Developmental Effects and Estrogenicity of Bisphenol A Alternatives in

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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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Environmental Science & Technology

ACS Paragon Plus Environment


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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]

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+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

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discussion, the occurrence of the specific abnormities indicated that the active

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pathways of bisphenol compounds in zebrafish embryos are not exactly the same.

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Estrogenic effects of bisphenol analogues

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The estrogenic potencies of bisphenol analogues have been the subject of many

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investigations. Reported in vitro data demonstrate that the potencies of BPS and BPF

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for estrogenic activities are similar to that of BPA.51 In addition, the

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endocrine-disrupting effects of BPAF, including thyroid disruption and estrogenic

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effects, have been observed in fish and mammals.52,53,35

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In silico

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ERs mediate critical physiological events in the endocrine and reproductive

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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

17


Page 18 of 42

Page 19 of 42


365

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)

18



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

19


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Page 21 of 42


408

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



Page 22 of 42

430

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)

21


Page 23 of 42


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

22



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.

23


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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

492

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steroidogenesis-related genes and their regulatory factors in response to bisphenol

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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

741

between treatments and the control (determined by a Dunnett post hoc comparison; *, p < 0.05; **, p < 0.01). Error bars indicate standard deviation.

36



742 743

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

744

significant differences between treatments and the control (determined by a Dunnett post hoc comparison; *, p < 0.05; **, p < 0.01). Error bars indicate standard

745

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,

748

spine and yolk sac decreased obviously with increasing exposure concentration. B) Rates of four typical deformities (yolk sac edema, spine deformation and

38



749

pericardial edema at 72 hpf and pigmentation reduction at 48 hpf) after exposure to the four bisphenol analogues. Asterisks denote significant differences between

750

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

753

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

755

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

756

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

759

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

760

indicate standard deviation.

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Developmental Effects and Estrogenicity of Bisphenol A Alternatives in

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