Toxicon 77 (2014) 16–25

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Early developmental toxicity of saxitoxin on medaka (Oryzias melastigma) embryos Li Tian a,1, Jinping Cheng b, c,1, Xueping Chen d, Shuk Han Cheng e, Yim Ling Mak b, Paul Kwan Sing Lam b, Leo Lai Chan b, **, Mingfu Wang a, * a

School of Biological Sciences, The University of Hong Kong, Pokfulam Road, 999077, Hong Kong, Special Administrative Region State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon 999077, Hong Kong, Special Administrative Region c State Key Laboratory in Estuarine and Coastal Research, East China Normal University, 200062 Shanghai, China d Vitargent (International) Biotechnology Limited, Hong Kong Science Park, Shatin 999077, Hong Kong, Special Administrative Region e Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon 999077, Hong Kong, Special Administrative Region b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 July 2013 Accepted 17 October 2013 Available online 30 October 2013

Saxitoxin (STX) is the most potent paralytic shellfish poisoning toxin in crustaceans and molluscs, and is known to cause intoxication to humans and marine animals due to its neurotoxicity. However, the extent of its early developmental toxicity to marine species remains unknown. In this study, we examined the early developmental toxicity of STX using marine medaka (Oryzias melastigma) embryos as model. The medaka embryos were exposed to STX for four days, from the early blastula stage onwards, and this exposure period covered the main developmental stage of the central nervous system and somites. After exposure, the treated medaka eleutheroembryos at 15 day post fertilization exhibited abnormal growth with longer body length and relatively smaller yolk sac size. High cell proliferation, neuron development, and metabolism were confirmed using whole-mount immunostaining and two-dimensional electrophoresis. In summary, STX disturbed the normal growth of medaka embryos probably by affecting the metabolic rate in the exposed medaka embryos. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Paralytic shellfish poisoning Saxitoxin Developmental toxicity Endocrine disruptor Proteomics

1. Introduction Paralytic shellfish poisoning (PSP) toxins, produced by harmful dinoflagellates, are a series of neurotoxins that accumulate mainly in shellfish and cause human intoxication through the intake of toxic mollusks. Vectors have been found involving clams, mussels, oysters, gastropods, cockles, fish, scallops, whelks, lobsters, copepods, crabs and so on, with a wide geographical distribution around the world (Kumar-Roiné et al., 2011). They are the commonest * Corresponding author. Tel.: þ852 2299 0338; fax: þ852 2299 0348. ** Corresponding author. E-mail addresses: [email protected] (L.L. Chan), [email protected] (M. Wang). 1 Li Tian and Jinping Cheng contributed equally to this work. 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.10.022

and the most lethal bio-toxins among the marine algal toxins, and pose serious public health threat in the affected area (Wong et al., 2011). Apart from humans, it affects marine fish and mammals as well, through either direct exposure or dietary uptake (Castonguay et al., 1997; Reyero et al., 1999; White, 1981). Among the PSP toxins, saxitoxin (STX) is the most potent. It is difficult to degrade and highly stable even after heating (Falconer and Humpage, 2005). The specific properties of STX make it a public health hazard. Pregnant women and young children are more vulnerable to STX. Various symptoms after direct exposure to STX, such as fever, eye irritation, abdominal pain, and skin rashes, have been found in infants and children (Rapala et al., 2005). When brain cells are destroyed and the endocrine is disrupted during the developmental stage

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in infants and children, the resulting dysfunction can be permanent and irreversible (Landrigan et al., 1999). Thus, it is important to study the early developmental toxicity of STX. Recent studies show that STX exposure results in edema and body curvature in zebrafish embryos, and sensorimotor impairments and paralysis in herring larvae (Lefebvre et al., 2004, 2005). STX is known to exert its neural toxicity by blocking the voltage-gated sodium channel in excitable cells, due to its high binding affinity to the soluble sites of sodium channels in the nervous system and muscles. Its neural toxicity may partly explain its toxic effects on the developing fish embryos and larvae, however, the knowledge on its developmental effects and relevant mechanisms is still limited. Marine medaka (Oryzias melastigma), a small teleost fish, is a popular model fish widely used for the study of human diseases and marine toxins. Their embryos are easy to culture and have a relatively short generation time, which make them an appropriate marine model for rapid toxicity screening. Medaka embryos are optically clear, and their developmental stages are thoroughly characterized, and thus, are convenient for the observation of organogenesis and the developmental process (Shi and Faustman, 1989). Based on their morphological and pathological changes, medaka embryos have been successfully used to study the teratogenic effects of azaspiracid, one novel marine phycotoxin, and the developmental toxicity of okadaic acid, a diarrhetic shellfish poisoning toxin (Colman et al., 2005; Escoffier et al., 2007). Usually combined with pathological observations in model animals, the twodimensional electrophoresis (2-DE) approach is widely used in toxicological studies to present a whole protein profile and elucidate molecular responses to toxicants. It has proved to be effective in identifying protein biomarkers with high resolution, statistical confidence and good compatibility with mass spectrometry. For example, in the study of the toxicity of freshwater microcystin-LR to zebrafish, 2-DE helped display diverse proteins responding to the toxin, and revealed the reactive oxygen species pathway might be the main toxic pathway (Wang et al., 2010). Therefore, to elucidate the molecular mechanism of STX on fish embryo development, we adopted 2-DE technique in this study. Currently, there are limited studies on the developmental toxicity of STX on children and marine species. In order to reveal the developmental toxicity of STX, medaka fish embryos are adopted as the developmental model, and investigated using various techniques. This study will contribute to the knowledge on STX intoxication during developmental stages. Furthermore, the unique protein patterns at different developmental stages provide potential biomarkers for the rapid screening of marine toxins.

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standard (65  3 mM) was purchased from the National Research Council Canada (CRM-STX-e, NRC Institute for Marine Biosciences, Canada) and stored at 20  C. The concentration of STX in the stock solution was quantified using high performance liquid chromatography (Waters) with a fluorescence detector (Waters 2475). 2.2. Medaka fish maintenance and four-day consecutive static exposure Adult medaka fish were maintained in a large aquarium (40 cm  60 cm  40 cm) filled with continuously aerated water at 26  C with a 14 h:10 h light–dark cycle. They were fed with commercial dry feed twice per day. The medaka embryos were directly collected from the females. The embryos were examined using a dissection microscope, and normal fertilized medaka eggs at Stage 10, early blastula stage, 0 day post fertilization (dpf) were cleaned and transferred to a sterile 96-well cell culture plate for subsequent exposure experiments. Each well, which contained 10 embryos, received either 840 or 1260 mg/L of STX, or none. The dose adopted was that suggested to induce abnormalities in zebrafish larvae (Lefebvre et al., 2004). Fifteen replicates were set up for each concentration. The control and exposure media were renewed every two days, and exposure lasted for four days. Following the period of exposure, the control and treated embryos were transferred to a clean 6-well cell culture plate, and the embryos were collected at different developmental time points: 4, 8, 12 and 16 dpf (newly hatched larvae, which are also named eleutheroembryos) for the subsequent proteomic study. 2.3. Body length measurement and morphological observations The measurement of body length was performed according to Cheng et al. (2007). The body length of the medaka embryos exposed to STX and control medaka embryos were measured at the 15th day of development. Ten embryos were randomly selected from each condition and anaesthetized with 0.016% (w/v) tricaine (Sigma). They were mounted in lateral view in a culture medium on clean slides, and then photographed with an Olympus disk scanning unit (Olympus, Tokyo, Japan). The pictures were then quantitatively analyzed with the public domain NIH Image program (developed by the US National Institutes of Health and publicly available on the Internet at http://rsb. info.nih.gov/nih-image/). Using this software, the overall body length of the embryos was manually quantified by point to point measurement. Meanwhile, early life parameters, such as the melanophores, dorsal fins and yolk sacs, were examined and photographed.

2. Materials and methods

2.4. Whole-mount immunostaining

2.1. Chemicals

Neurogenesis and cellular proliferation at 4 dpf in the embryos exposed to 840 mg/L of STX were examined using whole-mount immunostaining, and performed according to Chen et al. (2011). Primary antibodies used included a proliferating cell nuclear antigen (PCNA) (DAKO, 1:1000) and Zn-12 (DSHB, 1:100). AlexaFluor 488-conjugated goat

Purified STX powder (95% in purity) was obtained from Prof. H. N. Chou (Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan), and dissolved in Milli-Q water as the stock solution. An STX dihydrochloride

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anti-mouse IgG (Molecular Probes) was the secondary antibody. Photographs of the immunostained embryos were taken with a Leica camera (DFC310 FX) connected to a Leica fluorescence stereomicroscope (Leica M208 FA).

DFC310 FX). The induced green fluorescence intensity in the eleutheroembryo livers was measured using the MetaMorph imaging system (Molecular Device Corporation) and analyzed using one-way analysis of variance (ANOVA).

2.5. 2-DE proteomic analysis and protein identification

3. Results

At specific time points (4, 8, 12 and 16 dpf), the embryos and eleutheroembryos were collected and examined using a proteomic approach. The frozen embryos and eleutheroembryos were homogenized in 1 mL of 20% trichloroethane/acetone (w/v) with 20 mM dithiothreitol using a sonifier (Branson, US). The protein was extracted using a trichloroacetic acid/acetone precipitation method, and the final protein concentration was quantified using a 2-D Quant kit (GE Healthcare, US) with a SmartSpec 3000 spectrophotometer (Bio-Rad, US). The protein (450 mg) was loaded for the 2-DE study, using the method described in our previous study (Tian et al., 2011). Triplicates for both the exposure treatment (840 mg/L) and control were performed. The gels were visualized with colloidal Coomassie staining following an improved blue silver method as described by Candiano et al. (2004) and scanned using an ImageScanner (Amersham Biosciences). The gels were analyzed with ImageMaster 2D Platinum software (version 5.0, Amersham Biosciences). Spot intensities were normalized with total valid spot abundance in order to minimize non-expression related variations in spot intensity and hence accurately provide semi-quantitative information across different gels. Differences of 1.5 in expression (ratio %V) between matched spots were considered significant. An independent-samples t-test (p < 0.05) between the matched spots was carried out using SPSS 16.0 statistical software (Chicago, IL, USA). Protein spots were then manually excised from the 2-DE gels, and analyzed using a 4800 Plus MALDI TOF/TOF Analyzer (Applied Biosystems, Foster City, CA). For interpretation of the mass spectra, a combination of peptide mass fingerprints and peptide fragmentation patterns were used for protein identification from the NCBI database within the taxonomy of Actinopterygii (ray-finned fishes) using the Mascot search engine. The identified proteins were then matched to specific processes or functions by searching the European Bioinformatics Institute website (http://www.ebi.ac.uk/).

3.1. Exposure to STX induces abnormal development in medaka embryos

2.6. Endocrine-disrupting toxicity testing An estrogen responsive transgenetic choriogenin Hgreen fluorescent protein (ChgH-GFP) medaka fish line was established, as described in US patent application No. 12/ 730,956, (Cheng and Chen, 2010). Newly hatched transgenic fish larvae (eleutheroembryos) were exposed to estrogen E2 (3 ng/L), E2 (3 ng/L) þ STX (840 mg/L), or to the control of artificial seawater (with a salinity of 25 mg/L). Exposure was carried out using a 96-well plate. Each well contained three eleutheroembryos in 250 mL of the exposure solution. Each treatment was replicated seven times. After 24 h of exposure, the eleutheroembryos were imaged under a fluorescence microscope (Leica MZ10F) equipped with a GFP filter and a charge coupled device camera (Leica

The overall sub-chronic dose selected in our current study was based on previous studies, in which STX is reported to induce evident abnormalities, such as severe edema and neurobehavioral impairment, in zebrafish embryos and herring larvae (Lefebvre et al., 2004, 2005). In the present study, newly hatched medaka larvae (15 dpf) were examined after four days of exposure to STX, starting at the blastula stage. During the period tested, no significant mortality and abnormality were observed. However, abnormal development was observed following exposure to 840 and 1260 mg/L of STX, as measured in the body length, yolk sac size, dorsal fins and melanophores. Body length evidently increased in the embryos treated with 840 mg/L of STX, while it was slightly decreased with 1260 mg/L (p < 0.05) of STX (Fig. 1B). The dorsal fins grew larger and the melanophores grew darker as the concentrations increased compared to the control (Fig. 1A). Meanwhile, smaller yolk sac sizes were found in the STX treated embryos (Fig. 1A). Collectively, the altered features suggested accelerated growth in the treated embryos caused by the STX. 3.2. Neuron development and cellular proliferation in STXexposed embryos In order to investigate the accelerated development at the cellular and molecular levels, we examined medaka embryos (4 dpf) immediately after the STX exposure using whole-mount in situ hybridization and immunostaining methods. Zn-12 is the primary antibody that targets primary neurons and PCNA mainly presents the spatial differentiation and development in the embryonic body (Chen et al., 2011). Compared with the control, the dorsal views showed stronger primary neuron signals in the eyes, trigeminal ganglion and cranial neurons in the treated embryos, which indicated that increased neurogenesis started immediately after exposure to STX. As the embryo grew, proliferating cells were found in nearly all regions of the embryonic body, whereas the lateral view of the treated embryos revealed higher proliferation activity in the tail part (Fig. 2), which delineated the accelerated growth in the body of the treated embryos. 3.3. Characterization of proteins of medaka embryos after exposure to STX The 2-DE technique was combined with colloidal Coomassie blue staining to present the protein profiles of the medaka embryos at four different embryonic stages (4, 8, 12 and 16 dpf), in order to determine the proteins affected by STX during different developmental stages, and to elucidate the toxic effect of STX at the molecular level. The total

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Fig. 1. Lateral views of control and STX-exposed (840 mg/L, 1260 mg/L) embryos at 15 dpf (A). Exposure to dissolved STX induced the earlier formation of melanophores, accelerated dorsal fin growth, and smaller yolk sac volumes in the treated embryos. The black arrowheads indicate melanophores in both the control and STX treated embryos. The black asterisks indicate dorsal fins in the control and exposed embryos. The white asterisks indicate yolk sac in the control and exposed embryos. Scale bars, 500 mm. Body lengths (mm) of medaka embryos from the control group and different concentrations of STX treated groups at 15 dpf (B). Increased body lengths observed in STX treated embryos. All data are presented as mean  SEM (vertical bars) from 3 to 6 experiments. Significant differences between mean values have been determined using two-way ANOVA and Tukey’s post hoc test. The level of statistical significance in all cases was p < 0.05. The percentage data was log transformed before analysis. ANOVA was performed using SigmaStat software (SPSS Inc., Chicago, IL).

number of significantly altered protein spots was 9, 12, 13 and 15, respectively, in the gels of the four stages (1.5 fold; p < 0.05). Fig. 3 shows representative 2-DE gels of the medaka embryos at 4, 8, 12 and 16 dpf, and the successfully identified protein spots using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI TOF/TOF MS) which were labeled on each gel. A summary of the proteins with accession numbers, fold changes and Mascot scores is in Table 1 in the Supplementary data section. Among the altered proteins, two kinds of endoplasmic reticulum (ER) stress proteins,

calreticulins (CRTs) and protein disulfide-isomerases (PDIs), were up-regulated during the entire recovery phase after 4 days of exposure to STX (Fig. 4A). In addition, one predominant precursor of the yolk sac protein, vitellogenin 1 (Vtg1) was decreased, while Vtg (gij60101770), which shares a 90% identity in gene sequence in the EMBL CDS database, was speculated to be a fraction of Vtg 1, and immediately decreased after STX exposure, then increased during the recovery phase (Fig. 4B). Notably, at different stages, characteristic protein profiles were presented. In the 8 dpf gels, the proteins

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Fig. 2. Control and STX treated embryos (4 dpf) illustrated using PCNA and zn-12. PCNA stains spatial differentiation and development. Zn-12 stains primary neurons. Treated embryos stained with PCNA exhibited higher proliferation ability in the tail part in comparison to the control, and treated embryos stained with zn-12 exhibited higher neurogenesis in the eyes, trigeminal ganglion and cranial neurons in comparison to the control. Abbreviations: tl, tail; ey, eye; tg, trigeminal ganglion; cn, cranial neuron.

related to metabolism were mainly up-regulated, such as glyceraldehydes-3-phosphate dehydrogenase (Spot 6) and enolase (Spot 11) participating in carbohydrate metabolism, and lipocalin (Spot 7) and a fatty acidbinding protein (Spot 8) involved in the lipid metabolic process (Fig. 5A). In the 12 dpf gels, proteins related to protein phosphorylation were mainly up-regulated, such as serine/threonine-protein phosphatase (Spot 5), splicing factor arginine/serine rich 2 (Spot 6) and 14-3-3 protein beta/alpha (Spot 8) (Fig. 5B). In the 16 dpf gels, three heat shock proteins (HSPs) (Spots 13, 14 and 15) that belong to the heat shock 70 and 90 families were up-regulated in treated eleutheroembryos (Fig. 5C). The other increased proteins found were mainly involved in guanosine triphosphate (GTP) forming and GTPbinding, myosin and actin binding, proliferating and DNA binding.

3.4. Endocrine disruption of STX Estrogens are a primary factor for endocrine disruption. Transgenic marine medaka (ChgH-GFP medaka), which harbor the GFP gene and are regulated by the ChgH gene promoter, can be used to easily detect the estrogenic endocrine disruption activity of substances (Cheng and Chen, 2010). In order to investigate the estrogen-like disruption ability of STX, we adopted estrogen responsive transgenic ChgH-GFP marine medaka eleutheroembryos. Dose-dependent GFP signals would be induced in the liver of eleutheroembryos by estrogen. Fig. 6 shows the GFP intensities of the eleutheroembryos exposed to E2 (3 ng/L), E2 (3 ng/L) þ STX (840 mg/L), or the artificial seawater control. In the present study, no GFP was observed in the control eleutheroembryos without stimulation by estrogen, while clear GFP signals were found in the

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Fig. 3. Representative 2-DE gels from different stages (A: 4 dpf, B: 8 dpf, C: 12 dpf, D: 16 dpf) of medaka embryos after four days of exposure to STX (840 mg/L). Protein spots with fold difference 1.5 times between control and treated embryos were defined as significant changes and an independent t-test (p < 0.05) was carried out. Successfully identified protein spots through the use of MALDI TOF/TOF MS are labeled on each gel, which correspond to the spot numbers in Table 1 of the Supplementary data section. The molecular weights (MWs) and pI scales are indicated.

eleutheroembryos exposed to E2. A statistically significant reduction in fluorescence intensity was found in the eleutheroembryo livers exposed to estrogen together with STX (p < 0.05), which indicated that significant anti-estrogenic activity was shown by the STX. 4. Discussion STX is a known neurotoxin that has caused intoxication to humans and marine animals, however, its developmental toxicity remains unknown. The purpose of this study was to investigate the developmental toxicity of STX using medaka embryos as models. Medaka embryos were exposed to STX in the early blastula stage (0 dpf) for four consecutive days, which covered the main stage in the formation of the central nervous system and the development of organs and somites. Afterward, we transferred the

treated embryos to clean seawater until hatching, and observed the effects of STX on the embryos and eleutheroembryos. In the morphological observations, the newly hatched larvae (15 dpf) of the STX-exposed medaka fish exhibited abnormal growth with relatively longer body length, and the immunostaining results indicated that accelerated growth with high proliferation ability and neuron development could be traced back to 4 dpf, the period immediately after 4 days of consecutive exposure to STX. Moreover, according to the proteomic results, there were also increases in the proteins that participate in neural recovery and proliferation, such as nucleoside diphosphate kinase (NDK) and tubulin family proteins. The NDK catalyzes the synthesis of non-adenine-containing nucleoside triphosphates, for example, GTP from GDP, and GTP is essential for microtubule polymerization

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Fig. 4. Fold changes of ER proteins in STX treated (840 mg/L) embryos at 8, 12 and 16 dpf stages (A). CRT, like 2: calreticulin, like 2 (gij41054373); CRT: calreticulin (gij185134556); PDI: protein disulfide-isomerases (gij193788703); PDI-P5: protein disulfide-isomerases-related protein P5 (gij85719991). Fold changes of yolk sac precursor proteins in STX treated (840 mg/L) embryos at 4, 8, 12 and 16 dpf (B). VTG: vitellogenin (gij 60101770); VTG 1: vitellogenin 1 (gij157278094). Each bar represents the mean  SEM of fold changes.

(Biggs et al., 1990). The major components of microtubules, that is, tubulin proteins, which are found in the nerves of fish, also increased in the treated embryos (8 dpf). An increase in tubulin proteins means axon regeneration after nervous system injuries (Zupanc et al., 2006). Meanwhile, NDK plays a vital regulatory role in neural cell proliferation and differentiation in the embryogenesis process of fish as well (Murphy et al., 2000). The enhancement of NDK and tubulin proteins was in accordance with the high neuron development found in our immunostaining results. Apart from the increase in body length, we also found that the yolk sac in the treated embryos was smaller than that of the control. The yolk sac provides a variety of nutrients, such as amino acids, lipids, metal ions, phosphates and carbohydrates, for regular embryonic development (Gündel et al., 2007). The reduction in the yolk sac size might partly have been due to the need to satisfy a high consumption of nutrients and energy during abnormal growth in the treated embryonic body. Our proteomic results further showed up-regulation in glycolytic and lipid metabolic proteins (Fig. 5A) in the treated embryos, which helped provide sufficient energy to sustain growth. In addition, according to previous

studies, whole-body concentrations of the yolk sac protein are elevated or suppressed by the existence of estrogens or anti-estrogens in larval fish (Panter et al., 2002). In our study, a smaller yolk sac was observed in the STX treated embryos, and thus it was speculated that STX possesses an anti-estrogenic ability, and the reduction in the yolk sac size might also be caused by the suppression of yolk sac proteins by STX. In order to elucidate this question, estrogen responsive transgenic medaka fish was adopted, and the anti-estrogenic ability of STX was verified. This caused the reduction in Vtgs and further induced to a certain degree a smaller yolk sac in medaka embryos. As the yolk sac protein precursor, the content of Vtg 1 is usually adopted as a molecular biomarker for (anti-)estrogenic substances in fish embryos (Muncke and Eggen, 2006). According to our proteomic results, the content of Vtg 1 showed a decrease compared with that of the control during the whole embryogenesis process (Fig. 4B). Thus, a smaller yolk sac size might be affected by both the accelerated growth and the anti-estrogenic function of STX. In addition, anti-estrogenic substances are found which affect uterine peroxidase activity and uterine tissue weight, and even cause uterine atrophy (Astroff and Safe,

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Fig. 5. Spot volumes of metabolic proteins of both the control and STX treated (840 mg/L) embryos (8 dpf) (A). GAPDH: Glyceraldehyde-3phosphate dehydrogenase (gij124740); lipocalin (gij 225716896); H-FABP: heart-type fatty acid binding protein (H-FABP) (gij15072477); enolase (gij226441951). Spot volumes of phosphorylation related proteins of both the control and STX treated (840 mg/L) embryos (12 dpf) (B). PP2A: Serine/ threonine-protein phosphatase (gij146285377); SF-2: Splicing factor arginine/serine rich 2 (gij9837439); 14-3-3 beta/alpha: 14-3-3 protein beta/ alpha (gij225707890). Spot volumes of the HSPs of both the control and STX treated (840 mg/L) embryos (16 dpf) (C). HSP 90: heat shock 90 kDa protein 1 beta isoform b (gij185136252); HSP 70: Chromosome 4 SCAF15093, whole genome shotgun sequence (gij47218700); HSC70-1: Stress protein HSC70-1 (gij212274295). Each bar represents the mean  SEM of spot volumes.

1990; Dukes et al., 1994). Thus, the consumption of STX contaminated seafood, especially shellfish, should be noted and further studied in pregnant women in order to lower the risk of STX intake, and so prevent negative effects on the uterus and fetus. In addition, we examined the proteomic profiles at different developmental stages of the embryos in order to elucidate other toxic effects of STX from a molecular level, and characteristic patterns of altered proteins were found in each stage. First of all, two kinds of ER stress proteins, CRTs and PDIs were up-regulated during the entire recovery phase of the medaka embryos (Fig. 4A). CRTs are the major calcium-binding protein of the ER found in fish,

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and play important roles in calcium homeostasis and immune functions in embryonic development (Kales et al., 2007). Together with CRTs, PDIs are another type of major stress proteins in the ER, isomerizing the disulfide bonds of intramolecules and intermolecules of proteins (Tanaka et al., 2000). The up-regulation of CRTs and PDIs reveals ER stress, and they perform the functions of protection and wound healing by repairing the misfolding proteins (Michalak et al., 2009; Silvestre et al., 2006). For example, increases in CRTs and PDIs are found in rat brains after they suffered ischemia as protection against stress (Tanaka et al., 2000). In addition, ER damage caused by the injection of PSP (6.4 mg STXeq/kg) is reported in zebrafish (Zhang et al., 2011). In our study, HSPs, which play a similar role in wound healing as that of CRTs and PDIs by repairing misfolded and damaged proteins, were increased together with CRTs and PDIs in the 16 dpf eleutheroembryos (Fig. 5C). An increase in these stress proteins revealed that there was stress and damage imposed on the developing embryos by STX. Moreover, the patterns of the induced stress proteins were different under diverse conditions, and so certain patterns of the stress proteins could be used as potential biomarkers in identifying different toxins. Moreover, in the 12 dpf treated embryos, the proteins modulated by the phosphorylation and dephosphorylation processes were mainly increased (Fig. 5B). The 14-3-3 proteins regulate a very large number of protein kinases, phosphatases and other phosphoproteins, and are essential for the regulation of cell cycles and adaption abilities in organisms via the phosphorylation process (Kültz et al., 2001). Similar to 14-3-3 proteins, the phosphorylation of arginine/serine rich splicing factor 2 is also key to its splicing activity during stress (Metz et al., 2004; Yeakley et al., 1999). Together with these proteins, we also found an increase in Histone H2B and, according to Bungard et al. (2010), the phosphorylation of Histone H2B is related to cellular adaptation under stress. Unlike the proteins mentioned above, serine/threonine-protein phosphatase 2A (PP2A) is inactivated during the phosphorylation process; however, PP2A activity is also required for stress response and plays a different role in the pathway as opposed to 14-3-3 proteins (Santhanam et al., 2004). In our 12 dpf treated embryos, three proteins involved in signaling, cell growth and the phosphorylation process were mainly increased, which was also related to stress response. In conclusion, we examined morphological changes, neurogenesis, cellular proliferation and proteomic profiles of medaka embryos, and found that the treated embryos exhibited abnormal growth with high neurogenesis and cellular proliferation immediately after exposure to STX. The use of the proteomic method also revealed enhanced metabolism and stress response after STX exposure. Collectively, these indicated the developmental toxicity exhibited by STX on the fish embryos, and this is the first known study that focused on the developmental toxicity of STX apart from its neurotoxicity. The disruption ability of STX to normal growth rates in the model fish revealed a potential health hazard to fetuses and children.

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Fig. 6. Representative eleutheroembryo liver photos (A–C) and GFP signal intensity (D) of ChgH-GFP transgenic medaka fish exposed to E2 (3 ng/L), E2 (3 ng/ L) þ STX (840 mg/L) and the control. The scale bar is equal to 100 mm. GFP signal intensity in fish livers was significantly reduced when E2 was incubated together with STX (p < 0.05). The data were analyzed using one-way ANOVA, and are expressed as mean  SEM.

Acknowledgments This study was supported by the State Key Laboratory in Marine Pollution of City University of Hong Kong and grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (CityU3/CRF/08), and the program for New Century Excellent Talents in University (NCET-12-0181), with support from State Key Lab in Estuarine and Coastal Research (2012RCDW01). We thank Professor John Hodgkiss of The University of Hong Kong for polishing the English in this paper. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.toxicon.2013.10.022. Conflict of interest statement We confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. References Astroff, B., Safe, S., 1990. 2,3,7,8-Tetrachlorodibenzo-p-dioxin as an antiestrogen: effect on rat uterine peroxidase activity. Biochem. Pharmacol. 39, 485–488. Biggs, J., Hersperger, E., Steeg, P.S., Liotta, L.A., Shearn, A., 1990. A Drosophila gene that is homologous to a mammalian gene associated with tumor metastasis codes for a nucleoside diphosphate kinase. Cell 63, 933–940.

Bungard, D., Fuerth, B.J., Zeng, P.Y., Faubert, B., Maas, N.L., Viollet, B., Carling, D., Thompson, C.B., Jones, R.G., Berger, S.L., 2010. Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation. Science 329, 1201–1205. Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G.M., Carnemolla, B., Orecchia, P., Zardi, L., Righetti, P.G., 2004. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteomic analysis. Electrophoresis 25, 1327–1333. Castonguay, M., Levasseur, M., Beaulieu, J.L., Grégoire, F., Michaud, S., Bonneau, E., Bates, S.S., 1997. Accumulation of PSP toxins in Atlantic mackerel: seasonal and ontogenetic variations. J. Fish Biol. 50, 1203– 1213. Chen, X., Li, L., Cheng, J., Chan, L.L., Wang, D.Z., Wang, K.J., Baker, M.E., Hardiman, G., Schlenk, D., Cheng, S.H., 2011. Molecular staging of marine medaka: a model organism for marine ecotoxicity study. Mar. Pollut. Bull. 63, 309–317. Cheng, J., Flahaut, E., Cheng, S.H., 2007. Effect of carbon nanotubes on developing zebrafish (Danio Rerio) embryos. Environ. Toxicol. Chem. 26, 708–716. Cheng, S.H., Chen, X., 2010. Transgenic fish and uses thereof. US Patent application No. 12/730,956. Filed on March 24, 2010. IPC/PA/ 214/10/09. Colman, J.R., Twiner, M.J., Hess, P., McMahon, T., Satake, M., Yasumoto, T., Doucette, G.J., Ramsdell, J.S., 2005. Teratogenic effects of azaspiracid-1 identified by microinjection of Japanese medaka (Oryzias latipes) embryos. Toxicon 45, 881–890. Dukes, M., Chester, R., Yarwood, L., Wakeling, A.E., 1994. Effects of a nonsteroidal pure antioestrogen, ZM 189,154, on oestrogen target organs of the rat including bones. J. Endocrinol. 141, 335–341. Escoffier, N., Gaudin, J., Mezhoud, K., Huet, H., Chateau-Joubert, S., Turquet, J., Crespeau, F., Edery, M., 2007. Toxicity to medaka fish embryo development of okadaic acid and crude extracts of Prorocentrum dinoflagellates. Toxicon 49, 1182–1192. Falconer, I., Humpage, A., 2005. Health risk assessment of cyanobacterial (blue–green algal) toxins in drinking water. J. Environ. Res. Public Health 2, 43–50. Gündel, U., Benndorf, D., von Bergen, M., Altenburger, R., Küster, E., 2007. Vitellogenin cleavage products as indicators for toxic stress in zebrafish embryos: a proteomic approach. Proteomics 7, 4541– 4554.

L. Tian et al. / Toxicon 77 (2014) 16–25 Kales, S.C., Bols, N.C., Dixon, B., 2007. Calreticulin in rainbow trout: a limited response to endoplasmic reticulum (ER) stress. Comp. Biochem. Physiol. Part B: Biochem. Mol. Biol. 147, 607–615. Kültz, D., Chakravarty, D., Adilakshmi, T., 2001. A novel 14-3-3 gene is osmoregulated in gill epithelium of the euryhaline teleost Fundulus heteroclitus. J. Exp. Biol. 204, 2975–2985. Kumar-Roiné, S., Matsui, M., Pauillac, S., Laurent, D., 2011. Ciguatera fish poisoning and other seafood intoxication syndromes: a revisit and a review of the existing treatments employed in ciguatera fish poisoning. South Pac. J. Nat. Appl. Sci. 28, 1–26. Landrigan, P.J., Claudio, L., Markowitz, S.B., Berkowitz, G.S., Brenner, B.L., Romero, H., Wetmur, J.G., Matte, T.D., Gore, A.C., Godbold, J.H., Wolff, M.S., 1999. Pesticides and inner-city children: exposures, risks, and prevention. Environ. Health Perspect 107, 431–437. Lefebvre, K.A., Trainer, V.L., Scholz, N.L., 2004. Morphological abnormalities and sensorimotor deficits in larval fish exposed to dissolved saxitoxin. Aquat. Toxicol. 66, 159–170. Lefebvre, K.A., Elder, N.E., Hershberger, P.K., Trainer, V.L., Stehr, C.M., Scholz, N.L., 2005. Dissolved saxitoxin causes transient inhibition of sensorimotorfunction in larval Pacific herring (Clupea harengus pallasi). Mar. Biol. 147, 1393–1402. Metz, A., Soret, J., Vourc’h, C., Tazi, J., Jolly, C., 2004. A key role for stressinduced satellite III transcripts in the relocalization of splicing factors into nuclear stress granules. J. Cell Sci. 117, 4551–4558. Michalak, M., Groenendyk, J., Szabo, E., Gold, L.I., Opas, M., 2009. Calreticulin, a multi-process calcium-buffering chaperone of the endoplasmic reticulum. Biochem. J. 417, 651–666. Muncke, J., Eggen, R.I.L., 2006. Vitellogenin 1 mRNA as an early molecular biomarker for endocrine disruption in developing zebrafish (Danio rerio). Environ. Toxicol. Chem. 25, 2734–2741. Murphy, M., Harte, T., McInerney, J., Smith, T.J., 2000. Molecular cloning of an Atlantic salmon nucleoside diphosphate kinase cDNA and its pattern of expression during embryogenesis. Gene 257, 139–148. Panter, G.H., Hutchinson, T.H., Länge, R., Lye, C.M., Sumpter, J.P., Zerulla, M., Tyler, C.R., 2002. Utility of a juvenile fathead minnow screening assay for detecting (anti-)estrogenic substances. Environ. Toxicol. Chem. 21, 319–326. Rapala, J., Robertson, A., Negri, A.P., Berg, K.A., Tuomi, P., Lyra, C., Erkomaa, K., Lahti, K., Hoppu, K., Lepistö, L., 2005. First report of saxitoxin in Finnish lakes and possible associated effects on human health. Environ. Toxicol. 20, 331–340.

25

Reyero, M., Cacho, E., Martínez, A., Vázquez, J., Marina, A., Fraga, S., Franco, J.M., 1999. Evidence of saxitoxin derivatives as causative agents in the 1997 mass mortality of monk seals in the Cape Blanc Peninsula. Nat. Toxins 7, 311–315. Santhanam, A., Hartley, A., Düvel, K., Broach, J.R., Garrett, S., 2004. PP2A phosphatase activity is required for stress and Tor kinase regulation of yeast stress response factor Msn2p. Eukaryotic Cell 3, 1261–1271. Shi, M., Faustman, E.M., 1989. Development and characterization of a morphological scoring system for medaka (Oryzias latipes) embryo culture. Aquat. Toxicol. 15, 127–140. Silvestre, F., Dierick, J.F., Dumont, V., Dieu, M., Raes, M., Devos, P., 2006. Differential protein expression profiles in anterior gills of Eriocheir sinensis during acclimation to cadmium. Aquat. Toxicol. 76, 46–58. Tanaka, S., Uehara, T., Nomura, Y., 2000. Up-regulation of protein-disulfide isomerase in response to hypoxia/brain ischemia and its protective effect against apoptotic cell death. J. Biol. Chem. 275, 10388–10393. Tian, L., Wang, M., Li, X., Lam, P.K.S., Wang, M., Wang, D., Chou, H.N., Li, Y., Chan, L.L., 2011. Proteomic modification in gills and brains of medaka fish (Oryzias melastigma) after exposure to a sodium channel activator neurotoxin, brevetoxin-1. Aquat. Toxicol. 104, 211–217. Wang, M., Chan, L.L., Si, M., Hong, H., Wang, D., 2010. Proteomic analysis of hepatic tissue of zebrafish (Danio rerio) experimentally exposed to chronic microcystin-LR. Toxicol. Sci. 113, 60–69. White, A.W., 1981. Sensitivity of marine fishes to toxins from the red-tide dinoflagellate Gonyaulax excavata and implications for fish kills. Mar. Biol. 65, 255–260. Wong, C.K., Hung, P., Ng, H.C.C., Lee, S.Y., Kam, K.M., 2011. Cluster analysis of toxins profile pattern as a tool for tracing shellfish contaminated with PSP-toxins. Environ. Res. 111, 1083–1090. Yeakley, J.M., Tronchère, H., Olesen, J., Dyck, J.A., Wang, H.Y., Fu, X.D., 1999. Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors. J. Cell Biol. 145, 447–455. Zhang, D., Hu, C., Wang, G., Li, D., Li, G., Liu, Y., 2011. Zebrafish neurotoxicity from aphantoxinsdcyanobacterial paralytic shellfish poisons (PSPs) from Aphanizomenon flos-aquae DC-1. Environ. Toxicol.. http:// dx.doi.org/10.1002/tox.20714 (Advance Access published June 24, 2011). Zupanc, M.M., Wellbrock, U.M., Zupanc, G.K.H., 2006. Proteome analysis identifies novel protein candidates involved in regeneration of the cerebellum of teleost fish. Proteomics 6, 677–696.