- Email: [email protected]
PCB1254 exposure contributes to the abnormalities of optomotor responses and influence of the photoreceptor cell development in zebrafish larvae
Ecotoxicology and Environmental Safety 118 (2015) 133–138
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
PCB1254 exposure contributes to the abnormalities of optomotor responses and influence of the photoreceptor cell development in zebrafish larvae Xin Zhang a,b,1, Qin Hong b,1, Lei Yang b, Min Zhang a, Xirong Guo a,b, Xia Chi a,b,n, Meiling Tong a,b,n a
Department of Pediatrics, Nanjing Medical University, NO.140, Hanzhong Road, Nanjing 210029, China Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital of Nanjing Medical University, NO.123, Tianfei Road, Mochou Street, Nanjing 210004, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 December 2014 Received in revised form 18 April 2015 Accepted 21 April 2015
Polychlorinated biphenyls (PCBs), a group of highly toxic environmental pollutants, have been report to influence the visual system development in children. However, the underlying mechanism is unclear. The study was aim to investigate the effects of continuous PCBs exposure on optomotor response (OMR) and retinal photoreceptor cell development-related gene expression in zebrafish larvae. The fertilized zebrafish embryos were exposed to PCBs at concentrations of 0.125, 0.25, 0.5, and 1 mg/L until 7 days postfertilization. Control groups with blank and 0.01% methanol were also prepared. OMR test was used to detect the visual behavior. The mRNA expression of the CRX, RHO, SWS1, and SWS2 was assessed by the Quantitative Real-Time PCR. The OMR test showed that the visual behavior of the larvae was most sensitive when the grating spatial frequency was 0.20 LP/mm and the moving speed was 25 cm/s. Moreover, the proportion of positively swimming fish was significantly reduced in the 0.5 and 1 mg/L PCB1254 treatment group (Po 0.05) compared with the controls. In addition, the expression of SWS2 was significantly down-regulated in all PCB1254 treatment groups (P o0.05), whereas the decreased expression of the CRX, RHO and SWS1 was found in the 0.5 and 1 mg/L PCB1254 groups (Po 0.05). This is the first report to demonstrate that continue exposure of zebrafish larvae to PCBs causes photoreceptor cell development-related gene expression changes that lead to OMR behavioral alterations. Analysis of these visual behavioral paradigms may be useful in predicting the adverse effects of toxicants on visual function in fish. & 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Polychlorinated biphenyls Optomotor responses Zebrafish larvae
1. Introduction Nowadays, the occurrence of eye diseases in children has increased every year. According to an investigation on low vision and blindness in 0–6 years-old children in China, the overall incidence of visual impairment is 1.1%, including 0.33% blindness and 0.73% low vision (Fu et al., 2004). For these eye diseases, children's high susceptibility to environmental toxicants is an important factor (Landrigan and Miodovnik, 2011). Especially the polychlorinated biphenyls (PCBs), which are a group of highly toxic environmental n Corresponding authors at: Department of Pediatrics,Nanjing Maternity and Child Health Care Hospital of Nanjing Medical University, No. 123, Tianfei Road, Mochou Street, Nanjing 210004, China. E-mail addresses: [email protected] (X. Chi), [email protected] (M. Tong). 1 These authors contributed equally to this work.
pollutants in the world (Mamontova et al., 2013; Zhang et al., 2014), have attracted much attention because of the exceed standard levels in water bodies and soils in many regions of China (Gao et al., 2013; Huang et al., 2011; Jianying et al., 2009; Zheng et al., 2008), Experimental studies have indicated that PCBs have the ability of the environmental carcinogenesis and cause the impairment of neurobehavioral development, the endocrine, reproductive, and immune systems (Carpenter, 2006; McGlynn et al., 2009). Moreover, it can accumulate in the human body. In human, the adverse effects on visual system development such as neonatal periorbital edema and decreased visual acuity in infants have been associated with PCBs pollute. Further analysis has shown that exposure of the lactating women to PCBs can affects fetal neurodevelopment (including retinal development). The continue exposure to PCBs was reported to result in toxicity of visual system development in children (Gascon et al., 2013; Ribas-Fito et al., 2001; Tsukimori et al.,
http://dx.doi.org/10.1016/j.ecoenv.2015.04.026 0147-6513/& 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
134
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
2013), therefore, the effects of the PCBs on fetal development need particular concern, especially on the visual system. The study was conducted to explore the potential effect of PCBs exposure on visual function. However, due to children's low cognitive level and ethical limits, we selected the zebrafish model, zebrafish has been proved to be very useful for nervous system research because of the laminated structure similar to the human retina, especially the photoreceptor cells, which have a small body with relatively large eyes and highly developed vision. Moreover, zebrafish is highly sensitive to environmental pollutants. All above mentioned indicate that zebrafish is an ideal model animal for studying the effect of environmental toxicants on visual system development (Hao et al., 2012; Wang et al., 2012). The previous study had reported that continuous exposure to PCBs affected the arrangement of cells, the thickness of cell layers, and the number of cells in the retina of embryonic zebrafish Wang (2010). The objective of the present study was to further investigate the effects of continuous exposure to PCBs for 7 days post-fertilization (dpf) on visual behaviors and retinal photoreceptor cell development-related gene expression in zebrafish. Assays, measuring the optomotor response (OMR) on the zebrafish larvae, take advantage of visual reflexes and can be used to test visual function in zebrafish at an early stage. OMR test was used to evaluate visual behaviors in larval zebrafish. The expression of the photoreceptor cell development-related genes such as cone–rod homeobox (CRX), rhodopsin (RHO), short-wavelength-sensitive opsin (SWS1 and SWS2) were also detected. Our results showed that the exposure of zebrafish larvae to PBCs altered the expression of the photoreceptor cell development-related gene and led to disturbances in the visual system.
from the Model Animal Center of Nanjing University, Nanjing, China. Zebrafish was reared in circulating water at a temperature of 28 70.5 °C with a 14 h light/10 h dark cycle. Spawning was triggered under light and was completed within 30 min. Embryos were collected and immediately rinsed with distilled water. The selected embryos were randomly distributed into petri dishes containing Hank's balanced salt solution (0.10 g NaCl, 0.03 g KCl, 0.04 g CaCl2, and 0.16 g MgSO4 in 100 mL double-distilled H2O) (Penglase et al., 2012). All the animal experimental procedures were approved by the Animal Care and Use Committee of Nanjing Medical University, Nanjing, China. 2.2. PCB exposure
2. Materials and methods
In the study, PCB1254, a common industrial chemical of PCB mixture, was chosen as the exposed toxicant. The median lethal dose (LD50) of PCB1254 (4 99% purity, Sigma-Aldrich, US) for zebrafish is 1.082 mg/L (95% confidence interval 0.917–1.327 mg/L; unpublished data). PCB1254 was prepared at concentrations of 0.125, 0.25, 0.5, and 1 mg/L according to the toxicological principle and the LD50. As the stock PCB1254 solution (1 g/L) was prepared with methanol (analytical reagent, China) as a cosolvent, and methanol itself might interfere with the experimental results, a blank control group as well as a 0.01% methanol control group was set up for comparison to exclude the interference caused by the cosolvent. Six-well cell culture plates were used as the exposure apparatus, which contained 5 mL working solution per well. The collected embryos were randomly assigned to six experimental groups with 10 embryos per well. During the experimental period, the exposure solution was renewed daily. Until 7 days' continuous exposure, OMR experiment and gene expression analysis were performed, meanwhile, the larvae were immediately frozen in liquid nitrogen, and stored at 80 °C for RNA isolation.
2.1. Zebrafish maintenance
2.3. OMR test
Wild-type Tübingen zebrafish (Danio rerio) were purchased
To examine the effects of PCBs on visual behavior, the OMR test
Fig. 1. Apparatus and procedure for the OMR. (A) Apparatus used for measurement of the OMR. HDV refers to high-definition cameras, PC refers to computers. (B) the OMR test. There were four different behavioral responses of the larvae: (a) positive swimming, (b) opposite swimming, (c) hesitant swimming, and (d) immobility. ’refers to the direction of the grating.
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
was performed. OMR refers to visual behavioral responses (turning the head or the body). It is a rapid, simple, and efficient method for the visual function, and can effectively detect abnormalities of the visual function (Maaswinkel and Li, 2003; Najafian et al., 2014). Before OMR tests, larvae were all tested using escape reflex to select the individuals with normal movement abilities. A HD camera (Canon, HFG1, Japan) was placed 50 cm above a water tank (10 cm 5 cm 2 cm) that was above a viewing screen (Acer, V193WVCb, China) connected to a computer (Lenovo, E42, China). During our tests, the lights in the room were turned off and the intensity of light above the water surface was maintained at 180220CD/m2. All the tests were conducted between 14:00 and 18:00. A custom-designed grating software was used to produce black and white bars moving flatly at a constant speed. The initial speed of movement was set at 25 cm/s, and the spatial frequency was 0.04 LP/mm (Fig. 1A). Twenty selected larvae were randomly placed in the test tank with a water level of 1 cm. The fish was allowed to adapt for 2 min. Grating spatial frequency or speed of movement was changed to find the optimal conditions. Each grating was recorded for 1 min. Under optimal conditions, the number of sensitive fish from each group was counted and their percentage was calculated. The principle of double-blind testing was strictly followed during the experimental period. In each group, the test was repeated three times. 2.4. Gene transcription Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. Expression levels of CRX, RHO, SWS1 and SWS2 mRNA were detected, with actin as a Table 1 The primers of the real-time PCR. Names CRX
Primer sequences
Sense: 5´-cgtcgttgggcttcagtt-3´ antisense: 5´-cttcacgcatctttccttcc-3´ RHO Sense: 5´-cccctcaactacatcctgct-3´ Antisense: 5´-cgactttagccccatctcac-3´ SWS1 Sense: 5´-cacccagaaggcagagagag-3´ Antisense: 5´-tgagaagaaagcagggatgg-3´ SWS2 Sense: 5´-gctgagagggaggtgacaaa-3´ Antisense: 5´-tacaaaggcaggagggaatg-3´ β-Atcion Sense: 5´-cccagacatcagggagtgat-3´ Antisense: 5´-ctcgtggataccgcaagatt-3´
135
normalizing control. The primer sequences for the target genes were designed by Primer Premier 3.0 software (Table 1). The DNA was synthesized via Quantitative Real-Time PCR with an SYBR Green PCR kit (Roche, Switzerland) on the ABI 7500 RealTime PCR system (ABI, Carlsbad, CA, USA). Each target gene was tested using 3 replicates. Absorbance data were collected at the end of every extension, and cycle threshold (CT) values were used to analyze expression data using the 2-ΔΔCt method. 2.5. Statistical analysis All the data were analyzed by Levene's test for homogeneity of variance. Suppose the homogeneity of variances met, differences were assessed by one-way analysis of variance (ANOVA) and the post-hoc LSD test to determine the significant difference (P o0.05) between groups using the SPSS13.0 software (SPSS, USA). All the data were expressed as mean 7standard deviation.
3. Results 3.1. OMR behaviors in larval zebrafish Under different grating conditions, there were four different behavioral responses of the larvae: (a) positive swimming: fish swims along the direction of grating movement within 30 s, which indicates sensitive OMR (Maaswinkel and Li, 2003), (b) opposite swimming: fish swims in an opposite direction against the direction of grating movement within 30 s, (c) hesitant swimming: fish shows a delayed swimming response, and (d) immobility: fish do not move at all (Fig. 1B). The previous report demonstrated that the optimal test conditions were chosen when it has the highest proportion of positively swimming fish. In the experiment, when grating speed was set at 25 cm/s, the proportions of the positively swimming fish changed with the spatial frequency. As grating spatial frequency was progressively increased to 0.04, 0.05, 0.10, 0.20, and 0.80 LP/mm, the proportion of positively swimming fish first increased and then decreased. The result showed that when the spatial frequency reached 0.20 LP/mm, it has a peak value (73%) observed (Fig. 2 A). Later, grating spatial frequency was set at 0.20 LP/mm and the moving speed was changed. As grating speed was increased to 12.5, 25, 50, and 75 cm/s, the proportion of positively swimming fish first increased and then decreased. It is found that when the
Fig. 2. The erection of visual behavior experiments and PCBs can affect the characteristics of the zebrafish OMR. (A) The proportions of sensitive fish in the optomotor response (OMR) tests with varying grating width/speed. (B) The proportions of insensitive fish from the six groups in the optomotor response (OMR) tests when the grating spatial frequency was 0.20 LP/mm and the moving speed was maintained at 25 cm/s. Data are expressed as the means 7 standard deviations, n¼ 60. Asterisk (*) indicates a significant difference (P o0.05) between the six groups.
136
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
moving speed was 25 cm/s, a peak value (72.5%) was observed (Fig. 2B). 3.2. OMR behaviors after exposure to PCB1254 Spatial frequency of 0.20 LP/mm and speed of movement of 25 cm/s were taken as the optimal grating conditions for the OMR test in larval zebrafish. Under these optimal conditions, the OMR behavioral were compared among six groups exposed to different concentrations of PCB1254 (0.125, 0.25, 0.5, and 1 mg/L) until 7 dpf. Results showed that a significant decrease of the proportion of positively swimming fish was observed in the 0.5 and 1 mg/L PCB1254 groups, as compared with the control groups (Fig. 2C). 3.3. The expression of CRX, RHO, SWS1 and SWS2 after 7 days' exposure to PCB1254 In order to explore whether the abnormal OMR behavior was caused by the deficiency of photoreceptor cells, the developmentrelated gene expression level were tested by qRT-PCR. Results showed that the gene expression levels varied among different groups. After 7 days' continuous exposure to PCB1254, SWS2 mRNA expression level was significantly down-regulated in all groups, whereas CRX, RHO, and SWS1 mRNA expression levels were significantly down-regulated in the 0.5 and 1 mg/L PCB1254 groups (one-way ANOVA, P o0.05; Fig. 3A–D).
4. Discussion Environmental toxicants are one of the causes for eye diseases
in children (Tarczy-Hornoch et al., 2013). PCBs are persistent organic pollutants, which have stable chemical properties and can easily accumulate in the human body (Wong et al., 2013). Recent studies have found that PCBs can impair visual system development in fetuses and newborns through placenta or breast milk, thereby influencing the visual function (Asante et al., 2011; Pellacani et al., 2014). In order to investigate the effects of exposure to PCBs on children's visual system development, the model of the zebrafish was used. The similarities of the zebrafish visual system to human, as well as the improving of the methods for study OMR behavior in zebrafish, make this animal a valuable model in vision science. Moreover, embryos and larvae of zebrafish are highly sensitive to toxic substances (Sanden et al., 2012; Yin et al., 2014). At 7 dpf, retinal development is almost complete, which equivalent to 3 years old in children. Moreover, it has been reported that the OMR of zebrafish larvae peaks at about 7 dpf, so we chose this time point for our analyses (Najafian et al., 2014). The previous research has shown that embryonic exposure to PCB1254 can affect the retinal morphology in zebrafish at 3 dpf (Wang et al., 2012). However, the potential impact of PCB1254 on vision is virtually unknown. The present study demonstrated that continue exposure to PCB1254 altered the visual behavior. To our knowledge, this is the first study that investigated the effects of PCB1254 exposure on visual behavior in zebrafish. The OMR has been shown to be useful for toxicological research (Abdeljalil et al., 2005). In our research, the OMR test helped to select the fish that were insensitive to the changes of the grating and allowed us to screen for visual function defects (Chen et al., 2013). Therefore, OMR test, a good complement for morphological study and molecular experiments, can be used to identify the impairment of vision caused by PCB1254.
Fig. 3. Changes of the mRNA expressions. (A–D) The mRNA expression of the photoreceptor cell growth-related genes in the zebrafish larvae after exposed to PCB1254 for 7 days were detected. Data are expressed as the means7 standard deviations, n¼ 30. Asterisk (*) indicates a significant difference (Po 0.05) between the six groups.
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
OMR refers to a type of visual behavioral responses (turning the head or the body) with a black and white grating, the main detection tool used in the OMR tests, which can form visual stimuli of continuous tone changes. Because of their phototaxis (Mueller and Neuhauss, 2010), zebrafish will follow the light stripe and swim along the grating direction. The grating can be described in terms of speed and spatial frequency. Several studies have shown that the two parameters influence each other (Maaswinkel and Li, 2003). Therefore, when the tests were carried out, it was necessary for us to consider both the spatial frequency and speed, as they can have important implications on our OMR test results. It can be seen from our results that when the speed was constant, the proportions of sensitive fish changed with the spatial frequency. The result indicated that grating with a spatial frequency of 0.20 LP/mm and speed of movement of 25 cm/s were optimal for the OMR test in zebrafish larvae, which is similar to what has previously been reported for zebrafish (Maaswinkel and Li, 2003). Under optimal conditions, the results of our OMR test showed that following exposure for 7 days, there were less sensitive fish in the groups treated with 0.5 and 1 mg/L PCB1254 than in the control groups. These findings indicated that PCB1254 exposure during early development had a serious impact on the zebrafish visual system. Moreover, our previous studies found that exposure to PCB1254 caused increases in the total thickness of the retina, thickness of the ganglion cell layer, and thickness of the photoreceptor cell layer, as well as disorder of cell arrangement and reduction in cell number (Wang, 2010; Wang et al., 2012). Consequently, we can speculate that abnormal OMR in our study was caused by PCB1254 exposure and their interaction with the retina, especially, the photoreceptor cells. Retina has an important role in vision development. When ambient light is projected to the eyeball, it passes through the dioptric media of the retinal photoreceptor and bipolar cells to reach the retina, in which, retinal photoreceptor cells play an important role in this pathway (Zueva, 2012). Therefore, in the present study, the expression of relevant genes (CRX, RHO, SWS1, and SWS2), which is crucial in the development of photoreceptor cell, were investigated in zebrafish larvae exposed to PCB1254 for 7 dpf. The CRX gene is a transcription factor that affects the development of photoreceptor cell. It controls the early development and survival of photoreceptor cells. The RHO is an opsin of rod cells to receive weak light signals, whereas both SWS1 and SWS2 are opsins of cone cells to receive blue and ultraviolet signals, respectively (Tran et al., 2014; Yan et al., 2014). The result demonstrated that the expression level of SWS2 mRNA was significantly down-regulated in all groups, whereas the expression levels of other genes were significantly down-regulated in the 0.5 and 1 mg/L PCB1254 groups. Consistent with these results, reduction in the expression of CRX gene affects the expression of opsin and formation of rod outer segment (ROS) in the photoreceptor cells of zebrafish. Decrease in the expression of opsins (RHO, SWS1, and SWS2) affects the stability of cell membrane in photoreceptor cells and ultimately leads to the damage of the visual function (Suliman and Novales Flamarique, 2014). Combined with the results from the OMR test, it was found that despite a lack of abnormal OMR in the 0.125 and 0.25 mg/L PCB1254 groups, the corresponding expression level of SWS2 mRNA was significantly down-regulated, with no significant abnormalities of other gene expression. These results suggested that there may exist a compensatory mechanism so that down-regulation of expression of SWS2 gene is insufficient to cause complete damage to the photoreceptor cells. However, the result showed that PCB-induced abnormal visual behaviors in larval zebrafish were possibly related to impairment of photoreceptor cells.
137
5. Conclusions In summary, the present study demonstrated that the optimal grating conditions for the OMR test in zebrafish larvae are spatial frequency of 0.20 LP/mm and speed of 25 cm/s, which can provide us with a good experimental scheme for further study of the possible effects of environmental toxins on visual system. Moreover, it also provides the evidence that continue exposure to PCBs causes abnormalities of OMR behavior and down-regulation of retinal photoreceptor cell development-related genes expression in zebrafish larvae. However, the potential underlying mechanisms and implications of these OMR behavior changes need to be further investigated. In addition, it is necessary for us to strength the detection and control of environmental organic pollutants.
Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 81170889), Nanjing Medical Technology Development Fund of Jiangsu Province, China (YKK10032) and the Six Talents Peak Project of Jiangsu Province, China (2010YY001).
References Abdeljalil, J., et al., 2005. The optomotor response: a robust first-line visual screening method for mice. Vis. Res. 45, 1439–1446. Asante, K.A., et al., 2011. Human exposure to PCBs, PBDEs and HBCDs in Ghana: temporal variation, sources of exposure and estimation of daily intakes by infants. Environ. Int. 37, 921–928. Carpenter, D.O., 2006. Polychlorinated biphenyls (PCBs): routes of exposure and effects on human health. Rev. Environ. Health 21, 1–24. Chen, L., et al., 2013. Acute exposure to DE-71 causes alterations in visual behavior in zebrafish larvae. Environ. Toxicol. Chem. 32, 1370–1375. Fu, P., et al., 2004. A national survey on low vision and blindness of 0–6 years old children in China. Zhonghua yi xue za zhi 84, 1545–1548. Gao, S., et al., 2013. Seasonal and spatial distributions and possible sources of polychlorinated biphenyls in surface sediments of Yangtze Estuary, China. Chemosphere 91, 809–816. Gascon, M., et al., 2013. Evaluating the neurotoxic effects of lactational exposure to persistent organic pollutants (POPs) in Spanish children. Neurotoxicology 34, 9–15. Hao, Y., et al., 2012. Zebrafish-a new animal models of anterior segment embryonic development and diseases basic research. Zhonghua yan ke za zhi (Chin. J. Ophthalmol. 48, 76–79. Huang, H., et al., 2011. Residual characteristics and ecological risk assessment of polychlorinated biphenyls in surface sediments from Yangtze Estuary and nearshore of East China Sea. J. Tongji Univ. Nat. Sci. 39, 1500–1505. Jianying, Z., et al., 2009. The health risk assessment and bioaccumulation of PCBs in soil-vegetables nearby E-waste area. Acta Pedol. Sin. 46, 434–441. Landrigan, P.J., Miodovnik, A., 2011. Children's health and the environment: an overview. Mount Sinai J. Med.: J. Transl. Pers. Med. 78, 1–10. Maaswinkel, H., Li, L., 2003. Spatio-temporal frequency characteristics of the optomotor response in zebrafish. Vis. Res. 43, 21–30. Mamontova, E.A., et al., 2013. Polychlorinated biphenyls in surface soil in urban and background areas of Mongolia. Environ. Pollut. 182, 424–429. McGlynn, K.A., et al., 2009. Polychlorinated biphenyls and risk of testicular germ cell tumors. Cancer Res. 69, 1901–1909. Mueller, K.P., Neuhauss, S.C., 2010. Behavioral neurobiology: how larval fish orient towards the light. Curr Biol. 20, R159–R161. Najafian, M., et al., 2014. The effect of motion aftereffect on optomotor response in larva and adult zebrafish. Neurosci. Lett. 559, 179–183. Pellacani, C., et al., 2014. Synergistic interactions between PBDEs and PCBs in human neuroblastoma cells. Environ. Toxicol. 29, 418–427. Penglase, S., et al., 2012. Lab animals: standardize the diet for zebrafish model. Nature 491, 333. Ribas-Fito, N., et al., 2001. Polychlorinated biphenyls (PCBs) and neurological development in children: a systematic review. J. Epidemiol. Commun. Health 55, 537–546. Sanden, M., et al., 2012. Zebrafish (Danio rerio) as a model for investigating dietary toxic effects of deoxynivalenol contamination in aquaculture feeds. Food Chem. Toxicol. 50, 4441–4448. Suliman, T., Novales Flamarique, I., 2014. Visual pigments and opsin expression in the juveniles of three species of fish (rainbow trout, zebrafish, and killifish) following prolonged exposure to thyroid hormone or retinoic acid. J. Comp. Neurol. 522, 98–117.
138
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
Tarczy-Hornoch, K., et al., 2013. Prevalence and causes of visual impairment in Asian and non-Hispanic white preschool children: multi-ethnic Pediatric Eye Disease Study. Ophthalmology 120, 1220–1226. Tran, N.M., et al., 2014. Mechanistically distinct mouse models for CRX-associated retinopathy. PLoS Genet. 10, e1004111. Tsukimori, K., et al., 2013. Blood levels of PCDDs, PCDFs, and coplanar PCBs in Yusho mothers and their descendants: association with fetal Yusho disease. Chemosphere 90, 1581–1588. Wang, Y.-p, et al., 2010. Effects of embryonic exposure to polychlorinated biphenyls on zebrafish retinal morphology and the expression of CRX gene. Chin. J. Child Health Care 12, 021. Wang, Y.P., et al., 2012. Effects of embryonic exposure to polychlorinated biphenyls on zebrafish (Danio rerio) retinal development. J. Appl. Toxicol. 32, 186–193. Wong, T.W., et al., 2013. Levels of PCDDs, PCDFs, and dioxin-like PCBs in human milk among Hong Kong mothers. Sci. Total Environ. 463–464, 1230–1238. Yan, S., et al., 2014. The expression of three opsin genes from the compound eye of
Helicoverpa armigera (Lepidoptera: Noctuidae) is regulated by a circadian clock, light conditions and nutritional status. PLoS One 9, e111683. Yin, J., et al., 2014. Individual and joint toxic effects of cadmium sulfate and alphanaphthoflavone on the development of zebrafish embryo. J. Zhejiang Univ. Sci. B 15, 766–775. Zhang, Z., et al., 2014. Long term temporal and spatial changes in the distribution of polychlorinated biphenyls and polybrominated diphenyl ethers in Scottish soils. Sci. Total Environ. 468, 158–164. Zheng, G.J., et al., 2008. Polychlorinated dibenzo-p-dioxins and dibenzofurans pollution in China: sources, environmental levels and potential human health impacts. Environ. Int. 34, 1050–1061. Zueva, M.V., 2012. Maturation and plasticity of visual system: neurogenesis, synaptogenesis, and myelogenesis. Report I. Retina and retinogeniculate projections. Vestn. Oftalmol. 128, 37–41.
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
PCB1254 exposure contributes to the abnormalities of optomotor responses and influence of the photoreceptor cell development in zebrafish larvae Xin Zhang a,b,1, Qin Hong b,1, Lei Yang b, Min Zhang a, Xirong Guo a,b, Xia Chi a,b,n, Meiling Tong a,b,n a
Department of Pediatrics, Nanjing Medical University, NO.140, Hanzhong Road, Nanjing 210029, China Department of Pediatrics, Nanjing Maternity and Child Health Care Hospital of Nanjing Medical University, NO.123, Tianfei Road, Mochou Street, Nanjing 210004, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 23 December 2014 Received in revised form 18 April 2015 Accepted 21 April 2015
Polychlorinated biphenyls (PCBs), a group of highly toxic environmental pollutants, have been report to influence the visual system development in children. However, the underlying mechanism is unclear. The study was aim to investigate the effects of continuous PCBs exposure on optomotor response (OMR) and retinal photoreceptor cell development-related gene expression in zebrafish larvae. The fertilized zebrafish embryos were exposed to PCBs at concentrations of 0.125, 0.25, 0.5, and 1 mg/L until 7 days postfertilization. Control groups with blank and 0.01% methanol were also prepared. OMR test was used to detect the visual behavior. The mRNA expression of the CRX, RHO, SWS1, and SWS2 was assessed by the Quantitative Real-Time PCR. The OMR test showed that the visual behavior of the larvae was most sensitive when the grating spatial frequency was 0.20 LP/mm and the moving speed was 25 cm/s. Moreover, the proportion of positively swimming fish was significantly reduced in the 0.5 and 1 mg/L PCB1254 treatment group (Po 0.05) compared with the controls. In addition, the expression of SWS2 was significantly down-regulated in all PCB1254 treatment groups (P o0.05), whereas the decreased expression of the CRX, RHO and SWS1 was found in the 0.5 and 1 mg/L PCB1254 groups (Po 0.05). This is the first report to demonstrate that continue exposure of zebrafish larvae to PCBs causes photoreceptor cell development-related gene expression changes that lead to OMR behavioral alterations. Analysis of these visual behavioral paradigms may be useful in predicting the adverse effects of toxicants on visual function in fish. & 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Keywords: Polychlorinated biphenyls Optomotor responses Zebrafish larvae
1. Introduction Nowadays, the occurrence of eye diseases in children has increased every year. According to an investigation on low vision and blindness in 0–6 years-old children in China, the overall incidence of visual impairment is 1.1%, including 0.33% blindness and 0.73% low vision (Fu et al., 2004). For these eye diseases, children's high susceptibility to environmental toxicants is an important factor (Landrigan and Miodovnik, 2011). Especially the polychlorinated biphenyls (PCBs), which are a group of highly toxic environmental n Corresponding authors at: Department of Pediatrics,Nanjing Maternity and Child Health Care Hospital of Nanjing Medical University, No. 123, Tianfei Road, Mochou Street, Nanjing 210004, China. E-mail addresses: [email protected] (X. Chi), [email protected] (M. Tong). 1 These authors contributed equally to this work.
pollutants in the world (Mamontova et al., 2013; Zhang et al., 2014), have attracted much attention because of the exceed standard levels in water bodies and soils in many regions of China (Gao et al., 2013; Huang et al., 2011; Jianying et al., 2009; Zheng et al., 2008), Experimental studies have indicated that PCBs have the ability of the environmental carcinogenesis and cause the impairment of neurobehavioral development, the endocrine, reproductive, and immune systems (Carpenter, 2006; McGlynn et al., 2009). Moreover, it can accumulate in the human body. In human, the adverse effects on visual system development such as neonatal periorbital edema and decreased visual acuity in infants have been associated with PCBs pollute. Further analysis has shown that exposure of the lactating women to PCBs can affects fetal neurodevelopment (including retinal development). The continue exposure to PCBs was reported to result in toxicity of visual system development in children (Gascon et al., 2013; Ribas-Fito et al., 2001; Tsukimori et al.,
http://dx.doi.org/10.1016/j.ecoenv.2015.04.026 0147-6513/& 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
134
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
2013), therefore, the effects of the PCBs on fetal development need particular concern, especially on the visual system. The study was conducted to explore the potential effect of PCBs exposure on visual function. However, due to children's low cognitive level and ethical limits, we selected the zebrafish model, zebrafish has been proved to be very useful for nervous system research because of the laminated structure similar to the human retina, especially the photoreceptor cells, which have a small body with relatively large eyes and highly developed vision. Moreover, zebrafish is highly sensitive to environmental pollutants. All above mentioned indicate that zebrafish is an ideal model animal for studying the effect of environmental toxicants on visual system development (Hao et al., 2012; Wang et al., 2012). The previous study had reported that continuous exposure to PCBs affected the arrangement of cells, the thickness of cell layers, and the number of cells in the retina of embryonic zebrafish Wang (2010). The objective of the present study was to further investigate the effects of continuous exposure to PCBs for 7 days post-fertilization (dpf) on visual behaviors and retinal photoreceptor cell development-related gene expression in zebrafish. Assays, measuring the optomotor response (OMR) on the zebrafish larvae, take advantage of visual reflexes and can be used to test visual function in zebrafish at an early stage. OMR test was used to evaluate visual behaviors in larval zebrafish. The expression of the photoreceptor cell development-related genes such as cone–rod homeobox (CRX), rhodopsin (RHO), short-wavelength-sensitive opsin (SWS1 and SWS2) were also detected. Our results showed that the exposure of zebrafish larvae to PBCs altered the expression of the photoreceptor cell development-related gene and led to disturbances in the visual system.
from the Model Animal Center of Nanjing University, Nanjing, China. Zebrafish was reared in circulating water at a temperature of 28 70.5 °C with a 14 h light/10 h dark cycle. Spawning was triggered under light and was completed within 30 min. Embryos were collected and immediately rinsed with distilled water. The selected embryos were randomly distributed into petri dishes containing Hank's balanced salt solution (0.10 g NaCl, 0.03 g KCl, 0.04 g CaCl2, and 0.16 g MgSO4 in 100 mL double-distilled H2O) (Penglase et al., 2012). All the animal experimental procedures were approved by the Animal Care and Use Committee of Nanjing Medical University, Nanjing, China. 2.2. PCB exposure
2. Materials and methods
In the study, PCB1254, a common industrial chemical of PCB mixture, was chosen as the exposed toxicant. The median lethal dose (LD50) of PCB1254 (4 99% purity, Sigma-Aldrich, US) for zebrafish is 1.082 mg/L (95% confidence interval 0.917–1.327 mg/L; unpublished data). PCB1254 was prepared at concentrations of 0.125, 0.25, 0.5, and 1 mg/L according to the toxicological principle and the LD50. As the stock PCB1254 solution (1 g/L) was prepared with methanol (analytical reagent, China) as a cosolvent, and methanol itself might interfere with the experimental results, a blank control group as well as a 0.01% methanol control group was set up for comparison to exclude the interference caused by the cosolvent. Six-well cell culture plates were used as the exposure apparatus, which contained 5 mL working solution per well. The collected embryos were randomly assigned to six experimental groups with 10 embryos per well. During the experimental period, the exposure solution was renewed daily. Until 7 days' continuous exposure, OMR experiment and gene expression analysis were performed, meanwhile, the larvae were immediately frozen in liquid nitrogen, and stored at 80 °C for RNA isolation.
2.1. Zebrafish maintenance
2.3. OMR test
Wild-type Tübingen zebrafish (Danio rerio) were purchased
To examine the effects of PCBs on visual behavior, the OMR test
Fig. 1. Apparatus and procedure for the OMR. (A) Apparatus used for measurement of the OMR. HDV refers to high-definition cameras, PC refers to computers. (B) the OMR test. There were four different behavioral responses of the larvae: (a) positive swimming, (b) opposite swimming, (c) hesitant swimming, and (d) immobility. ’refers to the direction of the grating.
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
was performed. OMR refers to visual behavioral responses (turning the head or the body). It is a rapid, simple, and efficient method for the visual function, and can effectively detect abnormalities of the visual function (Maaswinkel and Li, 2003; Najafian et al., 2014). Before OMR tests, larvae were all tested using escape reflex to select the individuals with normal movement abilities. A HD camera (Canon, HFG1, Japan) was placed 50 cm above a water tank (10 cm 5 cm 2 cm) that was above a viewing screen (Acer, V193WVCb, China) connected to a computer (Lenovo, E42, China). During our tests, the lights in the room were turned off and the intensity of light above the water surface was maintained at 180220CD/m2. All the tests were conducted between 14:00 and 18:00. A custom-designed grating software was used to produce black and white bars moving flatly at a constant speed. The initial speed of movement was set at 25 cm/s, and the spatial frequency was 0.04 LP/mm (Fig. 1A). Twenty selected larvae were randomly placed in the test tank with a water level of 1 cm. The fish was allowed to adapt for 2 min. Grating spatial frequency or speed of movement was changed to find the optimal conditions. Each grating was recorded for 1 min. Under optimal conditions, the number of sensitive fish from each group was counted and their percentage was calculated. The principle of double-blind testing was strictly followed during the experimental period. In each group, the test was repeated three times. 2.4. Gene transcription Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. Expression levels of CRX, RHO, SWS1 and SWS2 mRNA were detected, with actin as a Table 1 The primers of the real-time PCR. Names CRX
Primer sequences
Sense: 5´-cgtcgttgggcttcagtt-3´ antisense: 5´-cttcacgcatctttccttcc-3´ RHO Sense: 5´-cccctcaactacatcctgct-3´ Antisense: 5´-cgactttagccccatctcac-3´ SWS1 Sense: 5´-cacccagaaggcagagagag-3´ Antisense: 5´-tgagaagaaagcagggatgg-3´ SWS2 Sense: 5´-gctgagagggaggtgacaaa-3´ Antisense: 5´-tacaaaggcaggagggaatg-3´ β-Atcion Sense: 5´-cccagacatcagggagtgat-3´ Antisense: 5´-ctcgtggataccgcaagatt-3´
135
normalizing control. The primer sequences for the target genes were designed by Primer Premier 3.0 software (Table 1). The DNA was synthesized via Quantitative Real-Time PCR with an SYBR Green PCR kit (Roche, Switzerland) on the ABI 7500 RealTime PCR system (ABI, Carlsbad, CA, USA). Each target gene was tested using 3 replicates. Absorbance data were collected at the end of every extension, and cycle threshold (CT) values were used to analyze expression data using the 2-ΔΔCt method. 2.5. Statistical analysis All the data were analyzed by Levene's test for homogeneity of variance. Suppose the homogeneity of variances met, differences were assessed by one-way analysis of variance (ANOVA) and the post-hoc LSD test to determine the significant difference (P o0.05) between groups using the SPSS13.0 software (SPSS, USA). All the data were expressed as mean 7standard deviation.
3. Results 3.1. OMR behaviors in larval zebrafish Under different grating conditions, there were four different behavioral responses of the larvae: (a) positive swimming: fish swims along the direction of grating movement within 30 s, which indicates sensitive OMR (Maaswinkel and Li, 2003), (b) opposite swimming: fish swims in an opposite direction against the direction of grating movement within 30 s, (c) hesitant swimming: fish shows a delayed swimming response, and (d) immobility: fish do not move at all (Fig. 1B). The previous report demonstrated that the optimal test conditions were chosen when it has the highest proportion of positively swimming fish. In the experiment, when grating speed was set at 25 cm/s, the proportions of the positively swimming fish changed with the spatial frequency. As grating spatial frequency was progressively increased to 0.04, 0.05, 0.10, 0.20, and 0.80 LP/mm, the proportion of positively swimming fish first increased and then decreased. The result showed that when the spatial frequency reached 0.20 LP/mm, it has a peak value (73%) observed (Fig. 2 A). Later, grating spatial frequency was set at 0.20 LP/mm and the moving speed was changed. As grating speed was increased to 12.5, 25, 50, and 75 cm/s, the proportion of positively swimming fish first increased and then decreased. It is found that when the
Fig. 2. The erection of visual behavior experiments and PCBs can affect the characteristics of the zebrafish OMR. (A) The proportions of sensitive fish in the optomotor response (OMR) tests with varying grating width/speed. (B) The proportions of insensitive fish from the six groups in the optomotor response (OMR) tests when the grating spatial frequency was 0.20 LP/mm and the moving speed was maintained at 25 cm/s. Data are expressed as the means 7 standard deviations, n¼ 60. Asterisk (*) indicates a significant difference (P o0.05) between the six groups.
136
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
moving speed was 25 cm/s, a peak value (72.5%) was observed (Fig. 2B). 3.2. OMR behaviors after exposure to PCB1254 Spatial frequency of 0.20 LP/mm and speed of movement of 25 cm/s were taken as the optimal grating conditions for the OMR test in larval zebrafish. Under these optimal conditions, the OMR behavioral were compared among six groups exposed to different concentrations of PCB1254 (0.125, 0.25, 0.5, and 1 mg/L) until 7 dpf. Results showed that a significant decrease of the proportion of positively swimming fish was observed in the 0.5 and 1 mg/L PCB1254 groups, as compared with the control groups (Fig. 2C). 3.3. The expression of CRX, RHO, SWS1 and SWS2 after 7 days' exposure to PCB1254 In order to explore whether the abnormal OMR behavior was caused by the deficiency of photoreceptor cells, the developmentrelated gene expression level were tested by qRT-PCR. Results showed that the gene expression levels varied among different groups. After 7 days' continuous exposure to PCB1254, SWS2 mRNA expression level was significantly down-regulated in all groups, whereas CRX, RHO, and SWS1 mRNA expression levels were significantly down-regulated in the 0.5 and 1 mg/L PCB1254 groups (one-way ANOVA, P o0.05; Fig. 3A–D).
4. Discussion Environmental toxicants are one of the causes for eye diseases
in children (Tarczy-Hornoch et al., 2013). PCBs are persistent organic pollutants, which have stable chemical properties and can easily accumulate in the human body (Wong et al., 2013). Recent studies have found that PCBs can impair visual system development in fetuses and newborns through placenta or breast milk, thereby influencing the visual function (Asante et al., 2011; Pellacani et al., 2014). In order to investigate the effects of exposure to PCBs on children's visual system development, the model of the zebrafish was used. The similarities of the zebrafish visual system to human, as well as the improving of the methods for study OMR behavior in zebrafish, make this animal a valuable model in vision science. Moreover, embryos and larvae of zebrafish are highly sensitive to toxic substances (Sanden et al., 2012; Yin et al., 2014). At 7 dpf, retinal development is almost complete, which equivalent to 3 years old in children. Moreover, it has been reported that the OMR of zebrafish larvae peaks at about 7 dpf, so we chose this time point for our analyses (Najafian et al., 2014). The previous research has shown that embryonic exposure to PCB1254 can affect the retinal morphology in zebrafish at 3 dpf (Wang et al., 2012). However, the potential impact of PCB1254 on vision is virtually unknown. The present study demonstrated that continue exposure to PCB1254 altered the visual behavior. To our knowledge, this is the first study that investigated the effects of PCB1254 exposure on visual behavior in zebrafish. The OMR has been shown to be useful for toxicological research (Abdeljalil et al., 2005). In our research, the OMR test helped to select the fish that were insensitive to the changes of the grating and allowed us to screen for visual function defects (Chen et al., 2013). Therefore, OMR test, a good complement for morphological study and molecular experiments, can be used to identify the impairment of vision caused by PCB1254.
Fig. 3. Changes of the mRNA expressions. (A–D) The mRNA expression of the photoreceptor cell growth-related genes in the zebrafish larvae after exposed to PCB1254 for 7 days were detected. Data are expressed as the means7 standard deviations, n¼ 30. Asterisk (*) indicates a significant difference (Po 0.05) between the six groups.
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
OMR refers to a type of visual behavioral responses (turning the head or the body) with a black and white grating, the main detection tool used in the OMR tests, which can form visual stimuli of continuous tone changes. Because of their phototaxis (Mueller and Neuhauss, 2010), zebrafish will follow the light stripe and swim along the grating direction. The grating can be described in terms of speed and spatial frequency. Several studies have shown that the two parameters influence each other (Maaswinkel and Li, 2003). Therefore, when the tests were carried out, it was necessary for us to consider both the spatial frequency and speed, as they can have important implications on our OMR test results. It can be seen from our results that when the speed was constant, the proportions of sensitive fish changed with the spatial frequency. The result indicated that grating with a spatial frequency of 0.20 LP/mm and speed of movement of 25 cm/s were optimal for the OMR test in zebrafish larvae, which is similar to what has previously been reported for zebrafish (Maaswinkel and Li, 2003). Under optimal conditions, the results of our OMR test showed that following exposure for 7 days, there were less sensitive fish in the groups treated with 0.5 and 1 mg/L PCB1254 than in the control groups. These findings indicated that PCB1254 exposure during early development had a serious impact on the zebrafish visual system. Moreover, our previous studies found that exposure to PCB1254 caused increases in the total thickness of the retina, thickness of the ganglion cell layer, and thickness of the photoreceptor cell layer, as well as disorder of cell arrangement and reduction in cell number (Wang, 2010; Wang et al., 2012). Consequently, we can speculate that abnormal OMR in our study was caused by PCB1254 exposure and their interaction with the retina, especially, the photoreceptor cells. Retina has an important role in vision development. When ambient light is projected to the eyeball, it passes through the dioptric media of the retinal photoreceptor and bipolar cells to reach the retina, in which, retinal photoreceptor cells play an important role in this pathway (Zueva, 2012). Therefore, in the present study, the expression of relevant genes (CRX, RHO, SWS1, and SWS2), which is crucial in the development of photoreceptor cell, were investigated in zebrafish larvae exposed to PCB1254 for 7 dpf. The CRX gene is a transcription factor that affects the development of photoreceptor cell. It controls the early development and survival of photoreceptor cells. The RHO is an opsin of rod cells to receive weak light signals, whereas both SWS1 and SWS2 are opsins of cone cells to receive blue and ultraviolet signals, respectively (Tran et al., 2014; Yan et al., 2014). The result demonstrated that the expression level of SWS2 mRNA was significantly down-regulated in all groups, whereas the expression levels of other genes were significantly down-regulated in the 0.5 and 1 mg/L PCB1254 groups. Consistent with these results, reduction in the expression of CRX gene affects the expression of opsin and formation of rod outer segment (ROS) in the photoreceptor cells of zebrafish. Decrease in the expression of opsins (RHO, SWS1, and SWS2) affects the stability of cell membrane in photoreceptor cells and ultimately leads to the damage of the visual function (Suliman and Novales Flamarique, 2014). Combined with the results from the OMR test, it was found that despite a lack of abnormal OMR in the 0.125 and 0.25 mg/L PCB1254 groups, the corresponding expression level of SWS2 mRNA was significantly down-regulated, with no significant abnormalities of other gene expression. These results suggested that there may exist a compensatory mechanism so that down-regulation of expression of SWS2 gene is insufficient to cause complete damage to the photoreceptor cells. However, the result showed that PCB-induced abnormal visual behaviors in larval zebrafish were possibly related to impairment of photoreceptor cells.
137
5. Conclusions In summary, the present study demonstrated that the optimal grating conditions for the OMR test in zebrafish larvae are spatial frequency of 0.20 LP/mm and speed of 25 cm/s, which can provide us with a good experimental scheme for further study of the possible effects of environmental toxins on visual system. Moreover, it also provides the evidence that continue exposure to PCBs causes abnormalities of OMR behavior and down-regulation of retinal photoreceptor cell development-related genes expression in zebrafish larvae. However, the potential underlying mechanisms and implications of these OMR behavior changes need to be further investigated. In addition, it is necessary for us to strength the detection and control of environmental organic pollutants.
Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (No. 81170889), Nanjing Medical Technology Development Fund of Jiangsu Province, China (YKK10032) and the Six Talents Peak Project of Jiangsu Province, China (2010YY001).
References Abdeljalil, J., et al., 2005. The optomotor response: a robust first-line visual screening method for mice. Vis. Res. 45, 1439–1446. Asante, K.A., et al., 2011. Human exposure to PCBs, PBDEs and HBCDs in Ghana: temporal variation, sources of exposure and estimation of daily intakes by infants. Environ. Int. 37, 921–928. Carpenter, D.O., 2006. Polychlorinated biphenyls (PCBs): routes of exposure and effects on human health. Rev. Environ. Health 21, 1–24. Chen, L., et al., 2013. Acute exposure to DE-71 causes alterations in visual behavior in zebrafish larvae. Environ. Toxicol. Chem. 32, 1370–1375. Fu, P., et al., 2004. A national survey on low vision and blindness of 0–6 years old children in China. Zhonghua yi xue za zhi 84, 1545–1548. Gao, S., et al., 2013. Seasonal and spatial distributions and possible sources of polychlorinated biphenyls in surface sediments of Yangtze Estuary, China. Chemosphere 91, 809–816. Gascon, M., et al., 2013. Evaluating the neurotoxic effects of lactational exposure to persistent organic pollutants (POPs) in Spanish children. Neurotoxicology 34, 9–15. Hao, Y., et al., 2012. Zebrafish-a new animal models of anterior segment embryonic development and diseases basic research. Zhonghua yan ke za zhi (Chin. J. Ophthalmol. 48, 76–79. Huang, H., et al., 2011. Residual characteristics and ecological risk assessment of polychlorinated biphenyls in surface sediments from Yangtze Estuary and nearshore of East China Sea. J. Tongji Univ. Nat. Sci. 39, 1500–1505. Jianying, Z., et al., 2009. The health risk assessment and bioaccumulation of PCBs in soil-vegetables nearby E-waste area. Acta Pedol. Sin. 46, 434–441. Landrigan, P.J., Miodovnik, A., 2011. Children's health and the environment: an overview. Mount Sinai J. Med.: J. Transl. Pers. Med. 78, 1–10. Maaswinkel, H., Li, L., 2003. Spatio-temporal frequency characteristics of the optomotor response in zebrafish. Vis. Res. 43, 21–30. Mamontova, E.A., et al., 2013. Polychlorinated biphenyls in surface soil in urban and background areas of Mongolia. Environ. Pollut. 182, 424–429. McGlynn, K.A., et al., 2009. Polychlorinated biphenyls and risk of testicular germ cell tumors. Cancer Res. 69, 1901–1909. Mueller, K.P., Neuhauss, S.C., 2010. Behavioral neurobiology: how larval fish orient towards the light. Curr Biol. 20, R159–R161. Najafian, M., et al., 2014. The effect of motion aftereffect on optomotor response in larva and adult zebrafish. Neurosci. Lett. 559, 179–183. Pellacani, C., et al., 2014. Synergistic interactions between PBDEs and PCBs in human neuroblastoma cells. Environ. Toxicol. 29, 418–427. Penglase, S., et al., 2012. Lab animals: standardize the diet for zebrafish model. Nature 491, 333. Ribas-Fito, N., et al., 2001. Polychlorinated biphenyls (PCBs) and neurological development in children: a systematic review. J. Epidemiol. Commun. Health 55, 537–546. Sanden, M., et al., 2012. Zebrafish (Danio rerio) as a model for investigating dietary toxic effects of deoxynivalenol contamination in aquaculture feeds. Food Chem. Toxicol. 50, 4441–4448. Suliman, T., Novales Flamarique, I., 2014. Visual pigments and opsin expression in the juveniles of three species of fish (rainbow trout, zebrafish, and killifish) following prolonged exposure to thyroid hormone or retinoic acid. J. Comp. Neurol. 522, 98–117.
138
X. Zhang et al. / Ecotoxicology and Environmental Safety 118 (2015) 133–138
Tarczy-Hornoch, K., et al., 2013. Prevalence and causes of visual impairment in Asian and non-Hispanic white preschool children: multi-ethnic Pediatric Eye Disease Study. Ophthalmology 120, 1220–1226. Tran, N.M., et al., 2014. Mechanistically distinct mouse models for CRX-associated retinopathy. PLoS Genet. 10, e1004111. Tsukimori, K., et al., 2013. Blood levels of PCDDs, PCDFs, and coplanar PCBs in Yusho mothers and their descendants: association with fetal Yusho disease. Chemosphere 90, 1581–1588. Wang, Y.-p, et al., 2010. Effects of embryonic exposure to polychlorinated biphenyls on zebrafish retinal morphology and the expression of CRX gene. Chin. J. Child Health Care 12, 021. Wang, Y.P., et al., 2012. Effects of embryonic exposure to polychlorinated biphenyls on zebrafish (Danio rerio) retinal development. J. Appl. Toxicol. 32, 186–193. Wong, T.W., et al., 2013. Levels of PCDDs, PCDFs, and dioxin-like PCBs in human milk among Hong Kong mothers. Sci. Total Environ. 463–464, 1230–1238. Yan, S., et al., 2014. The expression of three opsin genes from the compound eye of
Helicoverpa armigera (Lepidoptera: Noctuidae) is regulated by a circadian clock, light conditions and nutritional status. PLoS One 9, e111683. Yin, J., et al., 2014. Individual and joint toxic effects of cadmium sulfate and alphanaphthoflavone on the development of zebrafish embryo. J. Zhejiang Univ. Sci. B 15, 766–775. Zhang, Z., et al., 2014. Long term temporal and spatial changes in the distribution of polychlorinated biphenyls and polybrominated diphenyl ethers in Scottish soils. Sci. Total Environ. 468, 158–164. Zheng, G.J., et al., 2008. Polychlorinated dibenzo-p-dioxins and dibenzofurans pollution in China: sources, environmental levels and potential human health impacts. Environ. Int. 34, 1050–1061. Zueva, M.V., 2012. Maturation and plasticity of visual system: neurogenesis, synaptogenesis, and myelogenesis. Report I. Retina and retinogeniculate projections. Vestn. Oftalmol. 128, 37–41.