Fig. 1 Microscopic observations of zebrafish exposed to DBDPE-contaminated sediments from 4 to 120 hpf
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College of Materials Science and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018, China
1.College of Materials Science and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018, China
2.Shanghai Key Laboratory for Urban Ecological Processes and Eco-Restoration, School of Ecological and Environmental Science, East China Normal University, Shanghai 200062, China
3.College of Environment, Zhejiang University of Technology, Hangzhou 310014, China
Published: 2018-05 ,
Received: 18 January 2018 ,
Accepted: 20 March 2018
Cite this article
Mei-qing Jin, Dong Zhang, Ying Zhang, et al. Neurological responses of embryo-larval zebrafish to short-term sediment exposure to decabromodiphenylethane. [J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) 19(5):400-408(2018)
Mei-qing Jin, Dong Zhang, Ying Zhang, et al. Neurological responses of embryo-larval zebrafish to short-term sediment exposure to decabromodiphenylethane. [J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology) 19(5):400-408(2018) DOI: 10.1631/jzus.B1800033.
College of Materials Science and Environmental Engineering, Hangzhou Dianzi University, Hangzhou 310018, China
Decabromodiphenylethane (DBDPE) has been widely used as an alternative flame retardant due to the restriction or phase-out of traditional polybrominated diphenyl ethers (PBDEs), and is of increasing concern regarding its ubiquity, persistence, and potential adverse effects. In the present study, the toxicological effects of DBDPE were evaluated using zebrafish as an in vivo model. Upon being exposed to DBDPE-polluted sediments for a short term, it was found that the mortality and malformation of zebrafish (including edema, bent notochord, and bent tail) were not affected even at the highest concentration tested (1000.0 µg/kg dry sediment). Regarding behavioral responses, it was found that zebrafish larvae of 48 hours post fertilization (hpf) in all groups escaped successfully with a touch to the dorsal fin. However, when exposed to the highest DBDPE concentration, the larvae of 120 hpf exhibited significantly smaller distances as compared to the control. Moreover, the results of the acetylcholinesterase (AChE) activity, the expression levels of two important nerve-related genes, and the cell apoptosis all indicated that DBDPE posed low neurotoxicity in embryo-larval zebrafish. The results in this study shed some light on the potential risks of DBDPE in the real environment and highlight the application of the sediment exposure route in the future.
将受精后4小时(4 hpf)的斑马鱼胚胎置于对照底泥和染毒底泥(DBDPE系列浓度)中进行短期暴露,观察不同发育阶段的存活率、孵化率、畸形率以及行为(包括触碰反应和自由泳动)效应;并通过斑马鱼幼鱼的乙酰胆碱酶活性、神经系统的相关基因(α1-tubulin和gap43)的转录水平以及斑马鱼整体组织的细胞凋亡情况的检测探讨其神经毒性的潜在机制.
With the worldwide restriction on the use and production of polybrominated diphenyl ethers (PBDEs) due to their environmental and human health concerns, novel non-PBDE-halogenated flame retardants have been introduced into the market and used as replacements (Chen et al.,
As an additive flame retardant, DBDPE can be readily released into the environment during its production, use, recycle, and the disposal of its related products (Lee et al.,
Previous field studies have revealed a wide occurrence of DBDPE in biotic media, e.g. fish, birds, mammals, and even human hair and blood, indicating that DBDPE has the potential to biomagnify through the food webs and to exert harmful effects on the ecosystem and human health (He et al.,
According to previous studies, deca-BDE could exert neurological effects, for example, behavior changes in zebrafish (Garcia-Reyero et al.,
Therefore, the main objectives of this study were: (1) to investigate neurological responses to DBDPE by using zebrafish as an in vivo model; (2) to elucidate the potential mechanisms of DBDPE-induced neurotoxicity in embryo-larval zebrafish.
The artificial sediments were prepared according to previous described methods (EPA,
Embryos were obtained by natural mating of healthy adult zebrafish (wild type, AB strain) and maintained in petri dishes at 28.0 °C with a 12 h:12 h (light:dark) photoperiod in embryo water until chemical treatment. The conditions of the embryo water were as follows: 200 μg/ml of Instant Ocean (Aquarium Systems, Sarrebourg, France) was dissolved in reverse osmosis purified water, and the conductivity, pH, and hardness were kept in the range of 480–510 μS/cm, 6.9–7.2, and 53.7–71.6 mg/L CaCO3, respectively. All procedures were approved by the Association for Assessment and Accreditation of Laboratory Animal Care (Frederick, USA).
The petri dishes were used as the exposure vessels and 10 g spiked sediments were placed in each vessel, topped with oxygen saturated embryo water (4 ml/g). The vessels were then incubated at 28 °C in the darkness overnight for equilibrium before adding the embryos. On Day 2, 30 fertilized zebrafish embryos at 4 hours post fertilization (hpf) per vessel were transferred to the spiked sediments (or the control) and exposed from 4 to 120 hpf under semi-static conditions at 28.0 °C in the dark. Replacement of the topped embryo water was done daily, while the sediments remained unchanged.
Observations of zebrafish development were made during the exposure period using a dissecting microscope (SZX7, Olympus, Japan). Mortality and malformations were recorded in the control and spiked sediment groups every 24 h, and embryos and larvae were considered dead when no heartbeat was observed. Dead embryos or larvae were removed immediately at each observation time. From 48 hpf, embryos started to come out of the chorion asynchronously and the number of hatched embryos was recorded every 24 h.
To assess the effects of DBDPE on the motor behavior of zebrafish larvae, two types of behavior assay including touch-escape response and free-swimming activity, were conducted. For touch-escape response, 20 embryos at 48 hpf per group were randomly chosen and dechorionated. The embryos were touched dorsally near the tail fin area with a dissecting needle, and the numbers of the embryos that have escaping responses were recorded. For the free-swimming activity assessment, 10 normal larvae at 120 hpf per group were randomly chosen and individually transferred to a 24-well plate (one fish per well). The distances travelled during 1 h were achieved using the VideoTrack for Zebrafish™ (V3, ViewPoint Life Sciences, France) and analyzed based on previous studies with some modifications (Winter et al.,
A total of 90 zebrafish embryos per treatment were exposed to DBDPE (62.5, 125.0, 250.0, 500.0, 1000.0 µg/kg) and the control from 4 to 96 hpf. At the end of each exposure period (72, 96 hpf), the organisms from various concentration groups were collected for measurement of acetylcholinesterase (AChE) activity using an Amplite™ Fluorimetric Acetylcholinesterase Assay kit (AAT Bioquest®, Inc., USA) according to the manufacturer’s instructions. Five replicates were set for all the treatments.
At different sampling time points (48, 72, or 96 hpf), 30 fish per treatment were pooled together for the analyses of the selected gene expression levels by polymerase chain reaction (PCR). Total RNA in the embryos or larvae was extracted using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. The banding patterns on a 2% (0.02 g/ml) agarose gel were checked for the integrity of the total RNA and the purity was also analyzed using the Microvolume Spectrophotometers (Nanodrop 2000, Thermo, USA). Reverse transcription (RT) was carried out with a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). PCR was performed with the listed primers as shown in Table
Gene | Forward primer | Reverse primer |
---|---|---|
β-actin | CATCAGCATGGCTTCTGCTCTGTATGG | GACTTGTCAGTGTACAGAGACACCCT |
gap43 | GCAGCAGGAAGTGGAGAAGCCA | GGATTCCTCAGCAGCGTCTGGT |
α1-tubulin | AATCACCAATGCTTGCTTCGAGCC | TTCACGTCTTTGGGTACCACGTCA |
To estimate cell death due to apoptosis, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was conducted on whole-mounted zebrafish larvae at 96 hpf with a One Step TUNEL Apoptosis Assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). After exposure to various concentrations of DBDPE from 4 to 96 hpf, the larvae were fixed with 4% (w/v) paraformaldehyde, permeabilized, and incubated with the reaction mixture according to the assay protocol provided by the manufacturer’s instructions. The images of the apoptotic cells were taken under a fluorescent microscope (AZ100, Nikon, Japan).
All data were expressed as the mean±standard deviation (SD). Statistical analyses of the data were performed in the program package SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Data were analyzed for statistical significance with a one-way analysis of variance (ANOVA) followed by least-significant difference (LSD) (equal variances assumed) or Dunnett’s T3 (equal variances not assumed) test. In all cases, values were considered statistically different when P<0.05.
It was found that no zebrafish embryo or larva died even at the highest concentration tested (1000.0 µg/kg) during the exposure period. All the treatment groups were also examined from 4 to 120 hpf on the malformation effects of DBDPE on the zebrafish embryo-larval development, including edema, bent notochord, and bent tail. As shown in Fig.
Fig. 1 Microscopic observations of zebrafish exposed to DBDPE-contaminated sediments from 4 to 120 hpf
(a) Control; (b) 62.5 µg/kg; (c) 125.0 µg/kg; (d) 250.0 µg/kg; (e) 500.0 µg/kg; (f) 1000.0 µg/kg
The hatching rates of the tested groups were also recorded from 48 hpf, when the embryos started to come out from the chorions asynchronously. At 48 hpf, only a small portion of the embryos have hatched, with the hatching rates ranging from 3.3% to 16.7% (Table
Concentration (µg/kg) | Hatching rate (%) | ||
---|---|---|---|
48 hpf | 72 hpf | 96 hpf | |
Control | 6.7 | 96.7 | 100.0 |
62.5 | 16.7 | 100.0 | 100.0 |
125.0 | 6.7 | 93.3 | 100.0 |
250.0 | 6.7 | 96.7 | 100.0 |
500.0 | 3.3 | 100.0 | 100.0 |
1000.0 | 6.7 | 80.0 | 100.0 |
Two types of motor behavior, touch-escape response and free-swimming activity, were chosen to evaluate the effect of DBDPE exposure at different time points. At 48 hpf, the touch responses were examined and all the treated groups escaped immediately with the touch on the dorsal fin. At 120 hpf, free-swimming activity during a period of 1 h was detected, and the total distances travelled as well as the distances travelled at different speeds (low, <4 mm/s; medium, 4–20 mm/s; high, >20 mm/s) were recorded (Figs.
Fig. 2 Effects on the total distances of zebrafish by exposure to DBDPE-contaminated sediments from 4 to120 hpf
CTRL: control. ** Significant difference compared to the control at P≤0.01
Fig. 3 Effects on the distances travelled under different speeds
(a) Low, <4 mm/s; (b) Medium, 4–20 mm/s; (c) High, >20 mm/s; CTRL: control. * Significant difference compared to the control at P≤0.05
The effects of different DBDPE concentrations and time of exposure on AChE activity of zebrafish larvae were shown in Fig.
Fig. 4 Effects on the AChE activity of zebrafish
(a) 72 hpf; (b) 96 hpf. AChE: acetylcholinesterase; CTRL: control
In order to elucidate the potential mechanisms of the behavior alteration, the expression levels of two important nerve-related genes, α1-tubulin and gap43, were also detected in zebrafish larvae by RT-PCR. However, as shown in Fig.
Fig. 5 Expression levels of two nerve-related genes and β-actin detected by RT-PCR
The results for zebrafish sampled at 48, 72, and 96 hpf were listed from top to bottom; for each band, the control, 62.5, 125.0, 250.0, 500.0, and 1000.0 µg/kg groups were listed from left to right
Furthermore, in order to confirm whether exposure to DBDPE induces cell apoptosis, the TUNEL assay was then conducted in whole-mounted zebrafish larvae at 96 hpf. Nevertheless, as shown in Fig.
Fig. 6 Effects on the apoptotic cell death of zebrafish by exposure to DBDPE-contaminated sediments from 4 to 96 hpf
(a) Control; (b) 62.5.0 µg/kg; (c) 125.0 µg/kg; (d) 250.0 µg/kg; (e) 500.0 µg/kg; (f) 1000.0 µg/kg
In the present study, the embryo-larval zebrafish was used as a model for investigating the effect of DBDPE-contaminated sediment exposure on neurotoxicology, including teratology, motor behavior, AChE activity, and expression levels of nerve-related genes. As demonstrated in the results, exposure to DBDPE at the levels up to 1000.0 µg/kg of dry sediment during the embryo-larval stages did not cause any adverse effects on survival or malformation (including edema, bent notochord, and bent tail). The hatching process seemed to be slightly accelerated by exposure to the lowest concentration of DBDPE (62.5 µg/kg) and inhibited by the highest concentration of DBDPE (1000.0 µg/kg) at 72 hpf. Hatching is a process regulated by both the choriolytic enzyme and the physical movement of the embryo (Jin et al.,
Generally, low toxicity of DBDPE in our findings was consistent with those of previously reported studies (Hardy et al.,
The above data, however, were inconsistent with those reported by Nakari and Huhtala (
Many studies have found that BDE-209 can exert neurological effects, including changes in spontaneous behavior in the adult rat (Viberg et al.,
In this study, results indicated that a short-term exposure to DBDPE-polluted sediments could pose little risk to the gross morphology of embryo-larval zebrafish. The hatching process and the free-swimming activity seemed to be two sensitive endpoints, and responded positively at the highest concentration tested. Moreover, we tried to elucidate the potential mechanisms of DBDPE-induced neurotoxicity by examining the expression levels of two nerve-related genes, AChE activity, and the cell apoptosis. However, the mechanisms remained unclear and need to be explored in more details in the future. Furthermore, regarding the low concentration and short duration of exposure combined with the more realistic scenario we mimicked, the application of the sediment exposure route in the present study should be taken into consideration, especially for those hydrophobic contaminants.
Compliance with ethics guidelines
Mei-qing JIN, Dong ZHANG, Ying ZHANG, Shan-shan ZHOU, Xian-ting LU, and Hong-ting ZHAO declare that they have no conflict of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed.
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