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Bisphenol F disrupts thyroid hormone signaling and postembryonic development in Xenopus laevis Min Zhu, Xiaoying Chen, Yuanyuan Li, Nuoya Yin, Francesco Faiola, Zhanfen Qin, and Wuji Wei Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b06270 • Publication Date (Web): 11 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018
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Environmental Science & Technology
Bisphenol F disrupts thyroid hormone signaling and postembryonic development in Xenopus laevis Min Zhu †, ‡, Xiao-Ying Chen ‡, §, Yuan-Yuan Li ‡, §, Nuo-Ya Yin‡, §, Francesco Faiola‡, §, Zhan-Fen Qin *, ‡, §, Wu-Ji Wei *, † †
School of Environmental Sciences and Engineering, Nanjing Tech University,
Nanjing, 211816, China ‡
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research
Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China §
University of Chinese Academy of Sciences, Beijing 100049, China
Zhan-Fen Qin () · Wu-Ji Wei () Address: State Key Laboratory of Environmental Chemistry and Eco-toxicology, Research Centre for Eco-environmental Sciences, Chinese Academy of Sciences, 18 Shuangqing Road, P.O. Box 2871, Beijing 100085, China
Telephone: 86-10-62919177. Fax: 86-10-62923563.
E-mail:
[email protected] (Zhan-Fen Qin),
[email protected] (Wu-Ji Wei)
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Abstract
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The safety of bisphenol A (BPA) alternatives has attracted much attention due to their wide
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use. In this study, we investigated the effects of bisphenol F (BPF), an alternative to BPA, on
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thyroid hormone (TH) signaling and postembryonic development in vertebrates using
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T3-induced and spontaneous Xenopus metamorphosis as models. We found that in the
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T3-induced metamorphosis assay, higher concentrations of BPF (100-10000 nM) antagonized
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T3-induced TH-response gene transcription and morphological changes including intestinal
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remodeling in a concentration-dependent manner, whereas 10 nM BPF exerted stimulatory
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effects on T3-induced integral metamorphosis when inhibited T3-induced TH-response gene
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transcription, demonstrating TH signaling disrupting effects of BPF. In the spontaneous
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metamorphosis assay, correspondingly, BPF inhibited development at metamorphic climax (with
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high endogenous TH levels), but promoted pre- and pro-metamorphic development (with low
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endogenous TH levels), displaying a developmental stage-dependent manner. Importantly, we
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observed agonistic actions of BPF on Notch signaling in intestines, showing that BPF disrupts
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vertebrate development possibly via multi pathways besides TH signaling. Thus, we infer the
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biphasic concentration-response relationship between BPF exposure and T3-induced
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metamorphosis could result from the interactions of TH signaling with other signaling pathways
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such as Notch signaling. Our study highlights the adverse influences of BPF on vertebrate
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development.
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Introduction
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Bisphenol A (BPA) is a high production volume chemical widely used in plastics, food
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packages, receipts, etc.1 Numerous studies have demonstrated that BPA as an endocrine
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disruptor with thyroid disrupting, estrogenic and androgenic activities, might be harmful to
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human health.2, 3 Considering the known and potential adverse effects, BPA has begun to be
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removed from some of the consumer products. Alternatively, some BPA analogues, such as
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bisphenol F (BPF) and bisphenol S (BPS), have been used to replace BPA in some products.4
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BPF has been used for some consumer products such as paper products, personal care products,
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and food containers.5 Consequently, BPF has been found in environmental samples such as
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indoor dust, surface water, sediment and sewage sludge.6,7 Yamazaki et al.8 reported that BPF
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concentrations exceeded 1000 ng/L and even ranged up to 2850 ng/L (14 nM) in river and
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seawater. BPF has also been detected in human urine at frequencies and concentrations
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comparable to BPA.9 For example, Zhou et al.10 found BPF in 55% urine samples from 100
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American, with the highest concentration of 212 ng/mL (1.06 µmol/L).
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Due to the occurrence in the environment and human body, there is increasing concern about
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the safety of BPA alternatives including BPF.11 Several studies have shown that BPF, like BPA,
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has estrogenic or anti-androgenic activities,12 which is not surprising due to their similar
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chemical structure.13 Considering that BPA also has the potential to disrupt thyroid hormone (TH)
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signaling,14 we are interested in whether BPA alternatives have TH signaling disrupting activities.
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In the latest study, we found that BPF and BPS have the potencies to bind to TR and recruit
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coactivators and to affect TR-mediated gene transcription in vitro and in vivo, indicating they
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could interfere with TH signaling.15 Given important roles of THs in vertebrate development,
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there is a need to reveal effects of BPA alternatives on vertebrate development via TH signaling.
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It is well-known that TH actions via TH signaling are highly conserved across all vertebrate
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species.16 Most amphibians undergo TH-dependent metamorphosis with dramatic morphological
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and molecular changes,17,18 and even pre-metamorphic tadpoles can be induced to metamorphose
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precociously within several days.19,20 Therefore, amphibian metamorphosis serves as an ideal
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model to study TH actions on vertebrate development. Based on enough biological information,
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both T3-induced metamorphosis and spontaneous metamorphosis are also used to study TH
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signaling disrupting activities of chemicals. Heimeier et al.21 reported that BPA antagonized TH
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signaling pathway and inhibited T3-induced metamorphosis in Xenopus laevis. Our laboratory
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found that tetrabromobisphenol A (TBBPA) also had effects on T3-induced TH-response gene
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transcription, gross morphological changes and intestinal remodeling similar to BPA;
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correspondingly, it inhibited development at metamorphic climax, but promoted pre- and
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pro-metamorphic development, displaying a developmental stage-dependent manner.22 Recently,
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we developed the T3-induced Xenopus metamorphosis assay, which is able to easily detect TH
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signaling disrupting activities of chemicals using quantitative morphological endpoints as well as
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molecular endpoints.23,24
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The present study aimed to investigate whether BPF could disrupt TH signaling and
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postembryonic development in X. laevis. Firstly, we conducted the T3-induced Xenopus
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metamorphosis assay to detect TH signaling disruption using molecular and morphological
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endpoints. We particularly paid attention on intestinal remodeling, including morphological and
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histological changes and activation of Notch signaling, which is required for the formation of
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adult intestinal stem cells during TH-dependent intestinal remodeling.25-27 Additionally,
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considering the possible disruption of low concentrations of endogenous TH and the regulation
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of the hypothalamic-pituitary-thyroid (HPT) axis in the T3-induced metamorphosis assay, we
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further confirmed TH signaling disruption by BPF at the transcriptional level in in vitro cultured
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X. laevis tails, which is an ideal tissue for detecting TH signaling disruption.28,29 Corresponding
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to BPF exposure in the absence and presence of T3 in the T3-induced metamorphosis assay, we
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investigated the effects of BPF on pre- and pro-metamorphic development (with little
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endogenous TH) as well as metamorphosis at the climax (with high levels of endogenous TH),
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respectively. This study helps us further understand adverse influences of BPF on vertebrates and
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potential risks for humans.
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1 Materials and Methods
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1.1 Chemicals
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Dimethyl sulfoxide (DMSO) and 3-aminobenzoic acid ethyl ester (MS-222) were purchased
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from Sigma-Aldrich (St. Louis, MO, USA). T3 (3,3’,5-Triiodo-L-thyronine) was obtained from
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Geel Belgium (New Jersey, USA) and BPF was from Tokyo Chemical Industry Co., Ltd. (Tokyo,
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Japan). Stock solutions of T3 (7.06 mmol/L) and BPF (1 mol/L) were prepared by dissolving in
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ultrapure water and DMSO, respectively, and then were stored at －20°C. G-Red (Nucleic acid
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dye) and RNA Extraction kit were obtained from Bio Tekecorporation (Beijing, China). PCR
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primers were synthesized by Sangon Biotech (Beijing, China). RNase-free water, Quantscript RT
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Kit and Real Master Mix (SYBR Green) Kit were purchased from Tiangen (Beijing, China).
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Picric acid, streptomycin sulfate, penicillin G sodium salt, amphotericin B, gentamycin sulfate
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and other reagent were from Beijing Chemical Reagent Co., Ltd (Pure analysis, Beijing, China).
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Human chorionic gonadotropin (HCG, Yantai North Pharmaceutical Co. Ltd., Shandong, China)
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was dissolved in 0.6% NaCl.
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1.2 Animals
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Housing, feeding and breeding conditions for X. laevis (the offspring of adult frogs from
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Nasco, USA) were described in our previous study.30 Frogs are routinely raised in glass tanks
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containing charcoal-filtered tap water (20-21℃). On the fifth day post-fertilization, tadpoles
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were transferred into a flow through system (Esen, Beijing, China) and fed with Terzebrio
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molitor daily. Tadpoles were staged according to the Nieuwkoop and Faber system.31 All animal
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procedures were conducted according to Regulations for the Administration of Affairs
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Concerning Experimental Animals.32 Prior to experiment, all animal experimental protocols were
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approved by the Animal Ethics Committee at the Research Center for Eco-Environmental
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Sciences, Chinese Academy of Sciences.
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1.3 The T3-induced metamorphosis assay
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Tadpoles at stage 52 were randomly transferred into glass tanks containing 4 L water for
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treatment with a series of concentrations of BPF (10, 100, 1000, 10000 nM) in the absence or
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presence of 1 nM T3. Each treatment consisted of three replicate tanks with nine tadpoles per
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tank. DMSO concentration was 0.001 % (v/v) in all treatment groups including the control group.
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The test water (22±1℃) and chemical replacements were performed every day.
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After 24-hr exposure, three tadpoles from each tank were sampled and anesthetized in 100
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mg/L MS-222. The small intestines and tails were removed and immersed in RNA extraction kit
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separately for RNA extraction and subsequent transcriptional analysis for TH-response genes.
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After 96-hr exposure, the remaining six tadpoles in each tank were weighed and photographed
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under a stereoscopic microscope for gross morphological analysis. To study the effects of BPF
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on intestinal remodeling, then, three small intestines from each tank were fixed in Bouin’s
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solution for morphological and histological examination, and the other three small intestines
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were immersed in RNA extraction kit for transcriptional analysis of Notch-related genes.33 The
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experiment was repeated three times using tadpoles from different sets of adults.
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1.4 The in vitro tail assay
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Tails from tadpoles at stage 52 were cultured following the protocol reported by Chen et al.34
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Tails were amputated and washed with 0.6% NaCl several times. Groups of three tails were
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placed in each well and immersed in 3 mL 60-70% L-15 medium (Gibco-BRL, MA; pH 7.4) for
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24 hr at 21℃ as preincubation. Thereafter, tails were exposed to a series of concentrations of BPF
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(10, 100, 1000, 10000 nM) in the absence or presence of 5 nM T3. After 24 h, tails were
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immersed in RNA extraction kit separately for RNA extraction and subsequent analysis for
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TH-response gene transcription. The experiment was repeated three times using tadpoles from
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different sets of adults. The details of the experiment were described in Supporting Information
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(SI “Materials and methods”).
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1.5 The spontaneous metamorphosis assay
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Corresponding to exposure of tadpoles at stage 52 to BPF in the absence and presence of T3 in
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the T3-induced metamorphosis assay, pre- and pro-metamorphic tadpoles and tadpoles at
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metamorphic climax were treated with a series of concentrations of BPF, respectively. Firstly,
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tadpoles at stage 52 were exposed to 10-10000 nM BPF in glass tanks (10 L water and 10
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tadpoles per tank), with four replicate tanks for each treatment. Test water and chemical
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replacements were performed every 48 hr. The developmental stage, hindlimb length (HLL) and
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snout-vent length (SVL) of each tadpole were examined on day 7 and 14. Then, tadpoles at stage
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58, when the TH level increases dramatically, were exposed as described above. The
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developmental stage of each tadpole on day 7 as well as the time to reach stage 66 were
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examined. The spontaneous metamorphosis assay was repeated twice using tadpoles from
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different sets of adults.
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1.6 RNA extraction and RT-qPCR for gene transcriptional analysis
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RNA extraction and RT-qPCR for gene expression analysis were conducted as described in
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Supporting Information (SI “Materials and methods” and Table S1).
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1.7 Analysis for gross morphology
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We used the Image Processing Software of Chong Optec Instrument Co. for gross morphology
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analysis. According to the T3-induced metamorphosis assay described by Yao et al.,23 we
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measured morphological parameters as endpoints including head area (HA), mouth width (MW),
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unilateral brain width (ULBW), brain length (BL), HLL and SVL. Each parameter was
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normalized by the mean value for the control.
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1.8 Histological examination
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After morphological observation, the front end of small intestines (opalescent part close to the
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stomach) were dehydrated with a series of alcohol solutions (70-100%), cleared in xylene,
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embedded in paraffin, and sectioned at 6 µm thickness. Serial sections were collected on glass
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slides and stained with hematoxylin and eosin. Finally, the sections were observed under
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Axioskop standard microscope (Carl Zeiss, Germany).
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1.9 Analysis for integrated biomarker response
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Integrated biomarker response (IBR) is a good method to summarize multi biomarker responses
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into one general index.35,36 Following the method developed by Sanchez et al.,37 we calculated
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the IBR indexes for TH-response gene transcription, gross morphology and Notch-related gene
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transcription in the T3-induced metamorphosis assay, with the IBR indexes for TH-response
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gene transcription in tails cultured in vitro. Samples from the control group were considered as
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the reference for the IBR indexes of TH-response gene transcription and Notch-related gene
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transcription. The morphological biomarkers (BW, SVL, HLL, HA and BL) that characterize
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tadpole growth were integrated to calculate the IBR indexes for gross morphology for the
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treatments in the absence of T3, with samples from the control group as the reference; the
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biomarkers (BW, HA, MW, ULBW/BL and HLL/SVL) that characterize metamorphosis were
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integrated for the treatments in the presence of T3, with T3 treatment group as the reference.
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1.10 Data analysis
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Statistical analysis (the tank or culture well as the statistical unit) was performed using SPSS
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software version 16.0 (SPSS, USA). Considering the differences in the responsiveness to
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chemicals among the offspring from different sets of adult frogs, we run the T3-induced
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metamorphosis assay and the in vitro tail assay three times and the spontaneous metamorphosis
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assay twice using tadpoles from different sets of adults. We presented the results from one
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representative experiment rather than combining the replicate experiments because the results
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from the replicate experiments were consistent, and the data from one representative experiment
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was analyzed, following previous studies.22,38 The fold changes of relative gene expression data
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using qPCR were determined by the 2−ΔΔCt methods.39 Quantitative data were shown as mean ±
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standard error of the mean (SEM). Statistical differences in endpoints (except for IBR indexes
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for gross morphology) in the T3-induced metamorphosis assay and the in vitro tail assay were
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analyzed by two-way analysis of variance (ANOVA) followed by Tukey HSD test. One-way
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ANOVA followed by Dunnett’s test was also used to analyze the values for HLL, SVL and time
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from stage 58 to 66 in the spontaneous metamorphosis assay as well as IBR indexes for gross
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morphology in the T3-induced metamorphosis assay. Developmental stage was analyzed by
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Mann-Whitney U test according to OECD 231.40 Statistical significance was defined as p value
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