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Developmental Effects and Estrogenicity of Bisphenol A Alternatives in a Zebrafish Embryo Model Xiyan Mu, Ying Huang, Xuxing Li, Yunlei Lei, Miaomiao Teng, Xuefeng Li, Chengju Wang, and Yingren Li Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06255 • Publication Date (Web): 03 Feb 2018 Downloaded from http://pubs.acs.org on February 4, 2018
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Developmental Effects and Estrogenicity of Bisphenol A
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Alternatives in a Zebrafish Embryo Model
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Xiyan Mua*, Ying Huanga, Xuxing Lia, Yunlei Leia, Miaomiao Tengb, Xuefeng Lib,
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Chengju Wangb, Yingren Lia*
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a
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Sciences, Beijing, People’s Republic of China. 100141.
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b
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China. 100193.
Fishery Resource and Environment Research Center, Chinese Academy of Fishery
College of Sciences, China Agricultural University, Beijing, People’s Republic of
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*Corresponding authors:
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Xiyan Mu
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[email protected] 13
+86-010-68673951
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No.150 of Qingta, Yongding Road, Fengtai District, Beijing, People’s Republic of
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China. 100141
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ABSTRACT
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In order to understand the negative effects of bisphenol A (BPA) alternatives
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comprehensively, zebrafish embryos were used to assess the lethality, developmental
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effects and estrogenic activity of bisphenol analogues. The in silico estrogenic
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activities of bisphenol analogues were assayed by binding simulation. According to
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our results, the lethality of bisphenol analogues decreased in order of BPAF > BPA >
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BPF > BPS. BPAF and BPF induced significant effects on zebrafish embryos,
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including decreased heart rate, hatching inhibition and teratogenic effects. The
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binding potentials of bisphenol analogues towards zfERs decreased in the following
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order: BPAF > BPA > BPF > BPS. Among the three subtypes of zfERs, zfERβ2
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showed the highest binding activity towards the bisphenols, followed by zfERα and
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zfERβ1. In vivo estrogenic activity tests showed that BPAF, BPA and BPF
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significantly enhanced the protein levels of ERα along with the mRNA levels of esr1,
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esr2a, esr2b and vtg1 in zebrafish embryos. Esr2b showed the strongest response to
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BPAF and BPA exposure among the three esrs. In contrast, BPS did not significantly
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regulate ER protein level or ER transcription. In conclusion, BPAF showed the
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highest lethality, developmental effects and estrogenic activity (both in silico and in
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vivo) followed by BPA and BPF. BPS showed the weakest toxicity and estrogenic
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activity. zfERβ2 might act as the main target among the three ER subtypes of
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zebrafish after exposure to BPAF and BPA.
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KEYWORDS: Zebrafish embryos; Bisphenol analogues; Developmental effects;
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Lethality; Estrogenic activity 2
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INTRODUCTION
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Bisphenols are a family of compounds based on the bisphenol structure that are
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applied as important raw materials in epoxy resin and are widely used in the chemical,
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food and pharmaceutical industries. Bisphenol A (BPA), which is the most commonly
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used bisphenol compound, has been shown to have estrogenic effects and induce a
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variety of human diseases (e.g., obesity and reproductive diseases).1-5 Considering the
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adverse effects of BPA, several countries such as Canada and the European Union
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have banned the use of BPA.6,7 At the same time, the industrial use of BPA congeners
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has increased.8 Currently, bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF
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(BPAF) are frequently used as alternatives to BPA. BPF is applied in lining materials,
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flooring materials, coatings, plastics, pharmaceuticals and food packaging.9 BPS is
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mainly used in epoxy glue, can coatings and thermal paper.10 BPAF is used as a
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crosslinking agent for fluoroelastomers, electronic and optical fibers and as a
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copolymer of polyimides, polyamides and polyesters.11 With their increased
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application, the detection of bisphenol analogues has gradually increased in various
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products. In addition to commodities like cosmetics, canned foods and beverages,12,13
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bisphenol analogues have been detected in indoor dust,14 sediments,13 sewage
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sludge,15 surface water and the human body.16-18 While the detected levels of BPA
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alternatives are generally lower than those of BPA, some are on the same order of
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magnitude 19-21 or even higher.22,23 Ye et al. (2015) showed that the BPS and BPF
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levels in the urine of adults in the United States increased every year from 2010 to
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2014.24 3
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Theoretically, alternatives used to replace a chemical material of concern should
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be non-toxic or at least far less toxic than the original chemical. However, many
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chemical substitutes are unassessed before being placed on the market, and, in some
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cases, are similar enough to the original chemical to cause concern.5 BPF, BPS and
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BPAF are structurally similar to BPA; thus, they are likely to have similar
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physiological effects on organisms. Several in vitro tests demonstrated that both BPF
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and BPS had estrogenic and anti-androgenic activities. Rosenmai et al. (2014) found
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that BPS and BPF caused the same qualitative effects on estrogen receptor (ER) and
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androgen receptor activities in a human ovarian adenocarcinoma cell line, and they
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exhibited similar potencies as BPA.25 These findings indicate that the substitution of
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BPA with structural analogues should be carried out with caution. Kitamura et al.
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(2005) detected the estrogenic activities of several bisphenol compounds using human
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breast cancer cells and found that BPAF had the highest estrogenic activity (induction
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of ERE-luciferase activity) with an EC50 of 16.8 µg/L.2 They also reported that the
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EC50 values of BPF and BPS were 200.2 and 275.3 µg/L, respectively, which were
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close to that of BPA (143.8 µg/L). Despite the in vitro estrogenic activity of BPA
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substitutes, their in vivo impacts (e.g., lethality, developmental effects and immune
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response) are not completely clear. Therefore, it is necessary to identify and compare
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the hazards posed by BPA alternatives to screen for safe BPA replacements.
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Currently, zebrafish (Danio rerio) embryos are an important model in the risk
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assessment and toxicological investigation of hazardous chemicals.26-28 Zebrafish
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possess many advantages, including in vitro fertilization, high fecundity, rapid 4
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embryonic development and optical transparency, that make it easy to detect
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morphological endpoints and observe the development process in early life stages.29
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The aim of this study was to comprehensively identify the toxic effects of BPA and its
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three commonly used alternatives (BPS, BPF and BPAF) by comparing their lethality,
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developmental effects and estrogenic activity (both in vivo and in silico) using
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zebrafish embryo model.
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MATERIALS AND METHODS
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Zebrafish maintenance and embryo collection
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Adult wild-type zebrafish (strain AB; approximately 10 months old) were obtained
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from China Zebrafish Resource Center (Wuhan, China). All adult zebrafish were
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maintained in flow-through feeding equipment (Esen Corp. Beijing, China) at 26°C
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with a photoperiod of 14/10 (light/dark) and fed daily with live brine shrimp (Artemia
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salina). The preparation and collection of zebrafish embryos followed the procedure
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described in our previous work.30
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Chemicals and reagents
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The standard water was prepared in the lab with the formula of iso-7346-3.31 99%
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BPA (CAS: 80-05-7), 99% BPS (CAS: 80-09-1) and 99% BPAF (CAS: 1478-61-1)
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were purchased from J&K Scientific (Beijing, China). 99% BPF (CAS: 620-92-8) was
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purchased from Tokyo Chemical Industry (Tokyo, Japan). The stock solution of
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bisphenols used for exposure was prepared using acetone AR. All other reagents
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utilized were of analytical grade.
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Exposure and sample collection
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Experiments were performed in accordance with current Chinese legislation and
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were approved by the independent animal ethics committee at the Chinese Academy
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of Fishery Sciences.
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Lethality test
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Test solutions of four bisphenol analogues were prepared using standard water with
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different series of geometric concentrations on the basis of pre-experiments. Embryos
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during 16-cells stage (1.5-1.7 hours post-fertilization) were randomly transferred into
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test solutions in 24-well plates. Twenty wells were used in each plate, and each well
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contained 2 mL of exposure solution and one embryo. Both a blank control and
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solvent control were established. Each test concentration and control was replicated
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three times. The exposure lasted four days. The median lethal concentration (LC50)
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values and 95% confidence limits were calculated by probit regression analysis using
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SPSS 16.0 software. Please see the Supporting Information for exposure details.
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Developmental toxicity test
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Zebrafish embryos were exposed to solutions of bisphenol analogues at 5%, 25%
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and 50% of the LC50 concentration (0.1, 0.5 and 1.0 mg/L for BPAF; 0.5, 2.5 and 5
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mg/L for BPA; 1, 5 and 10 mg/L for BPF; and 2.5, 12.5 and 25 mg/L for BPS,
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respectively). Since the LC50 of BPS was not obtained, we used the highest
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concentration tested in the lethality test as the LC50 to calculate its concentrations for
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the developmental toxicity and estrogenic activity tests. The exposure method and
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quality control were the same as in the lethality test. Embryos during 16 cells-stage
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were randomly transferred into test solutions in 24-well plates. Twenty wells were
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used in each plate, and each well contained 2 mL of exposure solution and one
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embryo. Both a blank control and solvent control were established. Each test
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concentration and control was replicated 3 times.
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Estrogenic activity test
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Embryos were exposed to bisphenol analogue solutions at 1%, 10% and 50% of the
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LC50 concentration (0.02, 0.2 and 1.0 mg/L for BPAF; 0.1, 1 and 5 mg/L for BPA; 0.2,
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2 and 10 mg/L for BPF; and 0.5, 5 and 25 mg/L for BPS, respectively) in 1-L beakers.
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Each beaker contained 500 mL of exposure solution and about 100 embryos, and there
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were three beakers (replicate) in each treatment group. At 96 hours post fertilization
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(hpf), 60 hatched (or dechorionated) larvae from each replicate (beaker) were
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collected and washed twice with standard water. The embryo samples were averaged
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into two tubes (one for protein extraction and the other for RNA extraction) and stored
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at -80°C until analysis.
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Detection of morphological endpoints
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The number of dead individuals and the stage of embryonic development were
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determined daily. For embryos, death was judged using the lethal toxicological
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endpoints proposed by Nagel (2002).32 Larvae that had no heartbeat under
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micro-observation were considered to be dead. The hatching and development status
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of embryos were checked daily. The heart rate at 48 hpf and spontaneous movement
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(systemic twist of embryo) at 24 hpf were observed using a microscope. Five live
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embryos were selected from each replicate to count the times of heartbeat and
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spontaneous movement in 20s under microscope observation. Teratogenic effects
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were identified and recorded using a ZEISS Vert.A1 microscope (Jena, Germany).
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Docking studies
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The binding modes of the four bisphenol compounds to three ER subtypes (ERα,
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ERβ1 and ERβ2) were analyzed using homology modeling, molecular docking and
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molecular dynamics (MD) simulation methods. The sequences of the three ER
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subtypes were downloaded from the Universal Protein Resource (UniProt). The
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template for sequence alignment was identified through searching the Protein Data
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Bank (PDB) using the BLASTp program provided by UniProt with the default
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parameters. The three-dimensional (3D) structures of the ERs were downloaded from
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the RCSB PDB (ERα PDB ID: 1u9e; ERβ1 PDB ID: 1yye; ERβ2 PDB ID: 2yja) as
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the template structures. The 3D structures of the different ER subtypes were obtained
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using homology modeling with Modeller software. Subsequently, preliminary MD
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simulations were performed to refine the modeled protein structures. After that, four
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bisphenol ligands were docked into the three ER subtypes using Autodock tools, and
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the binding modes of 12 structurally diverse, representative binders were selected
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using molecular docking, (molecular mechanics-generalized born surface area,
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MM-GBSA) ranking and MD simulations. The MD simulations were carried out
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using AMBER11 software, and hydrogen bonds, hydrophobic interactions, free
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energy and other terms were analyzed to further understand the binding modes
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between ligands and ER subtypes. More details are provided in the Supporting
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Information.
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ELISA Tests
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The protein level of ERα was measured via ELISA. For each sample, 30 embryos
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were homogenized with saline on ice. The supernatant was collected for ELISA after
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centrifugation for 10 min at 3000 rpm/min and 4°C. ELISA was conducted according
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to the instructions provided by the ELISA Kit (Dongge Biotechnology Co., Ltd,
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Beijing, China). Details of the ELISA tests are provided in the Supporting
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Information.
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Gene expression analysis
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Total RNA was extracted from zebrafish embryos (30 embryos for each sample)
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using a spin column adsorption method following the manufacturer's protocols
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(Tiangen Biotech, Beijing, China). The concentration was measured based on the
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absorbance at 260 nm using a UV1240 spectrophotometer (PerkinElmer, Waltham,
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MA, USA). The purity was assessed by determining the A260/A280 ratio. RNA
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samples with A260/A280 ratio of 1.8-2.0 were selected for the next step. cDNA was
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then synthesized via reverse transcription using a quant RTase kit (Tiangen Biotech)
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in accordance with the manufacturer's recommendations. Real-time PCR reactions
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were performed using an ABI 7500 q-PCR system (Applied Biosystems, Carlsbad,
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CA, USA). SYBR Green PCR Master Mix reagent kits (Tiangen Biotech) were used
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for the quantification of gene expression. A 20-µL reaction system was used,
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according to the manufacturer's instructions (Table S1). Zebrafish-specific primers
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were designed for the genes of interest using Primer 5.0 software (Table S2). The
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housekeeping gene β-actin was used as an internal control. The stability of beta-actin
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after bisphenols exposure was validated using Bestkeeper and GeNorm.33,34 Please see
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Supporting Information for details. The PCR amplification procedure was as follows:
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95°C for 15 min followed by 40 cycles at 95°C for 10 s, 60°C for 20 s and 72°C for
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32 s. Quantification of the transcripts was performed using the 2-δδCt method. The
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amplification efficiencies of the housekeeping gene and target genes under the
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conditions above are given in the Supplementary Information (Table S3).
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Chemical confirmation of bisphenol analogues
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Exposure solutions in each replicate for all treatments were analyzed twice,
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respectively, at the beginning of exposure and after 24 h post-exposure (hpe) before
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the first replacement. Water samples (1 mL) were centrifuged at 4℃ under 12000
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rpm/min for 15 min and then the supernatant passed through a 0.22 µm filter. 1 µL
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filtrate was used for analysis. Samples were analyzed using the Dionex Ultimate 3000
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UPLC system which was coupled to a TSQ Quantiva Ultra triple-quadrupole mass
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spectrometer (Thermo Fisher, CA), and equipped with a heated electrospray
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ionization (HESI) probe in negative ion mode. Data analysis and quantitation were
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performed by the software Xcalibur 3.0.63 (Thermo Fisher, CA). Analysis results
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indicated that the deviations between nominal and actual bisphenol analogue
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concentrations were less than 20% (Table S4-S6). Since all test solutions in this
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research were renewed daily, thus the nominal dosage is able to represent the actual
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content in this work. Please see Supporting Information for details of
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chromatographic conditions and analytical data.
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Statistical analysis All statistical analyses were undertaken using SPSS 16.0 software. Differences
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were determined by one-way ANOVA and completed by Dunnett post hoc comparison.
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P < 0.05 was considered significant.
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RESULTS
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Solvent effect
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According to the statistics, there was no significant difference between the solvent
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and blank control for all tested indicators (data not shown). The data for the control
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shown in all figures (and tables) are the experimental data for the solvent control.
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Lethality of bisphenol analogues
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The 96h-LC50 values of BPAF, BPA and BPF towards zebrafish embryos were 1.95,
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10.43 and 19.59 mg/L, respectively (Table S7). No significant death was found in the
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group exposed to 50 mg/L BPS, indicating that the toxicity of BPS is much lower than
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those of other three bisphenols. In summary, the acute toxicity of the four bisphenol
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analogues towards zebrafish embryos decreased in the following order: BPAF >
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BPA > BPF > BPS.
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Bisphenol analogues induced developmental effects
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The developmental effects of the four bisphenols at dosages of 5%, 25% and 50%
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of LC50 were assessed. Significant hatch inhibition was observed in the groups
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exposed to BPAF and BPF (Figure 1). BPF significantly inhibited the embryo
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hatching ratio by 15% and 64% at the 25% and 50% LC50 concentrations (5 and 10
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mg/L), respectively, at 72 hpf. A 20% decrease in hatching ratio was observed in the
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group exposed to 50% LC50 (1 mg/L) BPAF. In contrast, BPA and BPS showed no
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apparent influence on hatching under the tested concentrations.
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BPAF, BPF and BPA produced significant reductions in the spontaneous
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movements of zebrafish embryos (Figure S1). The effective concentrations were 1.0
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(BPAF), 5.0 (BPA) and 5.0 (BPF) mg/L. There was no significant difference between
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the groups exposed to BPS and the control.
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BPAF apparently reduced the embryonic heart rate at 48 hpf. The heart rates of
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zebrafish embryos in the groups treated with 25% and 50% LC50 of BPAF were 88%
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and 77% those of the control group, respectively (Figure 2). Similarly, BPF
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significantly reduced heart rate, and the level of reduction increased with increasing
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dosage. No obvious effects on heart rate were observed in the BPA- and BPS-treated
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groups under the tested concentrations.
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Significant teratogenic effects, including yolk sac edema, pericardial edema and
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spine deformation, were induced by BPAF (Figure S2). The ratio and extent of
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deformity increased with increasing BPAF concentration. BPA also had the ability to
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cause malformation, although both the effective concentration and deformation ratio
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for BPA were lower than for BPAF. BPF produced significant pigmentation reduction
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in zebrafish embryos at 48 hpf. The pigmentations of the eyes, yolk sac and notochord
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of embryos in the BPF-exposure groups were significantly reduced compared to the
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control group (Figure 3A). The percentages of pigmentation reduction were 35% (1.0
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mg/L), 57% (5.0 mg/L) and 82% (10 mg/L; Figure 3B). In contrast, BPS did not
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generate any abnormities at the tested concentrations.
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Binding potential of bisphenol analogues towards zfERs
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Molecular binding between the four bisphenol analogues and three zebrafish ER
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subtypes was modeled. Figure 4A and Figure S3 illustrated the binding sites of
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bisphenols docked at the ERs subtypes. In the docked complexes, hydrogen bonds
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were formed between bisphenol analogues and Arg362, His492 and Glu321 of ERα.
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Similarly, a hydrogen bond was formed between bisphenol analogues and Arg379,
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His509 and Glu338 of ERβ1; and between bisphenol analogues and Arg364, His494,
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Glu323 of ERβ2. The four bisphenol compounds have the same binding sites towards
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the same subtype (Figure S3).
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The in silico results showed that the order of binding potentials was BPAF > BPA >
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BPF > BPS, and the corresponding binding energies were -16.86, -14.91, -12.42 and
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-10.05 kcal/mol for ERα, respectively, -13.77, -13.08, -11.49 and -11.23 kcal/mol for
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ERβ1, respectively, and -20.47, -18.59, -16.70 and -14.26 kcal/mol for ERβ2,
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respectively. Among the three zfERs subtypes, zfERβ2 showed the strongest binding
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potential towards the four bisphenol analogues (Figure 4C). The binding energies of
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zfERα towards the four bisphenol analogues were higher than those of zfERβ1, with
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the exception of the binding energy for BPS.
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Bisphenol analogues induced ERα protein level
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BPA, BPF and BPAF significantly enhanced the protein level of ERα after 96 h of 13
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exposure (Figure 4). BPAF showed the strongest induction of ERα, and the effective
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concentration was 1% LC50 (0.02 mg/L). The effective concentrations of BPA and
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BPF were 10% LC50 (1.0 mg/L) and 50% LC50 (10 mg/L), respectively. BPS did not
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induce ERα at the tested concentrations.
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Bisphenol analogues induced ERs-coding genes transcription
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The changes in the transcription of esr1, esr2a and esr2b were further assessed via
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q-PCR test. All bisphenol compounds significantly up-regulated the transcription
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levels of the three subtypes of ERs at concentrations of 50% LC50 with the exception
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of BPS, which showed no influence on ER gene expression (Figures S5A–C). The
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effective concentrations for the induction of ER transcription were 0.2, 1.0 and 10
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mg/L for BPAF, BPA and BPF, respectively. In contrast, 0.02 mg/L BPAF inhibited
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the transcriptions of esr2a and esr2b. In addition, the ER down-stream target gene
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vtg1 also showed significant up-regulation after bisphenol analogues exposure, and
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the effective concentration was 0.02 mg/L (BPAF), 0.1 (BPA) and 2 mg/L (BPF)
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respectively (Figure S5D).
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The mRNA levels of genes related to sex hormone homeostasis were measured
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after exposure to bisphenol analogues to identify the downstream effects of esr
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disruption. Significant elevation of CYP19a1 and HSD17b1 was observed in the
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groups treated with BPA, BPAF and BPF (Figures S6 A, C). The level of CYP17a1
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was significantly enhanced by 0.1 mg/L BPA and 10 mg/L BPF (Figure S6 B). No
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obvious modification of mRNA level was observed in the BPS-treated groups.
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DISCUSSION
With their increasing use in industry, BPA alternatives have become widely
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distributed in multiple environmental media, and their risks towards humans and the
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environment are drawing more and more attention. In vitro studies have identified
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various toxic effects of BPA substitutes, including endocrine disruption, oxidative
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stress, immune toxicity and reproductive toxicity.35-39 While the present study was
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intended to identify and compare the toxic effects of BPA and its alternatives using
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zebrafish embryo in vivo tests and in silico modeling.
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Lethality of bisphenol analogues
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According to the 96h acute toxicity test, all of the bisphenol analogues could induce
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significant lethality in zebrafish embryos under tested concentration except BPS
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which showed no significant effect on zebrafish survival at 2.5-50 mg/L. Among the
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four bisphenol analogues, BPAF showed the highest lethality towards zebrafish
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embryo with a LC50 value of 1.95 mg/L. Maćczak et al. (2017) investigated the in
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vitro oxidative damage capacities of BPA, BPAF, BPF and BPS and reported that
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BPAF induced the strongest alterations in ROS formation, lipid peroxidation and
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antioxidant enzyme activity. In contrast, BPS did not affect most of the studied
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parameters.37 Tisler et al. (2016) assessed the toxicities of three bisphenol compounds
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(BPAF, BPA and BPF) and found that BPAF was the most toxic towards both
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zebrafish and Daphnia magna.40 These results are in good agreement with the present
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study. 15
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Developmental effects of bisphenol analogues
According to the results of developmental toxicity tests, BPAF and BPF
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significantly influenced zebrafish embryo development, resulting in decreased heart
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rate and hatching inhibition. In contrast, BPA and BPS had no influence on heart rate
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and hatch time under the tested concentrations, which agrees with previous studies
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that demonstrated that BPA did not alter heart rate or hatch time at concentrations of
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0.2–1 mg/L.41-42 Similar with the lethality test, BPAF exhibited the strongest
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developmental effect towards zebrafish embryos, with an effective concentration of
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0.1 mg/L. BPS possessed the lowest developmental influence, as no apparent effects
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were observed in embryos treated with BPS (2.5–25 mg/L).
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The most obvious teratogenic effect induced by BPAF was yolk sac edema. Yolk
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sac is the nutrient reserve organ in zebrafish embryos and provides the nutrients
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needed for embryonic development. In fish eggs, the endogenous lipid reserves,
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which are mainly phospholipids and triacylglycerols, are in the form of yolk
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globules.43 The alteration of lipid metabolism may affect the yolk sacs.44,30 Previous
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studies demonstrated that BPA can increase adipocyte cell density and induce hepatic
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lipid accumulation in rats and mice, suggesting that lipid metabolism is a main
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pathway of BPA.45-48 Thus, the yolk sac abnormities induced by bisphenol analogues
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might originate from the disruption of lipid metabolism. BPF showed strong
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pigmentation inhibition under the tested concentrations (1–10 mg/L) at 48–96 hpf,
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while the other bisphenol analogues did not have similar effects. The mechanism of
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BPF-induced decreases in pigmentation needs to be further studied and
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tyrosinase-mediated melanin production is a possible target.49-50 Based on the above
347
discussion, the occurrence of the specific abnormities indicated that the active
348
pathways of bisphenol compounds in zebrafish embryos are not exactly the same.
349
Estrogenic effects of bisphenol analogues
350
The estrogenic potencies of bisphenol analogues have been the subject of many
351
investigations. Reported in vitro data demonstrate that the potencies of BPS and BPF
352
for estrogenic activities are similar to that of BPA.51 In addition, the
353
endocrine-disrupting effects of BPAF, including thyroid disruption and estrogenic
354
effects, have been observed in fish and mammals.52,53,35
355
In silico
356
ERs mediate critical physiological events in the endocrine and reproductive
357
systems.54 While mammals have been found to have two nuclear ER subtypes (ERα
358
and ERβ), teleost fish have at least three (ERα, ERβ1 and ERβ2).55 ERα and ERβ act
359
as ligand-inducible transcription factors upon binding estradiol (E2), which is the
360
primary endogenous ER ligand.56 To investigate the potentials of the four bisphenol
361
analogues as zfERs ligands, we performed a docking survey to verify the ER binding
362
modes of the bisphenols. According to our results, the binding modes of the four
363
bisphenols towards zfERs are similar (Figure 4, S1). The BP-binding cavity of zfERα
364
was located at the end of the zfERα molecule, resulting in stable interactions through
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hydrogen bonds with Arg362, Glu321 and His 492. This result is similar to that
366
reported for E2 binding with human ESR1 (hESR1),57 in which hydrogen bond
367
interactions were found between the E2 A-ring phenolic hydroxyl radical and the
368
side-chain residues of Glu353 (H3) and Arg394 (H6) at one end and between the E2
369
D-ring hydroxyl radical and His524 (H11) at the other end. Comparative analyses
370
indicated that the bisphenol analogues display similar ER binding modes with E2,
371
which may partly explain how bisphenols mimic endogenous ligands to disrupt the
372
endocrine system. The differences in receptor selectivity among the four bisphenol
373
compounds might be due to the different substitution patterns on the bisphenol
374
backbone structure (Figure 4B). According to the modeled ligand, the binding pocket
375
was surrounded by a hydrophobic cavity, which formed a strong hydrophobic area
376
(Figure 4D). BPAF showed the strongest effect because of its large hydrophobic
377
radicals (–CF3), which form a strong hydrophobic–hydrophobic interaction with the
378
hydrophobic cavity around the protein binding pocket. The ligand bridge radical of
379
BPA (–CH3) is less hydrophobic compared to –CF3, which makes its binding energy
380
towards ERs weaker than that of BPAF. Since there is no hydrophobic radical in the
381
ligand bridge chain of BPF, the BPF–zfERs binding energies are weaker than those of
382
BPA–zfERs and BPAF–zfERs. In contrast, the ligand bridge radical of BPS (O=S=O)
383
is hydrophilic and may exclude the amino acids of the hydrophobic pocket. Thus, BPS
384
had the weakest binding potential towards zfERs among the four bisphenol analogues.
385
Among the three subtypes of zfERs, zfERβ2 showed the highest binding potential
386
with bisphenol analogues followed by ERα and zfERβ1. Matsushima et al. (2010)
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387
reported that the binding activity of the BPAF receptor was three times higher for ERβ
388
(IC50 =18.9 nM) than for ERα (IC50 = 53.4 nM),58 which is consistent with our
389
results. However, Molina-Molina et al. (2013) also reported that BPA and BPF
390
activated both hERα and hERβ, whereas BPA and BPF were more active in the hERα
391
assay than in the hERβ assay,59 which contradicts the present study. The distinct
392
differences in binding potential might be due to the different amino acid sequences of
393
the ERs between species.
394
In vivo
395
To further understand the estrogenic activities of BPA and its alternatives, an in
396
vivo comparative study was conducted. According to our results, both BPF and BPAF
397
enhanced the protein level of ERα in zebrafish embryos, similar to BPA. In contrast,
398
BPS showed no significant effect on ERα under the tested concentrations. The
399
effective concentrations of BPAF, BPA and BPF were 0.02, 1.0 and 10 mg/L,
400
respectively. This is consistent with the in silico results, which indicated that the
401
binding potentials of the four bisphenol compounds towards zfERα decreased in the
402
following order: BPAF > BPA > BPF > BPS. Chen et al. (2016) summarized the
403
estrogenic potencies of bisphenol analogues based on the relative estrogenic potency
404
compared to BPA (the potency value of BPA was defined as 1). They reported that the
405
potency value in different studies ranged from 2.81 to 12.6 for BPAF (median = 6.67),
406
0.10 to 4.83 for BPF (median = 0.62), and 0.0001 to 0.90 for BPS (median = 0.16),
407
indicating that the estrogenic activities of the four bisphenols decreased in the order of
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BPAF > BPA > BPF > BPS.51 Rosenmai et al. (2014) determined the effects of BPF
409
and BPS on ER activity and found that the estrogenic potency of BPF was of the same
410
order of magnitude as that of BPA, while BPS was less estrogenic than BPA.25 The
411
results of the above studies are in good agreement with the results of the present
412
study.
413
Multiple ER genes are present in zebrafish, including esr1, esr2a and esr2b, which
414
code for ERα, ERβ1 and ERβ2 proteins, respectively.60 To compare the contributions
415
of the three subtypes of ERs in bisphenol-induced estrogenic activation, we further
416
assayed the disruption of the transcriptions of esr1, esr2a and esr2b by bisphenol
417
analogues. The transcription levels of the three types of ERs were enhanced by BPAF,
418
BPF and BPA; esr2b showed the strongest response to BPAF exposure with the lowest
419
LOEC value compared to esr1 and esr2a (Table 1). As shown in Figure 5, esr2b and
420
esr1 showed stronger responses to BPA exposure than esr2a. These results are
421
consistent with the computational results and suggest that among the three ER
422
subtypes in zebrafish, ERβ2 might be the main target of BPAF and BPA. However, no
423
apparent changes in transcription were observed for the three ER subtypes after
424
exposure to BPS. A previous study demonstrated that bisphenol compounds could
425
enhance the mRNA levels of ERs. Yamaguchi et al. (2015) reported that BPAF and
426
BPA could significantly increase the transcription level of esr1 in the livers of male
427
medaka (Oryzias latipes), and the LOEC values were 0.5 µM (≈ 0.168 mg/L) and 50
428
µM (≈ 11.4 mg/L), respectively.61 In this study, BPAF significantly inhibited the
429
mRNA levels of esr2a and esr2b at the lowest tested concentration (0.02 mg/L). This 20
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result is consistent with previous results indicating that BPAF acts as an agonist of
431
ERβ at higher concentrations and as an antagonist of ERβ at lower concentrations.62
432
Vtg1 is another important indicator for estrogenic activation. Previous studies have
433
shown that BPA and BPAF could significantly induce vtg1 level in adult zebrafish and
434
rare minnow (Gobiocypris rarus).52,63 In this study, BPA and BPAF significantly
435
elevated the transcription of vtg1 at 1% of LC50, and the effective concentrations are
436
lower than that of genes encoding ER, which is in consistent with a previous study.64
437
Furthermore, BPAF and BPF also significantly enhanced the transcription of genes
438
related to estradiol homeostasis, revealing their potential to disrupt sex hormone
439
levels.
440
Taking the results of modeling and in vivo tests together, BPAF showed the highest
441
estrogenic activity followed by BPA and BPF. However, BPS exhibited no effect on
442
the protein and mRNA levels of ERs, and its binding potentials with the three ER
443
subtypes were lower than those of the other bisphenol compounds. These results are
444
consistent with previous in vitro studies involving human breast cancer cells and
445
MCF-7 cells.65,59
446
Table 1. The effective concentrations (EC) of four tested bisphenol analogues for disrupting ER
447
transcription.
esr1
esr2a
esr2b
vtg1
BPA
1
1
1
0.1
BPF
10
10
10
2
BPS
>25
>25
>25
>25
EC (mg/L)
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BPAF
0.2
0.2
0.02
0.02
448
449
450
Risk identification of bisphenol analogues
Risk identification was conducted by calculation the risk ratio (Table S8) between
451
(1) MECs (Measured Environmental Concentrations) and the EC (effective
452
concentration) values obtained in estrogenic effect test (Table S9); (2) MECs and EC
453
values obtained in developmental toxicity test; (3) MECs and LC50 values.
454
The detected concentrations in surface waters are ranging from 0.0046 to 28 µg/L.
455
21, 22, 16, 66-68
456
however, the detected level of the three alternatives are close to BPA. The detected
457
range of BPF, BPS and BPAF was 0.0068-24.98 µg/L, 0.006-7.2 µg/L and
458
0.0082-15.3 µg/L respectively. According to Table S8, the risk ratio of BPF are
459
basically in the same order of magnitude as BPA, and the risk ratio of BPAF are even
460
higher than that of BPA. For BPS, the EC values are at least 3, 500 times higher than
461
the detected levels in environmental waters. Taken together, BPA alternatives (BPAF
462
and BPF) possess equivalent or stronger risk than BPA towards zebrafish based on the
463
results of the present study. As the environmental concentrations of BPA alternatives
464
will increase in the future based on their increasing application, much higher risk to
465
aquatic organisms should be predicted.
466
Although fewer MECs values exist for BPF, BPS and BPAF,15,16, 21,70
According to our results, BPS might be a relatively safe alternative for BPA
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467
because of its low acute lethality, developmental toxicity and estrogenic activity
468
towards zebrafish embryos compared with BPF and BPAF. However, the chronic
469
effects of long-term exposure to BPS, including sex hormone disruption and
470
reproductive alteration, are cause for concern.71,72
471
In this study, we compared the toxicity and estrogenic effects of BPA and its
472
alternatives using zebrafish embryos as model to reflect their environmental risks.
473
BPAF showed the highest lethality, developmental toxicity and estrogenic activity
474
(both in silico and in vivo), followed by BPA and BPF. BPS showed the weakest
475
toxicity and estrogenic activity. In addition, zfERβ2 might act as the main target
476
among the three ER subtypes of zebrafish after exposure to BPAF and BPA. To the
477
best of our knowledge, this is the first study to systematically compare and identify
478
the negative effects of the four most commonly used bisphenol compounds based on
479
lethality, developmental effects and estrogenic activity.
480
ACKNOWLEDGEMENTS
481
This research work was supported by the National Natural Science Foundation of
482
China (No. 21607173) and the Special Funds of Conservation of Species Resources
483
from Chinese Ministry of Agriculture “Ecological Environment Monitoring of
484
Chinese Fishery Water Areas”.
485
AUTHOR DECLARATION
486
We declare that we have no actual or potential competing financial interest and no part
487
of this paper has published or submitted elsewhere.
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488 489 490
SUPPORTING INFORMATION Details of exposure. Details in silico simulation. Details of qPCR. Details of ELISA test. Details of chemical analysis. Details of risk identification.
491
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REFERENCES
493
[1] Gould, J.; Leonard, L.; Maness, S.; Wagner, B.; Conner, K.; Zacharewski, T.; Safe,
494
S.; McDonnell, D.; and Gaido, K. Bisphenol A interacts with the estrogen receptor
495
alpha in a distinct manner from estradiol. Mol. Cell. Endocrinol. 1998, 142,
496
203-214.
497
[2] Kitamura, S.; Suzuki, T.; Sanoh, S.; Kohta, R.; Jinno, N.; Sugihara, K.; Yoshihara,
498
S.; Fujimoto, N.; Watanabe, H.; and Ohta, S. Comparative study of the
499
endocrine-disrupting activity of bisphenol A and 19 related compounds. Toxicol.
500
Sci. 2005, 84, 249-259.
501
[3] Yoshihara, S.; Mizutare, T.; Makishima, M.; Suzuki, N.; Fujimoto, N.; Igarashi, K.;
502
Ohta, S. Potent estrogenic metabolites of bisphenol A and bisphenol B formed by
503
rat liver S9 fraction: Their structures and estrogenic potency. Toxicol. Sci. 2004,
504
78 (1), 50−59.
505
[4] Li, Y.; Luh, C. J.; Burns, K. A.; Arao, Y.; Jiang, Z.; Teng, C. T.; Tice, R. R.; Korach,
506
K. S. Endocrine-Disrupting Chemicals (EDCs): In vitro mechanism of estrogenic
507
activation and differential effects on er target genes. Environ. Health. Perspect.
508
2013, 121 (4), 459-466.
24
ACS Paragon Plus Environment
Environmental Science & Technology
509
[5] Rochester, J. R.; Bolden, A. L. Bisphenol S and F: a systematic review and
510
comparison of the hormonal activity of bisphenol A substitutes. Environ. Health.
511
Perspect. 2015, 123(7), 643-650.
512
[6] Government of Canada. Order Amending Schedule I to the Hazardous Products
513
Act (Bisphenol A), Part II, 144, 7. 2010. Government of Canada. Available at:
514
http://www.chemicalsubstanceschimiques.gc.ca/challenge-defi/batch-lot-2/bisphen
515
ol-a/bpa-risk hazard-eng.php. Accessed February 21, 2014.
516
[7] The European Commission. Commission directive 2011/8/EU of 28 January 2011
517
amending Directive 2002/72/EC as regards the restriction of use of Bisphenol A in
518
plastic infant feeding bottles. 2011. Off. J. Eur. Union 10-05-2013.
519
[8] Danzl, E.; Sei, K.; Soda, S.; Ike, M.; Fujita, M. Biodegradation of bisphenol A,
520
bisphenol F and bisphenol S in seawater. Int. J. Environ. Res. 2009, Public Health,
521
6 (4), 1472-1484.
522
[9] Fiege, H.; Voges, H. W.; Hamamoto, T.; Umemura, S.; Iwata, T.; Miki, H. Phenol
523
derivatives. In: Ullmann’s Encyclopedia of Industrial Chemistry. 2000, Winheim,
524
Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 643-647.
525 526 527
[10] Clark, E. Sulfolane and sulfones. In: Kirk-Othmer Encyclopedia of Chemical Technology. 2012. New York, NY:John Wiley & Sons. [11] Song, S.; Ruan, T.; Wang, T.; Liu, R.; Jiang, G. Distribution and preliminary
528
exposure assessment of bisphenol AF (BPAF) in various environmental matrices
529
around a manufacturing plant in China. Environ. Sci. Technol. 2012, 46,
530
13136-13143.
25
ACS Paragon Plus Environment
Page 26 of 42
Page 27 of 42
Environmental Science & Technology
531
[12] Liao, C.; Kannan, K. A survey of alkylphenols, bisphenols, and triclosan in
532
personal care products from China and the United States. Arch. Environ. Contam.
533
Toxicol. 2014, 67, 50-59.
534
[13] Liao, C.; Liu, F.; Moon, H.; Yamashita, N.; Yun, S.; Kannan, K. Bisphenol
535
analogues in sediments from industrialized areas in the United States, Japan, and
536
Korea: spatial and temporal distributions. Environ. Sci. Technol. 2012a, 46,
537
11558-11565.
538
[14] Liao, C.; Liu, F.; Guo, Y.; Moon, H. B.; Nakata, H.; Wu, Q.; Kannan, K.
539
Occurrence of eight bisphenol analogues in indoor dust from the united states and
540
several asian countries: implications for human exposure. Environ. Sci. Technol.
541
2012b, 46, 9138-9145.
542
[15] Song, S.; Song, M.; Zeng, L.; Wang, T.; Liu, R.; Ruan, T.; Jiang, G. Occurrence
543
and profiles of bisphenol analogues in municipal sewage sludge in China. Environ.
544
Pollut. 2014, 186, 14-19.
545
[16] Jin, H.; Zhu, L. Occurrence and partitioning of bisphenol analogues in water and
546
sediment from Liaohe River Basin and Taihu Lake, China. Water Res. 2016, 103,
547
343-351.
548
[17] Xue, J.; Wu, Q.; Sakthivel, S.; Pavithran, P. V.; Vasukutty, J. R.; Kannan, K.
549
Urinary levels of endocrine-disrupting chemicals, including bisphenols, bisphenol
550
A diglycidyl ethers, benzophenones, parabens, and triclosan in obese and
551
non-obese Indian children. Environ. Res. 2015, 137, 120-128.
552
[18] Vandenberg, L. N.; Chahoud, I.; Heindel, J. J.; Padmanabhan, V.; Paumgartten, F.
26
ACS Paragon Plus Environment
Environmental Science & Technology
553
J.; Schoenfelder, G. Urinary, circulating, and tissue biomonitoring studies indicate
554
widespread exposure to bisphenol A. Environ. Health. Perspect. 2010, 118,
555
1055-1070.
556
[19] Yang, Y.; Lu, L.; Zhang, J.; Yang, Y.; Wu, Y.; Shao, B. Simultaneous
557
determination of seven bisphenols in environmental water and solid samples by
558
liquid chromatography electrospray tandem mass spectrometry. J. Chromatogr. A.
559
2014a, 1328, 26-34.
560
[20] Yang, Y.; Guan, J.; Yin, J.; Shao, B.; Li, H. Urinary levels of bisphenol analogues
561
in residents living near a manufacturing plant in south China. Chemosphere 2014b,
562
112, 481-486.
563
[21] Yan, Z.; Liu, Y.; Yan, K.; Wu, S.; Han, Z.; Guo, R.; Chen, M.; Yang, Q.; Zhang,
564
S.; Chen, J. Bisphenol analogues in surface water and sediment from the shallow
565
Chinese freshwater lakes: Occurrence, distribution, source apportionment, and
566
ecological and human health risk. Chemosphere 2017, 184, 318-328.
567
[22] Yamazaki, E.; Yamashita, N.; Taniyasu, S.; Lam, J.; Lam, P. K.; Moon, H. B.;
568
Jeong, Y.; Kannan, P.; Achyuthan, H.; Munuswamy, N.; Kannan, K. Bisphenol A
569
and other bisphenol analogues including BPS and BPF in surface water samples
570
from Japan, China, Korea and India. Ecotoxicol. Environ. Saf. 2015, 122,
571
565-572.
572
[23] Asimakopoulos, A. G.; Xue, J.; De Carvallo, B. P.; Iyer, A.; Abualnaja, K. O.;
573
Yaghmoor, S. S.; Kumosani, T. A.; Kannan, K. Urinary biomarkers of exposure to
574
57 xenobiotics and its association with oxidative stress in a population in Jeddah,
27
ACS Paragon Plus Environment
Page 28 of 42
Page 29 of 42
Environmental Science & Technology
575 576
Saudi Arabia. Environ. Res. 2015, 150, 573-581. [24] Ye, X.; Wong, L.; Kramer, J.; Zhou, X.; Jia, T.; Calafat, A. M. Urinary
577
concentrations of bisphenol A and three other bisphenols in convenience samples
578
of U.S. adults during 2000−2014. Environ. Sci. Technol. 2015, 49, 11834-11839.
579 580 581
[25] Rosenmai, A. K.; Dybdahl, M.; Pedersen, M. Are structural analogues to bisphenol A safe alternatives? Toxicol. Sci. 2014, 139(1), 35-47. [26] Zhang, X.; Zhou, Q.; Zou, W.; Hu, X. Molecular mechanisms of developmental
582
toxicity induced by graphene oxide at predicted environmental concentrations.
583
Environ. Sci. Technol. 2017, 51 (14), 7861-7871.
584
[27] Meredith, A. N.; Harper, B.; Harper, S. L. The influence of size on the toxicity of
585
an encapsulated pesticide: a comparison of micron- and nano-sized capsules.
586
Environ. Int. 2016, 86, 68-74.
587
[28] Lee, O.; Takesono, A.; Tada, M.; Tyler, C. R.; Kudoh, T. Biosensor Zebrafish
588
Provide New insights into potential health effects of environmental estrogens.
589
Environ Health Perspect. 2012, 120 (7), 1104433.
590
[29] Mu, X.; Chai, T.; Wang, K.; Zhu, L.; Huang, Y.; Shen, G.; Li, Y.; Li, X.; Wang, C.
591
The developmental effect of difenoconazole on zebrafish embryos: A mechanism
592
research. Environ. Pollut. 2016, 212, 18-26.
593
[30] Mu, X.; Pang, S.; Sun, X.; Gao, J.; Chen, J.; Chen, X.; Li, X.; Wang, C.
594
Evaluation of acute and developmental effects of difenoconazole via multiple
595
stage zebrafish assays. Environ. Pollut. 2013, 175, 147-157.
596
[31] ISO. Water Quality e Determination of the Acute Lethal Toxicity of Substances
28
ACS Paragon Plus Environment
Environmental Science & Technology
597
to a Freshwater Fish [Brachy danio rerio Hamiltone Buchanan (Teleostei,
598
Cyprinidae)]. In: Part 3: Flow-through Method. 1996.
599
[32] Nagel, R. DarT: the embryo test with the zebrafish Danio rerio-a general model
600
in ecotoxicology and toxicology. ALTEX, 2002, 19, 38-48.
601
[33] Pfaffl, M. W.; Tichopad, A.; Prgomet,C. Determination of Stable Housekeeping
602
Genes, Differentially Regulated Target Genes and Sample Integrity:
603
BestKeeper-Excel-Based Tool Using Pair-Wise Correlations. Biotechnol. Lett.
604
2004, 26(6), 509-515.
605
[34] Vandesompele, J.; Preter, K.; Pattyn, F.; Poppe, B.; Van Roy, N.; De Paepe, A.;
606
Speleman, F. Accurate normalization of real-time quantitative RT-PCR data by
607
geometric averaging of multiple internal control genes. Geno. Biol. 2002, 3,
608
research0034.1.
609
[35] Donoghue, L. J.; Neufeld, T. I.; Li, Y.; Arao, Y.; Coons, L. A.; Korach, K. S.
610
Differential activation of a mouse estrogen receptor β isoform (merβ2) with
611
endocrine-disrupting chemicals (EDCs). Environ. Health. Perspect. 2017, 125 (4),
612
634-642.
613
[36] Malaisé, Y.; Ménard, S.; Cartier, C.; Lencina, C.; Sommer, C.; Gaultier, E.;
614
Houdeau, E.; Guzylack-Piriou, L. Consequences of bisphenol a perinatal exposure
615
on immune responses and gut barrier function in mice. Arch. Toxicol. 2017, 92(1),
616
347-358.
617 618
[37] Maćczak, A.; Cyrkler, M.; Bukowska, B.; Michałowicz, J. Bisphenol A, bisphenol S, bisphenol F and bisphenol AF induce different oxidative stress and
29
ACS Paragon Plus Environment
Page 30 of 42
Page 31 of 42
Environmental Science & Technology
619
damage in human red blood cells (in vitro study). Toxicol. In Vitro 2017, 41,
620
143-149.
621
[38] Fic, A.; Mlakar, S. J.; Juvan, P.; Mlakar, V.; Marc, J.; Dolenc, M. S.; Broberg, K.;
622
Mašič, L. P. Genome-wide gene expression profiling of low-dose, long-term
623
exposure of human osteosarcoma cells to bisphenol A and its analogs bisphenols
624
AF and S. Toxicol. In Vitro 2015, 29(5), 1060-1069.
625
[39] Viñas, R.; Watson, C. S. Bisphenol S disrupts estradiol-induced nongenomic
626
signaling in a rat pituitary cell line: effects on cell functions. Environ. Health.
627
Perspect. 2013, 121(3), 352-358.
628
[40] Tisler, T.; Krel, A.; Gerzelj, U.; Erjavec, B.; Dolenc, M. S.; Pintar, A. Hazard
629
identification and risk characterization of bisphenols A, F and AF to aquatic
630
organisms. Environ. Pollut. 2016, 212, 472-479.
631
[41] Huang, Q.; Fang, C.; Chen, Y.; Wu, X.; Ye, T.; Lin, Y.; Dong, S. Embryonic
632
exposure to low concentration of bisphenol A affects the development of Oryzias
633
melastigma larvae. Environ. Sci. Pollut. Res. Int. 2011, 19(7), 2506-2514.
634
[42] Huang, Q.; Chen, Y.; Lin, L.; Liu, Y.; Chi, Y.; Lin, Y.; Ye, G.; Zhu, H.; Dong, S.
635
Different effects of bisphenol a and its halogenated derivatives on the
636
reproduction and development of Oryzias melastigma under environmentally
637
relevant doses. Sci. Total. Environ. 2017, 595, 752-758.
638 639 640
[43] Wiegand, M. D. Composition, accumulation and utilization of yolk lipids in teleost fish. Rev. Fish Biol. Fisheries 1996, 6, 259-286. [44] Hermsen, S. A. B.; Pronk, T. E.; Brandhof, E.; Ven, L. T. M.; Piersma, A. H.
30
ACS Paragon Plus Environment
Environmental Science & Technology
641
Chemical class-specific gene expression changes in the zebrafish embryo after
642
exposure to glycol ether alkoxy acids and 1,2,4-triazole antifungals. Reprod.
643
Toxicol. 2011, 32, 245-252.
644
[45] Lejonklou, M. H.; Dunder, L.; Bladin, E.; Pettersson, V.; Rönn, M.; Lind, L.;
645
Waldén, T. B.; Lind, P. M. effects of low-dose developmental bisphenol A
646
exposure on metabolic parameters and gene expression in male and female fischer
647
344 rat offspring. Environ. Health. Perspect. 2017, 125(6), 067018.
648
[46] Yang, S.; Li, T.; Zhang, A.; Gao, R.; Peng, C.; Liu, L.; Cheng, Q.; Mei, M.; Song,
649
Y.; Xiang, X.; Wu, C.; Xiao, X.; Li, Q. Dysregulated autophagy in hepatocytes
650
promotes bisphenol A (BPA)-induced hepatic lipid accumulation in male mice.
651
Endocrinology 2017, 158(9), 2799-2812.
652
[47] Lv, Q.; Gao, R.; Peng, C.; Yi, J.; Liu, L.; Yang, S.; Li, D.; Hu, J.; Luo, T.; Mei,
653
M.; Song, Y.; Wu, C.; Xiao, X.; Li, Q. Bisphenol A promotes hepatic lipid
654
deposition involving Kupffer cells M1 polarization in male mice. J Endocrinol.
655
2017, 234(2), 143-154.
656
[48] Susiarjo, M.; Xin, F.; Stefaniak, M.; Mesaros, C.; Simmons, R. A.; Bartolomei,
657
M. S. Bile acids and tryptophan metabolism are novel pathways involved in
658
metabolic abnormalities in BPA-exposed pregnant mice and male offspring.
659
Endocrinology 2017, 158(8), 2533-2542.
660 661 662
[49] Korner, A.; Pawelek, J. Mammalian tyrosinase catalyzes three reactions in the biosynthesis of melanin. Science 1982, 217, 1163-1165. [50] Lai, X.; Wichers, H. J.; Soler-Lopez, M.; Dijkstra, B. W. Structure of human
31
ACS Paragon Plus Environment
Page 32 of 42
Page 33 of 42
Environmental Science & Technology
663
tyrosinase related protein 1 reveals a binuclear zinc active site important for
664
melanogenesis. Angew. Chem. Int. Ed. Engl. 2017, 56(33), 9812-9815.
665
[51] Chen, D.; Kannan, K.; Tan, H.; Zheng, Z.; Feng, Y.; Wu, Y.; Widelka, M.
666
Bisphenol analogues other than BPA: environmental occurrence, human exposure,
667
and toxicity-a review. Environ. Sci. Technol. 2016, 50 (11), 5438-5453.
668
[52] Shi, J.; Jiao, Z.; Zheng, S.; Li, M.; Zhang, J.; Feng, Y.; Yin, J.; Shao, B.
669
Long-term effects of Bisphenol AF (BPAF) on hormonal balance and genes of
670
hypothalamus-pituitary-gonad axis and liver of zebrafish (Danio rerio), and the
671
impact on offspring. Chemosphere 2015, 128, 252-257.
672
[53] Yang, X.; Liu, Y.; Li, J.; Chen, M.; Peng, D.; Liang, Y.; Song, M.; Zhang, J.;
673
Jiang, G. Exposure to bisphenol af disrupts sex hormone levels and vitellogenin
674
expression in zebrafish. Environ. Toxicol. 2016, 31 (3), 285-294.
675
[54] Henley, D. V.; Korach, K. S. Physiological effects and mechanisms of action of
676
endocrine disrupting chemicals that alter estrogen signaling. Hormones 2010, 9
677
(3), 191-205.
678
[55] Yost, E.; Pow, C. L.; Hawkins, M. B.; Kullman, S. W. Bridging the gap from
679
screening assays to estrogenic effects in fish: potential roles of multiple estrogen
680
receptor subtypes. Environ. Sci. Technol. 2014, 48, 5211-5219
681 682 683 684
[56] Hall, J. M.; McDonnell, D. P. Coregulators in nuclear estrogen receptor action: from concept to therapeutic targeting. Mol. Interv. 2005, 5: 343-357. [57] Gao, Y.; Li, X.; Guo, L. H. Assessment of estrogenic activity of perfluoroalkylacids based on ligand-induced conformation state of human
32
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Environmental Science & Technology
685 686
estrogen receptor. Environ. Sci. Technol. 2013, 47 (1), 634-641. [58] Matsushima, A.; Liu, X.; Okada, H.; Shimohigashi, M.; Shimohigashi, Y.
687
Bisphenol AF is a full agonist for the estrogen receptor erα but a highly specific
688
antagonist for erβ. Environ. Health. Perspect. 2010, 118 (9), 1267-1272.
689
[59] Molina-Molina, J.; Amaya, E.; Grimaldi, M.; Sáenz, J.; Real, M.; Fernández, M.
690
F.; Balaguer, P.; Olea, N. In vitro study on the agonistic and antagonistic activities
691
of bisphenol-S and other bisphenol-A congeners and derivatives via nuclear
692
receptors. Toxicol. Appl. Pharmacol. 2013, 272, 127-136.
693
[60] Gorelick, D. A.; Iwanowicz, L. R.; Hung, A. L.; Blazer, V. S.; Halpern, M. E.
694
Transgenic Zebrafish Reveal Tissue-Specific Differences in Estrogen Signaling in
695
Response to Environmental Water Samples. Environ. Health. Perspect. 2014, 122
696
(4), 356-362.
697
[61] Yamaguchi, A.; Ishibashi, H.; Arizono, K.; Tominaga, N. In vivo and in silico
698
analyses of estrogenic potential of bisphenol analogs in medaka (Oryzias latipes)
699
and common carp (Cyprinus carpio). Ecotoxicol. Environ. Saf. 2015, 120,
700
198-205.
701
[62] Li, Y.; Burns, K. A.; Arao, Y.; Luh, C. J.; Korach, K. S. Differential estrogenic
702
actions of endocrine-disrupting chemicals bisphenol A, bisphenol AF, and
703
zearalenone through estrogen receptor α and β in vitro. Environ. Health. Perspect.
704
2012, 120 (7), 1029-1035.
705
[63] Zhang, Y.; Yuan, C.; Hu, G.; Li, M; Zheng, Y.; Gao, J.; Yang, Y.; Zhou, Y.; Wang,
706
Z. Characterization of four nr5a genes and gene expression profiling for testicular
33
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Page 34 of 42
Page 35 of 42
Environmental Science & Technology
707
steroidogenesis-related genes and their regulatory factors in response to bisphenol
708
A in rare minnow Gobiocypris rarus. Gen. Comp. Endocr. 2013, 194, 31–44
709
[64] Zhang, Y.; Gao, J.; Xu, P.; Yuan, C.; Qin, F.; Liu, S.; Zheng, Y.; Yang, Y.; Wang,
710
Z. Low-dose bisphenol A disrupts gonad development and steroidogenic genes
711
expression in adult female rare minnow Gobiocypris rarus. Chemosphere 2014,
712
112, 435-442.
713
[65] Mesnage, R.; Phedonos, A.; Arno, M.; Balu, S.; Christopher, C. J.; Antoniou, M.
714
N. Transcriptome profiling reveals bisphenol A alternatives activate estrogen
715
receptor alpha in human breast cancer cells. Toxicol. Sci. 2017, 158(2), 431-443.
716
[66] Gong, J.; Ran, Y.; Chen, D.; Yang, Y.; Ma, X. Occurrence and environmental risk
717
of endocrine disrupting chemicals in surface waters of the Pearl River, South
718
China. Environ. Monit. Assess. 2009, 156, 199-210.
719
[67] Kang, J. H.; Kondo, F. Bisphenol A in the surface water and freshwater snail
720
collected from rivers around a secure landfill. Bull. Environ. Contam. Toxicol.
721
2006, 76, 113-118.
722
[68] Bhandari, R. K.; Deem, S. L.; Holliday, D. K.; Jandegian, C. M.; Kassotis, C. D.;
723
Nagel, S. C.; Tillitt, D. E.; Vom Saal, F. S.; Rosenfeld, C. S. Effects of the
724
environmental estrogenic contaminants bisphenol A and 17a-ethinyl estradiol on
725
sexual development and adult behaviors in aquatic wildlife species. Gen. Comp.
726
Endocrinol. 2015, 214, 195-219.
727
[69] Yang, Y.; Guan, J.; Yin, J.; Shao, B.; Li, H. Urinary levels of bisphenol analogues
728
in residents living near a manufacturing plant in south China. Chemosphere 2014,
34
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729 730
112, 481-486. [70] Fromme, H.; Kuchler, T.; Otto, T.; Pilz, K.; Muller, J.; Wenzel, A. Occurrence of
731
phthalates and bisphenol A and Fin the environment. Water Res. 2002, 36,
732
1429-1438.
733
[71] Ji, K.; Hong, S.; Kho, Y.; Kyungho, C. Effects of bisphenol S exposure on
734
endocrine functions and reproduction of zebrafish. Environ. Sci. Technol. 2013,
735
47, 8793-8800.
736
[72] Naderi, M.; Wong, M. Y. L.; Gholami, F. Developmental exposure of zebrafish
737
(Danio rerio) to bisphenol-Simpairs subsequent reproduction potential and
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hormonal balancein adults. Aquat. Toxicol. 2014, 148, 195-203.
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Figure 1 Hatch ratio of zebrafish embryos at 48, 72 and 96 hpf after exposure of BPAF (A), BPF (B), BPA (C) and BPS (D). Asterisks denote significant differences
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between treatments and the control (determined by a Dunnett post hoc comparison; *, p < 0.05; **, p < 0.01). Error bars indicate standard deviation.
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Figure 2 Heart rate (number of heartbeats in 20 s) of zebrafish embryos at 48 hpf after exposure of BPAF (A), BPF (B), BPA (C) and BPS (D). Asterisks denote
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significant differences between treatments and the control (determined by a Dunnett post hoc comparison; *, p < 0.05; **, p < 0.01). Error bars indicate standard
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deviation.
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Figure 3 Teratogenic effects of bisphenol analogues towards zebrafish embryos. A) Reduction in pigmentation caused by BPF at 48 hpf; the pigmentations of eyes,
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spine and yolk sac decreased obviously with increasing exposure concentration. B) Rates of four typical deformities (yolk sac edema, spine deformation and
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pericardial edema at 72 hpf and pigmentation reduction at 48 hpf) after exposure to the four bisphenol analogues. Asterisks denote significant differences between
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treatments and the control (determined by a Dunnett post hoc comparison; *, p < 0.05; **, p < 0.01). Error bars indicate standard deviation.
751 752
Figure 4 A) The structures of BPA, BPAF, BPF and BPS. B) The binding complexes of three zfER subtypes with BPA. Hydrogen bond interactions were found
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between the phenolic hydroxyl groups of BPA and the side-chain residues of Glu and Arg at one end of the binding cavity in the zfERs and between the other
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hydroxyl groups of the BPA and His at the other end of the cavity. C) The calculated zfERs–bisphenols binding energies determined by modeling. D1) The binding
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pocket (red) surrounded by hydrophobic amino acids. D2) The amino acid residue (within 5 Å) around a small-molecule ligand. D3) The amino acid sequence around
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the small-molecule ligand within 5 Å.
757 758
Figure 5 Protein levels of ERα for the control and bisphenol-exposure groups. The relative protein level is represented as the fold change (the level of the control is
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1). Asterisks denote significant differences between treatments and the control (determined by a Dunnett post hoc comparison; *, p < 0.05; **, p < 0.01). Error bars
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indicate standard deviation.
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