(PBDEs) Results in Developmental Neurotoxicity in Zebrafish Larvae

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Prenatal Transfer of Polybrominated Diphenyl Ethers (PBDEs) Results in Developmental Neurotoxicity in Zebrafish Larvae Lianguo Chen,†,# Ke Yu,†,# Changjiang Huang,‡ Liqin Yu,† Bingqing Zhu,§,∥,⊥ Paul K. S. Lam,§,∥,⊥ James C. W. Lam,*,§,∥,⊥ and Bingsheng Zhou*,† †

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China ‡ Institute of Watershed Science and Environmental Ecology, Wenzhou Medical College, Wenzhou 325035, China § State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon, Hong Kong SAR, China ∥ Research Centre for the Oceans and Human Health, City University of Hong Kong, Shenzhen Research Institute Building, Shenzhen 518057, China ⊥ Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, China S Supporting Information *

ABSTRACT: Parental exposure to polybrominated diphenyl ethers (PBDEs) in animals has been found to be transferred to the offspring. The environmental health risk and toxicity to the offspring are still unclear. The objective of the present study was to identify environmentally relevant concentrations of PBDEs for parental exposure that would cause developmental neurotoxicity in the offspring. Adult zebrafish were exposed to environmentally relevant concentrations of DE-71 (0.16, 0.8, 4.0 μg/L) via water. The results showed that PBDE exposure did not affect larvae hatching, malformation, or survival. The residue of PBDEs was detected in F1 eggs upon parental exposure. Acetylcholinesterase (AChE) activity was significantly inhibited in F1 larvae. Genes of central nervous system development (e.g., myelin basic protein, synapsin IIa, α1-tubulin) were significantly downregulated in larvae. Protein levels of α1-tubulin and synapsin IIa were also reduced. Decreased locomotion activity was observed in the larvae. This study provides the first evidence that parental exposure to environmentally relevant concentrations of PBDEs could cause adverse effects on neurodevelopment in zebrafish offspring.



INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are additive flameretardants that have been extensively used in furniture, plastics, textiles, electronic enclosures, and circuitry. These compounds are routinely detected in samples of wild animals and humans.1 In addition, PBDEs are also detected in cord blood.2,3 Thus, PBDEs can be transferred across the placenta to the fetus and may cause latent adverse effects on neonates during lactation. In fish, the maternal transfer of PBDEs has previously been observed after dietary PBDE exposure,4,5 and via water in laboratory conditions.6 It has also been detected in striped bass (Morone saxatilis) eggs (100−150 ppb) in the San Francisco estuary.7 This transfer of PBDEs from parents could interfere with developmental effects on the progeny. The early life stages are more sensitive to the effects of environmental chemicals as they are undergoing critical developmental processes.8 It is generally recognized that the developing brain is extremely sensitive to many xenobiotics, and that certain chemicals can disrupt developmental processes in the general health of brain cells, and alter fundamental brain structures and functions, thereby leading to adverse impacts on learning, behavior, and health.9 Since the beginning of this century, great concerns have been raised about potentially © 2012 American Chemical Society

adverse effects of PBDEs related to their developmental neurotoxicity.10,11 Exposure to PBDEs can have adverse neurobehavioral effects, in particular during early neurodevelopment.11,12 Limited epidemiological studies on PBDEs in humans suggest that prenatal exposure may adversely impact cognitive and neuron developmental parameters in children. These effects are correlated with maternal serum levels of PBDEs13 and neuronal developmental effects were found to be related to cord blood PBDE concentrations during infant neurodevelopment.14 Despite several studies that studied the maternal transfer of PBDEs to fish eggs, the potential effects on developmental toxicity, in particular on neurodevelopment, in their offspring are virtually unknown. In the present study, we used zebrafish (Danio rerio) as a model to investigate the influence of parental PBDE exposure on developmental neurotoxicity in the offspring. The zebrafish is an attractive model for toxicological research.15 In addition, zebrafish have genetic properties similar Received: Revised: Accepted: Published: 9727

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skull by dissection. Five brains of zebrafish were pooled to be used as one replicate in each treatment. The brains were homogenized on ice in 60 vol (v/w) of Tris-citrate buffer (50 mM Tris, 2 mM EDTA, 2 mM EGTA, pH 7.4, with citric acid). For zebrafish larvae, 100 larvae were homogenized in the same buffer, centrifuged at 3000g for 10 min at 4 °C. AChE enzyme activity was measured by a commercial kit following the manufacturer’s instructions (Nanjing Keygen Biotech, Co, LTD). Protein was measured using the Bradford method. Quantitative Real-Time PCR Assay in Zebrafish Larvae. Total RNA was extracted from 30 homogenized zebrafish larvae by using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Total RNA extraction, purification, and quantification, and first-strand cDNA synthesis were performed as described previously by Yu et al.6 (see Text S1 in Supporting Information (SI)). The primer sequences of the selected genes were obtained by using the online Primer 3 program (http://frodo.wi.mit.edu/; see SI Table S1). The β-actin gene was selected as the internal standard. Quantitative real-time polymerase chain reaction (qRT-PCR) was analyzed on an ABI 7300 System (PerkinElmer Applied Biosystems, Foster City, CA, USA). Protein Extraction and Western Blot Analysis. A Western blotting assay was carried out using approximately 300 larvae from each replicate (n = 3). In total, 50 μg from each protein sample was separated by electrophoresis on a 12% SDS polyacrylamide gel. Following electrophoresis, proteins were then transferred to polyvinylidene difluoride membranes. The membrane was further blocked with 5% fat-free milk for 2 h in TBS (10 mM Tris, 150 mM NaCl, pH 8.0)/0.1% Tween 20, and incubated overnight at 4 °C with primary antibodies against α1-tubulin (1:500), SYN2a (1:1000), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1000). The rabbit α1tubulin antibody (Abcam, Cambridge, UK) has been verified to be reactive with zebrafish.27 The rabbit SYN2a-antibody (Synaptic Systems, Göttingen, Germany) was also suitable for use in zebrafish.28 After incubation with horseradish peroxidaseconjugated goat antimouse IgG (1:10 000) for 1 h at room temperature, the signal was detected by enhanced chemiluminescence, and the relative optical density of the bands was analyzed using Kodak film (Eastman Kodak Co., Rochester, NY) and Quantity One version 4.3 software (Bio-Rad, USA). Locomotor Activity Measurement. The locomotor activity was monitored with a Video-Track system (ViewPoint Life Sciences, Inc., Montreal, Canada) following previously described methods.29 Swimming behavior was monitored as the larval response to dark-to-light transition stimulation (10 min dark10 min light10 min dark). The dark−light stimulation test is a relatively simple and suitable behavioral assay for juvenile zebrafish, which are more likely to reflect changes in general locomotor activity.30 The data (frequency of movements, distances traveled, and total durations of movements) were collected every 60 s. Locomotor behavior was monitored in a total of 30 larvae per treatment (10 larvae per replicate and three replicates per treatment). The data were analyzed using custom Open Office.Org 2.4 software. Quantification of DE-71 in Adult Zebrafish and F1 Eggs. Concentrations of PBDEs were determined in adult fish (F0) and F1 eggs following a previously described method.31 The fish samples were weighed and then homogenized using a mixture of dichloromethane (DCM) and hexane (4:1 v/v). Then 5 ng of each internal standard (13C12-labeled BDE28,

to those of mammals related to nervous system development, and the fundamental processes of neurodevelopment in zebrafish are homologous to those that occur in humans.16,17 Zebrafish have proven to be very useful in research regarding the effects of toxins of developmental neurotoxicity in the vertebrate nervous system and are becoming increasingly commonly used in behavioral neuroscience and behavioral studies.18 Our objective was to characterize the effects of parental exposure to environmentally relevant concentrations of PBDEs (0.16, 0.8, and 4.0 μg/L)19 and to assess whether such exposures could cause developmental neurotoxicity in F1 larvae. Several genes related to neurodevelopment in zebrafish embryos/larvae were examined. These candidate genes can serve as biomarkers for developmental neurotoxicology by demonstrating their responsiveness to developmental neurotoxicants and by high expression levels during early developmental stages.20 We selected the following genes that are expressed exclusively in the nervous system during early development: myelin basic protein (MBP),21 growth associated protein 43 (GAP-43),22 α1-tubulin,23 synapsin IIa (SYN2a),24 and glial fibrillary acidic protein (GFAP).25 Acetylcholinesterase (AChE) enzyme activities were measured in the brain of F0 fish and F1 larvae and the locomotion behavior of larvae was monitored. The measurement of AChE activity is widely used as a sensitive biomarker for neurotoxicants.26 PBDEs were also measured in both parents and their eggs.



MATERIALS AND METHODS Reagents. A commercial PBDE mixture (DE-71, purity >99.9%) was obtained from Wellington Laboratory, Inc. (Ontario, Canada). TRIzol reagent and SYBR Green PCR kit were purchased from Invitrogen (New Jersey, USA) and Toyobo (Osaka, Japan), respectively. All other chemicals used in the present study were analytical grade standard. Zebrafish Maintenance and Experimental Design. Adult zebrafish (AB strain) (80 days postfertilization, dpf) were exposed to environmentally relevant concentrations of DE-71 (0.16, 0.8, and 4.0 μg/L) in 30-L glass tanks until 230 dpf. The control and exposure groups received 0.003% (v/v) dimethyl sulfoxide (DMSO). There were three replicate tanks, with each tank containing 20 males or females, respectively. During the experimental period, half of the exposure water in each tank was replaced daily with fresh solution at the appropriate concentration. After 150 days of exposure, their survival and growth (weight) were determined. The fish were paired and eggs were collected. The eggs were then rinsed thoroughly in freshwater five times to minimize PBDE contamination and to evaluate the parental transfer of PBDEs. A subset of eggs was placed in glass dishes containing freshwater without DE-71 exposure. The hatching, malformation, and survival in F1 were recorded after 4 dpf. The egg protein content and egg diameter were also recorded. The larvae (4 dpf) were randomly sampled, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent gene and protein expression analysis. A subset of the F1 larvae (4 dpf) was used for locomotor activity measurement. All animals were treated humanely and with the aim of alleviating any suffering. They were maintained in accordance with guidelines for the care and use of laboratory animals of the National Institute for Food and Drug Control of China. Acetylcholinesterase Activity Assay in Adult Zebrafish Brain and in Larvae. Adult fish were anesthetized in 0.03% MS-222, and their brains were removed from the cranial 9728

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Table 1. Gene Transcription Levels in Zebrafish Larvaea gene name α1-tubulina MBPa SYN2aa GAP-43a GFAPa

0 μg/L 1.0 1.0 1.0 1.0 1.0

± ± ± ± ±

0.0 0.1 0.1 0.1 0.1

0.16 μg/L −1.2 −1.3 −1.5 1.0 1.0

± ± ± ± ±

0.0* 0.1* 0.0* 0.0 0.0

0.8 μg/L −1.4 −1.5 −1.5 1.0 0.9

± ± ± ± ±

0.1** 0.0*** 0.0** 0.0 0.0

4.0 μg/L −1.7 −1.7 −1.7 1.2 1.1

± ± ± ± ±

0.0*** 0.0*** 0.1** 0.0* 0.1

Values represent the mean ± SEM of three replicates (30 larvae per replicate) and are expressed as fold change relative to control. *P < 0.05. **P < 0.01. ***P < 0.001 indicate a significant difference between the exposure groups and the control group. a

AChE activity in the zebrafish brain (data not shown). However, this enzyme activity was significantly inhibited in larvae (F3,8 = 14.24, P = 0.0014) derived from the adult fish exposed to 0.16 μg/L (20.1 ± 4.6%, P = 0.003), 0.8 μg/L (25.8 ± 3.2%, P = 0.001), and 4.0 μg/L DE-71 (29.2 ± 2.2%, P = 0.0003) compared with controls. AChE gene expression was significantly downregulated in the larvae (F3,8 = 15.14, P = 0.0012) from the 0.16 μg/L (22.4 ± 2.3%, P = 0.0055), 0.8 μg/ L (27.1 ± 2.6%, P = 0.0014), and 4.0 μg/L (37.2 ± 5.4%, P = 0.0002) exposed groups. Gene and Protein Expressions in Zebrafish Larvae. The genes of central nervous system development, differentiation, and growth were evaluated. Table 1 shows the relative expressions of significantly up- or downregulated genes in larvae derived from adults exposed to 0.16, 0.8, or 4.0 μg/L DE-71, respectively. The gene expression levels of myelin basic protein (MBP) (F3,8 = 14.30, P = 0.0003; 0.16 μg/L, P = 0.0104; 0.8 μg/L, P = 0.0003; 4.0 μg/L, P < 0.0001) and synapsin IIa (SYN2a) (F3,8 = 10.10, P = 0.0043; 0.16 μg/L, P = 0.0027; 0.8 μg/L, P = 0.0028; 4.0 μg/L, P = 0.0013) in larvae were significantly downregulated. The expression of α1-tubulin was also downregulated (F3,8 = 14.03, P = 0.0003; 0.16 μg/L, P = 0.0041; 0.8 μg/L, P = 0.0057; 4.0 μg/L, P < 0.0001), while a small but significant upregulation of GAP-43 was observed in the 4.0 μg/L exposure group (F3,8 = 5.50, P = 0.024; 4.0 μg/L, P = 0.0129). Glial fibrillary acidic protein (GFAP) gene expression was not changed. Expression of α1-tubulin and synapsin II a (SYN2a) proteins was examined by Western blotting, and the results showed a concentration-dependent reduction of α1-tubulin (11.2%, 15.4%, 32.5%) (F3,8 = 21.16, P = 0.0029; 0.16 μg/L, P = 0.0604; 0.8 μg/L, P = 0.0209; 4.0 μg/ L, P = 0.0006) (Figure 1A) and SYN2a proteins (15.8%, 34.8%, 57.7%) (F3,8 = 616.47, P < 0.0001; 0.16 μg/L, P = 0.0004; 0.8 μg/L, P < 0.0001) (Figure 1B). Locomotor Activity. The locomotor traces of the F1 larvae (4 dpf) were monitored during the dark−light−dark stimulation period (Figure 2A). The average swimming speed is summarized in Figure 2B. Average swim speed of larvae indicated that adult exposure to 0.16, 0.8, and 4.0 μg/L DE-71 resulted in significantly slower speed than the control during the first dark period (F3,116 = 26.32, P < 0.0001; 0.16 μg/L, P = 0.0082; 0.8 μg/L, P = 0.0009; 4.0 μg/L, P < 0.0001), the light period (F3,116 = 81.35, P < 0.0001; 0.16−4.0 μg/L, P < 0.0001), and the second dark period (F3,116 = 12.19, P < 0.0001; 0.16 μg/L, P = 0.403; 0.8 μg/L, P = 0.0005; 4.0 μg/L, P = 0.0006) (Figure 2B). PBDEs Content in F0 Adult Fish and F1 Eggs. Seven congeners were detected in exposed F0 zebrafish, where BDE47 contributed to most of the total PBDE body burden, followed by BDE-100 and 28, in both females and males (Figure 3A). In the females, the detected total contents of PBDEs were 1669 ± 159, 6086 ± 294, and 10 368 ± 791 ng/g

BDE47, BDE99, BDE153, BDE154, and BDE183) was added to the samples. 13C12-labeled BDE139 was added as the recovery spike. The quantification of PBDEs was performed using a GC (Agilent 7890A) equipped with a mass-selective detector (Agilent 5975C) using the electron impact (EI) mode. PBDE congeners (BDE28, BDE47, BDE99, BDE100, BDE153, BDE154, and BDE183) were quantified using the isotope dilution method to their corresponding 13C12-labeled congeners. The recovery of 13C12-labeled BDE ranged from 85% to 98%. The detection limit was calculated as three times the procedural blank (0.1 ng/g for tetra-BDE, 0.05 ng/g for tri- and penta- to hepta-BDEs). Quality Assurance and Quality Control (QA/QC). Procedural blanks were analyzed simultaneously in every batch of five samples to check for interference or contamination from the solvent or glassware. The detection limit was calculated as three times the procedural blank (0.1 ng/g for tetra-BDE, 0.05 ng/g for tri- and penta- to hepta-BDEs).6,31 Statistical Analysis. All data are expressed as the mean ± standard error (SEM). The normality of the data was verified using the Kolmogorov−Smirnov test. The homogeneity of variance was analyzed by Levene’s test. Differences between the control and each exposure group were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s to test determine significant differences between DE-71 exposure groups and the control group with SPSS 13.0 software (SPSS, Chicago, IL, USA). A P < 0.05 was considered statistically significant.



RESULTS Toxicological End Points. After 150 days of exposure, there were no significant differences in survival in the adult fish (F0). Growth (body weight) was inhibited in the males at the DE-71 treatments (F3,68 = 3.091, P = 0.0355) of 0.16 μg/L (13.7%, P = 0.020), 0.8 μg/L (11.8%, P = 0.033), and 4.0 μg/L (13.7%, P = 0.010) relative to the control, respectively. The gonadal somatic index (GSI) was significantly increased at 0.8 μg/L (F3,68 = 3.71, P = 0.0217; 51.1%, P = 0.004) and 4.0 μg/L (37.4%, P = 0.019) DE-71 exposure groups in the females. The brain somatic index (BSI) was increased in the females in the 4.0 μg/L DE-71 exposure group (F3,68 = 2.938, P = 0.0481; 17.5%, P = 0.0250). No effects were found on the hepatic somatic index (HSI) (SI Table S2). However, long-term exposure caused a significant reduction of eggs produced in the 4.0 μg/L exposure group (F3,8 = 3.214, P = 0.0476; 43.6%, P = 0.0410). The egg protein contents were also reduced in the 4.0 μg/L exposure group (F3,8 = 4.269, P = 0.0447; 10.9%, P = 0.0110). There was also no significant difference in the rates of hatching, malformation, and survival in the F1 generation (see SI Table S3). AChE Activity in F0 Brain and F1 Larvae. The exposure of zebrafish to 0.16, 0.8, and 4.0 μg/L DE-71 did not alter 9729

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Figure 2. Locomotor behavior of the zebrafish larvae after parental exposure to DE-71 (0, 0.16, 0.8, or 4.0 μg/L) for 150 days. (A) Locomotor traces of the larvae; (B) average swimming speed of the larvae during the dark−light−dark photoperiod stimulation test. Data are expressed as the mean ± SEM of three replicates (ten larvae per replicate) in 60-s intervals. *Designates significant differences compared to the control. **P < 0.01; ***P < 0.001.

Figure 1. Western blot analysis of protein levels in the larvae after parental exposure to DE-71 (0, 0.16, 0.8, or 4.0 μg/L) for 150 days. (A) α1-Tubulin protein levels in zebrafish larvae; (B) protein levels of SYN2a in zebrafish larvae. GAPDH was used as an internal control. The data represented similar results from three replicate samples. The bands from three replicate samples were quantified by densitometry, and the results were normalized to GAPDH expression in each sample as the mean ± SEM fold change relative to the control. *Designates significant differences compared to the control. *P < 0.05; ***P < 0.001.

relevant concentrations of PBDEs in F1 zebrafish caused developmental neurotoxicity in their offspring. Thus, our results emphasize the significance of environmental risk assessment in terms of the transfer of toxins from exposed parents to their offspring. In our study, significant levels of DE-71 were detected in the eggs, indicating the transfer of DE-71 from exposed adult fish to their offspring. Among the detected PBDEs congeners in both the adult fish and eggs, the concentration of BDE 47 was higher than BDE 99. BDE 99 is metabolized rapidly in common carp (Cyprinus carpio), where it is converted to BDE 47.32 Thus, the finding that BDE47 was the most abundant congener in our study could be a result of the metabolism of BDE 99. The evaluation of cholinergic system parameters has emerged as an important strategy to assess neurochemical, behavioral, and toxicological phenotypes in zebrafish larvae and adults.18,33 We found no significant effects on this enzyme activity in adult fish brains. Few studies have addressed the effects of PBDEs on AChE activity in fish brains and the mechanisms of toxicity are not known. A previous study showed no significant effects of exposure to DE-71 (0.1, 0.5, or 2.5 μg DE-71/g, fed for 10 weeks) on ChE activity in the cerebral cortex of adult female ranch minks (Mustela vison).34

wet weight in the 0.16, 0.8, and 4.0 μg/L exposure groups, respectively. In the males, the detected total PBDE contents were 3421 ± 235, 9420 ± 1490, and 15 142 ± 768 ng/g in the 0.16, 0.8, and 4.0 μg/L exposure groups, respectively. The detected total contents of PBDEs were 8.9 ± 1.5 ng/g in the control females and 9.6 ± 2.9 ng/g in the control males, respectively. In the F1 eggs, six congeners were detected, where BDE-47 was the predominant congener, followed by BDE-100 (Figure 3B). The total body burden showed a concentration-dependent relationship between the parental exposure concentrations of 0.16, 0.8, and 4.0 μg/L DE-71 with the values of 250 ± 11, 2384 ± 316, and 6366 ± 648 ng/g wet weight, respectively. PBDEs were not detected in the control eggs.



DISCUSSION By performing enzyme measurement, gene and protein expression studies, and larval locomotor behavior, we demonstrated that maternal exposure to environmentally 9730

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Figure 3. PBDE contents in zebrafish after exposure to DE-71 (0, 0.16, 0.8, or 4.0 μg/L) for 150 days. (A) PBDEs content in F0 adult zebrafish; (B) PBDEs in the F1 eggs derived from parental zebrafish exposure to DE-71. For adult fish, the values represent the mean of three individual replicate fish. For eggs, PBDEs were measured in 100 eggs, with three replicate samples. (a),(b),(c),(d) Represent the eggs derived from parental exposure of 0, 0.16, 0.8, and 4.0 μg/L DE-71, respectively.

is known that AChE is required for neuronal development in the zebrafish embryo.35 To understand the primary damaging events that occur in the developing larval brain, we investigated genes and proteins that are exclusively expressed in the brain during early developmental stages. We found that the expression of most genes was significantly downregulated. In our study, the expression levels of α1-tubulin at the gene and protein level were significantly downregulated. The gene α1-tubulin encodes an intermediate filament protein that forms an essential part of the

The results suggest that brain ChE enzyme is not a major target for PBDEs during long-term exposure to low concentrations. However, in our study, we found a significant inhibition of AChE activity in fish larvae after PDBE exposure, which suggested that developmental effects in offspring are more sensitive than those of their respective parents. Decreased AChE activity could result in less acetylcholine (Ach), which is important for cholinergic neurotransmission; this may impair acetylcholine-mediated neurotransmission in zebrafish larvae. It 9731

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Although the mechanisms of the effects of PBDE exposure on gene and protein levels in larvae are not fully understood, alterations in the thyroid hormone (TH) regulation may play a critical role in the onset of these effects.45 TH plays a crucial role during brain development, and disturbances in TH homeostasis can cause serious impairment in neurological development.46,47 Therefore, it has been proposed that a disruption of TH homeostasis may explain the neurotoxicity of PBDEs.12 Our previous study demonstrated that 150 days of exposing zebrafish embryos to DE-71 (1, 3, 10 μg/L) can elevate TH levels in adults and that increased TH levels are also transferred to F1 eggs.6 In the present study, our results showed again that significant levels of DE-71 are transferred from exposed adult fish to their offspring, resulting in an increased prevalence of impaired health in the offspring. A 21-day dietary PBDE-47 treatment (2.4 or 12.3 μg/pair/day) in adult fathead minnows (Pimephales promelas) resulted in depressed plasma thyroxine (T4) and changed brain mRNA levels of THresponsive genes.45 The authors suggested that TH-responsive pathways in the brain could be particularly sensitive to disruption by PBDEs. However, exactly how PBDEs influence TH-mediated neural functions remains to be investigated. In the present study, we confirmed that exposure to environmentally relevant concentrations of PBDEs in F0 zebrafish can alter neurobehavior in their offspring. Several previous studies have shown neurobehavioral alterations in rodent models upon exposure to PBDEs.48,49 Recently, locomotor behavior approaches have also been performed to study the neurobehavioral effects of PBDEs in zebrafish.29,50 Neurobehavior is an integrated response of many physiological and biochemical processes. Thus, alternations of locomotor behavior could be due to downregulation of AChE gene transcription, the inhibition of AChE activity, alterations of the natural function of genes and proteins, or by direct neurotoxic effects on neuronal cells. A previous study demonstrated that the lack of AChE activity in zebrafish results in an increase of muscular activity and can cause a progressive myopathy.35 This report is the first study to demonstrate that parental exposure of PBDEs could result in adverse neurodevelopmental effects in offspring in a model fish. It should be noted that in developing countries (such as China), where most informal and primitive e-waste recycling occurs, environmental exposure to PBDEs is prevalent at high concentrations in local human residents and bioaccumulation in wild animals occurs.51 Higher concentrations of PBDEs have been measured in human umbilical cord blood (up to 505 ng/g lipid)3 from Guiyu, and in frog eggs (Rana limnocharis) (up to 125 ng/g) in Qingyuan, South China,52 but studies regarding the adverse health effects in wild animals and humans are scarce. Our study suggests the need for additional work to advance our understanding of the effects of perinatal exposure to PBDEs on neurodevelopment.

microtubule cytoskeleton in developing or regenerating axons and dendrites.36 A proteomics study showed a single dose of BDE-99 (12 mg/kg body weight) given to male mice at day 10 postnatally caused changes in brain protein expression after 24 h, and that one-third of those proteins were decreased and were related to the cytoskeleton in the striatum.37 A recent proteomics study showed that exposure to low-dose BDE-99 (0.3 μM, 3 μM) in cultured cortical cells isolated from rat fetuses induced marked effects on cytoskeletal proteins.38 These results suggest that the cytoskeleton system is sensitive to PBDE stress. Given the functional importance of cytoskeletal organization for neurodevelopment, the proper interaction of the actin cytoskeleton and related proteins controls neuronal development. This includes mediating axonal branching as well as dendritic spine and synapse formation. Hence, alternations of the cytoskeleton are likely to have dramatic effects on both brain architecture and function. Myelin basic protein (MBP), a biomarker of myelination that is expressed in oligodendrocytes, is a major component of myelin sheath. MBP is required for the myelination of axons in the developing central nervous system in zebrafish.21 Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is highly expressed in astrocytes and radial glial cells of the central nervous system and is considered a marker of astroglia in the brain.25 In our study, MBP gene transcription levels were downregulated, but GFAP was not affected. Although no studies have analyzed the impact of PBDEs on MBP and GFAP in rodents or fishes, an earlier study showed that PCB exposure resulted in reduced MBP, but did not affect GFAP levels in the corpus callosum white matter tissue of male offspring.39 A recent study also showed that developmental exposure to PCB mixture in rats resulted in a significant reduction in MBP protein levels in female offspring.40 SYN2a is a biomarker of synapse formation in mammals, which plays an important role in both synaptogenesis and neurotransmitter release.24 The function of SYN2a has not been studied in zebrafish. We observed the downregulation of SYN2a gene and protein expression. This may affect synaptogenesis and neurotransmitter release, and lead to neurobehavioral impairments. Growth associated protein (GAP-43) is expressed at high levels in zebrafish neurons during development.22 GAP-43 is also frequently used as a marker for modulating synapse formation and in reinducing axonal growth for regeneration after damage.41 In our study, GAP-43 gene expression was upregulated in larval fish after parental exposure to higher DE71 concentration (4.0 μg/L). In mice, the increased expression of GAP-43 protein (1.25-fold) was found in developing mouse brains after 24 h exposure to BDE-99 (12 mg/kg body weight).42 Following neonatal exposure to BDE-209 (20.1 mg/ kg body) in mice, GAP-43 was significantly increased in the hippocampus after 7 days.43 However, neonatal exposure to BDE-203 and -206 (21 μmol/kg) for 24 h in mice during rapid brain growth and development did not affect GAP-43.44 Lower (0.3 μM and 3 μM), but not higher, concentrations (10 μM or 30 μM) of BDE-99 have been shown to increase the expression of GAP-43 in cultured cortical cells isolated from rat fetuses.38 It is tempting to speculate that the upregulation of GAP-43 is likely to represent an adaptive elevation that maintains overall brain growth required to offset the direct effects of toxicants.37 Our results are consistent with previous reports that showed the upregulation of GAP-43 expression in the brain, which most likely reflects a compensatory mechanism.



ASSOCIATED CONTENT

S Supporting Information *

Text S1 outlines gene transcription; Table S1 shows the primer sequences; Table S2 contains growth and somatic index in F0 zebrafish; Table S3 shows the reproductive end points in F1 zebrafish. This information is available free of charge via the Internet at http://pubs.acs.org. 9732

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; phone: 852 3442 4126; fax: 852 2194 2281 (J.C.W.L.); E-mail: [email protected]; phone: 86-27-68780042; fax: 86-27-68780123 (B.Z.). Author Contributions #

Co-first authors.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (20890113), the Hong Kong Research Grants Council (CityU 160610), and the State Key Laboratory of Freshwater Ecology and Biotechnology (2011FBZ13)



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