TBBPA and Its Alternatives Disturb the Early Stages of Neural

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Ecotoxicology and Human Environmental Health

TBBPA and Its Alternatives Disturb the Early Stages of Neural Development by Interfering with the NOTCH and WNT Pathways Nuoya Yin, Shaojun Liang, Shengxian Liang, Renjun Yang, Bowen Hu, Zhanfen Qin, Aifeng Liu, and Francesco Faiola Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00414 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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Environmental Science & Technology

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TBBPA and Its Alternatives Disturb the Early Stages of Neural Development by Interfering with the NOTCH and WNT Pathways

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Nuoya Yin1,2,#, Shaojun Liang1,2,#, Shengxian Liang1,2, Renjun Yang1,2,

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Bowen Hu1,2, Zhanfen Qin1,2, Aifeng Liu3, Francesco Faiola1,2,*

1 2

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1

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for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China

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2

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Beijing, 100049, China

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center

College of Resources and Environment, University of Chinese Academy of Sciences,

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3

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Bioprocess Technology, Chinese Academy of Science, Qingdao 266101, China

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#

Co-first author

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*

Corresponding Author: Francesco Faiola, E-mail: [email protected], Tel/Fax: 86

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10-62917609

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Keywords: TBBPA, TBBPS, TCBPA, neural development, embryonic stem cells,

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NOTCH, WNT.

CAS Key Laboratory of Bio-based Materials, Qingdao Institute of Biomass Energy and

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ACS Paragon Plus Environment


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Abstract

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Tetrabromobisphenol A (TBBPA), as well as its alternatives Tetrabromobisphenol S

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(TBBPS) and Tetrachlorobisphenol A (TCBPA), are widely used halogenated flame

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retardants. Their high detection rates in human breast milk and umbilical cord serum have

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raised wide concerns about their adverse effects on human fetal development. In this

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study, we evaluated the cytotoxicity and neural developmental toxicity of TBBPA,

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TBBPS and TCBPA with a mouse embryonic stem cell (mESC) system, at human body

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fluid and environmental relevant doses. All the three compounds showed similar trends in

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their cytotoxic effects. However, while TBBPA and TBBPS stimulated ESC neural

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differentiation, TCBPA significantly inhibited neurogenesis. Mechanistically, we

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demonstrated that, as far as the NOTCH (positive regulator) and WNT (negative

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regulator) pathways were concerned, TBBPA only partially and slightly disturbed them,

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whereas TBBPS significantly inhibited the WNT pathway, and TCBPA down-regulated

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the expression of NOTCH effectors but increased the WNT signaling, actions which both

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inhibited neural specification. In conclusion, our findings suggest that TBBPS and

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TCBPA may not be safe alternatives to TBBPA, and their toxicity need to be

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comprehensively evaluated.

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Introduction

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Flame retardants are used in paints, building materials, synthetic textiles, and plastic


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products, including electronic circuit boards and other electronic equipment, and so on, to

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prevent fires. Tetrabromobisphenol A (TBBPA) is the most widely employed brominated

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flame-retardant (BFR), accounting for about 60% of the total BRF applications.1 As

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alternatives to TBBPA, Tetrabromobisphenol S (TBBPS) and Tetrachlorobisphenol A

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(TCBPA), have been also extensively used in recent years.2 According to previous

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studies, TBBPA, TBBPS and TCBPA can be considerably detected in the environment,

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in water, soil, dust and air, as well as aquatic organisms.3-5 Most importantly, the

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presence of these three pollutants has been measured in human body fluid samples, up to

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649.45 ng/g lipid (equal to 4.8 nM) in umbilical cord serum,6 and 11 ng/g in breast milk.7

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These high detection rates have prompted the evaluation of the health risks associated

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with TBBPA and its derivatives. Owing to the high concentrations detected in the human

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umbilical cord and newborn sera, and the fact that even a slight perturbation in the

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embryonic developmental process could cause severe defects at the postnatal stage,8 it is

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urgent to investigate the potential developmental toxicity of TBBPA, TBBPS and

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TCBPA.

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in vitro cytotoxicity studies demonstrated TBBPA enhanced the production of

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intracellular reactive oxygen species9 and calcium concentrations, and inhibited viability

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of cerebellar granule cells.10-12 In addition, it exerted several adverse effects on

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neurotransmission.13, 14 Animal tests have already showed that TBBPA impaired neural

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development15-17. For instance, exposure to TBBPA in adult Wistar rats affected the

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nervous system in both male and female offspring.18 Moreover, TBBPA was found to



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accumulate in brain regions, including the striatum, which might account for behavioral

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alterations.19 However, there is still controversy about TBBPA potential developmental,

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especially neural, toxicity in vivo. Limited knowledge is available on TBBPS and

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TCBPA health risks. Nonetheless, due to the similarity in their structures, it is reasonable

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to assume similar TBBPA, TBBPS and TCBPA potential adverse effects on human

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health.1, 20-22

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Embryonic stem cells (ESCs) provide a powerful in vitro model to comprehensively

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evaluate

chemical

toxicity,

including

developmental,

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cytotoxicity.23-27 Stem cells applications in toxicology have been gradually increasing and

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toxicity results with ESC systems in vitro have already been shown to be consistent with

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clinic reports.24, 25 In the present study, we evaluated the potential toxicity of TBBPA,

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TBBPS and TCBPA at environmental and human relevant doses, with a mouse ESC

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(mESC) model. We demonstrated that, at concentrations from 1 to 1000 nM, which did

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not interfere with ESC self-renewal and proliferation, the three pollutants exerted

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neurodevelopmental toxicity: TBBPA and TBBPS abnormally promoted neural

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development while TCBPA inhibited it. More specifically, alterations in the NOTCH and

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WNT signal transduction pathways were involved in the toxicity effects and imply the

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necessity for further risk assessments for TBBPA, TBBPS and TCBPA.

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Materials and methods

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Compound preparation. TBBPA (TCI, Shanghai Development Co., Ltd.; purity >


organ,

functional

and

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98.0%), TBBPS (Hwrk Chem Co., Ltd. China; purity > 98.0%) and TCBPA (J & K

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Scientific., Ltd. China; purity > 98.0%) were dissolved in dimethyl sulfoxide (DMSO,

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Amresco, USA, cat. # 0231).

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Cell culture. J1 mESCs (Cell Bank/Stem Cell Core Facility, SIBCB, CAS) were cultured

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in 0.1% gelatin (Sigma-Aldrich, USA, cat. # G2500) coated plates and ESC complete

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medium (KnockOut™ Dulbecco's Modified Eagle Medium (Gibco, USA, cat. #

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10829-018) supplemented with 15% fetal bovine serum (FBS, Corning, USA, cat. #

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35-01-CV), 1% (v/v) GlutaMax (Gibco, USA, cat. # 35050), 1% (v/v) nucleosides

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(Millipore, USA cat. # ES-008-D), 1% (v/v) nonessential amino acids (Gibco, USA, cat.

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# 11140), 10−4 M β-mercaptoethanol (Solarbio, China, cat. # M8210), 100 U/mL

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penicillin and 100 µg/mL streptomycin (Gibco, USA, cat. # 15140-122), and 1000 U/mL

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leukemia inhibitory factor (LIF, Merck Millipore, Darmstadt, Germany)) at 37°C in

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humidified air and 5% CO2. mESCs were passaged with 0.05% trypsin-EDTA (Gibco,

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USA, cat. # 25300-062) every three days and the medium replaced daily.

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AlamarBlue cell viability assay and IC50 calculation. mESCs were seeded (5000 cells

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per well for 72 h cell viability test, and 200 cells per well for 7-day sub-lethal toxicity test)

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on 0.1% gelatin coated 96-well plates, in 100 µL ESC complete medium. Four hours after

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seeding, media were replaced with fresh ESC complete medium containing different

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concentrations of TBBPA, TBBPS and TCBPA (or 0.1% DMSO vehicle control). Media

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were refreshed every other day. At the designated time points, cells were incubated with

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fresh media containing AlamarBlue (Thermo Fisher Scientific, USA, cat. # DAL1025)



100

for 4 h at 37°C. Then, fluorescence values were measured with a Varioskan LUX

101

(Thermo, USA) plate reader, at the excitation wavelength of 530 nm and the emission

102

wavelength of 590 nm. The IC50 (half maximal inhibitory concentration) values were

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calculated using IBM SPSS Statistics version 20.

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Alkaline phosphatase (AP) staining. 300 cells per well were seeded onto 0.1% gelatin

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coated 12-well plates and cultured for 7 days in ESC complete medium containing

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different doses of TBBPA, TBBPS and TCBPA (or 0.1% DMSO vehicle control). Media

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were changed every other day. At Day 7, an AP staining kit (Sigma-Aldrich, USA. cat. #

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86R) was used according to the manufacturer's instructions. Briefly, cells were fixed for

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30 s with a Citrate-Acetone-Formaldehyde solution and then stained for 15 min with

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fresh alkaline-dye solution.


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Intracellular reactive oxygen species9 measurement. Formation of oxidative stress was

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measured with the fluorescent probe DCFH-DA (2′,7′-Dichlorofluorescin diacetate) as

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previously described.28 Briefly, mESCs grown on 96-well plates were loaded with 100

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µM DCFH-DA (Sigma-Aldrich, USA, cat. # D6883) in culture media for 30 min at 37 °C,

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then the cells were treated for 2, 4, 8, 24 h with 0-200 µM TBBPA, TBBPS and TCBPA

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(or 0.1% DMSO vehicle control). The fluorescence values were measured using a

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Varioskan LUX plate reader at excitation and emission wavelengths of 485 and 530 nm,

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respectively.

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Intracellular free calcium ion level detection. Intracellular free calcium ion levels were

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determined by using the fluorescent probe Fura-2/AM (Sigma, USA, cat. # 47989).

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Briefly, mESCs growing in 24-well plates were loaded with 5 µM Fura-2/AM for 30 min

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in Hank’s buffer (Solarbio, China, cat. # H1025) at 37°C. Then, the cells were incubated

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with 0 to 200 µM TBBPA, TBBPS and TCBPA (or 0.1% DMSO vehicle control). The

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increase in intracellular Ca2+ was expressed as the fluorescence intensity ratio measured

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at 340 and 380 nm excitation wavelengths (F340/F380), which is proportional to the

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calcium ion concentration. One F340/F380 emission measurement at 510 nm was

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obtained every 50 s.

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Embryoid Body (EB)-based neural progenitor cell (NPC) induction. ESCs were

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differentiated into NPCs following a published protocol.29 Briefly, for EB formation,



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4×106 mESCs were seeded in suspension onto 10-cm petri dishes (NEST, China, cat. #

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752001) in 15 mL EB medium (ESC complete medium deprived of LIF), supplemented

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with TBBPA, TBBPS and TCBPA (or 0.1% DMSO vehicle control). Media were

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replaced every other day. At the fourth day, 5 µM retinoic acid (RA, Sigma, USA, cat. #

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R2526) was added to the medium to specifically induced neuroectoderm from which

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NPCs are derived, for additional 4 days. Then, EBs were dissociated at Day 8 with 0.05%

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trypsin-EDTA and 2×106 single cells per well were plated onto poly-l-lysine/laminin

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(Sigma-Aldrich, USA, cat. # P7890 and Roche, USA, cat # 11243217001, respectively)

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coated 6-well plates in N2 medium (DMEM/F12 basal medium (Gibco, USA, cat. #

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C11330500BT), 1X N2 supplement (Gibco, USA, cat. # 17502-048), 1% GlutaMAX,

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and 2% Pen/Strep antibiotics). 2 h later, media were replaced with fresh N2 media

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supplemented with TBBPA, TBBPS and TCBPA (or 0.1% DMSO vehicle control). After

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one additional day, samples were collected for RNA extraction and quantitative reverse

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transcription PCR (qRT-PCR) analyses.

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ESC monolayer neural differentiation. ESC neural differentiation was performed as

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previously described.30, 31 Briefly, 1×105 cells per well were seeded onto 0.1% gelatin

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coated 6-well plates and cultured in N2B27 medium (50% DMEM/F12 and 50%

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Neurobasal medium (Gibco, USA, cat. # 21103-049), supplemented with 1X N2

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supplement, 1X B27 supplement (Gibco, USA, cat. # 17504-044), 1% bovine serum

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albumin fraction V (Beyotime, China, cat. # st023), 1% GlutaMax and 0.1 mM

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β-mercaptoethanol) containing different concentrations of TBBPA, TBBPS and TCBPA


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(or 0.1% DMSO vehicle control). Media were changed every other day. Samples were

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collected at Days 0, 6, 9 and 12, for RNA extraction and qRT-PCR analyses.

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Immunostaining. Cells differentiated with the monolayer neural induction protocol were

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re-plated at Day 6, onto poly-l-lysine/laminin coated 12-well plates and cultured for six

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additional days. Then, cells were fixed and stained with a MAP2 antibody at Day 12.

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Detailed information can be found in the Supporting Information.

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RNA extraction and qRT-PCR analyses. RNA extraction and gene expression analysis

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were conducted as described in the Supporting Information (see also Table S1).

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Western-blot. Western-blot was conducted as described in the Supporting Information.

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Statistical Analysis. Statistical analyses were performed with an unpaired, two-tailed

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Student’s t-test. Significance values and biological independent repeat numbers are

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indicated in the figure legends.

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Results

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Evaluation of TBBPA, TBBPS and TCBPA cytotoxicity in mESCs

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As an initial evaluation of the toxicity of TBBPA, TBBPS and TCBPA in mESCs, we

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determined their cytotoxicity at concentrations from 1 to 300 µM, upon 24 and 72 h

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exposures. As shown in Figure 1A, the three chemicals did not produce any detectable

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cell viability changes at concentrations ≤ 1 µM, for up to 72 h. For the longest treatment

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(72 h), we were also able to calculate the IC50 values (Figure 1D), which were in the

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same order or magnitude, with TBBPA slightly more cytotoxic than TBBPS and TCBPA.



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To investigate whether doses lower than 1 µM did not only alter mESC viability, but

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also their self-renewal, at incubations longer than 72 h, we incubated proliferating ESCs

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with 1-1000 nM TBBPA, TBBPS and TCBPA, for 7 days. ESC viability and self-renewal

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abilities were assessed by AlamarBlue assay and AP staining, respectively. As depicted

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in Figures 1 B-C, viability was not affected, and similar numbers of mESC colonies were

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stained dark red for AP in each well, indicating TBBPA and its derivatives did also not

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affect mESC self-renewal, upon 7 days’ exposure, compared to solvent DMSO control.

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These results imply that TBBPA, TBBPS and TCBPA environmental and human body

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fluid concentrations, which are in the nM range, are far from being lethal to mESCs.

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To analyze the effects of cytotoxic concentrations of TBBPA and its two derivatives,

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we determined the intracellular calcium influx, since Ca2+-dependent depolarization of

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mitochondria has been suggested to contribute to cell stress. 10 µM TBBPA and TCBPA

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provoked an increase in intracellular calcium ions in mESCs around 7-8-min exposure,

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but those perturbations returned to normal levels after about 35 min, suggesting that 10

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µM TBBPA and TCBPA could perturb the cellular calcium ion levels but the cells still

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recovered due to the anti-stress machinery (Figure 2A). However, 100 and 200 µM

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TBBPA and TCBPA increased the free calcium ion levels consistently and at relatively

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high levels, relative to controls, confirming that those two compounds increased the

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cellular free calcium ion levels in a dose-dependent manner. Interestingly, TBBPS did not

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show any perturbation of the calcium influx, even at the highest concentration tested

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(Figure 2A).


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Since ROS formation is another early indicator of cell metabolism dysfunction and

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stress, we assessed intracellular ROS levels upon TBBPA, TBBPS and TCBPA exposure

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for 2, 4, 8 and 24 h. As shown in Figure 2B, the three chemicals significantly increased

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ROS accumulation in a time and dose dependent manner. More specifically, TBBPA and

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TBBPS stimulated ROS production at an early time point (4 h) than TCBPA did.

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However, at concentrations below 1 µM, which include environmental and human body

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fluid relevant doses, the three compounds did not stimulate any oxidative stress under the

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conditions tested, in mESCs.

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TBBPA, TBBPS and TCBPA significantly impaired EB-based neural differentiation

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of mESCs

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A few animal studies with rats and zebrafish have already reported TBBPA might

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interfere with embryo development, especially for the neural lineages,18,

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consistent conclusions about its developmental toxicity have not been reached. As far as

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TBBPS and TCBPA are concerned, they are widely used as TBBPA substitutes, but the

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evaluation of their potential developmental neural toxicity is still elusive. Thus, to

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appraise the effects of TBBPA, TBBPS and TCBPA on the early stages of embryonic

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neural development in vitro, we induced NPC specification in ESCs differentiated via EB

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formation (see material and methods), a process known to mimic, in vitro, the early

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stages of embryonic development, in vivo. We collected Day 9 NPCs and analyzed

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specific marker gene expression changes, upon pollutant treatment at environmental and

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human relevant doses, by qRT-PCR. As depicted in Figure 3A, TBBPA and TBBPS


32, 33

yet


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overall enhanced the expression of the neural progenitor markers Pax6, Sox1 and Sox3,

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and neurogenesis genes Map2 and NeuroD, as compared to DMSO solvent control. In

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contrast, TCBPA consistently down-regulated all the neural lineage specific markers

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checked.

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To assess whether those gene expression changes were the consequence of abnormal

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ESC differentiation and not cytotoxic affects in NPCs, we measured NPC viability in N2

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medium upon 24 h incubation with the three chemicals or DMSO vehicle control (Figure

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3B). We did not detect any cytotoxic effects, except when NPCs were incubated with 500

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nM TBBPS (Figure 3B), implying that the abnormal expression patterns of the marker

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genes were the results of developmental toxicity independent of cytotoxicity. The

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obvious cytotoxicity of 500 nM TBBPS to NPCs in N2 medium, could also explain the

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opposite trend of expression of the genes checked as compared to the other doses (500

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nM vs. 1-100 nM) and control.

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We confirmed the toxic effects of 10-100 nM TBBPA, TBBPS and TCBPA by

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assessing the protein level changes for MAP2 by Western-blot (Figure 3C; note the same

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trend of expression for MAP2 at mRNA and protein levels upon pollutant treatments,

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compared to control), suggesting the three compounds could impair neuronal

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morphogenesis, cytoskeleton dynamics, and organelle trafficking in axons and dendrites.

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Comprehensively, our results imply abnormal stimulation, for TBBPA and TBBPS, and

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repression, for TCBPA, of neurogenesis during embryonic development.

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TBBPA, TBBPS and TCBPA affected the monolayer neural differentiation of


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mESCs

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To substantiate the effects of TBBPA, TBBPS and TCBPA on neural development

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observed in EB-based ESC differentiation, we stimulated the generation of NPCs from

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ESCs with an alternative procedure: adherent monolayer neural differentiation. First, we

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assessed whether the concentrations chosen could cause acute cytotoxicity under N2B27

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culture conditions. We decided to look at ROS level changes, upon 24 h chemical

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treatment, as an early indication of acute cytotoxicity. As shown in Figure S1 we did not

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detect any ROS formation after 1-1000 nM of TBBPA, TBBPS and TCBPA treatments,

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as compared to control, indicating acute cytotoxicity could be ruled out under those

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conditions.

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Subsequently, we performed mESC monolayer neural differentiation for 12 days,

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upon constant exposure to 1-500 nM TBBPA, TBBPS and TCBPA. Samples were

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collected at Days 0 (undifferentiated ESCs not exposed to any pollutants), 6, 9 and 12,

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and neural progenitor (Pax6, Sox1, Sox3,), neurogenesis related (Map2, NeuroD, Dcx)

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and astrocyte related (Gfap), marker expression measured by qRT-PCR. TBBPA and

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TBBPS treatments showed a general up-regulation in all the markers analyzed, with a

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more significant dose-dependent increase for TBBPS at Day 12 (Figures 4 and S2).

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Conversely, TCBPA inhibited the expression of those genes (Figures 4 and S2).

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To further determine the repercussions of TBBPA, TBBPS and TCBPA treatments

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on NPC formation, we seeded equal amounts of Day 6 differentiating cells onto

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poly-l-lysine/laminin coated plates and stained them at Day 12 with an antibody against



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MAP2. Only 100 nM treated samples were selected for immunostaining. Consistent with

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the qRT-PCR (Figures 3A and 4) and Western-blot results (Figure 3C), TBBPA and

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TBBPS slightly increased the number of MAP2 positive (MAP2+) cells, compared with

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control group, whereas TCBPA diminished it (Figure 5). We also employed Western-blot

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to determine the changes in MAP2 protein levels upon pollutants’ exposure, in the

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monolayer neural induction conditions. Figure S3 shows that TBBPA and TBBPS

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up-regulated, while TCBPA repressed, MAP2 protein levels. Overall these results

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remarkably implicate a potential developmental neurotoxicity for TBBPA, TBBPS and

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TCBPA even at low doses.

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TBBPA, TBBPS and TCBPA perturbed neural specification by altering the

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NOTCH and WNT pathways

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To understand the molecular mechanisms behind the three pollutants’ different manners

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of affecting neural specification, we turn to the NOTCH and canonical WNT signaling

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pathways, which play critical roles in the early stages of neural development. Thus, we

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investigated the expression levels of the NOTCH effectors Hes1 and Hes5, as well as the

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WNT target genes Lef1 and Axin2, by qRT-PCR, during monolayer neural induction and

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upon pollutant exposure. TBBPA treatment seemed to slightly up-regulate the expression

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of NOTCH effectors (Figures 6A and B), while did not interfere much with the WNT

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cascade (Figure 6C). This implies TBBPA may stimulate neural specification via

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up-regulation of the NOTCH signaling. Conversely, TBBPS significantly down-regulated

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the expression of the two WNT target genes, Axin2 and Lef1, but did not alter


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consistently the NOTCH pathway (Figures 6A-C), suggesting that TBBPS abnormally

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promoted neural development by weakening the inhibitory effects of the WNT pathway.

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Interestingly, TCBPA treatment notably impaired the NOTCH pathway (Figures 6A-B),

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and slightly stimulated WNT signaling, particularly at Days 6 and 9 of the neural

280

differentiation. These data revealed that TCBPA may compromise NPC maintenance and

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proliferation via the NOTCH pathway and repress neural development at early stages

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through activating the canonical WNT pathway.

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Discussions

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The high frequency of detection for TBBPA and its derivatives, TBBPS and TCBPA,

285

in the environment and human body, has triggered concerns about their potential adverse

286

effects on health. For instance, recent studies have reported that TBBPA might influence

287

embryonic development32, 34-36 and the central nervous system.18, 33, 37-39 However, no

288

consensus has been reached yet. In the present study, we evaluated the cytotoxicity and,

289

most importantly, neural developmental toxicity of TBBPA, TBBPS and TCBPA, at

290

concentrations comprising environmental and human body fluid relevant doses, with a

291

mESC system, and explored the probable molecular mechanisms behind their toxicity.

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TBBPA and its derivatives decreased cell viability of proliferating mESCs at

293

concentrations ≥ 100 µM (Figure 1). Although TBBPA showed slightly higher

294

cytotoxicity than TBBPS and TCBPA, their IC50 values were in the same order of

295

magnitude, similar as in other immortalized cell lines40, 41, but higher than in primary



296

cells like rat cerebellar granule cells (12.5 µM).10, 11, 42, 43 However, the behavior of the

297

three compounds was different in stimulating intracellular ROS accumulation and

298

calcium ion flux, which are early indicators of cell metabolism dysfunctions. In fact,

299

TBBPA and TBBPS stimulated ROS accumulation faster than TCBPA (Figure 2A).

300

Moreover, TBBPA and TCBPA increased intracellular calcium ions, but TBBPS did not

301

(Figure 2B). These phenomena may be attributed to their different chemical structures.

302

TBBPA and TCBPA share the same alkyl bridge between the phenyl rings, but those are

303

bridged by a sulfunyl group in TBBPS. In addition, TBBPA and TBBPS contain

304

bromines while TCBPA chlorines (Figure S4).

305

Contrary to cytotoxicity, even low concentrations of TBBPA, TBBPS and TCBPA,

306

which included human body fluid and environmental doses,6, 44, 45 showed deleterious

307

effects on neural development in vitro, which we assessed by measuring changes in

308

neural progenitor markers (Pax6, Sox1 and Sox3)30, 46 and neurogenesis genes (Map2,

309

NeuroD and Dcx),46, 47 and confirmed with two different induction protocols (Figures

310

3-5). The different behavior of TCBPA, compared to TBBPA and TBBPS, in altering

311

neural specification (repression vs. activation) may also be explained by discrepancy in

312

3- and 5-substituents in the bisphenol structure. Our TBBPA neural development toxicity

313

data are consistent with phenotypes Saegusa and colleagues reported.37 They found that

314

after rats were exposed to low TBBPA dosages in their diet from gestational Day 10 to

315

Day 20, their offspring manifested an increase in interneurons and NeuN-positive mature

316

neurons in the dentate hilus at the postnatal Days 20 and 77, respectively. Moreover,


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Chen et al. confirmed TBBPA low concentrations affected the development and behavior

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in zebrafish.33

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To dissect potential TBBPA, TBBPS and TCBPA molecular mechanisms during

320

neural development, we took into consideration the fact that the WNT canonical cascade

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pathway is closely associated with the NOTCH signaling in regulating cell fate decisions

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during the early stage of embryonic development.48 The NOTCH pathway is essential for

323

the maintenance and proliferation of NPCs and regulates the neural lineage

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specification.49, 50 For example, impaired NOTCH pathway decreased the neural sphere

325

sizes in an in vitro culture and caused defects in neural development.51 Hes1 and Hes5 are

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effectors of the NOTCH pathway in the development of the central nervous, as well as

327

peripheral nervous systems.50, 52 It has already been proved that down-regulation of Hes1

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and Hes5 impairs NPC maintenance and delays neurogenesis and gliogenesis.53,

329

Conversely, canonical WNT pathway has been reported to inhibit the early stages of

330

neural development.55, 56 De Wit and colleagues found that both genders of zebrafish

331

exposed to TBBPA displayed interference in the WNT signaling pathway,57 which is

332

consistent with our findings (Figure 6). However, the perturbations we observed in Figure

333

6 were slightly, implying that besides the WNT and NOTCH pathways, there could be

334

other pathways contributing to the abnormal neural development caused by TBBPA.

335

Interestingly, TBBPS inhibited Lef1 and Axin2 more noteworthily than it up-regulated

336

Hes1 and Hes5, meaning it might promote neural development mostly by inhibiting the

337

WNT signaling pathway (Figure 6). On the contrary, TCBPA repressed the NOTCH


54


338

pathway significantly, which might account, for the most part, for the observed

339

repression of neural development. Nevertheless, it also moderately enhanced the WNT

340

pathway, which probably also contributed to TCBPA-dependent repression of the neural

341

development (Figure 6).

342

Since the genes and pathways we analyzed in our mouse stem cell toxicology

343

system are conserved in humans,58 we speculate that TBBPA, TBBPS and TCBPA

344

deleterious effects could occur during human embryonic development as well. Our

345

findings also prove that TBBPS and TCBPA may exhibit sever developmental toxicity

346

and are not safe substitutes for TBBPA.

347


Page 18 of 36

Page 19 of 36


348

Acknowledgements

349

This work was supported by the Chinese Academy of Sciences Strategic Priority

350

Research Program (XDB14040301); the National Natural Science Foundation of China

351

(21577166 and 21461142001); the Chinese Academy of Sciences Hundred Talent

352

Program (29[2015]30); and the Key Research Program of Frontier Sciences, CAS

353

(QYZDJ-SSW-DQC017).

354

Conflicts of Interest

355

The authors declare that they have no conflict of interest.

356

Supporting Information. Brief descriptions of additional methods, Figures S1-4,

357

and Table S1 are supplied as Supporting Information.

358 359

References

360 361 362 363 364 365 366 367 368 369 370 371 372 373 374

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528

Figures

529

530 531

Figure 1. TBBPA, TBBPS and TCBPA caused cytotoxicity in proliferating mESCs

532

only at concentrations much higher than 1 µM. A) Cell viability assessment by

533

AlamarBlue assay after 3 day exposure. B) Cell viability assessment by AlamarBlue

534

assay, and C) AP staining, upon seven day exposure. – indicates no treatment; 0 nM

535

refers to 0.1% DMSO only. Values in A) and B) are means ± SEM for three independent


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536

experiments. *p ≤ 0.01. D) IC50 values in mESCs upon 72-h exposure to TBBPA, TBBPS

537

and TCBPA. Shown are means ± SEM of three independent experiments.

538



539

540 541

Figure 2. TBBPA, TBBPS and TCBPA perturbed intracellular calcium levels and

542

stimulated ROS formation in different fashions. A) The alteration of intracellular

543

calcium ions in mESCs within 4000 s upon treatment with TBBPA, TBBPS and TCBPA,

544

respectively. Shown are the ratios of the fluorescence values at 340 over 340 nM which

545

represent the intracellular free calcium ion levels. The results are from one representative


Page 26 of 36

Page 27 of 36


546

experiment. B) The relative ROS levels compared to vehicle control. Data are calculated

547

as fold changes relative to control, and presented as mean ± SD of three independent

548

experiments. *p ≤ 0.01.

549



550 551


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Page 29 of 36


552 553

Figure 3. TBBPA, TBBPS, and TCBPA significantly affected the EB-based neural

554

differentiation of mESCs. A) Neural progenitor (Pax6, Sox1 and Sox3) and



555

neurogenesis (NeuroD and Map2) markers, analyzed by qRT-PCR. Data are normalized

556

to Gapdh and the expression levels in DMSO control (ctrl). B) Cell viability assessment

557

for NPCs incubated with 1-500 nM TBBPA, TBBPS and TCBPA for 24 h in N2 medium.

558

C) Western-blot analysis of MAP2. Shown is one representative experiment and GAPDH

559

serves as loading controls. The values in A) and B) are means ± SD (n=3) for

560

representative experiments. #p ≤ 0.0005, relative to DMSO controls.

561


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562



563

Figure 4. TBBPA, TBBPS, and TCBPA affected the neural monolayer

564

differentiation of mESCs with different behaviors. Neural progenitor (Pax6, Sox1 and

565

Sox3) and neurogenesis (Map2, NeuroD and Dcx) markers, measured by qRT-PCR. Data

566

are normalized to Gapdh and the expression levels in mESCs at Day 0. The values shown

567

are means ± SD for one representative experiment in triplicates. #p ≤ 0.0005, relative

568

to DMSO controls; statistical significance for Map2, NeuroD and Dcx was only analyzed

569

at Day12.

570

571 572

Figure 5. TBBPA, TBBPS and TCBPA altered NPC MAP2+ populations. Cells were

573

immunostained with a MAP2 antibody. The scale bar in the top panels is 200 µM, while

574

the one in the bottom panels 100 µM. Shown is one representative experiment.

575


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576



577


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Page 35 of 36


578

Figure 6. TBBPA, TBBPS and TCBPA altered neural specification via the NOTCH

579

and canonical WNT pathways. A) Gene expression levels for Hes1 and Hes5, analyzed

580

by qRT-PCR. B) HES1 protein levels measured by Wester-blot. Shown is one

581

representative experiment. C) mRNA expression levels for Lef1 and Axin2 detected by

582

qRT-PCR. Data in A) and C) are normalized to Gapdh and the expression levels in

583

undifferentiated mESCs. Values are shown as mean ± SD (n=3) for one representative

584

experiment. #p ≤ 0.0005, relative to DMSO controls.




Page 36 of 36

TBBPA and Its Alternatives Disturb the Early Stages of Neural

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