Mechanisms of hexabromocyclododecanes induced developmental toxicity in marine medaka (Oryzias melastigma) embryos

Aquatic Toxicology 152 (2014) 173–185

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Mechanisms of hexabromocyclododecanes induced developmental toxicity in marine medaka (Oryzias melastigma) embryos Haizheng Hong, Dongmei Li, Rong Shen, Xinhong Wang, Dalin Shi ∗ State Key Laboratory of Marine Environmental Science and Key Laboratory of the Ministry of Education for Coastal and Wetland Ecosystems, Xiamen University, Xiamen 361102, China

a r t i c l e

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Article history: Received 17 January 2014 Received in revised form 8 April 2014 Accepted 9 April 2014 Available online 18 April 2014 Keywords: Hexabromocyclododecanes Developmental toxicity Oryzias melastigma Oxidative stress

a b s t r a c t Hexabromocyclododecanes (HBCDs) are widely used as additive brominated flame retardants, and are now ubiquitous contaminants in the environmental media and biota, including the marine environment and marine organisms. However, the impacts of HBCDs on marine fish are not well known. In this study the embryos of marine medaka (Oryzias melastigma) were used to assess the developmental toxicity of HBCDs. Freshly fertilized marine medaka embryos were exposed to various concentrations of technical HBCD (tHBCD, 0, 5, 20 and 50 ␮g/L) until the first fry stage, and hatch success, morphology and cardiac function were examined. In all the exposure groups (5, 20 and 50 ␮g/L) tHBCD significantly increased the embryo heart beats. The measurement of sinus venosus–bulbus arteriosus (SV–BA) distance indicated that tHBCD significantly enlarged the SV–BA distance at exposure concentrations of 20 and 50 ␮g/L. The malformation rate at the first fry stage was also induced by tHBCD in a dose dependent manner, with the formation of pericardial edema and yolk sac edema as the most frequently observed malformation. In addition, the concentrations of total HBCD isomers (HBCDs) in embryos in the current study were comparative with environmental levels and increased with increasing exposure duration. Furthermore, exposure to tHBCD also induced the level of 8-oxodG, a representative oxidative DNA damage. The mechanisms of HBCD-induced developmental toxicity were further explored by TUNEL assay, gel-based quantitative proteomic approach and measurement of the expression of several stress responsive genes, such as p53, TNF-␣, IL-1␤, CYP1A, COX-1 and COX-2, together with the activities of caspases. The results suggested that HBCDs exposure at environmentally realistic concentrations induced oxidative stress and apoptosis, and suppressed nucleotide and protein synthesis, which all together resulted in developmental toxicity, particularly in the cardiovascular system, in the embryos of O. melastigma. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Hexabromocyclododecanes (HBCDs) are additive brominated flame retardants, primarily used in polystyrene insulation foam boards, textile products and electronic products. The commercially available HBCD (technical HBCD, tHBCD) is often characterized as a mix of mainly three diastereoisomers, ␣-, ␤- and ␥-HBCD, and the distribution of these diastereoisomers in tHBCD varies in a range of 1–12% ␣-HBCD, 10–13% ␤-HBCD and 75–89% ␥-HBCD (Koeppen et al., 2007). As the third most widely used brominated flame retardants in the world, after tetrabromobisphenol A and polybrominated diphenyl ethers, HBCDs are now ubiquitous

∗ Corresponding author at: Room A418, College of the Environment and Ecology, Xiamen University, South Xiangan Rd, Xiangan District, Xiamen, Fujian 361102, China Tel.: +86 592 2182322; fax: +86 592 2184101. E-mail address: [email protected] (D. Shi). http://dx.doi.org/10.1016/j.aquatox.2014.04.010 0166-445X/© 2014 Elsevier B.V. All rights reserved.

environmental contaminants, and are found in sediment, soil, air, animal tissue and even in human blood and milk (Yu et al., 2008; Sellstrom et al., 1998). HBCDs have been classified as persistent organic pollutants (POPs) and recently have been included in the Stockholm Convention for consideration of global elimination (www.pops.int). HBCDs have been found in the aquatic environment, and their levels in river sediments are in the ␮g/kg (dry weight) range, with exceptionally high levels (1680 ␮g/kg dry weight) recorded in the river Skerne in England (Morris et al., 2004). The concentrations of HBCDs in marine sediments are relatively lower, in the range of sub␮g/kg to ␮g/kg (dry weight), e.g. 3.4–6.9 ␮g/kg dry weight of HBCDs were detected in Scheldt/Rhine/Meuse estuaries of the North Sea continental shelf (Klamer et al., 2005). Furthermore, HBCDs are readily bioavailable and can be bioaccumulated in the aquatic organisms through the food web (Tomy et al., 2004). Relatively high concentrations of HBCDs were detected in several freshwater and marine fish species downstream of the source of the chemical and in

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Table 1 HBCDs concentrations in freshwater and marine fish collected from polluted areas as reported in the literature. Species

Freshwater fish Barbel Eels and trout Pike Crucian carp, mud carp and northern snakehead Crucian carp, and goby Goby Silver carp, big head carp, grass carp, common carp, white amur bream, mandarin fish, brass gugdgeon and snake head Common carp, crucian carp, white amur bream and mandarin fish Marine fish Mackerel, cod, thorny skate, pollock and flounder Sole, Plaice, bib and whiting a b

Sample location

Tissue

HBCDs concentrations

Reference

ng/g ww

ng/g lw

Muscle Liver Muscle Muscle Muscle

89–750 432–554 21–10,275 21.2–39.2a –

– –

Muscle Eggs Muscle

14–18a 16a 0.3–7.7a

174–194 203 11–330

Egg

0.6–15a

11–240

Etnefjorden, Norway

Muscle

2–300b

219–30,316

Koppen et al. (2010)

Western Scheldt, Spain

Muscle

0.4–11a

40–1113

Janak et al. (2005)

Cinca River, Spain Rivers Skerne and Tees, UK Rivers Viskan and Häggån, Sweden Qingyuan, China Laizhou bay, China Yangtze River

4000–8000 11–2370

Eljarrat et al. (2004) Allchin and Morris (2003) Sellstrom et al. (1998) Wu et al. (2010) Li et al. (2012) Xian et al. (2008)

The unit of the concentrations were converted from ng/g lw to ng/g ww based on the reported lipid content (%lipids) in the same literature. The unit of the concentrations were converted from ng/g lw to ng/g ww assuming lipid content was 1%.

highly industrialized areas in Europe and in China (Xian et al., 2008; Allchin and Morris, 2003; Koppen et al., 2010) (Table 1). For example, 10,275 ng/g wet weight of HBCDs were found in the eels caught from the polluted river Skerne in England, and 300 ng/g wet weight of HBCDs were detected in the marine cod fish sampled from Etnefjorden, Norway. Laboratory study demonstrated that HBCDs were maternally transferred to the zebrafish eggs after parental exposure (Nyholm et al., 2008). Meanwhile, measurement of the field samples also found that the concentrations of HBCDs in fish eggs were either higher than or similar to those found in fish muscles (Xian et al., 2008; Li et al., 2012) (Table 1). It is noted that the fish embryonic development stage shows great sensitivity to environmental contaminants (McKin, 2011), and therefore it is important to assess the impacts of HBCDs on fish embryo development. Previous research using freshwater zebrafish embryos as a model demonstrated that HBCDs significantly increased the malformation rate and reduced survival at relatively high exposure concentrations (up to 1 mg/L) (Deng et al., 2009; Du et al., 2012). Among the HBCD diastereoisomers, the order of developmental toxicity in zebrafish is ␥-HBCD > ␤-HBCD > ␣-HBCD (Du et al., 2012). In addition, HBCDs induce the generation of reactive oxygen species (ROS) and caspase-3 and -9 mediated apoptosis (Deng et al., 2009; Du et al., 2012). HBCDs also induce hepatic enzymes (EROD) and oxidative stress in another freshwater species, the Chinese rare minnow (Zhang et al., 2008). However, the long term exposure of an euryhaline species, the European flounder (Platichthys flesus), to HBCDs in seawater (salinity 32‰) does not affect the survival and growth, nor are the hepatic microsomal enzymes affected (Kuiper et al., 2007). The sensitivity of freshwater and seawater species to environmental pollutants is affected by various biological and physicochemical factors, and is also strongly influenced by the chemical’s mode of toxic action (Wheeler et al., 2002; Leung et al., 2001). Therefore, in order to understand the toxicity of HBCDs to marine fish species more thoroughly, it is important to choose a marine fish model for toxicity investigation. For this purpose, marine medaka (Oryzias melastigma), which is adopted by ILSI Health and Environmental Science Institute for embryo toxicity testing, is becoming a very useful model for estuarine and marine ecotoxicological studies (Chen et al., 2011). In this study, embryos of O. melastigma were used to assess the potential developmental toxicity of HBCDs. The concentrations of total HBCDs accumulated in the embryos were measured and the

developmental toxicity endpoints were examined. In addition, the gene expression patterns related to oxidative stress and apoptosis, as well as protein expression profiles upon HBCD exposure, were also examined to shed light on the potential mechanism of action induced by HBCDs. 2. Materials and methods 2.1. Chemicals and reagents Technical HBCD (tHBCD) was purchased from Tokyo Chemical Industry Co. (Tokyo, Japan); ␣-HBCD, ␤-HBCD and ␥-HBCD were ordered from AccuStandard, Inc. (New Haven, CT, USA); 13 C-␣-HBCD, 13 C-␤-HBCD and 13 C-␥-HBCD were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA); and the organic solvents (acetonitrile, hexane and methanol) used for sample extraction and LC–MS/MS analysis were obtained from Tedia Inc. (Fairfield, OH, USA). All other reagents were purchased from Sigma–Aldrich Chemical Co. (St. Louis, Washington, USA) except where indicated. 2.2. Toxicity endpoints for measurement To assess the developmental toxicity, the malformation of the embryos was observed everyday under the microscope and the survival rate, hatching success rate and hatch-out time were recorded. The heart rate and the distance between the sinus venosus (SV) and bulbus arteriosus (BA) region of the heart (SV–BA distance) were measured as described previously (Huang et al., 2011), to evaluate the effect of tHBCD on embryo cardiac development. SV–BA distance was presented as the length of a straight line connecting the centers of the two structures under the same amplification factor (Fig. 2C). To assess whether tHBCD exposure induced the generation of reactive oxygen species (ROS) and further led to oxidative DNA damages in O. melastigma embryos, the levels of 8-oxo-7,8dihydro-2 -deoxyguanosine (8-oxodG), a widely used biomarker of oxidative stress (Ravanat et al., 2002), were measured in the genomic DNA of the embryos. To evaluate whether tHBCD exposure induced the gene expression, which may lead to the generation of ROS, the gene expression levels of TNF-␣, IL-1␤, COX-1, COX-2 and CYP1A were monitored by real-time quantitative PCR (qPCR).

H. Hong et al. / Aquatic Toxicology 152 (2014) 173–185

A

B

5

20

50

200

(μg/L)

310

((nM)

0

7.8

31

78

Malformation observation (n=4)

√

√

√

√

Heart beat (n=20)

√

√

√

√

SV-BA distance (n=20)

√

√

√

√

ΣHBCDs concentration (n = 4)

√

√

√

√

dG ((n=4) 4) 88-oxodG

√

√

√

√

Gel-based proteomics (n=3)

√

√

√

√

Gene expression (n=4)

√

√

√

√

Caspase activities (n=4)

√

√

√

√

TUNEL (n=20)

√

√

√

√

ΣHBCDs conc.

( p)0 (dpf)

0

1

ΣHBCDs conc.

4

6

Heart beat SV-BA Gene expression Caspase activities

For assessment of gene and protein expression levels, measurement of caspase activities and TUNEL assay, the embryos were cultured under the same conditions as described above except that the exposure concentrations were 0, 20, 50 and 200 ␮g/L (corresponding to 0, 31, 78 and 310 nM, respectively). 2.4. Sample extraction and LC–MS/MS quantification of HBCD isomers

Gene expression TUNEL

8-oxodG

5

175

8

1st fryy

ΣHBCDs conc. Heart beat SV-BA Gene expression Caspase activities G lb Gel-base d protteomics i

Scheme 1. Exposure concentrations (A) and sampling schedule (B) for the measurement of individual endpoints.

To evaluate whether tHBCD induced p53, an important oxidative stress responsive gene, and further led to apoptosis via the activation of caspase-dependent pathway, the gene and protein expression level of p53 and the activities of caspase-3, caspase-8 and caspase-9 were measured. Furthermore, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was also conducted to detect DNA fragmentation which results from apoptotic signaling cascades. In addition, gel-based quantitative proteomic approach was applied to reveal differentially expressed proteins upon tHBCD exposure, which would help to further understand the mechanisms of tHBCD-induced developmental toxicity in O. melastigma embryos. 2.3. Medaka maintenance and embryo toxicity test Marine medaka (O. melastigma) were maintained following the previously described conditions (Bo et al., 2012). Briefly, the O. melastigma were maintained at 28 ± 2 ◦ C in a 14-h light:10-h dark photoperiod in aerated artificial seawater at a salinity of 30‰ and a dissolved oxygen concentration of 5.8 ± 0.2 mg/L. The fish were fed with Artemi anauplii twice daily. The freshly fertilized eggs of O. melastigma were collected daily and visually sorted under a dissecting microscope to isolate embryos at developmental stages 12–15 (Iwamatsu, 2004). The sorted embryos were randomly distributed into several groups and the exposure was started immediately after the sorting. The exposure concentrations, toxicity endpoints selected for measurements, sample collecting time points and sample replication numbers were indicated in Scheme 1. For the assessment of developmental toxicity (e.g. malformation, heart beat and SV–BA distance, etc.), quantitative proteomic analysis and measurement of 8-oxodG and HBCD isomers, the embryos were exposed to 0, 5, 20 and 50 ␮g/L (corresponding to 0, 7.8, 31 and 78 nM, respectively) of tHBCD in seawater. The solvent control group received the same concentration of DMSO as the treated groups (0.01%, v/v). Each exposure group was cultured in 60-mm crystallizing dishes containing 20 mL of exposure media. The exposure solution was freshly prepared and renewed daily, and the exposure was terminated on 17 days post fertilization (dpf).

The embryos were first spiked with the 13 C-labeled ␣-HBCD, ␤HBCD and ␥-HBCD isomers, and then homogenized and extracted in 5 mL hexane:acetone (1:1, v/v) by ultrasonication for 30 min. After centrifugation, the supernatant was collected and the pellet was extracted again using 5 mL of hexane:acetone (1:1, v/v) by ultrasonication for 30 min. The resulting supernatant was combined and concentrated to 0.5 mL under N2 flow. The quantification of the HBCD isomers was carried out on an Agilent 1290-6490 UPLC-triple quadruple mass spectrometry system (Agilent Technologies, Palo Alto, CA, USA). The separation of ␣-HBCD, ␤-HBCD and ␥-HBCD isomers was achieved on an Agilent XDB C18 HPLC column (2.1 mm × 150 mm, particle size 3.5 ␮m) at a flow rate of 0.3 mL/min. The mobile phase consisted of two solvents: mobile phase A (30% methanol in water, v/v) and mobile phase B (30% methanol in acetonitrile, v/v). The linear gradient program used for HPLC separation was as follows: linear gradient from 70 to 100% B (0–10 min); hold isocratic at 100% B (10–15 min); and re-equilibrate at 70% B for 10 min. The LC eluate was directed to a mass spectrometer equipped with an electro-spray ionization (ESI) probe operated in negative ion-mode. The ␣-, ␤- and ␥-HBCD isomers and their corresponding 13 C-labeled internal standards were monitored in multiple reaction monitoring mode (MRM) with transition events of m/z 640.8 > 80.8 and m/z 652.8 > 80.8, respectively. 2.5. Quantification of 8-oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG) The genomic DNA was isolated using a DNA Extractor® WB kit (Wako Pure Chemical Industries, Osaka, Japan) following the manufacture’s protocol, except that 0.1 mM of desferrioxamine was included in the solutions used for DNA extraction to minimize the artifactual oxidation of DNA (Ravanat et al., 2002). The extracted DNA was hydrolyzed using the enzymatic digestion method described previously (Hong et al., 2006). Briefly, the DNA was first digested by 2 units for nuclease P1 and 0.025 unit of calf spleen phosphodieserase in 15 mM sodium acetate buffer (pH 5.0) containing 1 mM zinc acetate. The digestion was carried out at 37 ◦ C for 1.5 h. To the digestion mixture were then added 0.003 units of snake venom phosphodiesterase, 20 units of alkaline phosphates and 10 ␮L of 0.5 M Tris–HCl buffer (pH 8.9). The digestion was continued at 37 ◦ C for another 1.5 h, and the solution was neutralized by adding 6 ␮L of 0.5 M HCl. To the mixture was then added isotopically labeled 8-oxodG as the internal standard and the resulting samples were subjected to LC–MS/MS analysis. A 2.1 mm × 100 mm Kinetex C18 column (particle size 1.7 ␮m, Phenomenex, Torrance, CA, USA) was used for the separation of the DNA hydrolysis samples, and the flow rate was 300 ␮L/min, which was delivered by an Agilent 1290 UPLC pump. A 12 min gradient of 0–30% acetonitrile in 20 mM ammonium acetate was employed for the separation of 8-oxodG from other deoxynucleosides, including deoxyguanosine (dG) in the DNA digestion mixture. The effluent from the LC column was delivered to an Agilent 6490 triple-quadruple mass spectrometer, which was equipped with an electrospray ionization source operating in positive-ion mode. The concentration of 8-oxodG was quantified using the isotope-dilution method with the labeled 8-oxodG as the internal standard and the dG concentration was quantified using the external calibration

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method. The selected MRM transitions for the quantification of 8oxodG, labeled 8-oxodG and dG were m/z 284 > 168, 289 > 173 and 268 > 152, respectively. 2.6. Protein extraction Proteins were extracted from medaka embryos following the trichloroacetic acid (TCA)/acetone precipitation method (Wang et al., 2009). Briefly, frozen embryos were first washed with cold 10% TCA/acetone (v/v), and then homogenized in 1.0 mL of 20% TCA/acetone (v/v) lysis buffer using a glass homogenizer followed by an ultrasonic disrupter. After centrifugation at 18,000 × g for 30 min at 4 ◦ C, the pellet was washed twice with ice-cold 80% acetone (v/v) and twice with pure acetone. The residual acetone was evaporated in a SpeedVac for about 5 min. Subsequently, the protein pellet was dissolved in 120 ␮L rehydration buffer containing 30 mM Tris, 7 M urea, 2 M thiourea, and 4% CHAPS. The protein concentrations were quantified using the 2-D Quant kit (GE Healthcare Life Sciences, PA, USA). 2.7. 2-D electrophoresis Onto each gel 100 ␮g of protein was loaded and separated using 2-D gel electrophoresis. First dimension gel electrophoresis was carried out using an 18 cm Immobiline Dry Strip with a linear pH 4–7 gradient (Bio-Rad Laboratories, CA, USA) and an Ettan IPGphor 3 Isoelectric Focusing System (GE Healthcare Life Sciences) set at 20 ◦ C. Strips were rehydrated in a solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT and 0.5% (v/v) IPG buffer 4–7. Isoelectric focusing was performed in the following steps: at 40 V for 6 h, at 100 V for 6 h, at 500 V for 30 min, at 1000 V for 1 h, at 2000 V for 1 h, and at 10,000 V for 7.5 h. After equilibration in a solution containing 6 M urea, 2% SDS, 50 mM Tris–HCl (pH 8.8), 30% glycerol, 1% DTT and a trace amount of bromophenol blue, the strips were further alkylated in an alkylation buffer (2.5% iodoacetamide instead of 1% DTT in the equilibration buffer). Subsequently the second dimension separation was performed in a 12.5% SDS-PAGE with a Protean II Xi Cell System (Bio-Rad Laboratories). Electrophoresis was carried out at 12.5 mA/gel for 30 min, followed by a 6 h run at 25 mA/gel until the bromophenol blue front reached the very bottom of the gel. The protein spots were visualized using silver staining. Two-dimensional gel electrophoresis was performed in triplicate and from three independent protein extractions for each exposure group. 2.8. Image acquisition and analysis The gel images were captured on a scanner (EPSON V700) and Image Master 2D Platinum 7 (GE Healthcare Life Sciences) software was used to match and analyze the image. The density of the protein spots was expressed as the volume of the spots, which was determined in comparison with the total volume of all the spots within the same gel. Only more than 1.5-fold changes between the tHBCD-treated and control groups were accepted. 2.9. Protein identification and data analysis The proteins of interest were manually excised from the 2-D gels. The gel pieces were washed with Milli-Q H2 O for 10 min at 50 ◦ C, destained with 1% (w/v) potassium ferricyanide and 1.6% (w/v) sodium thiosulfate for 10 min at 50 ◦ C, and washed again with Milli-Q H2 O for 10 min at 50 ◦ C. After dehydration with 100% acetonitrile, the dry gel pieces were digested by adding 10.0 ng/␮L trypsin (Promega, WI, USA) in 10 mM ammonium bicarbonate and incubated at 37 ◦ C for 4–16 h. The samples were further analyzed using MALDI-TOF/TOF mass spectrometry or LC–MS/MS.

For MALDI-TOF/TOF analysis, 1 ␮L of the digest mixture was mixed with 0.5 ␮L of 8 mg/ml ␣-cyano-4-hydroxy-cinnamic acid (in 50% acetonitrile/0.1% tri-fluoroacetic acid) and spotted on the MALDI target plate. MALDI-TOF and tandem TOF/TOF analysis were performed on an AB Sciex MALDI-TOFTM 5800 analyzer (Applied Biosystems, Foster City, CA). The data were acquired in the positive MS reflector mode with a scan range from 900 to 4000 m/z, and the five most abundant monoisotopic precursors (S/N > 200) were selected for MS/MS analysis. For LC–MS/MS analysis, the peptides were separated on a fused silica capillary column packed with 5 ␮m C18 resin (75 ␮m × 15 cm; New Objective, Woburn, MA) with mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). A gradient (300 nL/min) was run over 40 min from 3 to 40% mobile phase B. The HPLC eluent was then subjected to an AB Sciex TripleTOF 5600 system (Applied Biosystems). For information dependent acquisition, survey scans were acquired for 250 ms and 20 product ion scans were collected at 50 ms/scan. The acquired mass spectrometry raw data files were searched against the NCBI database for fish species with Protein Pilot software V.4.2 (Applied Biosystems). One missed cleavage site was allowed for trypsin digestion; cysteine carbamidomethylation was assumed as a fixed modification; and methionine was assumed to be partially oxidized. The tolerances were specified as ±0.05 Da for MS and ±0.05 Da for MS/MS fragments. Results with Confidence Interval (CI) values greater than 95% were considered to be a positive identification. The identified proteins were then matched to specific processes or functions by searching Gene Ontology (http://www.geneontology.org/).

2.10. Real-time quantitative PCR (qPCR) Total RNA was extracted from ten 5- and 8-dpf embryos or the 1st fry stage larvae per replicate. An equal amount of RNA was then reverse-transcribed using mixtures of oligo (dT) primer (5 -TTTTTTTTTTTTTTTTTTTT-3 ) and random primers by M-MLV reverse transcriptase (BGI, Shenzhen, China) to generate cDNA. qPCR was carried out on a CFX96TM Real-Time System (Bio-Rad Laboratories) using SYBR Green I. The primers for qPCR of the TNF-␣, IL-1␤, COX-1, COX-2 and 18s rRNA genes were based on other published papers (Huang et al., 2011, 2012b). The primers for p53 (F:5 -TAAACGGCAAAGTGTTACGG3 , R: 5 -CCATGCACGAGCTATTACACA-3 ) and CYP1A (F: 5 CCGAAATCCTACCCTGTCTG-3 , R: 5 -TGGGATTGTGAATGGAAGAA3 ) were designed at the Genscript website and checked for validity using the Primer-Blast tool in NCBI. The thermal cycle program consisted of an initial denaturation step at 95 ◦ C for 3 min, followed by 50 cycles of 95 ◦ C for 10 s, 60 ◦ C for 35 s (18 s, COX-1, COX-2 and CYP1A) or 65 ◦ C for 35 s (p53) or 70 ◦ C for 35 s (TNF-␣ and IL-1␤). Dissociation curve analysis was done to confirm that only the targeted PCR product was amplified and detected. Gene expression levels were normalized to 18s rRNA expression levels. Two replicates of qPCR were performed for each sample and four samples were performed for each tHBCD exposure group. The fold change of the tested genes was analyzed using the 2−Ct method.

2.11. Caspase activity measurement The measurement of caspase-3, caspase-8 and caspase-9 activities was performed using caspase-3, caspase-8 and caspase-9 colorimetric protease assay kits (Keygene Biotech Co., Nanjing, China) following the manufacturer’s instruction (Deng et al., 2009). The protein concentration was determined with reference to standards of bovine serum albumin using BCA assay (Thermo Fisher

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Scientific, MA, USA). The enzyme activity was expressed as the fold change over the control samples. 2.12. TUNEL assay The fragmented DNA of apoptotic cells was identified by the TUNEL method (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling), using the fluorescent in situ cell death detection kit (Roche diagnostic, Switzerland) following the manufacturer’s instruction. Briefly, twenty larvae at 1st fry stage were fixed for 2 h at room temperature in 4% paraformaldehyde in phosphate buffer saline (PBS, freshly prepared). The samples were then washed twice in PBS and further permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate (freshly prepared) for 10 min on ice, followed by two rinses in PBS. The samples were incubated with the label solution mixture overnight at 4 ◦ C and then shaken for 1 h at 37 ◦ C. The labeling reaction was stopped by washing the samples in PBS three times (5 min each) at room temperature to remove excessive TdT and deoxynucleotides. After TUNEL staining, the larvae were photographed by a fluorescent microscope with an excitation wavelength of 485 nm and emission wavelength of 510 nm. 2.13. Western blotting Total protein was extracted from 10 larvae per replicate. The protein concentration was determined with reference to standards of bovine serum albumin using BCA assay (Thermo Fisher Scientific, MA, USA). An equal amount of protein was used for analysis. Pre-stained molecular weight markers (Bio-Rad Laboratories) were run as standards on each gel. The protein was fractionated on SDS-PAGE and transferred to PVDF membranes at 300 mA for 1 h. The membrane was blocked with 5% non-fat dry milk and incubated with the primary anti-p53 antibody (Bioss, Beijing, China) and anti-MDM2 antibody (Keygene Biotech Co., Nanjing, China) for 1 h and washed three times. After incubating with an antirabbit IgG-alkaline phosphatase conjugated secondary antibody (Sigma–Aldrich Chemical Co.) at room temperature for 1 h, the membrane was washed three times again, then rinsed twice with a buffer containing 100 mM Tris (pH 9.5), 100 mM NaCl, and 10 mM MgCl2 and incubated with NBT/BCIP solution (Roche, Indianapolis, USA) until dark bands appeared. 3. Results

Fig. 1. tHBCD exposure induced malformation in O. melastigma embryos. (A) Cumulative malformation rate in O. melastigma embryos exposed to different tHBCD concentrations (0, 5, 20 and 50 ␮g/L). The values are presented as the mean ± SD (n = 4). The value that is significantly different from the control is indicated by an asterisk (one-way ANOVA, followed by LSD tests: *p < 0.05); (B) hatched 1st fry stage larvae developed normally in the control group; (C) representative example of pericardial edema and yolk sac edema developed in 5 and 20 ␮g/L tHBCD exposure groups; (D) representative example of spinal curvature developed in the 50 ␮g/L tHBCD exposure group.

3.1. Developmental toxicity tHBCD exposure did not have significant effects on O. melastigma embryo hatch success, survival rate or hatch-out time, while it caused malformation of the hatched larvae in a concentrationdependent manner, with malformation rates of 9.2 ± 10.7%, 6.4 ± 9.4%, 19.7 ± 15.4% and 40.1 ± 16.7% at the exposure concentrations of 0, 5, 20 and 50 ␮g/L, respectively (Fig. 1A). Particularly, at the highest exposure concentration (i.e., 50 ␮g/L), the malformation rate was significantly higher than that in the solvent control group (p < 0.05). The developmental abnormalities included yolk sac edema, pericardial edema and spinal curvature (Fig. 1B–D). At the exposure concentrations of 0, 5 and 20 ␮g/L, only yolk sac edema and pericardial edema were observed in the abnormal embryos, while at 50 ␮g/L, 15% of the abnormal embryos showed all the three malformation syndromes and the rest 85% of the abnormal embryos only had yolk sac and pericardial edemas. The heart rate of the O. melastigma embryos was recorded on both 5 dpf and 8 dpf, in that these two time points were within the critical period for medaka embryo heart development (Huang et al., 2011). The results showed that the heart rate of medaka embryos was induced by exposure to tHBCD in a concentration dependent

manner, which indicated that tHBCD had significant tachycardia effects on marine medaka embryos (Fig. 2A). For instance, on 8 dpf, the heart rate of the embryos significantly increased from 145 ± 15 to 163 ± 15, 170 ± 16, and 174 ± 21 beats/min, upon exposure to tHBCD at the concentrations of 5, 20, and 50 ␮g/L, respectively. To further examine the effects of tHBCD on the cardiac development of the O. melastigma, the SV–BA distance, which is used as an important index for the evaluation of the cardiac development (Huang et al., 2011), was measured in the developing embryos. The results showed that the SV–BA distance of embryos was induced by exposure to tHBCD in a dose dependent manner (Fig. 2B). Compared to the controls, the SV–BA distance on 8 dpf significantly increased after treated with two high doses, 20 and 50 ␮g/L (p < 0.05). 3.2. Increased levels of 8-oxodG To assess whether tHBCD exposure induced oxidative stress and further led to oxidative DNA damages in O. melastigma embryos, the levels of 8-oxodG were measured after 6 days of exposure. 8-oxodG is the oxidative product of deoxyguanine induced by

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25

* 8-oxodG G/106 dG

20

*

15 10 5 0 control

5 μg/L μg

20 μg/L μg

50 μg μg/L

tHBCD concentraon in the medium Fig. 3. tHBCD exposure induced the formation of 8-oxodG in O. melastigma embryos on 6 dpf in a dose dependent manner. The values are presented as the mean ± SD (n = 4). The values that are significantly different from the control are indicated by asterisks (one-way ANOVA, followed by LSD tests: *p < 0.05).

indicated that tHBCD exposure induced oxidative stress, which led to the formation of higher levels of oxidative DNA damages in O. melastigma embryos. If the oxidative lesions were not repaired in time and properly, they might induce mutation and cause genotoxicity effects in the embryos. 3.3. Induction of caspase activity To evaluate whether tHBCD induced apoptosis via the caspasedependent pathway, the activities of caspase-3, caspase-8 and caspase-9 were evaluated on 5 and 8 dpf after exposure. In the 200 ␮g/L treatment group, the activity of caspase-3 was significantly induced on 5 dpf, and the activities of all the three caspases were significantly induced on 8 dpf (Fig. 4). Although the activities of caspase-8 and caspase-9 were not significantly induced on 5-dpf, the caspase-8 activity on 8 dpf was increased by 105 ± 8%, 107 ± 5, and 117 ± 4%, and caspase-9 activity was increased by 101 ± 6%, 103 ± 11% and 114 ± 5% at the exposure concentrations of 20, 50 and 200 ␮g/L, respectively. 3.4. Detection of apoptosis by whole mount TUNEL assay After exposure to tHBCD at the concentrations of 0, 20, 50 and 200 ␮g/L, the larvae at the 1st fry stage were fixed, stained using fluorescent TUNEL staining method and then visualized under fluorescent microscope. No obvious apoptotic cells were observed in control group (Fig. 5A), while starting from the 20 ␮g/L treatment group the apoptotic cells appeared, mainly in the heart area. Particularly, the larvae in the 200 ␮g/L treatment group had very obvious apoptotic hearts (Fig. 5D). Fig. 2. The measurement of heart rate (A) and SV-BA distance (B) in O. melastigma embryos on 5 dpf and 8 dpf after exposure to different tHBCD concentrations (0, 5, 20 and 50 ␮g/L). The values are presented as the mean ± SD (n = 20 for each replicate). The values that are significantly different from the control are indicated by asterisks (one-way ANOVA, followed by LSD tests: *p < 0.05; **p < 0.01). (C) The heart structure of the embryos showing the sinus venosus (SV), atrium (A), ventricle (V) and bulbus arteriosus (BA). The SV–BA distance is the direct distance between the centers of SV and BA.

various types of ROS, which is highly mutagenic and results in G to T transversion (Loft and Poulsen, 1996). Levels of 8-oxodG are associated with high cancer risk and are used as a robust biomarker for risk assessment of various cancers induced by oxidative stress (Ravanat et al., 2002; Sova et al., 2010). The levels of 8-oxodG were 6.9, 10.8, 14.9 and 16.9 lesions/106 deoxyguanine for the control, 5, 20 and 50 ␮g/L treatment groups, respectively (Fig. 3). These results

3.5. Induction of stress-responsive genes and proteins The expression profile of several heart-related genes (COX-1 and COX-2), oxidative stress related genes (CYP1A, IL-1␤ and TNF-␣) and apoptotic gene p53 were analyzed on 5, 8 dpf and at the 1st fry stages. The mRNA expression levels of the p53, IL-1␤ and TNF-␣ genes were significantly up-regulated at the exposure concentration of 50 or 200 ␮g/L on the three selected developmental stages (Fig. 6), while the expression of other selected genes was not significantly changed compared to the control group (data not shown). Furthermore, the expression levels of p53, IL-1␤ and TNF-␣ genes were induced in a dose-dependent manner at most of the selected developmental stages (Fig. 6). In addition, the level of p53 protein, which was quantified using western blotting, was up-regulated by 1.29 ± 0.04 folds (p < 0.05) compared with the control at the 1st fry

H. Hong et al. / Aquatic Toxicology 152 (2014) 173–185

179

Cas spase -3 activity (%co ontrol)

200 180

A

**

60 160 140

*

control 20 μg/L 50 μg/L 200 μg/L

120 100 80 60 40 20 0

Ca aspase-8 activity (%co ontrol)

140 120

B

*

C

*

100 80 60 40 0 20 0

Caspase-9 activity (% %control)

140 120 100 80 60

Fig. 5. Induction of apoptosis in the heart region of O. melastigma at the 1st fry stage detected by TUNEL assay. The embryos were exposed to 0 (A), 20 (B), 50 (C) and 200 ␮g/L (D) of tHBCD and the larvae at the 1st fry stage were fixed for TUNEL staining. Red arrows indicate apoptotic hearts of the larvae. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

40 20 0

5 dpf

8 dpf

Fig. 4. Induction of the activities of caspase-3 (A), caspase-8 (B) and caspase-9 (C) in O. melastigma embryos on 5 and 8 dpf after exposure to various concentrations of tHBCD (0, 20, 50 and 200 ␮g/L). The values are presented as the mean ± SD (n = 4). The values that are significantly different from the control are indicated by asterisks (one-way ANOVA, followed by LSD tests: *p < 0.05; **p < 0.01).

binding and signaling (spots 4 and 5), and two proteins in proteolysis (spots 7 and 10). The other four proteins were categorized into the oxidation-reduction process, regulation of cell morphology and other functions. 3.7. Concentrations of HBCDs in the embryos

stage upon 200 ␮g/L tHBCD exposure. However, the level of MDM-2 protein, which plays an important role in p53 protein stability, was not significantly changed upon tHBCD exposure (data not shown). 3.6. Changes of protein expression profiles Representative 2-DE gels of proteins extracted from the control and tHBCD-treated medaka embryos are shown in Fig. 7. On average, more than 1500 protein spots were detected in each gel using silver staining and the image analysis software. Compared with the 2-DE gels of the control group, a total of 17 protein spots from the tHBCD-exposed medaka embryos were significantly down-regulated (≥1.5-fold, p < 0.05). No up-regulated protein spots were found in the tHBCD treated groups. These altered protein spots were excised, trypsin digested and submitted for identification using MALDI-TOF/TOF or LC–MS/MS analysis. After searching against the NCBI database for fish species, 15 protein spots were successfully identified with CI values greater than 95% (Table 2). Among them, two proteins (spots 1 and 12) were engaged in gene transcription and translation, five proteins were involved in metabolism (spots 3, 11, 6,14 and 15), two proteins in calcium

All three diastereoisomers, ␣-, ␤- and ␥-HBCD were detected in the O. melastigma embryos from all the tHBCD exposure groups. The concentrations of total HBCD isomers (HBCDs) increased over time during the exposure period up to 8 dpf, indicating continuous accumulation of HBCD isomers in medaka embryos (Fig. 8). The isomer compositions in the embryos were 6%, 5% and 89%, respectively for the ␣-, ␤- and ␥-HBCD isomers, which were almost identical to those in the tHBCD used for exposure. 4. Discussion 4.1. Comparison of HBCDs levels in the wild fish and in the O. melastigma embryos used in this study HBCDs are widely used as additive brominated flame retardants and have been detected in various environmental media, such as air, sediment, animal tissues and human bodies. HBCDs are highly bioaccumulative and high levels of HBCDs have been detected in freshwater and marine fish species. A general but not exhaustive summary of relatively high levels of HBCDs in the fish caught

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H. Hong et al. / Aquatic Toxicology 152 (2014) 173–185

Table 2 The differentially altered proteins identified in O. melastigma embryos upon exposure to tHBCD (5 ␮g/L, 20 ␮g/L and 50 ␮g/L) for 8 days. No. on gels

Identity

Regulation of transcription and translation Uncharacterized: 1 polycomb protein SFMBT2 12 Uncharacterized: arginine/serine-rich coiled-coil 1 RSRC1 Metabolism 3

UniProt accession no.

H2TYJ2

H2UGI9

Ribose-phosphate pyrophosphate kinase

H2TMA8

40S ribosomal protein like

I3JN34

Q2PHF0

14

Brain-type fatty acid binding protein (FABP7) Vitellogenin II

15

Vitellogenin-like

H2LQ37

Functional description

Regulation of transcription, DNA dependent mRNA splicing

Organism

No. of peptidesa

Folds down-regulatedb 5 ␮g/L

20 ␮g/L

50 ␮g/L

Takifugu rubripes

1

–

–

3.26 (2.74)*

Takifugu rubripes

1

2.25 (1.40)*

3.08 (1.27)*

6.32 (1.92)***

Takifugu rubripes

1

1.75 (0.51)*

1.54 (0.23)

3.56 (0.97)**

Nucleotide biosynthetic process Ribosomal small subunit assembly, translation Lipid binding, transporter activity

Oreochromis 3 niloticus

–

–

2.13 (1.17)*

Oryzias latipes

4

–

–

1.97 (0.71)*

lipid transporter activity lipid transporter activity

Oryzias latipes Oryzias latipes

2

–

2.12 (0.20)**

1.86 (1.19)*

1

1.66 (0.60)

2.91 (2.02)

2.67 (2.12)

H2SHJ9

Calcium ion binding

Takifugu rubripes

3

1.68 (0.70)

1.98 (1.36)*

1.65 (0.45)

H2M0U7

Calcium ion binding

Oryzias latipes

4

1.87 (1.54)

1.92 (1.65)

4.60 (2.16)**

Regulation of proteolysis Ubiquitin-conjugating 7 protein (E2)-like

I3K0Z8

Oreochromis 1 niloticus

3.34 (1.98)

2.13 (0.98)

3.29 (1.16)*

10

H2SP96

Ubiquitin conjugation pathway Ubiquitin protein degradation

Takifugu rubripes

2

2.96 (2.93)

2.34 (2.22)*

4.36 (3.35)*

H2L585

Oxidationreduction process

Oryzias latipes

1

–

–

1.98 (0.77)*

H2UBH6

Regulation of cell morphology, cytoskeletal organization

Takifugu rubripes

1

–

2.31 (2.70)

4.05 (2.13)**

Q8JI23

Egg shell protein

Oryzias latipes Takifugu rubripes

2

2.15 (0.64)*

2.04 (0.42)

2.21 (1.30)

11

6

Calcium binding and signaling Uncharacterized: 4 myosin regulatory light chain Uncharacterized: 5 parvalbumin beta-like

Coiled coil domain containing 106 protein (CCDC106)

Oxidation-reduction process 18 Uncharacterized: short-chain dehydrogenase/reductase family Regulation of cell morphology Uncharacterize: 16 Bromodomain and WD repeat domain containing 3 (BRWD3) Others 13 17

Choriogenin L Uncharacterized: transmembrane and coiled-coil domain family 3 (TMCC3)

Q8UW61

H2V2L0

Unknown

1

1.80 (0.75)

*

2.26 (1.02)

4.38 (2.28)*

a

No. of peptides presented with the CI value higher than 95%. Only more than 1.5-fold decrease between the HBCD-treated and the control groups are presented. The folds down-regulated are presented as mean (S.D.). The folds significantly down-regulated are indicated by asterisks (one-way ANOVA followed by LSD tests): * p < 0.05. ** p < 0.01. *** p < 0.001. b

from both freshwater and marine ecosystems is shown in Table 1. According to these studies, high levels of HBCDs were accumulated in the muscles and other organs (including eggs) of many fish species (Eljarrat et al., 2004; Allchin and Morris, 2003; Wu et al., 2010; Sellstrom et al., 1998; Li et al., 2012; Xian et al., 2008). For instance, an individual value in excess of 10,000 ng/g ww of HBCDs was found in the muscle of edible fish from Rivers Skerne/Tees, UK (Allchin and Morris, 2003). The quantification of

HBCDs in a group of marine fish from a Norwegian fjord (Etnefjorden) revealed that HBCDs in the marine fish filet can reach 300 ng/g ww (Koppen et al., 2010). In addition, a few field studies shown that HBCD concentrations in the fish eggs were higher than or similar to those found in the fish muscles (Xian et al., 2008; Li et al., 2012). Furthermore, a laboratory study reported that HBCDs were maternally transferred to the fish eggs, in which the concentrations of HBCDs were higher than those measured in the adults

H. Hong et al. / Aquatic Toxicology 152 (2014) 173–185

181

4

A) p53

**

Folds cha ange

3

* 2

control 20 μg/L 50 μg/L 200 μg/L

*

*

1

0

5

B) IL-1β **

Folds change

4

**

3

*

2 1 0

2.5 2 5

C) TNF-α

*

Follds change

2.0

* * 1.5 1.0 10 0.5 0.0

5 dpf

8 dpf

1st fry

Fig. 6. Induction of the gene expression of p53 (A), IL-1␤ (B) and TNF-␣ (C) in O. melastigma embryos on 5 dpf, 8 dpf and 1st fry stage after exposure to 0, 20, 50 and 200 ␮g/L of tHBCD. The values are presented as the mean ± SD (n = 4). The values that are significantly different from the control are indicated by asterisks (one-way ANOVA, followed by LSD tests: *p < 0.05; **p < 0.01).

(Nyholm et al., 2008), further supporting that HBCDs were able to be present in the fish eggs. In our study, on 8 dpf when significant tachycardial effects and increase of SV–BA distances were induced by HBCDs, the HBCDs in the O. melastigma embryos were measured to be 527, 4077 and 8950 ng/g ww for the exposure concentrations of 5, 20 and 50 ␮g/L, respectively. These results suggested that the levels of HBCDs accumulated in the O. melastigma embryos upon exposure to 5–50 ␮g/L tHBCD for up to 8 days were comparable to those in the fish samples caught at the highly polluted area (Koppen et al., 2010; Allchin and Morris, 2003). It was therefore conceivable that environmentally realistic concentrations of HBCDs were likely to cause developmental toxicity in fish in both the marine and freshwater environment. The diastereoisomer composition in the O. melastigma embryos showed that the ␣-, ␤- and ␥-HBCD accounted for 5%, 6% and 89% of HBCDs, respectively. These isomer compositions were very similar to those of tHBCD used for exposure, suggesting that medaka embryo had limited bioconversion activity for HBCD isomers. Nevertheless, in the field samples, the percentage of ␣-HBCD was much higher than those of ␥-HBCD and ␤-HBCD (Allchin and Morris, 2003; Wu et al., 2010; Sellstrom et al., 1998; Li et al., 2012; Xian

Fig. 7. Representative 2-DE gels of the proteins extracted from O. melastigma embryos on 8 dpf. The protein spots were visualized using silver staining. The circle and number indicated spots with significant change in intensity (p < 0.05, n = 3, Turkey’s tests). (A) Control; (B) 50 ␮g/L.

et al., 2008). Therefore, it is important to understand the difference in toxicity of HBCD isomers. In this context, it was reported that the developmental toxicity of HBCD isomers in zebrafish was in the order of ␥-HBCD > ␤-HBCD > ␣-HBCD (Du et al., 2012). In order to better evaluate the potential environmental risk of HBCDs to marine fish embryos, the toxicity of different diastereoisomers of HBCDs in O. melastigma embryos should be evaluated in future studies. 4.2. HBCD induced developmental toxicity in O. melastigma embryos The O. melastigma embryo can serve as an excellent model for estuarine and marine ecotoxicological studies (Chen et al., 2011). Our results showed that waterborne exposure of O. melastigma embryos to tHBCD caused significant developmental toxicity.

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10000 5 μg/L 20 μg/L 50 μg/L

ng ΣHBCD/g wt

8000

6000

4000

2000

0 0

2

4

6

8

10

Exposure duration (days) Fig. 8. Concentrations of HBCDs in O. melastigma embryos after exposure to various concentrations of tHBCD (5, 20, 50 ␮g/L) for 1, 4 and 8 days. The values are presented as the mean ± SD (n = 4).

The major tHBCD-induced malformation in the marine medaka included pericardial edema, yolk sac edema and spinal curvature, which were very similar to those observed in the freshwater zebra fish embryos (Du et al., 2012; Deng et al., 2009). The development of the cardiovascular system in vertebrates such as fish is very complex and very often is the important target of a variety of different types of environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), perfluorooctanesulfonate (PFOS) and polybrominated diphenyl ether (PBDE) (Huang et al., 2011, 2012a; Lema et al., 2007; Antkiewicz et al., 2005). Besides the appearance of pericardial edema, tHBCD changed the heart structure in the O. melastigma embryos, as indicated by the increase in SV–BA distance. Additionally, upon exposure to tHBCD at the concentrations up to 1000 ␮g/L for 48 h or 96 h, the heart rate of zebra fish embryos was also significantly changed (either decreased or increased) (Deng et al., 2009; Du et al., 2012). Meanwhile our study indicated that tHBCD induced significant tachycardial effects in the embryos of O. melastigma at the exposure concentrations of 5–50 ␮g/L. Taken together, it is suggested that tHBCD at environmentally realistic concentrations might change the heart rate and heart structure of fish embryos, and therefore might pose a potential risk to the aquatic organisms. 4.3. HBCDs induced oxidative stress, apoptosis and stress-related gene expression in O. melastigma Waterborne tHBCD is able to induce oxidative stress and apoptosis through involvement of the caspases pathway in zebrafish embryo development (Deng et al., 2009). Particularly, the tHBCDinduced apoptotic cells are mainly found in the heart region of zebrafish, which may be one of the reasons for tHBCD-induced heart malformation (Deng et al., 2009). Likewise, our study revealed that tHBCD induced the formation of 8-oxodG, a robust biomarker for oxidative stress (Valavanidis et al., 2009), in O. melastigma embryos, suggesting that tHBCD also promoted the formation of ROS and subsequently caused oxidative DNA damages in the embryos. 8-oxodG is mutagenic and is one of the most important DNA oxidative products; if not repaired timely and properly by the base-excision DNA repair pathway, it will lead to G to T transversion (Sova et al., 2010; Ravanat et al., 2002). In addition, the apoptotic cells were detected mainly in the hearts of the 1st fry stage larvae as revealed by TUNEL staining, which further supported that tHBCD induced oxidative stress, resulted in apoptosis and finally led to cardiovascular toxicity in the O. melastigma embryos.

Most of the toxic endpoints which appear in TCDD and PAHexposed vertebrates are thought to be mediated by the AhR/ARNT (aryl hydrocarbon receptor/AHR nuclear translocation protein) pathway. The activated AhR increases expression of CYP1 family genes, shifts the cellular redox state toward oxidizing conditions and results in an oxidative stress (Dalton et al., 2002). Among the known AhR target genes, cytochrome P450 1A (CYP1A) is the best characterized one and induction of CYP1A expression has been used as a marker of AhR/ARNT pathway activation (Antkiewicz et al., 2005; Huang et al., 2012a). In this regard, we did not see the induction of CYP1A expression in O. melastigma upon tHBCD exposure (data not shown), indicating HBCDs did not activate AhR/ARNT pathway or other possible CYP1A-activation pathways (Delescluse et al., 2000) to induce oxidative stress in O. melastigma embryos. This result was not surprising since HBCDs do not possess the planar aromatic structure, which is usually a requirement for binding to AhR (Delescluse et al., 2000). COX-1 and COX-2 genes are well known for their involvement in inflammatory processes promoting the formation of ROS. COX2 is transcriptionally induced in Oryzias latipes (Japanese medaka) embryos treated with AhR ligand TCDD, which then leads to the inflammatory mediated pericardial edema and heart dysmorphogenesis (Dong et al., 2010). Furthermore, COX-2 expression is also induced by non-AhR ligands, such as arachidonic acid and PFOS, resulting in inflammatory mediated heart failure in fish embryos (Huang et al., 2007, 2011). However, HBCDs exposure did not change the expression of either COX-1 or COX-2 genes in O. melastigma embryos (data not shown), which suggested that HBCDs induced cardiac toxicity was unlikely through the activation of the COX genes to trigger the inflammatory formation of ROS. TNF-␣ (tumor necrosis factor-␣) mediated signaling pathway initiates and accelerates vascular inflammation, oxidative stress and apoptosis, and plays an important role in cardiovascular dysfunction (Zhang et al., 2009). TNF-␣ is reported to stimulate ROS production by several mechanisms, including its effect as an agonist to increase the product of NAD(P)H-oxidase and O2 and thus promote the formation of ROS, and its role in the changes of mitochondria redox state (Madamanchi et al., 2005; Mariappan et al., 2007). Another inflammatory cytokine, interleukin-1␤ (IL1␤), also has been implicated in playing a role in ROS generation (Hensley et al., 2000). We found that tHBCD induced the expression levels of both TNF-␣ and IL-1␤ in O. melastigma on 5 dpf, 8 dpf and the 1st fry stage (Fig. 6), suggesting that inflammatory mediators, for example TNF-␣ and IL-1␤, might contribute to HBCDs-induced oxidative stress. In addition to its role in oxidative stress, TNF-␣ exserts pro-apoptotic effects via activation of the caspase-8 and caspase -3 (Wang et al., 2008; Elmore, 2007). The measurement of caspase-8 and caspase-3 activities indeed showed that tHBCD exposure enhanced both the caspase-8 and caspase3 activities (Fig. 4), implying that HBCD might induce apoptosis through the activation of the TNF-␣-caspase-8 pathway.p53 gene is one of the critical genes that responds to a variety of stresses, including oxidative stress. The ROS can alter DNA structure and generates oxidative DNA damages, for example 8-oxodG, which will be detected and repaired by certain DNA repair enzymes, and in the meantime activate p53 pathway to promote cell cycle arrest or apoptosis (Harris and Levine, 2005). Our results indeed showed that in corresponding to the increased level of 8-oxodG, the expression of both p53 gene and protein was induced upon exposure to tHBCD (Fig. 6). Furthermore, it is known that activation of p53 triggers the mitochondria-mediate apoptotic pathway and then results in cytochrome c release and activation of caspase-9 and caspase-3 (Elmore, 2007). In this regard, the HBCDs-induced expression of p53 and the activation of caspase 3 and caspase9 were observed using zebrafish (Deng et al., 2009) and marine medaka (in this study) as model fish. In addition, Deng et al. (Deng

H. Hong et al. / Aquatic Toxicology 152 (2014) 173–185

et al., 2009) showed that upon HBCDs exposure and induction of p53 expression, the expression of Bax and Puma genes, two key players in the mitochondria-mediate apoptotic pathway, were also upregulated. In response to a stress signal, such as ROS, the activated p53 proteins are also autoregulated by a variety of negative and positive feedback loops, including several ubiquitin ligases responsible for p53 ubiquitination and degradation, such as MDM-2 (Harris and Levine, 2005). Deng et al. showed that the expression of MDM2 gene was down regulated upon exposure to 1 mg/L tHBCD in zebrafish embryos (Deng et al., 2009). However, owing to lack of gene sequence information on MDM-2 in O. melastigma, the present study did not investigate MDM-2 expression at gene expression level. Nevertheless, our immunoblotting results indicated that the level of MDM-2 protein in marine medaka did not change significantly upon tHBCD exposure (data not shown). It is possible that if higher exposure concentrations were chosen, more significant changes in the level of MDM-2 protein might have been observed in O. melastigma. It is also likely that the correlation between the gene expression and protein levels of MDM-2 in fish embryos was not very strong. Anyhow, it remains unclear whether MDM2 gene takes part in the HBCDs-induced toxicity in O. melastigma embryos.

183

4.4. Protein responses revealed by gel-based proteomics The gel-based quantitative proteomic study identified several down regulated proteins upon tHBCD exposure, including proteins involved in proteolysis, transcription, translation, nucleotides and lipid metabolism, calcium binding and signaling and other cellular processes (Table 2). CCDC106 is recently characterized as a p53 interacting partner and promotes p53 degradation in cells (Zhou et al., 2010). The other protein involved in proteolysis is an ubiquitin-conjugating protein (E2) like protein. E2 proteins are a family of ubiquitin-conjugating proteins involved in ubiquitin-mediated proteolysis, which are characterized by a conserved ubiquitin-conjugating domain (van Wijk and Timmers, 2010). They receive ubiquitin from E1 protein and selectively interact with ubiquitin ligases to facilitate the transfer of ubiquitin to ubiquitin ligase. It is reported that a few E2 proteins, such as UbcH5B/C and Rad6 are involved in the negative regulation of p53 (Lyakhovich and Shekhar, 2003; Saville et al., 2004). Although it has been well recognized that MDM-2 plays an important role in p53 regulation under a variety of stress, it is conceivable that suppressed expression of E2 and CCDC106 protein could also contribute to HBCDs-induced upregulation of p53 protein, and this deserves further investigation.

Scheme 2. Proposed mechanism for tHBCD induced toxicity in O. melastigma embryos. It is hypothesized that tHBCDs-induced the expression of TNF-␣ and IL-1␤, which stimulated the formation of ROS and subsequently induced the formation of DNA oxidative damages, which then activated the p53 signaling pathway. At the mean time, the suppression of the ubiquitin-conjugating protein (E2) and CCDC106 might help to stabilize the p53 protein and resulted in the elevation of p53 protein level in the embryos. Subsequently, p53 triggered cell-cycle check points and other repair mechanisms to repair damaged DNA, and also promoted apoptosis through the mitochondria-mediate pathway, which led to the activation of caspase-3 and caspase-9. In addition, TNF-␣ also activated caspase-8 and subsequently caspase-3. Furthermore, tHBCD might also induce the embryonic malformation through inhibition of protein and nucleotide synthesis. The suppression of ccdc106, E2, ribose-phosphate pyrophosphate kinas and 40S ribosomal protein was suggested by quantitative proteomic approach and had not been confirmed by immunoblotting due to the unavailability of related antibodies, therefore is indicated by dashed lines.

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Nucleotide biosynthesis, transcription and translation are considered to be essential for the development of embryos. During embryo development, the cells are active in transcribing and maintaining high levels of mRNA, and are also active in the synthesis of ribosomal protein to increase the ribosomal number for massive protein production (Clegg, 1982; Smith et al., 1970). Our results showed that tHBCD exposure significantly suppressed the expression of several proteins involved in these key cellular processes. Ribose-phosphate pyrophosphate kinase converts ribose 5-phosphate into phosphoribosyl pyrophosphate, which provides a phospho-ribose group for synthesis of nucleotides as building blocks for RNA synthesis. 40S ribosomal protein is involved in the ribosomal small subunit assembly and thus is essential for protein synthesis. SFMBT2 and RSRC1 proteins both play an important role in the regulation of transcription. It is therefore conceivable that the significant down regulation of these proteins might lead to embryo developmental toxicity, including malformation. Brain-type fatty acid binding protein (FABP7) is expressed in the central nervous system (CNS) of vertebrates, including mammals and fish. The amino sequence of FABP7 in Oryzias latipes (Japanese medaka) Exhibits 80% identity with those of other higher vertebrates (Maruyama et al., 2008). The expression of FABP7 gene in Japanese medaka showed that it is distributed in the entire CNS including the retina and the spinal cord. Furthermore, the disruption of FABP7 gene expression in mice affects the brain functions, including emotion, learning and memory (Owada et al., 2006). Likewise, exposure of HBCDs to rats causes impaired behavior, learning and memory as well (Eriksson et al., 2006). The underlying mechanism is still not clear, although it is suggested that HBCDs may interfere with the thyroid hormone level and affect the brain development (Ibhazehiebo et al., 2011). Out results showed that HBCDs suppressed the expression of FABP7 protein, which thus might be one of the reasons for HBCD-induced neurotoxicity. Since we were unable to measure the neurotoxicity index in the current study, it is worthwhile to investigate the HBCDs-induced neurotoxicity in model fish in the future.

5. Conclusions Waterborne tHBCD exposure at environmentally realistic concentrations induced malformation, increase of heart rate and changes of the heart structures in O. melastigma embryo development. It was hypothesized that HBCDs induced the expression of TNF-␣ and IL-1␤, which then stimulated the formation of ROS. The excessive amount of ROS induced the formation of DNA oxidative damages, which were sensed by various DNA repair enzymes, followed by the activation of p53 signaling pathway (Scheme 2). Meanwhile, the suppression of E2 and CCDC106, which might take part in the proteolysis of p53, might help to stabilize the p53 protein and elevate the p53 at protein level in the embryos. Subsequently, p53 triggered cell-cycle check point for repairing DNA oxidative damages and apoptosis through mitochondriamediated apoptosis, which led to the activation of caspase-9 and caspase-3. In addition, TNF-␣ promoted apoptosis via activation of caspase-8 and subsequently caspase-3. Our quantitative proteomics study also revealed that exposure to tHBCD affected the nucleotide and protein biosynthesis, which might result in harmful effects in embryo development. In addition, there might be other unrevealed molecular mechanisms for HBCDs-induced toxicity, such as what was the molecular receptor of HBCDs; how HBCDs activated expression of TNF-␣ and IL-1␤; and whether there were other mechanisms for promoting the formation of ROS.

Acknowledgments We thank three anonymous reviewers for constructive and helpful comments on this work. We also thank Prof. Kejian Wang for providing O. melastigma embryos for exposure and Prof. Yinsheng Wang for providing isotopically labeled 8-oxodG. The work was supported by the National Science Foundation of China (No. 41206090), Research Fund for the Doctoral Program of Higher Education of China (No. 20120121120032) and the Recruitment Program of Global Youth Experts. Prof. John Hodgkiss is thanked for his assistance with the English. References Allchin, C.R., Morris, S., 2003. Hexabromocyclododedane (HBCD) diasterioisomers and brominated diphenyl ether congener (BDE) residues in edible fish fro the Rivers Skerne and Tees, UK. Organohalogen Compd. 61, 41–44. Antkiewicz, D.S., Burns, C.G., Carney, S.A., Peterson, R.E., Heideman, W., 2005. Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol. Sci. 84, 368–377. 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