dual-luciferase Reporter assays for hGRα. CHO-K1 cells were transfected with pF25GFP-hGRα,

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pMMTV-luc, and pRL-TK using Lipofectamine 2000 (Invitrogen, CA). In order to measure the

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agonistic activity of hGRα, cells were treated with either 10-5 or 10-6 M of test compounds or 0.1%

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DMSO (vehicle control). While for the measurement of antagonistic activity, the cells were exposed to

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10-7 M cortisol in combination with test compounds at doses of 10-9 M-10-6 or 10-5 M after 30 min

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pre-treatment with the test compound alone at corresponding concentrations. After an exposure period

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of 24h, firefly luciferase and renilla luciferase activities were measured with fluorescence

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spectrophotometer (Tecan) using the Dual-luciferase Reporter Assay Kit (Promega, Madison, WI) as

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previously described.17 The relative transcriptional activity was presented as the ratio of firefly to

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renilla luciferase activity.

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Real-Time Quantitative PCR. H4IIE and J774A.1 cells were treated with the test chemical at 10-5 or

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10-6 M for 24 h and cells were collected for RNA isolation. Total RNA was extracted using TRIzol®

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Reagent (Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer’s instruction. The quality

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and purity of RNA was determined using spectrophotometer (Tecan). Real-time quantitative PCR was

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performed using the SYBR Green PCR master mix (Toyobo) on Mx3000P Real-time PCR system

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(Agilent Technologies, Palo Alto, CA) as previously described.18 The primer sequences of the

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concerning genes were validated and presented as follows: forward 5’-GGG AAA TCG TGC GTG

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ACA TT-3’and reverse 5’-GCG GCA GTG GCC ATC TC-3’ for rat β-actin; forward 5’-GCC GAG

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GGC CCA CTA AAG-3’and reverse 5’-AGC ATC AAA GGT GGA AGA ATG G-3’ for rat GAPDH;

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forward 5’-CCA GTA TTC ACC CCG GCT AC-3’ and reverse 5’-TTT GCT GTG ATG CCC CAG

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AT-3’ for rat Arg; forward 5’-CCA AGA GCA GAG AGA CAC CG-3’ and reverse 5’-CAC CAC

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ATA GGG CGA GTC TG-3’for rat PEPCK; forward 5’-CAA ACC TCA CTC CTC GTG GT-3’ and

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reverse 5’-CGG TCC CTT CTT TCT CCT TCA-3’ for rat TAT; forward 5’-CCC ACT CCT AAG

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AGG AGG ATG-3’ and reverse 5’-AGG GAG ACC AAA GCC TTC AT-3’ for mouse β-actin;

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forward 5’- TGA CTG CAA CGC CAA AGC-3’ and reverse 5’-CTG ATA CAT TTC GGT GTT CAT

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GGT T-3’ for mouse GILZ . Relative mRNA levels were calculated using the ∆∆ threshold cycle (Ct)

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method and normalized to the endogenous reference gene β-actin as previously described.16

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Molecular Docking and Molecular Dynamic Simulation. Molecular docking software Molegro 7



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Virtual Docker (MVD, version 4.5.0) was used to describe the possible intermolecular binding modes

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of GR with the tested chemicals.The crystal structure of the antagonist form of GR in complex with

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RU486 (entry code 3H52, Brookhaven Protein Data Bank) was chosen as the template.19 The

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antagonists including RU486 were automatically docked into the GR ligand binding pocket, which was

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defined as a sphere with a user-defined origin and a radius of 10Å. Thirty independent MolDock SE

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searching algorithm runs were carried out, and the energetic evaluations of the complexes were

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implemented with the MolDock Score function. Thirty poses at most for each ligand were finally

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generated and saved. Based on the ranking scores and visual inspection, the preferable output

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conformations were selected for furtherstructural analysis.

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Statistical Analysis. The statistical analysis was carried out using SPSS version 16.0 (SPSS,

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Chicago, IL) and Origin 8.0 (OriginLab, Northampton, MA). Data for all experiments were presented

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as the mean ± SD (standard deviation) of at least three independent assays with triplicates. Statistical

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analysis was carried out using one-way ANOVA followed by Dunnett's post hoc test, and differences

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were considered significant for p < 0.05.

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RESULTS

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Agonistic effects of tested chemicals in GR assays. Cortisol, a typical glucocorticoid hormone that

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binds to GR, was used as the positive control for the transactivation of GR. The concentration

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-response curve of GR transactivation by cortisol indicated that the maximal GR transcriptional activity

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was reached at 10-7 M cortisol or higher (Figure S1). Thus, in the subsequent experiments, the agonistic

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effects of tested chemicals were represented as the relative induction rate compared with the GR

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activity induced by 10-7 M cortisol. The results of MTS assay showed that at the concentration of 10-6

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M for resmethrin, carbendazim or tolylfluanid, and 10-5 M for the other compounds, no cytotoxic

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effects were observed (Figure S2). Glucocorticoid-like effects of 34 pesticides and metabolites at

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non-cytotoxic doses were subsequently examined by the dual luciferase reporter gene assay. None of

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the 34 chemicals showed a significant induction at concentrations of 10-5 M or 10-6 M, indicating that

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the tested compounds did not exhibit agonistic effects on GR (Figure S3).

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Antagonistic effects of tested chemicals in GR assays. RU486, a GR antagonist, was used as the

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positive control for evaluating antagonistic effects in GR assays. Among the 34 tested pesticides and

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metabolites, 5 pyrethroids (bifenthrin, λ-cyhalothrin, cypermethrin, resmethrin and 3-PBA), 4 8


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organochlorines (o,p’-DDT, p,p’-DDT, p,p’-DDE and methoxychlor), carbamate insecticide

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ethiofencarb, triazine herbicide atrazine and tolylfluanid fungicide, significantly attenuated

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cortisol-induced GR transactivation at the concentration of 10-5 M or 10-6 M (Figure 1). Luciferase

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activities did not significantly change in the cells lysis after direct incubation with these compounds,

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suggesting that these potential antagonists did not directly inhibit luciferase activities in transactivation

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assays (Figure S4). Therefore, a total of 12 test compounds exhibited GR antagonistic properties under

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the experimental conditions.

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The concentration-dependent GR antagonistic activities of these 12 pesticides and metabolites

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were further determined at doses of 10-9 to10-5 M, except for resmethrin and tolylfluanid where a range

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of 10-9 - 10-6 M was used (Figure 2). RU486 at the concentration of 10-7M or higher markedly reduced

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the response to 10-7M cortisol. The 20% relative inhibitory concentration (RIC20) and the 50% relative

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inhibitory concentration (RIC50), which is the concentrations of the test compounds reducing 20% and

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50% of 10-7 M cortisol-induced GR activity, respectively, were estimated from the concentration

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-response curves for the 12 candidate pesticides (Table 2). The relative inhibition rate (RIR) of these 12

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chemicals at the highest tested concentration (10-5 M or 10-6 M), represented as % decrease of cortisol

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response, is also given in Table 2. Among these GR antagonists, the organochlorine p,p´-DDT,

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pyrethroids resmethrin, and carbamate insecticide ethiofencarb were distinctly more potent than the

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others, with RIC20 of 1.05×10-8 M, 7.77×10-8 and 8.43×10-8 M, respectively (Table 2). Nine of the test

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chemicals, i.e., bifenthrin, λ-cyhalothrin, cypermethrin, resmethrin, o,p´-DDT, p,p´-DDT, methoxychlor,

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ethiofencarb and tolylfluanid, exhibited a potent antagonistic activity against GR with RIC20 lower than

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10-6 M (Table 2). The remaining three compounds, i.e., the pyrethroid metabolite 3-PBA,

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organochlorine metabolite p,p´-DDE, as well as atrazine, showed week antagonistic effects with RIC20

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higher than 10–6 M (Table 2).

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Disruption of glucocorticoid-responsive gene expression. To further validate the anti-glucocorticoid

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potency of the 12 candidate pesticide compounds via GR, the mRNA levels of the liver specific

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glucocorticoid-responsive genes Arg, PEPCK and TAT were determined in the rat hepatoma H4IIE

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cells that express endogenous GR.7,20,21 Dose-dependent gene regulation by cortisol indicated that the

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zenith of Arg, PEPCK and TAT gene expression was achieved at 10-7 M cortisol or higher (data not

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shown). The results of MTS assay showed that resmethrin, o,p’-DDT or tolylfluanid at the

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concentration of 10-6 M, and the other 9 compounds at the concentration of 10-5 M, had no cytotoxic 9



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effects on H4IIE cells (Figure S5). Thus, H4IIE cells were treated with 10-7 M cortisol in combination

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with the tested chemicals at non-cytotoxic concentrations (10-5 M or 10-6 M) and the expression of Arg,

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PEPCK and TAT genes were subsequently examined (Figure 3). As shown in Figure 3A, the

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organochlorine insecticides o,p’-DDT and methoxychlor antagonized cortisol up-regulated Arg gene

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expression by 58.7% and 24.7% compared to the cortisol-treated positive control, respectively. A total

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of 6 test compounds, including bifenthrin, 3-PBA, o,p’-DDT, p,p’-DDT, p,p’-DDE and methoxychlor

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significantly inhibited the cortisol-induced PEPCK expression (Figure 3B). A total of 4 pesticides, i.e.

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cypermethrin, o,p’-DDT, methoxychlor and tolylfluanid, showed a dominant inhibition in TAT

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expression as compared to the cortisol control (Figure 3C). In comparison, ethiofencarb or atrazine did

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not influence the expression of these glucocorticoid-induced genes (Figure 3).

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Furthermore, the mouse macrophage cell line J774A.1 that also expresses endogenous GR22,23 was

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recruited to assess the antagonistic effects of 12 candidate pesticides by measuring the expression of

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GILZ, which is an anti-inflammatory protein induced by glucocorticoid action.24 The results of MTS

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assay indicated that 12 chemicals at the concentration of 10-5 M did not affect the viability of J774A.1

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cells (data not show). J774A.1 cells were treated with the tested chemicals at non-cytotoxic

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concentration of 10-5 M in presence of 10-7 M cortisol and the expression of GILZ was assessed. As

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shown in Figure 3 D, a total of 8 tested pesticide compounds, i.e., λ-cyhalothrin, resmethrin, 3-PBA,

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o,p´-DDT, p,p´-DDT, p,p´-DDE, methoxychlor and tolylfluanid, remarkably reduced

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cortisol-stimulated GILZ expression in J774A.1 cells.

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The expression of all genes was also normalized to GAPDH and exhibited a similar pattern with

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that of normalization to β-actin (data not show). In addition, the relative expression levels of GAPDH

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had no changes after normalization to β-actin, indicating that the reference genes were not influenced

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by chemical treatments (Figure S6).

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Molecular Docking. To better understand the capacity of the 12 pesticide compounds to repress

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glucocorticoid signaling, the interactions between the candidate compounds and the ligand binding

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pocket of the human GR were analyzed using Molegro Virtual Docker. The crystal structure of GR

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with RU486 was used as the initial template for molecular docking. The superimposition of the crystal

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and docked pose of RU486 gives a RMSD of 0.73 Å compared with the original structure, indicating

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that this method with Molegro Virtual Docker had a reasonable repeatability and accuracy (Figure S7). 10


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All 12 pesticides are located deep in the hydrophobic cavity surrounded by the following amino acid

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residues: Leu563, Asn564, Leu566, Gly567, Gly568, Gln570, Trp600, Met601, Met604, Leu608,

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Arg611, Phe623, Gln642, Leu732, Cys736, Met752, Leu753 and Ile756. The association between

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antagonistic rate and docking scores of two major groups of active candidate pesticides, pyrethroids

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and organochlorines, was further evaluated (Figure 4). A simple linear relationship was observed

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between RIR and docking score, with R2 equals to 0.99 and 0.80 for pyrethroids and organochlorines,

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respectively (Figure 4). It suggests that the binding of chemicals to GR is the key step for the

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antagonistic effects. The docking view of 4 active candidate pesticides (3-PBA, methoxychlor,

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tolylfluanid and atrazine) and 2 inactive pesticides (β-BHC and Carbaryl) was showed in Figure S8. In

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comparison with the inactive chemicals, the antagonistic pesticides had lower docking score and some

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of them had hydrogen bonds (Figure S8). However, for hydrogen bonds, there are also large differences

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among the active pesticides with methoxychlor having a polar surface area of 18.5 Å and 2 hydrogen

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acceptor sites, whereas tolyfluanid has a polar surface area of 74.3 Å and 6 hydrogen acceptor sites.

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Thus different chemicals may bind to GR through different interactions.

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DISCUSSION A large number of man-made chemicals, including pesticides, were previously evaluated for

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sex-steroid disrupting activities.3 For example, screening of 200 pesticides for their estrogenicity via

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hERα/β and anti-androgenicity via hAR identified a number of xenoestrogens and antiandrogens

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among pesticides.3 However, to our knowledge, there is little information on effects of these chemicals

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on non-sexual hormonal functions, such as the GR-mediated glucocorticoidic activity. In the present

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study, we evaluated 34 pesticide compounds from 8 groups, including both legacy and current-use

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pesticides. Surprisingly, none of the test chemicals showed GR agonistic activity, but 12 of the 34

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candidate chemicals exhibited apparent antagonistic effects. For these compounds, with the exception

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of tolylfluanid,6,7,8 this study represented the first time that their GR antagonistic effects have been

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observed.

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Organochlorine insecticides are of global concern and have been extensively studied as potential

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EDCs due to their persistence and ubiquitous occurrence.25,26 Here we demonstrated that the four BHC

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isomers did not show agonistic or antagonistic activity toward GR. However, the DDT analogues, 11



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including o,p´-DDT, p,p´-DDT, and p,p´-DDE, and methoxychlor, were found to exert

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anti-glucocorticoidic activity through GR. The internal exposure levels of total DDT in the general

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population of developed countries were reported to be from 10 to 117 ppb.27 In some developing

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countries where DDT is still used for malaria control, including several African countries, the median

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levels of p,p’-DDT and p,p’-DDE in human plasma were reported to be 530 ppb and 1259 ppb,

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respectively.28 In a downstream DDT-sprayed area in South Africa, extremely high concentrations of

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p,p´-DDT and p,p´-DDE were detected in freshwater fish Oreochromis mossambicus with mean levels

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reaching 5.9 ppm and 7.6 ppm, respectively.29 The RIC20 of the anti-glucocorticoidic activities of

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p,p’-DDT and p,p’-DDE found in this study were 3.7 ppb (1.05×10-8 M) and 352.5 ppb (1.11×10-6 M),

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respectively. Therefore, in areas where DDT products are still used, the residue levels of DDT and

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DDE in human or wildlife may approach or exceed these RIC20, suggesting potential adverse effects on

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human or wildlife through the disruption of GR functions. Although the serum levels of DDT

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analogues in populations of developed countries are considerably lower than these RIC20 values,

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evaluation of potential risks is still warranted because these compounds are persistent and highly

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bioaccumulative.30 Notably, all DDT analogues tested in this study significantly inhibited the

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expression of PEPCK gene in rat hepatic cells. PEPCK is a glucocorticoid-regulated enzyme that

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catalyzes the rate-controlling step of gluconeogenesis.31 Animal studies showed that liver-specific

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PEPCK null mice developed dramatic hepatic steatosis.32 In a recent study, significantly higher DDT

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levels were found in the glaucous gulls (Larus hyperboreus) with hepatic steatosis, as compared to the

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gulls without hepatic steatosis in Svalbard.33 The anti-glucocorticoidic activities of DDT may be one of

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the possible causes for the DDT-induced hepatic steatosis in wildlife. Meanwhile, all tested DDT

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analogues reduced the cortisol-stimulated GILZ expression, which suggesting that DDT may also

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disrupt the glucocorticoid related anti-inflammatory and immunosuppressive response.

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Pyrethroids have become a major class of insecticides with an increasingly heavy use in

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agricultural and residential areas.34,35 The common urine metabolite of most pyrethroids, 3-PBA, has a

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detection frequency > 70% in the general U.S. population,34 and > 80% in Australia, China and

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Japan .36,37,38 The urine concentrations of 3-PBA observed in the general populations ranged from 0.01

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to 89.7 ppb,36,39,40 while higher levels were found in an occupationally exposed population with levels

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up to 261 ppb.41 For the first time, we recognized pyrethroids and their metabolite 3-PBA as potential

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anti-glucocorticoid compounds. Our results showed that 4 of the 6 pyrethroids considered in this study 12


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exhibited a potent antagonistic activity against GR, especially cypermethrin and resmethrin, with RIC20

310

of 43.9 ppb and 26.3 ppb (1.05×10–7 M and 7.77×10–8), respectively. Additionally, 3-PBA was also an

311

anti-glucocorticoid compound with a RIC20 of 384.7 ppb (1.80×10–6 M). Although the single RIC20 of

312

each pyrethroid is higher than the reported internal exposure levels, further research is merited given

313

the additive or synergistic effects of the cumulative exposure to mixtures of pyrethroids and/or their

314

metabolites.

315

Carbamate insecticides are yet another heavily used group of pesticides.1 In this study we tested

316

three carbamates and found that ethiofencarb showed a low RIC20 (19.0 ppb). Although ethiofencarb

317

has been banned in the United States because of its hazardous properties, it is still used in Asia, Europe

318

and some developing countries in America with detectable residues in agricultural products42.

319

Additional screening of other commonly used carbamate pesticides may be also necessary.

320

Atrazine has been one of the most widely used herbicides for the last few decades and is one of the

321

most frequently detected pesticide contaminants in groundwater and surface water43,44. The urinary

322

atrazine has also been found in agricultural workers with the maximum level up to 68 ppb.45 In this

323

study, atrazine was found to possess weak GR antagonistic activity with a RIC20 of 530.5ppb. However,

324

atrazine did not have significant effects on the expression of GR downstream genes. The effects of

325

atrazine on the glucocorticoid homeostasis need to be further validate through future research.

326

Tolylfluanid, an active fungicide ingredient, is widely applied in Europe, Australia and New

327

Zealand for agricultural disease control and also used in antifouling paints.46 A previous study

328

suggested that tolylfluanid acted as a GR antagonist, and it could bind to GR competitively and

329

diminish glucocorticoid-induced TAT activity in rat hepatoma cells.7 In subsequent studies, however, it

330

was observed that tolylfluanid activated GR and modulated adipogenesis of mice adipocyte-like cells

331

and primary adipocytes.6,8 In the present study, tolylfluanid suppressed the cortisol-induced GR

332

transactivation and the expression of glucocorticoid-responsive gene TAT in rat H4IIE cells. The

333

reduction of cortisol-stimulated GILZ expression in macrophage cells also supported this result.

334

Molecular docking also suggested hydrogen bonding between the sulfonyl group of tolylfluanid and the

335

γ-amide of Gln570, which concurs with the previous hypothesis that the methyl sulfonyl group is an

336

important structure associated with the ligand binding domain of GR.7 Our findings support the notion

337

that tolylfluanid is an antagonist against GR. The inconsistent observations in published studies on

338

tolylfluanid could be explained by the differential presence/absence of specific GR 13



339

co-activator/repressors in specific cell types that may impact ligand specificity.

340

Four DDT analogues tested in this study showed relatively potent and consistent GR antagonistic

341

activity in different assessments when compared to other class of pesticides. All these DDT analogues

342

not only repressed GR transactivation, but also inhibited PEPCK expression in hepatocyte and

343

down-regulated GILZ expression in macrophage. Although four pyrethroids showed

344

anti-glucocorticoid activity in GR transactivation assays, each pyrethroid only inhibited one target of

345

the cortisol-induced gene induction in the hepatocyte. Pyrethroids are all esters and are rapidly

346

degraded by hydrolytic and oxidative metabolism.47 It can be speculated that the diminished

347

antagonistic effects for pyrethroids in hepatocyte may be attributed to the degradation of pyrethroids.

348

However, the underlying mechanisms are not completely understood and need to be fully clarified. The

349

results also indicate that multiple assessments should be used to accurately assess the effects of EDCs.

350

More than a third of pesticides and metabolites considered in this study exhibited

351

anti-glucocorticoidic activity. However, the list of test compounds represented only a small sample size

352

of the great number of pesticide active ingredients. Further research should consider an expanded list of

353

pesticides and other man-made chemicals for GR antagonist/agonist screening. Even though these

354

pesticides are generally weak antagonists against GR, it should be emphasized that humans and wildlife

355

are constantly exposed to numerous environmental stressors including pesticides and their metabolites.

356

Therefore, there exists a likelihood that additive or synergistic effects from multiple EDCs may occur,

357

and such effects should be evaluated under relevant environmental conditions.

358 359 360

ACKNOWLEDGMENTS

361

This work was supported by the National Nature Science Foundation of China (21377113 and

362

21320102007) and Zhejiang Provincial Natural Science Foundation of China (LR15B070001).

363 364

Supporting Information Available

365

Additional experimental information contains 1table and 8 figures. This information is available free of

366

charge via the Internet at http://pubs.acs.org

367 368 14


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535

FIGURE LEGENDS

536

Figure 1. Antagonistic effects of pesticides in the hGR transactivation assays. CHO-K1 cells

537

transfected with pF25GFP-hGRα, pMMTV-luc and pRL-TK were incubated with positive control (10-7

538

M cortisol), vehicle control (0.1% DMSO), antagonist control (10-7 M RU486) in the presence of 10-7

539

M cortisol or pesticides at highest non-cytotoxic concentration in the presence of 10-7 M cortisol. (A)

540

Antagonistic effects of 10-5 M of bifenthrin, λ-cyhalothrin, cypermethrin, fenvalerate, permethrin,

541

3-PBald, 3-PBA, 3-PBalc and 10-6 M of resmethrin. (B) Antagonistic effects of 10-5 M ofα-BHC,

542

β-BHC, δ-BHC, λ-BHC, o,p´-DDT, p,p´-DDT, p,p´-DDE and methoxychlor. (C) Antagonistic effects of

543

10-5 M of chlorpyrifos, diazinon, parathion-ethyl, carbaryl, ethiofencarb, fenobucarb, atrazine, DEA and

544

DIA. (D) Antagonistic effects of 10-5 M of metolachlor, S-metolachlor, benomyl, fipronil, fomesafen,

545

paraquat and 10-6 M of carbendazim and tolylfluanid. Values represent the mean ± SD of triplicate

546

measurements in three independent experiments and are presented as percent induction, with 100%

547

activity defined as the activity achieved with 10-7 M cortisol (positive control).

548

compared with positive control (=100%).

*p < 0.05, **p

Endocrine-Disrupting Effects of Pesticides through Interference with

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