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Tris(1,3-dichloro-2-propyl) phosphate disrupts axonal growth, cholinergic system and motor behavior in early life zebrafish
Accepted Manuscript Title: Tris(1,3-dichloro-2-propyl) Disrupts Axonal Growth, Cholinergic System and Motor Behavior in Early Life Zebrafish Authors: Rui Cheng, Yali Jia, Lili Dai, Chunsheng Liu, Jianghua Wang, Guangyu Li, Liqin Yu PII: DOI: Reference:
S0166-445X(17)30241-2 http://dx.doi.org/10.1016/j.aquatox.2017.09.003 AQTOX 4735
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
7-6-2017 30-8-2017 2-9-2017
Please cite this article as: Cheng, Rui, Jia, Yali, Dai, Lili, Liu, Chunsheng, Wang, Jianghua, Li, Guangyu, Yu, Liqin, Tris(1,3-dichloro-2-propyl) Disrupts Axonal Growth, Cholinergic System and Motor Behavior in Early Life Zebrafish.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2017.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Tris(1,3-dichloro-2-propyl) Disrupts Axonal Growth, Cholinergic System and
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Motor Behavior in Early Life Zebrafish
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Rui Chenga, Yali Jiaa, Lili Daib, Chunsheng Liua,c,d, Jianghua Wanga, Guangyu
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Lia, Liqin Yua,*
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a
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
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b
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences
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c
Collaborative Innovation Center for Efficient and Health Production of Fisheries in
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Hunan Province, Hunan Changde 415000
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d
Hubei Provincial Engineering Laboratory for Pond Aquaculture
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* Adress correspondence to
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College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China. Tel:
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86 27 87282113. Fax: 86 27 87282114. Email: [email protected]
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Highlights
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1. TDCIPP altered motor behaviors in zebrafish larvae.
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2. TDCIPP decreased neuron-specific GFP expression in Τg (HuC- GFP) zebrafish
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larvae. 3. TDCIPP inhibited axonal growth of secondary motor neurons and altered expressions of genes related to axonal growth. 4. TDCIPP inhibited the cholinergic system in zebrafish larvae.
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5. TDCIPP induced neurobehavioral alterations may result from combined effects of altered motor neuron structure and inhibition of cholinergic system.
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Abbreviation List Abbreviation
Full Name
TDCIPP
tris(1,3-dichloro-2-propyl) phosphate
PBDEs
polybrominated diphenyl ethers
BDE-47
2,2',4,4'-tetrabrominated biphenyl ether
BPA
25
bisphenol A
DP
dechlorane plus
CPF
chlorpyrifos
DE-71
a mixture of polybrominated diphenyl ethers
MC-LR
microcystin-LR
ACh
acetylcholine
AChE
acetylcholinesterase
Tg
transgenic
gapdh
glyceraldehyde-3-phosphate dehydrogenase
shha
sonic hedgehog a
gap43
growth associated protein 43
elavl3
elav like neuron-specific RNA binding protein 3
ngn1
neurogenin 1
mbp
myelin basic protein
syn2a
synapsinII a
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Abstract：TDCIPP could have neurotoxic effects and alter motor behaviors in zebrafish
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(Danio rerio) larvae, however, the underlying mechanisms are still unknown. In this
28
study, zebrafish embryos were subjected to waterborne exposure of TDCIPP at 100,
29
300, 600, 900 μg/L from 2 to 120-hour post-fertilization (hpf). Behavioral
30
measurements indicate that TDCIPP exposure significantly elevated spontaneous
31
movement, and altered swimming behavior response of larvae to both light and dark
32
stimulation. Interestingly, in accordance with these motor effects, TDCIPP significantly
33
decreased expression of the neuron-specific GFP in transgenic (HuC-GFP) zebrafish
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larvae as well as decreased expression of the neural marker genes elavl3 and ngn1,
35
inhibited the axonal growth of the secondary motoneurons and altered the expressions
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of axon-related genes (α1-tubulin, shha and netrin2) in zebrafish larvae. Furthermore,
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TDCIPP
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acetylcholinesterase (AChE) enzyme, and decreased the total acetylcholine (ACh)
39
concentration. Our data indicate that the alteration in motor neuron and inhibition of
40
cholinergic system could together lead to the TDCIPP induced motor behavior
41
alterations in zebrafish larvae.
42
Keywords：TDCIPP; zebrafish larvae; motor behavior; axonal growth
exposure
at
900
μg/L
significantly
increased
the
activity
of
43
1. Introduction
44
Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been used as an
45
organophosphate flame retardant for decades in various products (e.g. plastics, textiles,
46
foams, electronic equipment, varnishes, and furniture), with an estimated annual
47
production ranged from 4500 to 22700 tones between 1998 and 2006 in the United
48
States (Stapleton et al., 2009; van der Veen and de Boer, 2012). Like polybrominated
49
diphenyl ethers (PBDEs), this chemical can leach out of materials and can now be
50
detected in various environments and biota samples (Cao et al., 2014; Meeker et al.,
51
2013; Meeker and Stapleton, 2010; Wei et al., 2015; Yang et al., 2014). In surface water,
52
TDCIPP concentrations have been generally reported at ng/L levels (Cao et al., 2012;
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Hu et al., 2014; Shi et al., 2016; Wang et al., 2011), but it is not readily degraded in
54
water and could accumulate in aquatic organisms. Indeed, it has been reported that
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TDCIPP in freshwater fish could reach up to several hundred μg/kg lipid weight (e.g.
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140 μg/kg in perch) (Sundkvist et al., 2010).
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Recently, numerous in vitro and in vivo studies highlighted the neurotoxic effects
58
of the flame retardant TDCIPP. For instance, in vitro studies have shown that TDCIPP
59
decreased cell viability, increased apoptosis in various neuronal cell types (Li et al.,
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2017a, b; Ta et al., 2014), and interfered with cell differentiation and cell migration in
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PC12 cells (Dishaw et al., 2011). An in vivo study has shown that TDCIPP extensively
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affected the neurotrophic factors as well as their receptors in adult Chinese rare minnow
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(Gobiocypris rarus) (Yuan et al., 2016). In zebrafish (Danio rerio), TDCIPP exposure
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could reduce levels of neurotransmitter (e.g. serotonin and dopamine), downregulate
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the developmental gene expressions in nervous system (e.g., myelin basic protein (mbp)
66
and synapsinII a (syn2a)) in adults (Wang et al., 2015b) and 5dpf F1 larvae derived
67
from exposed parents (Wang et al., 2015a). In addition to these biochemical effects,
68
several studies in early life zebrafish have shown that acute or developmental exposure
69
to TDCIPP caused significant behavior alterations, particularly in larval swimming
70
activity (Dishaw et al., 2014; Jarema et al., 2015; Noyes et al., 2015; Oliveri et al.,
71
2015), indicating that changes in behavior may be a sensitive endpoint to TDCIPP
72
exposure. However, the possible mechanisms underlying the observed motor behavior
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changes following TDCIPP exposure have not been explored.
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In zebrafish, motor activities in larvae are mainly generated in the hindbrain and
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the spinal cord (Drapeau et al., 2002). Several types of neuronal cells are thought to be
76
involved in the control of locomotion behavior, and motoneurons are responsible for
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excitatory synaptic activity as well as firing patterns with respect to swimming
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(Brustein et al., 2003). Abnormalites in axnoal growth of motor neurons have been
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suggested to be correlated with motor behavior changes in zebrafish larvae after some
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chemicals exposures (e.g., chlorpyrifos (CPF), 2,2',4,4'-tetrabrominated biphenyl ether
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(BDE47), bisphenol A (BPA) and dechlorane plus (DP )) (Yang et al., 2011; Chen et al.,
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2012b; Wang et al., 2013; Chen et al., 2017). In zebrafish, several genes are related to
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axonogenesis (Zhang et al., 2011). The α1-tubulin gene is essential for development
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and regeneration of axons and dendrites (Baas et al., 1997). Sonic hedgehog a (Shha)
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is an axonal cue on retinal ganglion cell (RGC) and spinal cord commissural axons
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(Charron et al., 2003; Kolpak et al., 2005). Netrins (netrin1b and netrin2) are
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demonstrated to stimulate axonal outgrowth in zebrafish embryos (Lauderdale et al.,
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1997). And growth associated protein 43 (gap43) is expressed in zebrafish neurons at
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high levels during axonal development and regeneration. Recent studies have reported
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that alterations of the expressions of these genes may be associated with the inhibition
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of axonal growth in zebrafish larvae exposed to some chemicals (Zhang et al., 2011;
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Chen et al., 2012b; Chen et al., 2017). Thus, neuroactive chemicals that affect motor
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neurons development, at morphology or/and molecular level, may affect fish swimming
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activity.
95
Motor behavior not only relies on normal neuronal innervations, but also
96
associates with the cholinergic system (Rico et al., 2011). In zabrafish, the
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neurotransmitter acetylcholine (ACh) is a signaling molecule that elicits several actions
98
at neuromuscular junctions (NMJ) (Behra et al., 2002). It has been reported that a
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mutation in zebrafish acetylcholinesterase (ache) caused severe impairment of motility
100
(Behra et al., 2002). Although recent studies reported that exposure to TDCIPP resulted
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in no effects on AChE activities or Ach contents in fish, the concentrations are relative
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low at which no behavior alterations were observed or evaluated (Wang et al., 2015b;
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Yuan et al., 2016). It was still unknown whether alterations in motor activity induced
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by TDCIPP were associated with the cholinergic system.
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The objective of the present study was to verify whether TDCIPP developmental
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exposure induces motor behavior changes and elucidate the potential mechanisms
107
underlying the neurobehavioral alterations. In this study, we evaluated alterations in
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motor behaviors, which was a confirmatory of previous studies (Dishaw et al., 2014;
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Jarema et al., 2015; Noyes et al., 2015; Oliveri et al., 2015), and worked as a basis for
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studying the underlying mechanisms. We also evaluated the effects of TDCIPP on
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neuron development, axonal growth of motor neurons as well as expressions of genes
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related to axonal growth, and cholinergic system, which may help to elucidate the
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neurobehavioral alterations.
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2. Materials and methods
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2.1. Chemicals and reagents
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Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP, purity > 95%) was obtained
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from TCI Tokyo Chemical Industry Company (Tokyo, Japan). Dimethyl sulfoxide
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(DMSO, purity > 99%) used for storing and diluting TDCIPP, and MS-222 (3-
119
aminobenzoic acid ethyl ester, methane sulfonate salt) were purchased from Sigma-
120
Aldrich (St. Louis, MO, USA). The TRIzol reagent, PrimeScript Reverse Transcription
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(RT) Reagent kits and SYBR Green kits were obtained from TaKaRa (TaKaRa, Dalian,
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China). All the other reagents used in our research were of analytical grade.
123
2.2. Zebrafish maintenance and embryo exposure to TDCIPP
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Adult zebrafish (Danio rerio; AB strain) maintenance as well as embryo exposure
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were carried out according to previous protocol (Yu et al., 2010). Briefly, normally
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developed zebrafish embryos that reached blastula stage (2 h post fertilization; hpf)
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were visually selected. The embryos were randomly mixed and distributed in glass
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beakers (200 embryos per beaker) containing 500 mL TDCIPP of different nominal
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exposure concentrations (0, 100, 300, 600, and 900 μg/L) for 120 hours. The lowest
130
exposure concentration (100 μg/L) was selected based on a pilot range-finding study,
131
in which we found this concentration to be the maximum no observed effect
132
concentration on behavior alteration. Additionally, we also noticed in a recent study
133
that zebrafish larvae exposed to TDCIPP less than 100 μg/L for 120 hours did not
134
significantly differ from control larvae in locomotion (Wang et al., 2015b). Both the
135
control and the TDCIPP-treated embryos were added 0.001% (v/v) DMSO. A total of
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three replicates were used for each treatment, and the embryos were cultured at 28 ±
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0.5 °C with illumination of 14 h light/10 h dark (L/D) cycles. During the experimental
138
period, 50% of the TDCIPP solution was daily renewed to maintain the appropriate
139
concentration. After behavior analysis at 120 hpf, larvae were sampled, frozen with
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liquid nitrogen immediately, and stored at -80 °C until subsequent analysis. Acute
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endpoints, including mortality, hatching, and malformation were recorded. The body
142
length was determined by measuring from the anteriormost part of the head until the tip
143
of the tail along the body axis (10 larvae/replicate) with Image Pro Plus software (Media
144
Cybernetics). All studies were conducted in accordance with the guidelines for the care
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and use of laboratory animals of the National Institute for Food and Drug Control of
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China.
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2.3. Locomotor behavior measurement of zebrafish larvae
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The spontaneous movement test was videotaped via a CCD camera mounted to a
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dissection microscope (Leica, Germany), as depicted in a recent study (Chen et al.,
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2017). Embryos were exposed to TDCIPP in 6-well plate beginning at 2 hpf. Starting
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from 18 hpf, alternating tail coiling or bending was recorded with the videotape as
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spontaneous movement for 1 min every two hours until 28 hpf. All recordings on the
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recording station started after 5 min adaptation. For each treatment group, totally 30
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embryos from three replicates were used for analysis.
155
Larval locomotor activity at 120 hpf was quantified according to Wang et al. (2014)
156
using a Video-Track system (ViewPoint Life Sciences, Montreal, Canada). Larvae that
157
were dead or showed deformity were excluded from the assay. The swimming speed in
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response to dark and light transition (5 min dark and 5 min light) was monitored. Data
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on distance traveled, movement frequency, and movement duration were recorded
160
every 30 s, and each assay was repeated 4 times. Data were analyzed with custom Open
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Office Org 2.4 software. The net speed changes in response to the switch of light state
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were recorded as the differences between the average swimming speeds at the end of
163
one light state (for instance, the light period) and the beginning of the following light
164
state (for instance, the dark period).
165
2.4. Transgenic zebrafish larvae assay
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Adult transgenic (HuC-GFP) zebrafish (Danio rerio, AB strain) were purchased
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from the China Zebrafish Resource Center at the Institute of Hydrobiology, Chinese
168
Academy of Sciences (Wuhan, China). The protocol of adult fish maintenance and
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embryo exposure were the same as described above in section 2.2. After 120 hpf
170
exposure of TDCIPP (0, 100, 300, 600, and 900 μg/L), images of the Tg (HuC-GFP)
171
embryos were acquired using a fluorescence microscope (Leica M205 FA, Leica
172
Microsystems, Germany). GFP fluorescence was measured for Tg (HuC-GFP) larvae
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with Image J software (http://rsbweb.nih.gov/ij/) based on 2D images.
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2.5. Gene expression
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At 120 hpf, 30 larvae were randomly sampled from each beaker (n=3) and
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preserved with RNAiso Plus following the manufacturer’s instructions (Takara, Dalian,
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China). RNA extraction, and first-strand cDNA synthesis were conducted according to
178
our previous study (Yu et al., 2010). Primers used in this study were designed with the
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online Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/; sequences were
180
provided in the supporting information as Table A1). Expression of glyceraldehyde 3-
181
phosphate dehydrogenase (GAPDH) did not change after various concentrations of
182
TDCIPP exposure and was thus adopted as an internal control. The quantitative real-
183
time polymerase chain reaction (qRT-PCR) was done using SYBR Green PCR kits
184
(Takara, Dalian, Liaoning, China) on an ABI Step One Plus RT-PCR (Applied
185
Biosystem, Foster City, CA) system, and the relative expressions of genes were
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determined by the 2-ΔΔCt method.
187
2.6. Whole-mount immunohistochemistry
188
At 120 hpf, the larvae were sampled and anesthetized in 0.03% tricaine
189
methanesulfonate (MS-222), and then were fixed in 4% paraformaldehyde. Well-
190
characterized antibodies were used to perform the assay of whole-mount
191
immunohistochemistry in both control and TDCIPP-exposed larvae to visualize subsets
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of neurons as well as their axons, according to previously described methods (e.g. Zhu
193
et al., 2016) (for more details, see supporting information, Text A1). Secondary motor
194
neurons (SMNs) were immunolabeled with ZN-5 (1:200, Zebrafish International
195
Resource Center, University of Oregon). Samples were imaged with an inverted
196
fluorescent microscope (Leica DMI 6000B, Leica Microsystems, Germany). Axon
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length was quantified with Image J software for the larvae (10, n = 3 replicates), and
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the average value of axon length of each larva was then normalized to the body width.
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The body width was determined by drawing a straight line across the trunk region to
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the above of the terminal of the yolk extension, according to a previously described
201
method (Yang et al., 2011).
202
2.7. Acetylcholine concentration measurement
203
Measurement of acetylcholine concentration were according to a method
204
previously described (Wang et al., 2015a). Quantification of ACh was performed using
205
high-performance liquid chromatography equipped with a Colochem 5600 A
206
electrochemical detector (ESA). The analytical procedure is described in detail in the
207
Supporting Information (Text A2).
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2.8. Acetylcholinesterase activity measurement
209
At 120 hpf, 100 larvae were randomly selected for per beaker (n = 3 replicates)
210
and homogenized on ice with 500 μL of tris-citrate buffer (containing 50mM tris, 2mM
211
EDTA, 2mM EGTA; pH adjusted to 7.4 with citric acid) (Chen et al., 2012a), and the
212
homogeneous liquid was then centrifuged at 3000 × g for 10 min at 4 °C. The
213
supernatant was then transferred to a new tube. The enzyme activity was measured
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using an AChE assay kit according to the manufacturer’s instructions (Nanjing
215
JianCheng, Co, Ltd.). The protein concentration was also measured through the
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Bradford method using bovine serum albumin (BSA) as a standard.
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2.9. Statistical analysis
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Normality and homogeneity of variance were tested using Kolmogorov-Smirnov
219
test and Levene's test, respectively for all data. All data are reported as means ± standard
220
deviation of the mean (SD). The differences of the solvent control and exposure groups
221
were analyzed by one-way analysis of variance (ANOVA) and Tukey's test with SPSS
222
19.0 (SPSS, Chicago, IL). A P value less than 0.05 was considered to be statistically
223
significant.
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3. Results
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3.1. Mortality and effects of TDCIPP on early developing zebrafish
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The effects of different concentrations of TDCIPP (0, 100, 300, 600, and 900 μg/L)
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exposure on the survival, hatching, and malformation rate, and body length of the
228
zebrafish larvae were measured (Table 1). Results show that the hatching rate at 96 hpf
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and survival rate at 120 hpf decreased only in the highest exposure group (Table 1). The
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malformation rate (trunk curvature) at 120 hpf was significantly increased to 7.0 % ±
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2.0 after exposure 900 μg/L TDCIPP. Significantly lower body length was found in 120
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hpf larvae which had been exposed to 600 (3.42 ± 0.18 mm) and 900 μg/L TDCIPP
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(3.32 ± 0.24 mm) compared to the control (3.72 ± 0.11 mm).
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3.2. TDCIPP exposure altered spontaneous movement
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Spontaneous movement was evaluated from 18 to 28 hpf. In control embryos, side-
236
to-side alternating contractions initiated at 18 hpf, and gradually reached a peak of 6
237
bends/min at 22 hpf, but decreased to 4 bends/min at 24 hpf, and then fluctuated in this
238
way for nearly 4 hours. In TDCIPP treated embryos, spontaneous movement initiated
239
at similar time (∼18 hpf), but exhibited increased bending frequency from 18 to 22 hpf,
240
with peak frequencies appeared at 22 hpf (Fig. 1). Significantly increased spontaneous
241
movement was observed at 22 and/or 24 hpf in TDCIPP-treated larvae (300, 600 and
242
900 μg/L), and the highest frequency of 9.1 ± 1.0 bends/min appeared in 900 μg/L
243
TDCIPP treated larvae (Fig. 1).
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3.3. TDCIPP caused significant reduction in neuron-specific GFP expression in
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transgenic zebrafish
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In order to intuitively evaluate the effect of TDCIPP on the nervous system of
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developing zebrafish, we used a transgenic zebrafish line (HuC-GFP), whose neurons
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could be specifically identified through the expression of GFP. Compared with the
249
control, exposure of this transgenic zebrafish to TDCIPP with lower concentrations
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(100, 300 and 600 μg/L) for 120 h caused no significantly differences, while the 900
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μg/L TDCIPP treated embryos showed a significant reduction (45.2%) in GFP
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expressions in the brain as well as the spinal cord (Fig. 3B).
253
3.4. TDCIPP inhibited expressions of early neuronal maker genes
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In order to confirm the neural abnormalities caused by TDCIPP, we examined the
255
early neuronal maker genes (elavl3 and ngn1) at mRNA level in TDCIPP-exposed
256
zebrafish larvae at 120 hpf. The expression of the elavl3 gene was decreased by 1.23-,
257
1.31- and 1.27-fold after exposure to TDCIPP of 300, 600 and 900 μg/L, respectively.
258
For ngn1, the expression was significantly downregulated by 1.51-fold after exposure
259
to TDCIPP of 900 μg/L (Fig. 4).
260
3.5 TDCIPP altered swimming activities in 120 hpf larvae
261
Locomotion activities in larvae at 120 hpf were further evaluated under alternating
262
light-dark stimulation. Locomotor traces used for the larvae are presented in Fig. 2A.
263
In all groups, the swimming speed usually decreased rapidly when photoperiod was
264
switched from dark to light and reversed vice versa. Embryos exposed to TDCIPP of
265
300 and 600 μg/L exhibited significantly higher swimming speed than the controls
266
during the 5-min dark period, however, significantly decreased swimming speed was
267
observed in embryos exposed to TDCIPP of 900 μg/L during the 5-min light period
268
(Fig. 2B).
269
3.6. TDCIPP inhibited axonal growth and altered axonal growth-related gene
270
expression
271
Fig.5A shows representative immunofluorescence micrographs of the axons of the
272
secondary motor neurons in ventral and dorsal trunk regions of the TDCIPP-treated
273
group and the solvent control group. Morphological analyses revealed that 900 μg/L
274
TDCIPP exposure significantly reduced the length of the dorsal and ventral axon from
275
secondary moto neuron (Fig. 5B, C). At mRNA level, the expression of five genes
276
associated with axonal growth (α1-tubulin, shha, netrin1b, nertin2 and gap43) were
277
further assessed by qRT-PCR. The gene expression of α1-tubulin, shha and nestrin2
278
were significantly up-regulated at 120 hpf in 900 μg/L TDCIPP treated group (Fig. 5D).
279
For gap43 and netrin1b, there was no significant difference in expression of these genes.
280
3.7. TDCIPP decreased larval ACh content and increased AChE activity
281
In the solvent control group, the ACh content in the whole body was measured to
282
be 5063.0 ng/g wet mass (wm). No significant difference was observed in the ACh
283
contents between the embryos exposed to 100, 300 and 600 μg/L TDCIPP and those in
284
the control group. However, exposure to 900 μg/L TDCIPP resulted in significantly
285
lower (45.6%) ACh content relative to the solvent controls (Fig. 6A). Additionally,
286
significant increase of the total AChE activity was observed in 600 and 900 μg/L
287
TDCIPP exposure groups (24.5% and 29.8%, respectively; Fig. 6B)
288
4. Discussion
289
In accordance with similar studies of TDCIPP, we found that TDCIPP treatment
290
at several hundred μg/L resulted in developmental toxicity, including reduced survival
291
and hatching rates, decreased body length and increased malformation (spinal curvature)
292
in zebrafish embryos and larvae (see also McGee et al., 2012; Fu et al., 2013; Wang et
293
al., 2013). In addition, we also observed elevated spontanoues movemoment and altered
294
swimming behaviors in TDCIPP-treated zebrafish larvae, which evoke us to explore
295
the possible mechanisms underlying the neurobehavior effects induced by TDCIPP.
296
Spontaneous movement is a transient motor behavior that occurs from late-
297
segmentation (~17–19 hpf) through early-pharyngula (~27–29 hpf), which represents
298
an early, primitive form of motor activity within zebrafish embryos (Vliet et al., 2017).
299
For the first time, our data show that TDCIPP exposure significantly elevated frequency
300
of spontaneous movement from 22 hpf to 24 hpf，but at the final period (~28hpf), the
301
group exposed to TDCIPP presented similar low frequency to control group. Similar
302
effects were also observed in other flame retardants, such as BDE-47 and DP (Chen et
303
al., 2012b; Chen et al., 2017). It has been reported that spontaneous movement
304
originates from spinal neuron innervations and is not myogenic (Brustein et al., 2003).
305
We thus evaluated the effects of TDCIPP exposure on neuron development in Tg (HuC-
306
GFP) zebrafish, in which altered GFP expression indicates that chemicals caused
307
abnormalities in the spinal and the brain (Chen et al., 2014; Kim et al., 2016). In this
308
study, significantly decreased GFP expression was observed in TDCIPP-treated Tg
309
zebrafish larvae, which probably means that TDCIPP disturbed the brain and spinal
310
neurons. Moreover, we observed significantly downregulation of expressions of
311
neuronal maker genes (elavl3 (encoding HuC) and neurogenin1 (ngn1)) (Kim et al.,
312
1996; Ma et al., 1996) in the TDCIPP-exposed larvae, which may also indicate the
313
neuron abnormalities. Although a direct link between the disturbed spinals and altered
314
spontaneous movement has not been reported, several studies speculated that some
315
chemicals (e.g. BDE-47, DP) may interfere with spinal neurons and contribute to the
316
increased spontaneous movement (Chen et al., 2012b; Chen et al., 2017). We thus
317
speculate that the disturbance in spinal neurons caused by TDCIPP in this study may
318
be a possible factor to the increased elevated spontaneous movement.
319
As a more complex behavior, swimming arises later in development and require
320
chemical transmission and hindbrain inputs (Drapeau et al., 2002). In the present study,
321
swimming activity was evaluated in larvae at 120 hpf using alternating light-dark
322
stimulation. Hypoactivity was observed in the highest exposure group (900 μg/L)
323
during the light period, however, exposure to TDCIPP of lower doses (300 and 600
324
μg/L) produced hyperactivity during the dark period. The hyperactivity with lower
325
exposure concentration but hypoactivity with higher exposure concentration have also
326
been reported in previous TDCIPP studies (Jarema et al., 2015；Noyes et al., 2015),
327
but the concentrations therein were different from those used in our study, specifically,
328
the lower concentrations inducing hyperactivity used by Jarema et al. (2015) and Noyes
329
et al. (2015) (517~1637 μg/L and 276~2757 μg/L, respectively) include our high
330
concentrations inducing hypoactivity. This discrepancy may result from the actual
331
exposure concentrations, but unfortunately, all these studies did not determine the
332
TDCIPP content. Another possible reason could be the different exposure time adopted
333
between the above studies and our study. Jarema et al. (2015) and Noyes et al. (2015)
334
used exposure times ranged from 6 to 120 hpf, but we used exposure times from 2 to
335
120 hpf in our study. Indeed, by testing a series of exposure scenarios, McGee et al.
336
(2012) suggested that early zebrafish embryonic stages (0.75-2 hpf, 0.75-96 hpf) were
337
much more sensitive toTDCIPP than later-stage embryos (5.25-96hpf). The low-dose
338
stimulatory effect may be considered to be an adaptive response for the body to protect
339
itself from toxicant stress (Calabrese, 2005). For the high-dose inhibitory effect, the
340
neural abnormalities as we observed may be a contributor.
341
As motor neuron is the major cell type that regulates swimming behavior at early
342
stage of zebrafish (Brustein et al., 2003), the effect of TDCIPP exposure on axonal
343
growth of secondary motor neuron was examined in the present study. Morphometric
344
analysis showed a significant reduction of both ventral and dorsal axon length in
345
zebrafish larvae exposed to 900 μg/L TDCIPP. Additionally, upregulated expressions
346
of axon growth related genes (α1-tubulin, shha and netrin2) was observed in the same
347
exposure group. In fact, upregulation of genes of α1-tubulin and/or gap43 has been
348
suggested as a compensatory feedback in response to inhibition of axonal growth in
349
zebrafish larvae following other chemical exposure (e.g., DP and BDE-47) (Chen et al.,
350
2017; Chen et al., 2012b). Therefore, the upregulated gene expressions may further
351
indicate that TDCIPP exposure perturbed secondary motor neuron axonal growth.
352
Although no relationships have been reported so far between the axonal growth and the
353
motor behavior for TDCIPP, a link between aberrant axonal growth and deficient in
354
swimming behavioral after exposure to other chemicals (e.g. CPF and BDE47) has been
355
reported in developing zebrafish (Chen et al., 2012b; Yang et al., 2011). We thus suggest
356
that structure disruptions of secondary motor neurons could cause alteration in
357
swimming behavior in zebrafish larvae.
358
Our results confirmed that relatively lower concentration of TDCIPP do not alter
359
the cholinergic system in fish (see also Wang et al., 2015b; Yuan et al., 2016). However,
360
for the first time, we found that higher concentration of TDCIPP significantly increased
361
the AChE activity and reduced the Ach content in 120 hpf zebrafish larvae. ACh plays
362
a critical role in modulating motor and cognitive functions in the cholinergic system
363
(Driscoll et al., 2009). The ACh level in zebrafish is specifically maintained through
364
the hydrolysis of AChE (Behra et al., 2002). Therefore, the decline of ACh content may
365
be due to over-hydrolysis of AChE, and could probably cause altered neuromuscular
366
junction as well as behavioral response. Inhibited ACh content accompanied by adverse
367
effects on swimming behavior has been observed in zebrafish larvae when they were
368
exposed to other chemicals, such as PBDE mixture DE-71 (Chen et al., 2012a),
369
microcystin-LR (MC-LR) (Wu et al., 2016). Consequently, another contributor to the
370
inhibition in locomotor behavior at the highest does in this study could be the
371
interference of the cholinergic system.
372
The present study confirmed that TDCIPP exposure induces locomotor behavior
373
alterations in zebrafish larvae. More importantly, for the first time, we investigated the
374
possible mechanisms of motor behavior alterations induced by TDCIPP in zebrafish
375
larvae. The results suggest that the behavioral changes induced by TDCIPP may result
376
from the combined effects of altered motor neuron structure and inhibited cholinergic
377
system. However, it should be noted that motor behavior is an integrated result of
378
different physiological and biochemical processes, and might be also influenced by
379
other factors (e.g. aberrant somatic muscle differentiation, calcium channels and signals)
380
(see Drapeau et al., 2002). Further studies will be needed to explore these possibilities.
381
Acknowledgments
382
The work in this study was supported by the National Natural Science Foundations
383
of China (21677057, 21307162 and 21622702), and Huazhong Agricultural University
384
Scientific & Technological Self-innovation Foundation (Program 2662014BQ035).
385
Appendix A. Supplementary Data
386
Procedure for immunohistochemistry staining (Text A1).
Analytical procedure
387
for acetylcholine concentration measurement (Text A2). Primer sequences used in the
388
present study (Table A1).
389
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390
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546
Table 1. Development parameters of zebrafish larvae exposed to 0, 100, 300, 600, 900 μg/L TDCIPP until 120 h post fertilizationa TDCIPP (μg/L) Malformation rate (%) Survival rate (%) Hatching rate (%) Body length (mm)
547 548
a
*
0
100
300
1.0 ± 1.0
1.3 ± 1.1
3.0 ± 2.0
95.0 ± 1.0 91.7 ± 2.1 3.71 ± 0.11
90.0 ± 4.6 88.3 ± 2.1 3.61 ± 0.19
88.7 ± 3.2 88.3 ± 3.8 3.60 ± 0.18
600 5.0 ± 2.0 84.0 ± 2.7 82.3 ± 5.1 3.42 ± 0.18***
900 7.0 ± 2.0** 74.3 ± 8.3*** 72.3 ± 2.0*** 3.32 ± 0.24***
Data are expressed as mean ± SD of three replicates (30 larvae per replicate).
P < 0.01, **P < 0.01, ***P < 0.001 indicate significant difference detected between control and TDCIPP exposed group
549
550 551
Figure 1. Spontaneous movement in zebrafish larvae from the control group and
552
exposure groups (100, 300, 600, 900 μg/L) was recorded every two hours from 18 to
553
28 hpf. The data are expressed as mean ± SD of three replicates (10 larvae per replicate).
554
*
555
exposed group.
556
557
P < 0.05,
**
P < 0.01 indicate significant difference between control and TDCIPP
558 559
Figure 2. (A) Locomotor patterns in response to an alternating light change. (B) The
560
average swimming speed of 5-min intervals for each light state (light or dark). The data
561
are expressed as mean ± SD of three replicates (10 larvae per replicate). *P < 0.05, **P
562
< 0.01 and
563
TDCIPP exposed group.
564 565 566
***
P < 0.001 indicate significant difference detected between control and
567 568
Figure 3. Effect of TDCIPP on Tg (HuC-GFP) zebrafish larvae at 120hpf. (A)
569
Representative images from control group and highest TDCIPP (900 μg/L) exposure
570
group; (B) Relative GFP expression in larvae after various concentrations of TDCIPP
571
(0, 100, 300, 600, 900 μg/L) at 120 hpf. The data are expressed as mean ± SD of three
572
replicates (10 larvae per replicate). *P < 0.05, **P < 0.01 indicate significant difference
573
detected between control and TDCIPP exposed group.
574
575 576
Figure 4. The mRNA levels of neuron marker genes (elavl3 and ngn1) in zebrafish
577
larvae at 120 hpf. The data are expressed as mean ± SD of three replicates (30 larvae
578
per replicate). *P < 0.05, **P < 0.01 indicate significant difference detected between
579
control and TDCIPP exposed group.
580 581
582
583 584
585 586
Figure 5. TDCIPP exposure affected axonal growth of secondary motoneuron and
587
axonal growth-related gene expression. (A) Representative photomicrographs
588
illustrating ZN-5 immunopositive axons in dorsal and ventral trunk regions. (B)
589
Quantitative measurements of dorsal length (10 larvae each replicate, n=3 replicates).
590
(C) Quantitative measurements of ventral axon length (10 larvae each replicate, n=3
591
replicates). (D) Gene expression profiles in zebrafish larvae at 120 hpf (30 larvae each
592
replicate, n=3 replicates). *P < 0.05, **P < 0.01 indicate significant difference detected
593
between control and TDCIPP exposed group.
594
595 596
Figure 6. Acetylcholine (ACh) content (A) and acetylcholinesterase (AChE) activity
597
(B) in zebrafish larvae following exposure to various concentrations of TDCIPP (0, 100,
598
300, 600, 900 μg/L) at 120 hpf. The data are expressed as mean ± SD of three replicates
599
(100 larvae per replicate). *P < 0.05, **P < 0.01 indicate significant difference detected
600
between control and TDCIPP exposed group.
S0166-445X(17)30241-2 http://dx.doi.org/10.1016/j.aquatox.2017.09.003 AQTOX 4735
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
7-6-2017 30-8-2017 2-9-2017
Please cite this article as: Cheng, Rui, Jia, Yali, Dai, Lili, Liu, Chunsheng, Wang, Jianghua, Li, Guangyu, Yu, Liqin, Tris(1,3-dichloro-2-propyl) Disrupts Axonal Growth, Cholinergic System and Motor Behavior in Early Life Zebrafish.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2017.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Tris(1,3-dichloro-2-propyl) Disrupts Axonal Growth, Cholinergic System and
2
Motor Behavior in Early Life Zebrafish
3
Rui Chenga, Yali Jiaa, Lili Daib, Chunsheng Liua,c,d, Jianghua Wanga, Guangyu
4
Lia, Liqin Yua,*
5
a
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China
6
b
Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences
7
c
Collaborative Innovation Center for Efficient and Health Production of Fisheries in
8
Hunan Province, Hunan Changde 415000
9
d
Hubei Provincial Engineering Laboratory for Pond Aquaculture
10
11
* Adress correspondence to
12
College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China. Tel:
13
86 27 87282113. Fax: 86 27 87282114. Email: [email protected]
14
Highlights
15
1. TDCIPP altered motor behaviors in zebrafish larvae.
16
2. TDCIPP decreased neuron-specific GFP expression in Τg (HuC- GFP) zebrafish
17 18 19 20
larvae. 3. TDCIPP inhibited axonal growth of secondary motor neurons and altered expressions of genes related to axonal growth. 4. TDCIPP inhibited the cholinergic system in zebrafish larvae.
21 22 23
5. TDCIPP induced neurobehavioral alterations may result from combined effects of altered motor neuron structure and inhibition of cholinergic system.
24
Abbreviation List Abbreviation
Full Name
TDCIPP
tris(1,3-dichloro-2-propyl) phosphate
PBDEs
polybrominated diphenyl ethers
BDE-47
2,2',4,4'-tetrabrominated biphenyl ether
BPA
25
bisphenol A
DP
dechlorane plus
CPF
chlorpyrifos
DE-71
a mixture of polybrominated diphenyl ethers
MC-LR
microcystin-LR
ACh
acetylcholine
AChE
acetylcholinesterase
Tg
transgenic
gapdh
glyceraldehyde-3-phosphate dehydrogenase
shha
sonic hedgehog a
gap43
growth associated protein 43
elavl3
elav like neuron-specific RNA binding protein 3
ngn1
neurogenin 1
mbp
myelin basic protein
syn2a
synapsinII a
26
Abstract：TDCIPP could have neurotoxic effects and alter motor behaviors in zebrafish
27
(Danio rerio) larvae, however, the underlying mechanisms are still unknown. In this
28
study, zebrafish embryos were subjected to waterborne exposure of TDCIPP at 100,
29
300, 600, 900 μg/L from 2 to 120-hour post-fertilization (hpf). Behavioral
30
measurements indicate that TDCIPP exposure significantly elevated spontaneous
31
movement, and altered swimming behavior response of larvae to both light and dark
32
stimulation. Interestingly, in accordance with these motor effects, TDCIPP significantly
33
decreased expression of the neuron-specific GFP in transgenic (HuC-GFP) zebrafish
34
larvae as well as decreased expression of the neural marker genes elavl3 and ngn1,
35
inhibited the axonal growth of the secondary motoneurons and altered the expressions
36
of axon-related genes (α1-tubulin, shha and netrin2) in zebrafish larvae. Furthermore,
37
TDCIPP
38
acetylcholinesterase (AChE) enzyme, and decreased the total acetylcholine (ACh)
39
concentration. Our data indicate that the alteration in motor neuron and inhibition of
40
cholinergic system could together lead to the TDCIPP induced motor behavior
41
alterations in zebrafish larvae.
42
Keywords：TDCIPP; zebrafish larvae; motor behavior; axonal growth
exposure
at
900
μg/L
significantly
increased
the
activity
of
43
1. Introduction
44
Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been used as an
45
organophosphate flame retardant for decades in various products (e.g. plastics, textiles,
46
foams, electronic equipment, varnishes, and furniture), with an estimated annual
47
production ranged from 4500 to 22700 tones between 1998 and 2006 in the United
48
States (Stapleton et al., 2009; van der Veen and de Boer, 2012). Like polybrominated
49
diphenyl ethers (PBDEs), this chemical can leach out of materials and can now be
50
detected in various environments and biota samples (Cao et al., 2014; Meeker et al.,
51
2013; Meeker and Stapleton, 2010; Wei et al., 2015; Yang et al., 2014). In surface water,
52
TDCIPP concentrations have been generally reported at ng/L levels (Cao et al., 2012;
53
Hu et al., 2014; Shi et al., 2016; Wang et al., 2011), but it is not readily degraded in
54
water and could accumulate in aquatic organisms. Indeed, it has been reported that
55
TDCIPP in freshwater fish could reach up to several hundred μg/kg lipid weight (e.g.
56
140 μg/kg in perch) (Sundkvist et al., 2010).
57
Recently, numerous in vitro and in vivo studies highlighted the neurotoxic effects
58
of the flame retardant TDCIPP. For instance, in vitro studies have shown that TDCIPP
59
decreased cell viability, increased apoptosis in various neuronal cell types (Li et al.,
60
2017a, b; Ta et al., 2014), and interfered with cell differentiation and cell migration in
61
PC12 cells (Dishaw et al., 2011). An in vivo study has shown that TDCIPP extensively
62
affected the neurotrophic factors as well as their receptors in adult Chinese rare minnow
63
(Gobiocypris rarus) (Yuan et al., 2016). In zebrafish (Danio rerio), TDCIPP exposure
64
could reduce levels of neurotransmitter (e.g. serotonin and dopamine), downregulate
65
the developmental gene expressions in nervous system (e.g., myelin basic protein (mbp)
66
and synapsinII a (syn2a)) in adults (Wang et al., 2015b) and 5dpf F1 larvae derived
67
from exposed parents (Wang et al., 2015a). In addition to these biochemical effects,
68
several studies in early life zebrafish have shown that acute or developmental exposure
69
to TDCIPP caused significant behavior alterations, particularly in larval swimming
70
activity (Dishaw et al., 2014; Jarema et al., 2015; Noyes et al., 2015; Oliveri et al.,
71
2015), indicating that changes in behavior may be a sensitive endpoint to TDCIPP
72
exposure. However, the possible mechanisms underlying the observed motor behavior
73
changes following TDCIPP exposure have not been explored.
74
In zebrafish, motor activities in larvae are mainly generated in the hindbrain and
75
the spinal cord (Drapeau et al., 2002). Several types of neuronal cells are thought to be
76
involved in the control of locomotion behavior, and motoneurons are responsible for
77
excitatory synaptic activity as well as firing patterns with respect to swimming
78
(Brustein et al., 2003). Abnormalites in axnoal growth of motor neurons have been
79
suggested to be correlated with motor behavior changes in zebrafish larvae after some
80
chemicals exposures (e.g., chlorpyrifos (CPF), 2,2',4,4'-tetrabrominated biphenyl ether
81
(BDE47), bisphenol A (BPA) and dechlorane plus (DP )) (Yang et al., 2011; Chen et al.,
82
2012b; Wang et al., 2013; Chen et al., 2017). In zebrafish, several genes are related to
83
axonogenesis (Zhang et al., 2011). The α1-tubulin gene is essential for development
84
and regeneration of axons and dendrites (Baas et al., 1997). Sonic hedgehog a (Shha)
85
is an axonal cue on retinal ganglion cell (RGC) and spinal cord commissural axons
86
(Charron et al., 2003; Kolpak et al., 2005). Netrins (netrin1b and netrin2) are
87
demonstrated to stimulate axonal outgrowth in zebrafish embryos (Lauderdale et al.,
88
1997). And growth associated protein 43 (gap43) is expressed in zebrafish neurons at
89
high levels during axonal development and regeneration. Recent studies have reported
90
that alterations of the expressions of these genes may be associated with the inhibition
91
of axonal growth in zebrafish larvae exposed to some chemicals (Zhang et al., 2011;
92
Chen et al., 2012b; Chen et al., 2017). Thus, neuroactive chemicals that affect motor
93
neurons development, at morphology or/and molecular level, may affect fish swimming
94
activity.
95
Motor behavior not only relies on normal neuronal innervations, but also
96
associates with the cholinergic system (Rico et al., 2011). In zabrafish, the
97
neurotransmitter acetylcholine (ACh) is a signaling molecule that elicits several actions
98
at neuromuscular junctions (NMJ) (Behra et al., 2002). It has been reported that a
99
mutation in zebrafish acetylcholinesterase (ache) caused severe impairment of motility
100
(Behra et al., 2002). Although recent studies reported that exposure to TDCIPP resulted
101
in no effects on AChE activities or Ach contents in fish, the concentrations are relative
102
low at which no behavior alterations were observed or evaluated (Wang et al., 2015b;
103
Yuan et al., 2016). It was still unknown whether alterations in motor activity induced
104
by TDCIPP were associated with the cholinergic system.
105
The objective of the present study was to verify whether TDCIPP developmental
106
exposure induces motor behavior changes and elucidate the potential mechanisms
107
underlying the neurobehavioral alterations. In this study, we evaluated alterations in
108
motor behaviors, which was a confirmatory of previous studies (Dishaw et al., 2014;
109
Jarema et al., 2015; Noyes et al., 2015; Oliveri et al., 2015), and worked as a basis for
110
studying the underlying mechanisms. We also evaluated the effects of TDCIPP on
111
neuron development, axonal growth of motor neurons as well as expressions of genes
112
related to axonal growth, and cholinergic system, which may help to elucidate the
113
neurobehavioral alterations.
114
2. Materials and methods
115
2.1. Chemicals and reagents
116
Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP, purity > 95%) was obtained
117
from TCI Tokyo Chemical Industry Company (Tokyo, Japan). Dimethyl sulfoxide
118
(DMSO, purity > 99%) used for storing and diluting TDCIPP, and MS-222 (3-
119
aminobenzoic acid ethyl ester, methane sulfonate salt) were purchased from Sigma-
120
Aldrich (St. Louis, MO, USA). The TRIzol reagent, PrimeScript Reverse Transcription
121
(RT) Reagent kits and SYBR Green kits were obtained from TaKaRa (TaKaRa, Dalian,
122
China). All the other reagents used in our research were of analytical grade.
123
2.2. Zebrafish maintenance and embryo exposure to TDCIPP
124
Adult zebrafish (Danio rerio; AB strain) maintenance as well as embryo exposure
125
were carried out according to previous protocol (Yu et al., 2010). Briefly, normally
126
developed zebrafish embryos that reached blastula stage (2 h post fertilization; hpf)
127
were visually selected. The embryos were randomly mixed and distributed in glass
128
beakers (200 embryos per beaker) containing 500 mL TDCIPP of different nominal
129
exposure concentrations (0, 100, 300, 600, and 900 μg/L) for 120 hours. The lowest
130
exposure concentration (100 μg/L) was selected based on a pilot range-finding study,
131
in which we found this concentration to be the maximum no observed effect
132
concentration on behavior alteration. Additionally, we also noticed in a recent study
133
that zebrafish larvae exposed to TDCIPP less than 100 μg/L for 120 hours did not
134
significantly differ from control larvae in locomotion (Wang et al., 2015b). Both the
135
control and the TDCIPP-treated embryos were added 0.001% (v/v) DMSO. A total of
136
three replicates were used for each treatment, and the embryos were cultured at 28 ±
137
0.5 °C with illumination of 14 h light/10 h dark (L/D) cycles. During the experimental
138
period, 50% of the TDCIPP solution was daily renewed to maintain the appropriate
139
concentration. After behavior analysis at 120 hpf, larvae were sampled, frozen with
140
liquid nitrogen immediately, and stored at -80 °C until subsequent analysis. Acute
141
endpoints, including mortality, hatching, and malformation were recorded. The body
142
length was determined by measuring from the anteriormost part of the head until the tip
143
of the tail along the body axis (10 larvae/replicate) with Image Pro Plus software (Media
144
Cybernetics). All studies were conducted in accordance with the guidelines for the care
145
and use of laboratory animals of the National Institute for Food and Drug Control of
146
China.
147
2.3. Locomotor behavior measurement of zebrafish larvae
148
The spontaneous movement test was videotaped via a CCD camera mounted to a
149
dissection microscope (Leica, Germany), as depicted in a recent study (Chen et al.,
150
2017). Embryos were exposed to TDCIPP in 6-well plate beginning at 2 hpf. Starting
151
from 18 hpf, alternating tail coiling or bending was recorded with the videotape as
152
spontaneous movement for 1 min every two hours until 28 hpf. All recordings on the
153
recording station started after 5 min adaptation. For each treatment group, totally 30
154
embryos from three replicates were used for analysis.
155
Larval locomotor activity at 120 hpf was quantified according to Wang et al. (2014)
156
using a Video-Track system (ViewPoint Life Sciences, Montreal, Canada). Larvae that
157
were dead or showed deformity were excluded from the assay. The swimming speed in
158
response to dark and light transition (5 min dark and 5 min light) was monitored. Data
159
on distance traveled, movement frequency, and movement duration were recorded
160
every 30 s, and each assay was repeated 4 times. Data were analyzed with custom Open
161
Office Org 2.4 software. The net speed changes in response to the switch of light state
162
were recorded as the differences between the average swimming speeds at the end of
163
one light state (for instance, the light period) and the beginning of the following light
164
state (for instance, the dark period).
165
2.4. Transgenic zebrafish larvae assay
166
Adult transgenic (HuC-GFP) zebrafish (Danio rerio, AB strain) were purchased
167
from the China Zebrafish Resource Center at the Institute of Hydrobiology, Chinese
168
Academy of Sciences (Wuhan, China). The protocol of adult fish maintenance and
169
embryo exposure were the same as described above in section 2.2. After 120 hpf
170
exposure of TDCIPP (0, 100, 300, 600, and 900 μg/L), images of the Tg (HuC-GFP)
171
embryos were acquired using a fluorescence microscope (Leica M205 FA, Leica
172
Microsystems, Germany). GFP fluorescence was measured for Tg (HuC-GFP) larvae
173
with Image J software (http://rsbweb.nih.gov/ij/) based on 2D images.
174
2.5. Gene expression
175
At 120 hpf, 30 larvae were randomly sampled from each beaker (n=3) and
176
preserved with RNAiso Plus following the manufacturer’s instructions (Takara, Dalian,
177
China). RNA extraction, and first-strand cDNA synthesis were conducted according to
178
our previous study (Yu et al., 2010). Primers used in this study were designed with the
179
online Primer 3 software (http://bioinfo.ut.ee/primer3-0.4.0/primer3/; sequences were
180
provided in the supporting information as Table A1). Expression of glyceraldehyde 3-
181
phosphate dehydrogenase (GAPDH) did not change after various concentrations of
182
TDCIPP exposure and was thus adopted as an internal control. The quantitative real-
183
time polymerase chain reaction (qRT-PCR) was done using SYBR Green PCR kits
184
(Takara, Dalian, Liaoning, China) on an ABI Step One Plus RT-PCR (Applied
185
Biosystem, Foster City, CA) system, and the relative expressions of genes were
186
determined by the 2-ΔΔCt method.
187
2.6. Whole-mount immunohistochemistry
188
At 120 hpf, the larvae were sampled and anesthetized in 0.03% tricaine
189
methanesulfonate (MS-222), and then were fixed in 4% paraformaldehyde. Well-
190
characterized antibodies were used to perform the assay of whole-mount
191
immunohistochemistry in both control and TDCIPP-exposed larvae to visualize subsets
192
of neurons as well as their axons, according to previously described methods (e.g. Zhu
193
et al., 2016) (for more details, see supporting information, Text A1). Secondary motor
194
neurons (SMNs) were immunolabeled with ZN-5 (1:200, Zebrafish International
195
Resource Center, University of Oregon). Samples were imaged with an inverted
196
fluorescent microscope (Leica DMI 6000B, Leica Microsystems, Germany). Axon
197
length was quantified with Image J software for the larvae (10, n = 3 replicates), and
198
the average value of axon length of each larva was then normalized to the body width.
199
The body width was determined by drawing a straight line across the trunk region to
200
the above of the terminal of the yolk extension, according to a previously described
201
method (Yang et al., 2011).
202
2.7. Acetylcholine concentration measurement
203
Measurement of acetylcholine concentration were according to a method
204
previously described (Wang et al., 2015a). Quantification of ACh was performed using
205
high-performance liquid chromatography equipped with a Colochem 5600 A
206
electrochemical detector (ESA). The analytical procedure is described in detail in the
207
Supporting Information (Text A2).
208
2.8. Acetylcholinesterase activity measurement
209
At 120 hpf, 100 larvae were randomly selected for per beaker (n = 3 replicates)
210
and homogenized on ice with 500 μL of tris-citrate buffer (containing 50mM tris, 2mM
211
EDTA, 2mM EGTA; pH adjusted to 7.4 with citric acid) (Chen et al., 2012a), and the
212
homogeneous liquid was then centrifuged at 3000 × g for 10 min at 4 °C. The
213
supernatant was then transferred to a new tube. The enzyme activity was measured
214
using an AChE assay kit according to the manufacturer’s instructions (Nanjing
215
JianCheng, Co, Ltd.). The protein concentration was also measured through the
216
Bradford method using bovine serum albumin (BSA) as a standard.
217
2.9. Statistical analysis
218
Normality and homogeneity of variance were tested using Kolmogorov-Smirnov
219
test and Levene's test, respectively for all data. All data are reported as means ± standard
220
deviation of the mean (SD). The differences of the solvent control and exposure groups
221
were analyzed by one-way analysis of variance (ANOVA) and Tukey's test with SPSS
222
19.0 (SPSS, Chicago, IL). A P value less than 0.05 was considered to be statistically
223
significant.
224
3. Results
225
3.1. Mortality and effects of TDCIPP on early developing zebrafish
226
The effects of different concentrations of TDCIPP (0, 100, 300, 600, and 900 μg/L)
227
exposure on the survival, hatching, and malformation rate, and body length of the
228
zebrafish larvae were measured (Table 1). Results show that the hatching rate at 96 hpf
229
and survival rate at 120 hpf decreased only in the highest exposure group (Table 1). The
230
malformation rate (trunk curvature) at 120 hpf was significantly increased to 7.0 % ±
231
2.0 after exposure 900 μg/L TDCIPP. Significantly lower body length was found in 120
232
hpf larvae which had been exposed to 600 (3.42 ± 0.18 mm) and 900 μg/L TDCIPP
233
(3.32 ± 0.24 mm) compared to the control (3.72 ± 0.11 mm).
234
3.2. TDCIPP exposure altered spontaneous movement
235
Spontaneous movement was evaluated from 18 to 28 hpf. In control embryos, side-
236
to-side alternating contractions initiated at 18 hpf, and gradually reached a peak of 6
237
bends/min at 22 hpf, but decreased to 4 bends/min at 24 hpf, and then fluctuated in this
238
way for nearly 4 hours. In TDCIPP treated embryos, spontaneous movement initiated
239
at similar time (∼18 hpf), but exhibited increased bending frequency from 18 to 22 hpf,
240
with peak frequencies appeared at 22 hpf (Fig. 1). Significantly increased spontaneous
241
movement was observed at 22 and/or 24 hpf in TDCIPP-treated larvae (300, 600 and
242
900 μg/L), and the highest frequency of 9.1 ± 1.0 bends/min appeared in 900 μg/L
243
TDCIPP treated larvae (Fig. 1).
244
3.3. TDCIPP caused significant reduction in neuron-specific GFP expression in
245
transgenic zebrafish
246
In order to intuitively evaluate the effect of TDCIPP on the nervous system of
247
developing zebrafish, we used a transgenic zebrafish line (HuC-GFP), whose neurons
248
could be specifically identified through the expression of GFP. Compared with the
249
control, exposure of this transgenic zebrafish to TDCIPP with lower concentrations
250
(100, 300 and 600 μg/L) for 120 h caused no significantly differences, while the 900
251
μg/L TDCIPP treated embryos showed a significant reduction (45.2%) in GFP
252
expressions in the brain as well as the spinal cord (Fig. 3B).
253
3.4. TDCIPP inhibited expressions of early neuronal maker genes
254
In order to confirm the neural abnormalities caused by TDCIPP, we examined the
255
early neuronal maker genes (elavl3 and ngn1) at mRNA level in TDCIPP-exposed
256
zebrafish larvae at 120 hpf. The expression of the elavl3 gene was decreased by 1.23-,
257
1.31- and 1.27-fold after exposure to TDCIPP of 300, 600 and 900 μg/L, respectively.
258
For ngn1, the expression was significantly downregulated by 1.51-fold after exposure
259
to TDCIPP of 900 μg/L (Fig. 4).
260
3.5 TDCIPP altered swimming activities in 120 hpf larvae
261
Locomotion activities in larvae at 120 hpf were further evaluated under alternating
262
light-dark stimulation. Locomotor traces used for the larvae are presented in Fig. 2A.
263
In all groups, the swimming speed usually decreased rapidly when photoperiod was
264
switched from dark to light and reversed vice versa. Embryos exposed to TDCIPP of
265
300 and 600 μg/L exhibited significantly higher swimming speed than the controls
266
during the 5-min dark period, however, significantly decreased swimming speed was
267
observed in embryos exposed to TDCIPP of 900 μg/L during the 5-min light period
268
(Fig. 2B).
269
3.6. TDCIPP inhibited axonal growth and altered axonal growth-related gene
270
expression
271
Fig.5A shows representative immunofluorescence micrographs of the axons of the
272
secondary motor neurons in ventral and dorsal trunk regions of the TDCIPP-treated
273
group and the solvent control group. Morphological analyses revealed that 900 μg/L
274
TDCIPP exposure significantly reduced the length of the dorsal and ventral axon from
275
secondary moto neuron (Fig. 5B, C). At mRNA level, the expression of five genes
276
associated with axonal growth (α1-tubulin, shha, netrin1b, nertin2 and gap43) were
277
further assessed by qRT-PCR. The gene expression of α1-tubulin, shha and nestrin2
278
were significantly up-regulated at 120 hpf in 900 μg/L TDCIPP treated group (Fig. 5D).
279
For gap43 and netrin1b, there was no significant difference in expression of these genes.
280
3.7. TDCIPP decreased larval ACh content and increased AChE activity
281
In the solvent control group, the ACh content in the whole body was measured to
282
be 5063.0 ng/g wet mass (wm). No significant difference was observed in the ACh
283
contents between the embryos exposed to 100, 300 and 600 μg/L TDCIPP and those in
284
the control group. However, exposure to 900 μg/L TDCIPP resulted in significantly
285
lower (45.6%) ACh content relative to the solvent controls (Fig. 6A). Additionally,
286
significant increase of the total AChE activity was observed in 600 and 900 μg/L
287
TDCIPP exposure groups (24.5% and 29.8%, respectively; Fig. 6B)
288
4. Discussion
289
In accordance with similar studies of TDCIPP, we found that TDCIPP treatment
290
at several hundred μg/L resulted in developmental toxicity, including reduced survival
291
and hatching rates, decreased body length and increased malformation (spinal curvature)
292
in zebrafish embryos and larvae (see also McGee et al., 2012; Fu et al., 2013; Wang et
293
al., 2013). In addition, we also observed elevated spontanoues movemoment and altered
294
swimming behaviors in TDCIPP-treated zebrafish larvae, which evoke us to explore
295
the possible mechanisms underlying the neurobehavior effects induced by TDCIPP.
296
Spontaneous movement is a transient motor behavior that occurs from late-
297
segmentation (~17–19 hpf) through early-pharyngula (~27–29 hpf), which represents
298
an early, primitive form of motor activity within zebrafish embryos (Vliet et al., 2017).
299
For the first time, our data show that TDCIPP exposure significantly elevated frequency
300
of spontaneous movement from 22 hpf to 24 hpf，but at the final period (~28hpf), the
301
group exposed to TDCIPP presented similar low frequency to control group. Similar
302
effects were also observed in other flame retardants, such as BDE-47 and DP (Chen et
303
al., 2012b; Chen et al., 2017). It has been reported that spontaneous movement
304
originates from spinal neuron innervations and is not myogenic (Brustein et al., 2003).
305
We thus evaluated the effects of TDCIPP exposure on neuron development in Tg (HuC-
306
GFP) zebrafish, in which altered GFP expression indicates that chemicals caused
307
abnormalities in the spinal and the brain (Chen et al., 2014; Kim et al., 2016). In this
308
study, significantly decreased GFP expression was observed in TDCIPP-treated Tg
309
zebrafish larvae, which probably means that TDCIPP disturbed the brain and spinal
310
neurons. Moreover, we observed significantly downregulation of expressions of
311
neuronal maker genes (elavl3 (encoding HuC) and neurogenin1 (ngn1)) (Kim et al.,
312
1996; Ma et al., 1996) in the TDCIPP-exposed larvae, which may also indicate the
313
neuron abnormalities. Although a direct link between the disturbed spinals and altered
314
spontaneous movement has not been reported, several studies speculated that some
315
chemicals (e.g. BDE-47, DP) may interfere with spinal neurons and contribute to the
316
increased spontaneous movement (Chen et al., 2012b; Chen et al., 2017). We thus
317
speculate that the disturbance in spinal neurons caused by TDCIPP in this study may
318
be a possible factor to the increased elevated spontaneous movement.
319
As a more complex behavior, swimming arises later in development and require
320
chemical transmission and hindbrain inputs (Drapeau et al., 2002). In the present study,
321
swimming activity was evaluated in larvae at 120 hpf using alternating light-dark
322
stimulation. Hypoactivity was observed in the highest exposure group (900 μg/L)
323
during the light period, however, exposure to TDCIPP of lower doses (300 and 600
324
μg/L) produced hyperactivity during the dark period. The hyperactivity with lower
325
exposure concentration but hypoactivity with higher exposure concentration have also
326
been reported in previous TDCIPP studies (Jarema et al., 2015；Noyes et al., 2015),
327
but the concentrations therein were different from those used in our study, specifically,
328
the lower concentrations inducing hyperactivity used by Jarema et al. (2015) and Noyes
329
et al. (2015) (517~1637 μg/L and 276~2757 μg/L, respectively) include our high
330
concentrations inducing hypoactivity. This discrepancy may result from the actual
331
exposure concentrations, but unfortunately, all these studies did not determine the
332
TDCIPP content. Another possible reason could be the different exposure time adopted
333
between the above studies and our study. Jarema et al. (2015) and Noyes et al. (2015)
334
used exposure times ranged from 6 to 120 hpf, but we used exposure times from 2 to
335
120 hpf in our study. Indeed, by testing a series of exposure scenarios, McGee et al.
336
(2012) suggested that early zebrafish embryonic stages (0.75-2 hpf, 0.75-96 hpf) were
337
much more sensitive toTDCIPP than later-stage embryos (5.25-96hpf). The low-dose
338
stimulatory effect may be considered to be an adaptive response for the body to protect
339
itself from toxicant stress (Calabrese, 2005). For the high-dose inhibitory effect, the
340
neural abnormalities as we observed may be a contributor.
341
As motor neuron is the major cell type that regulates swimming behavior at early
342
stage of zebrafish (Brustein et al., 2003), the effect of TDCIPP exposure on axonal
343
growth of secondary motor neuron was examined in the present study. Morphometric
344
analysis showed a significant reduction of both ventral and dorsal axon length in
345
zebrafish larvae exposed to 900 μg/L TDCIPP. Additionally, upregulated expressions
346
of axon growth related genes (α1-tubulin, shha and netrin2) was observed in the same
347
exposure group. In fact, upregulation of genes of α1-tubulin and/or gap43 has been
348
suggested as a compensatory feedback in response to inhibition of axonal growth in
349
zebrafish larvae following other chemical exposure (e.g., DP and BDE-47) (Chen et al.,
350
2017; Chen et al., 2012b). Therefore, the upregulated gene expressions may further
351
indicate that TDCIPP exposure perturbed secondary motor neuron axonal growth.
352
Although no relationships have been reported so far between the axonal growth and the
353
motor behavior for TDCIPP, a link between aberrant axonal growth and deficient in
354
swimming behavioral after exposure to other chemicals (e.g. CPF and BDE47) has been
355
reported in developing zebrafish (Chen et al., 2012b; Yang et al., 2011). We thus suggest
356
that structure disruptions of secondary motor neurons could cause alteration in
357
swimming behavior in zebrafish larvae.
358
Our results confirmed that relatively lower concentration of TDCIPP do not alter
359
the cholinergic system in fish (see also Wang et al., 2015b; Yuan et al., 2016). However,
360
for the first time, we found that higher concentration of TDCIPP significantly increased
361
the AChE activity and reduced the Ach content in 120 hpf zebrafish larvae. ACh plays
362
a critical role in modulating motor and cognitive functions in the cholinergic system
363
(Driscoll et al., 2009). The ACh level in zebrafish is specifically maintained through
364
the hydrolysis of AChE (Behra et al., 2002). Therefore, the decline of ACh content may
365
be due to over-hydrolysis of AChE, and could probably cause altered neuromuscular
366
junction as well as behavioral response. Inhibited ACh content accompanied by adverse
367
effects on swimming behavior has been observed in zebrafish larvae when they were
368
exposed to other chemicals, such as PBDE mixture DE-71 (Chen et al., 2012a),
369
microcystin-LR (MC-LR) (Wu et al., 2016). Consequently, another contributor to the
370
inhibition in locomotor behavior at the highest does in this study could be the
371
interference of the cholinergic system.
372
The present study confirmed that TDCIPP exposure induces locomotor behavior
373
alterations in zebrafish larvae. More importantly, for the first time, we investigated the
374
possible mechanisms of motor behavior alterations induced by TDCIPP in zebrafish
375
larvae. The results suggest that the behavioral changes induced by TDCIPP may result
376
from the combined effects of altered motor neuron structure and inhibited cholinergic
377
system. However, it should be noted that motor behavior is an integrated result of
378
different physiological and biochemical processes, and might be also influenced by
379
other factors (e.g. aberrant somatic muscle differentiation, calcium channels and signals)
380
(see Drapeau et al., 2002). Further studies will be needed to explore these possibilities.
381
Acknowledgments
382
The work in this study was supported by the National Natural Science Foundations
383
of China (21677057, 21307162 and 21622702), and Huazhong Agricultural University
384
Scientific & Technological Self-innovation Foundation (Program 2662014BQ035).
385
Appendix A. Supplementary Data
386
Procedure for immunohistochemistry staining (Text A1).
Analytical procedure
387
for acetylcholine concentration measurement (Text A2). Primer sequences used in the
388
present study (Table A1).
389
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390
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546
Table 1. Development parameters of zebrafish larvae exposed to 0, 100, 300, 600, 900 μg/L TDCIPP until 120 h post fertilizationa TDCIPP (μg/L) Malformation rate (%) Survival rate (%) Hatching rate (%) Body length (mm)
547 548
a
*
0
100
300
1.0 ± 1.0
1.3 ± 1.1
3.0 ± 2.0
95.0 ± 1.0 91.7 ± 2.1 3.71 ± 0.11
90.0 ± 4.6 88.3 ± 2.1 3.61 ± 0.19
88.7 ± 3.2 88.3 ± 3.8 3.60 ± 0.18
600 5.0 ± 2.0 84.0 ± 2.7 82.3 ± 5.1 3.42 ± 0.18***
900 7.0 ± 2.0** 74.3 ± 8.3*** 72.3 ± 2.0*** 3.32 ± 0.24***
Data are expressed as mean ± SD of three replicates (30 larvae per replicate).
P < 0.01, **P < 0.01, ***P < 0.001 indicate significant difference detected between control and TDCIPP exposed group
549
550 551
Figure 1. Spontaneous movement in zebrafish larvae from the control group and
552
exposure groups (100, 300, 600, 900 μg/L) was recorded every two hours from 18 to
553
28 hpf. The data are expressed as mean ± SD of three replicates (10 larvae per replicate).
554
*
555
exposed group.
556
557
P < 0.05,
**
P < 0.01 indicate significant difference between control and TDCIPP
558 559
Figure 2. (A) Locomotor patterns in response to an alternating light change. (B) The
560
average swimming speed of 5-min intervals for each light state (light or dark). The data
561
are expressed as mean ± SD of three replicates (10 larvae per replicate). *P < 0.05, **P
562
< 0.01 and
563
TDCIPP exposed group.
564 565 566
***
P < 0.001 indicate significant difference detected between control and
567 568
Figure 3. Effect of TDCIPP on Tg (HuC-GFP) zebrafish larvae at 120hpf. (A)
569
Representative images from control group and highest TDCIPP (900 μg/L) exposure
570
group; (B) Relative GFP expression in larvae after various concentrations of TDCIPP
571
(0, 100, 300, 600, 900 μg/L) at 120 hpf. The data are expressed as mean ± SD of three
572
replicates (10 larvae per replicate). *P < 0.05, **P < 0.01 indicate significant difference
573
detected between control and TDCIPP exposed group.
574
575 576
Figure 4. The mRNA levels of neuron marker genes (elavl3 and ngn1) in zebrafish
577
larvae at 120 hpf. The data are expressed as mean ± SD of three replicates (30 larvae
578
per replicate). *P < 0.05, **P < 0.01 indicate significant difference detected between
579
control and TDCIPP exposed group.
580 581
582
583 584
585 586
Figure 5. TDCIPP exposure affected axonal growth of secondary motoneuron and
587
axonal growth-related gene expression. (A) Representative photomicrographs
588
illustrating ZN-5 immunopositive axons in dorsal and ventral trunk regions. (B)
589
Quantitative measurements of dorsal length (10 larvae each replicate, n=3 replicates).
590
(C) Quantitative measurements of ventral axon length (10 larvae each replicate, n=3
591
replicates). (D) Gene expression profiles in zebrafish larvae at 120 hpf (30 larvae each
592
replicate, n=3 replicates). *P < 0.05, **P < 0.01 indicate significant difference detected
593
between control and TDCIPP exposed group.
594
595 596
Figure 6. Acetylcholine (ACh) content (A) and acetylcholinesterase (AChE) activity
597
(B) in zebrafish larvae following exposure to various concentrations of TDCIPP (0, 100,
598
300, 600, 900 μg/L) at 120 hpf. The data are expressed as mean ± SD of three replicates
599
(100 larvae per replicate). *P < 0.05, **P < 0.01 indicate significant difference detected
600
between control and TDCIPP exposed group.