Developmental Toxicity, Oxidative Stress, and Related Gene Expression Induced by Dioxin-Like PCB 126 in Zebrafish (Danio rerio) Han Liu,1† Fang-Hong Nie,2† Hong-Ying Lin,1 Yi Ma,1 Xiang-Hong Ju,1 Jin-Jun Chen,1* Ravi Gooneratne3* 1
Department of Veterinary Medicine, Agricultural College, Guangdong Ocean University, Zhanjiang 524088, China
2
College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China
3
Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, New Zealand
Received 31 March 2014; revised 17 August 2014; accepted 23 August 2014 ABSTRACT: 3,30 ,4,40 ,5-Pentachlorobiphenyl (PCB126) cause multiple adverse effects in organisms including animals and humans. Although PCB toxicities are linked to oxidative damage in rodents, the mechanism in early life stages of zebrafish is not clear. To explore the developmental toxicity mechanism of PCB126, three paradigms (toxicological phenotypes, biochemical changes, and molecular changes) were studied in 3-h postfertilization (hpf) zebrafish (Danio rerio) embryos exposed to different PCB126 concentrations (0, 16, 32, 64, and 128 lg/L) until 168 hpf. Developmental malformations, including pericardial and yolk sac edema, impaired lower jaw growth, spinal curvature, head edema and failure to inflate the swim bladder were observed, some as early as 72 hpf. Mortality was not apparent in early stages but significantly increased in a dose-dependent manner from 144 hpf onward. A dose-dependent significant increase in malformation rate was observed from 72 hpf onward with up to 100% at 132 hpf in embryos exposed to 128 lg/L of PCB126. Higher doses of PCB126 significantly decreased the copperzinc superoxide dismutase (CuZn-Sod), catalase (Cat), and glutathione peroxidase (Gpx) enzyme activities at 96, 132 hpf, but markedly declined from thereafter. PCB126 at 128 lg/L significantly increased the malondialdehyde content at 72, 96, and 132 hpf. The transcriptional gene expression of antioxidant enzymes Cat and Gpx was upregulated in embryos exposed to 64 lg/L of PCB126 at 24 and 96 hpf. Sod1 messenger RNA (mRNA) was low in embryos exposed to 32 lg/L at 72 and 96 hpf but was induced in embryos exposed to 64 and 128 lg/L doses at 132 hpf. Collectively, the results suggest oxidative stress as a major factor in the induction of multiple developmental abnormalities in early life stages of zebrafish exposed to PCB126. However, the relationship between the antioxidant enzyme activity and the C 2014 Wiley Periodicals, mRNA expression was not clear and the potential reasons for this are discussed. V Inc. Environ Toxicol 00: 000–000, 2014.
Keywords: PCB126; zebrafish embryo; developmental toxicity; antioxidant enzymes; mRNA
Corresponding to: R. Gooneratne; e-mail: Ravi.Gooneratne@lincoln. ac.nz and J.-J.Chen; e-mail: [email protected] Contract grant sponsor: International Science and Technology Cooperation Project of Guangdong Province, China.
Contract grant number: 2010B050600004. † These authors contributed equally to this work. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22044
C 2014 Wiley Periodicals, Inc. V
1
2
LIU ET AL.
INTRODUCTION Dioxin-like polychlorinated biphenyls (DlPCBs) are ubiquitous, lipophilic environmental contaminants responsible for persistence and biomagnification in the global ecosystem (Nfon and Cousins, 2006; Micheletti et al., 2007). 3,30 ,4,40 ,5-pentachlorobiphenyl (PCB126), the most representative coplanar congeners of DIPCBs, has similar structure and biological effects to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), and therefore is considered the most toxic PCB congener. Some DIPCBs have been found in a variety of foodstuffs, including seafood and meat products, and also in breast milk (Baars et al., 2004; Sasamoto et al., 2006; Domingo and Bocio, 2007). Adverse effects of exposure to DIPCBs include reproductive toxicity, immunotoxicity, neurotoxicity, carcinogenesis, and developmental toxicity (Schantz, 1996; Gutleb et al., 2000; Bunaciu et al., 2007; Nagayama et al., 2007). Previous studies have demonstrated that TCDD and PCB126 exposure induce a suite of developmental abnormalities in chicken eggs (Jin et al., 2001; Blankenship et al., 2003) and fish embryos (Teraoka et al., 2002; Antkiewicz et al., 2005; Yamauchi et al., 2006). There is some evidence that both TCDD and PCBs induced toxicity is related to oxidative stress as indicated by increased production of reactive oxygen species (ROS), lipid peroxidation, and associated decline in antioxidant enzyme activities in chicks (Jin et al., 2001; Hilscherova et al., 2003), rats (Latchoumycandane et al., 2002a, 2002b, 2003; Murugesan et al., 2008), and fish (Schlezinger et al., 2006). Under normal physiological conditions, there is a balance between ROS production and activity of a family of antioxidant enzymes (Hilscherova et al., 2003). However, when the generation of ROS exceeds beyond the protective capacity of these enzymes, oxidative damage occurs (Lassen et al., 2008). Exposure of fish to PCB126 or PCB169 significantly increased ROS production in liver microsomes thereby inducing oxidative stress (Schlezinger et al., 2006). Interestingly, coexposure of zebrafish embryos to antioxidant vitamin E and PCB126 effectively protects against PCB126 induced morphological changes (Na et al., 2009). All these studies suggest
Abbreviations AHR Cat DlPCBs Gpx hpf h MDA NAC PCB ROS Sod TCDD
aryl hydrocarbon receptor catalase dioxin-like polychlorinated biphenyls glutathione peroxidase postfertilization malondialdehyde N-acetylcysteine 126 3,30 ,4,40 ,5-pentachlorobiphenyl reactive oxygen species superoxide dismutase 2,3,7,8-tetrachlorodibenzo-p-dioxin
Environmental Toxicology DOI 10.1002/tox
that PCB126 induced developmental toxicity is associated with oxidative stress. However, it is not known whether exposure of zebrafish embryos to PCB126 affects the antioxidant enzymes activities and related gene expression. The antioxidant enzymatic protection against ROS include superoxide dismutase (Sod), catalase (Cat), glutathione-Stransferase (Gst), and glutathione peroxidase (Gpx) enzymes (Lassen et al., 2008). Acute doses of TCDD or PCBs cause lipid peroxidation and significant oxidative damage to peritoneal and adhesion fibroblasts in the human (Saed et al., 2010) testicular and Sertoli cell (Krishnamoorthy et al., 2005). Twaroski et al. (2001) demonstrated that PCB77 decreased hepatic Gpx activity and Gpx1 messenger RNA (mRNA) gene expression in rats. However, Kern et al. (2002) showed that although TCDD induced oxidative stress enzymes in both adipocyte and liver of rats, the mRNA expression in these tissues was unaffected. Many studies have demonstrated that developmental and reproductive toxicities of TCDD and PCBs in mammal are associated with oxidative stress followed by changing the antioxidant enzymes activity and related gene expression, but the mechanism underlining cellular oxidative damage on developmental toxicity of DIPCBs in zebrafish is not clear. Monitoring and assessing the effects of aquatic contaminants is critical for protection of human health and the environment. Zebrafish is an established vertebrate model for pharmacology, ontogenetic development, and toxicology studies because of its small size, low cost, diverse adaptability, short breeding cycles, high fecundity, and transparent embryos (Dai et al., 2014). Zebrafish embryos are more sensitive than mammals to certain dioxin-like compounds, and early organogenesis is easy to observe (McGrath and Li, 2008; Yang et al., 2009). To better understand the mechanism of developmental toxicity induced by PCB126 including oxidative stress in zebrafish embryos, three paradigms, toxicological phenotypes (mortality and malformation rates), biochemical parameters [(CuZn-Sod, Cat, Gpx enzyme activities, and malondialdehyde (MDA) measurement], and molecular changes (Sod1, Cat, and Gpx mRNA expression) were studied from 3- to 168-h postfertilization (hpf), following exposure to PCB126.
MATERIALS AND METHODS Fish Maintenance and Embryos Collection Juvenile AB strain zebrafish (Danio rerio) were purchased from China Zebrafish Resources Center and cultured in an aquarium until sexual maturation. Fish were kept at 28 C in a 14:10 h light:dark cycle and maintained in a recirculation system to supply sufficient oxygen. Zebrafish were fed a tropical fish commercial flake food twice a day. Embryos were obtained from healthy adult fish with a ratio of 1:2 for
PCB126 INDUCED OXIDATIVE STRESS IN ZEBRAFISH EMBRYO
3
TABLE I. Sequences of primer pairs used in the real-time quantitative PCR Target Gene Sod1 Cat Gpx b-Actin
Primer Sequences F: 50 -GGCCAACCGATAGTGTTAGA-30 R: 50 -CCAGCGTTGCCAGTTTTTAG-30 F: 50 -CCAGCCACGCTTCCTTGAGT-30 R: 50 -TCTCTCGGCTTCATTTAGCACCT-30 F: 50 -GCCCGCATTCAGATTCCTCA-30 R: 50 -AACGCACCACTTGGCCCTC-30 F: 50 -GATGCGGAAACTGGCAAAGG-30 R: 50 -GAGGAGGGCAAAGTGGTAAACG-30
female to male within 2 h of spawning, and cultured using a standard procedure described by Westerfield (1995).
PCB Exposure for Deformity Screening 3,30 ,4,40 ,5-Pentachlorobiphenyl (PCB126, purity > 99%), obtained from Dr. Ehrenstorfer (GmbH Germany) was dissolved in dimethylsulfoxide (DMSO). Six hundred healthy developing embryos at 3 hpf were selected (using a stereo microscope) and randomly divided into five groups (n 5 120 per treatment). The embryos were exposed to PCB126 concentrations of 0, 16, 32, 64, and 128 lg/L in 0.01% DMSO (v/ v) in 6 mL solution (containing 38.7 mM NaCl, 1.0 mM KCl, 1.7 mM HEPES-NaOH, pH 7.2, 2.4 mM CaCl2; Dong et al., 2002), and incubated in 6-well clear polystyrene multiwell culture plates (Corning, NY) at 28 C. In deformity screening, each dose exposure was performed in triplicate and each replicate contained 40 embryos per well. During the study, dead embryos were removed immediately and 50% of the test medium changed each day until the end of the experiment at 168 hpf. Developmental toxicity was assessed using two end points, malformation rates and mortality. Cumulative mortality was recorded every 12 h from 12 to 168 hpf. The dead identified as embryo condensation, missing heartbeat, or failure to develop somites, were removed immediately from the multiwell plates. The number of developmental malformations including pericardial and yolk sac edema, spinal curvature, and head edema, were recorded every 12 h and imaged using a Nikon microscope (Tokyo, Japan) from 12 to 144 hpf.
Determination of CuZn-Sod, Cat, Gpx Activity, and MDA Content One thousand and five hundred healthy embryos were exposed to five concentrations of PCB126 (0, 16, 32, 64, and 128 lg/L) as above in triplicate (n 5 300 per treatment). At four time points, 24, 72, 96, and 132 hpf, 15–20 embryos/larvae from each treatment in triplicate (n 5 45–60 per timepoint) were collected and homogenized with 10 volumes of cold buffer [consisting of 10 mmol/L sucrose, 10 mmol/L Tris–HCl, and 0.1 mmol/L EDTA-2Na (pH 7.4)]. The homogenate was centrifuged at 2000 3 g for 10 min at 4 C,
Product Length (bp)
Accession Number
205
Y12236.1
127
AF170069.1
95
BC046044.1
116
AF057040
and the supernatant used for determination of CuZn-Sod, Cat, Gpx activities, and MDA and protein content, using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on the method described by Liu et al. (2008). Of the 300 embryos, only 180–240 embryos (45–60 3 4 time points) were used in the analysis. The excess was to allow for the deaths that occurred during the experiment. MDA was quantified by the thiobarbituric acid (TBA) method according to the manufacturer’s instructions, and the red color produced by MDA reacting with TBA was measured spectrophotometrically at 532 nm.
Gene Expression Studies RNA Extraction and Reverse Transcription Nine hundred embryos (used in excess to allow for any deaths during the experiment) were used in triplicate with 180 per treatment (0, 16, 32, 64, and 128 lg/L). At four time points, 24, 72, 96, and 132 hpf, 10 embryos/larvae of each treatment in triplicate (n 5 30) were collected. Total RNA was extracted by Trizol Reagent (Invitrogen, Carlsbad, CA). The quality of RNA was evaluated based on the integrity of 28s and 18s by 2% agarose gel electrophoresis. First-strand complementary DNA (cDNA) was synthesized from 3 lg of total RNA using a reverse transcriptase kit of TaKaRa Biochemicals (Dalian, China).
Quantitative Real-Time Polymerase Chain Reaction The quantitative real-time polymerase chain reaction (qPCR) was performed using the SYBR green Premix Ex TaqTM kit (TaKaRa, Dalian, China), and the reaction measured using a Chromo4TM detector (Bio-rad, Hercules, CA). The gene-specific primers were designed with the Primer Premier 5.0 software. Primers sequences, amplicon sizes, and annealing temperatures are shown in Table I. The housekeeping gene b-actin of zebrafish was used as an internal standard to eliminate variations in mRNA and cDNA quantity and quality. 20 lL qPCR reaction was performed with 10 lL 2 3 SYBR green Premix, 0.2 lM of each target genespecific primer, and 1lL cDNA template. The housekeeping gene and three target genes were evaluated by three-step
Environmental Toxicology DOI 10.1002/tox
4
LIU ET AL.
activities and related gene mRNA expression were carried out with SPSS 13.0 (SPSS, Chicago, IL). Statistical differences were determined by one-way ANOVA followed by a post hoc Tukey’s HSD test. All data were expressed as mean 6 standard error of the mean (SEM). Values were considered statistically significant when P < 0.05 and highly significant when P < 0.01.
RESULTS Developmental Toxicity
Fig. 1. Dose-dependent increase in mortality of embryos at different PCB126 concentrations from 24- to 168-h postfertilization (hpf). Values are shown as mean 6 SEM of three replicates each with 40 embryos. Asterisk(s) represent statistically significant differences from the control at P < 0.05 (*) and P < 0.01 (**).
qPCR with the same thermal cycling conditions: denaturation for 45 s at 95 C, 40 cycles of 15 s at 95 C, annealing step of 15 s at 61 C, an elongation step of 30 s at 72 C, and a melting curve analysis performed to demonstrate the specificity of the PCR product as a single peak. The relative quantification of specific gene expression normalized for the bactin expression in each sample was expressed as a percentage of the control value as described by Livak and Schmittgen (2001).
Statistical Analysis Mortality and malformation rates data were analyzed using Excel (Microsoft 2003). Analysis of antioxidant enzyme
Fig. 2. Dose-dependent increase in malformation rates (% affected) of embryos exposed to different PCB126 concentrations from 48- to 144-h postfertilization (hpf). Values are shown as mean 6 SEM of three replicates each with 40 embryos. Asterisk(s) represent statistically significant differences from the control at P < 0.05 (*) and P < 0.01 (**).
Environmental Toxicology DOI 10.1002/tox
A large suite of developmental abnormalities and mortality induced by PCB126 were observed for 168 hpf. Although there was no obvious difference in mortality between PCB126treated groups and control group from 12 to 132 hpf, malformations increased significantly (P < 0.05) in the treated groups from 144 hpf onward (Fig. 1). As expected, the highest mortality was in the embryos exposed to 128 lg/L of PCB126 and was 100% at 168 hpf. Malformation rates were most pronounced at 72, 96, and 132 hpf exposure to 64 and 128 lg/L PCB126 and significantly increased (P < 0.01) from 132 hpf in all PCB126 treatments groups (Fig. 2), up to 100% at 132 hpf in 128 lg/L of PCB126-treated group. In general, both malformation rates and mortality increased in a dose- and timedependent manner from 60 hpf onward. Minor changes were detected in the highest treatment group until 60 h exposure to PCB126 but from 72 hpf onward, more and marked multiple deformities including pericardial edema and spinal curvature appeared and were most evident at 96 and 132 hpf (Fig. 3).
Antioxidant Enzyme Activity and MDA Content In general, compared to the control group, CuZn-Sod, Cat, and Gpx activities decreased in all groups during the experiment (Fig. 4). CuZn-Sod activity in PCB126-treated groups at 24 hpf was similar to the controls but was significantly lower (P < 0.01) in 128 lg/L PCB126-treated group at 72, 96, and 132 hpf [Fig. 4(A)]. CuZn-Sod activity decreased significantly (P < 0.01) by 29, 20, 37, and 28% on exposure to 16, 32, 64, and 128 lg/L PCB126, respectively, at 96 and132 hpf. Similarly, Cat activity also significantly decreased (P < 0.05) at 72 hpf in the 64 and 128 lg/L PCB126 groups [Fig. 4(B)]. At 96 and 132 hpf, Cat activity significantly decreased (P < 0.01) in all PCB126-treated groups and a marked decline to
Department of Veterinary Medicine, Agricultural College, Guangdong Ocean University, Zhanjiang 524088, China
2
College of Food Science and Technology, Guangdong Ocean University, Zhanjiang 524088, China
3
Faculty of Agriculture and Life Sciences, Lincoln University, Lincoln 7647, New Zealand
Received 31 March 2014; revised 17 August 2014; accepted 23 August 2014 ABSTRACT: 3,30 ,4,40 ,5-Pentachlorobiphenyl (PCB126) cause multiple adverse effects in organisms including animals and humans. Although PCB toxicities are linked to oxidative damage in rodents, the mechanism in early life stages of zebrafish is not clear. To explore the developmental toxicity mechanism of PCB126, three paradigms (toxicological phenotypes, biochemical changes, and molecular changes) were studied in 3-h postfertilization (hpf) zebrafish (Danio rerio) embryos exposed to different PCB126 concentrations (0, 16, 32, 64, and 128 lg/L) until 168 hpf. Developmental malformations, including pericardial and yolk sac edema, impaired lower jaw growth, spinal curvature, head edema and failure to inflate the swim bladder were observed, some as early as 72 hpf. Mortality was not apparent in early stages but significantly increased in a dose-dependent manner from 144 hpf onward. A dose-dependent significant increase in malformation rate was observed from 72 hpf onward with up to 100% at 132 hpf in embryos exposed to 128 lg/L of PCB126. Higher doses of PCB126 significantly decreased the copperzinc superoxide dismutase (CuZn-Sod), catalase (Cat), and glutathione peroxidase (Gpx) enzyme activities at 96, 132 hpf, but markedly declined from thereafter. PCB126 at 128 lg/L significantly increased the malondialdehyde content at 72, 96, and 132 hpf. The transcriptional gene expression of antioxidant enzymes Cat and Gpx was upregulated in embryos exposed to 64 lg/L of PCB126 at 24 and 96 hpf. Sod1 messenger RNA (mRNA) was low in embryos exposed to 32 lg/L at 72 and 96 hpf but was induced in embryos exposed to 64 and 128 lg/L doses at 132 hpf. Collectively, the results suggest oxidative stress as a major factor in the induction of multiple developmental abnormalities in early life stages of zebrafish exposed to PCB126. However, the relationship between the antioxidant enzyme activity and the C 2014 Wiley Periodicals, mRNA expression was not clear and the potential reasons for this are discussed. V Inc. Environ Toxicol 00: 000–000, 2014.
Keywords: PCB126; zebrafish embryo; developmental toxicity; antioxidant enzymes; mRNA
Corresponding to: R. Gooneratne; e-mail: Ravi.Gooneratne@lincoln. ac.nz and J.-J.Chen; e-mail: [email protected] Contract grant sponsor: International Science and Technology Cooperation Project of Guangdong Province, China.
Contract grant number: 2010B050600004. † These authors contributed equally to this work. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22044
C 2014 Wiley Periodicals, Inc. V
1
2
LIU ET AL.
INTRODUCTION Dioxin-like polychlorinated biphenyls (DlPCBs) are ubiquitous, lipophilic environmental contaminants responsible for persistence and biomagnification in the global ecosystem (Nfon and Cousins, 2006; Micheletti et al., 2007). 3,30 ,4,40 ,5-pentachlorobiphenyl (PCB126), the most representative coplanar congeners of DIPCBs, has similar structure and biological effects to 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), and therefore is considered the most toxic PCB congener. Some DIPCBs have been found in a variety of foodstuffs, including seafood and meat products, and also in breast milk (Baars et al., 2004; Sasamoto et al., 2006; Domingo and Bocio, 2007). Adverse effects of exposure to DIPCBs include reproductive toxicity, immunotoxicity, neurotoxicity, carcinogenesis, and developmental toxicity (Schantz, 1996; Gutleb et al., 2000; Bunaciu et al., 2007; Nagayama et al., 2007). Previous studies have demonstrated that TCDD and PCB126 exposure induce a suite of developmental abnormalities in chicken eggs (Jin et al., 2001; Blankenship et al., 2003) and fish embryos (Teraoka et al., 2002; Antkiewicz et al., 2005; Yamauchi et al., 2006). There is some evidence that both TCDD and PCBs induced toxicity is related to oxidative stress as indicated by increased production of reactive oxygen species (ROS), lipid peroxidation, and associated decline in antioxidant enzyme activities in chicks (Jin et al., 2001; Hilscherova et al., 2003), rats (Latchoumycandane et al., 2002a, 2002b, 2003; Murugesan et al., 2008), and fish (Schlezinger et al., 2006). Under normal physiological conditions, there is a balance between ROS production and activity of a family of antioxidant enzymes (Hilscherova et al., 2003). However, when the generation of ROS exceeds beyond the protective capacity of these enzymes, oxidative damage occurs (Lassen et al., 2008). Exposure of fish to PCB126 or PCB169 significantly increased ROS production in liver microsomes thereby inducing oxidative stress (Schlezinger et al., 2006). Interestingly, coexposure of zebrafish embryos to antioxidant vitamin E and PCB126 effectively protects against PCB126 induced morphological changes (Na et al., 2009). All these studies suggest
Abbreviations AHR Cat DlPCBs Gpx hpf h MDA NAC PCB ROS Sod TCDD
aryl hydrocarbon receptor catalase dioxin-like polychlorinated biphenyls glutathione peroxidase postfertilization malondialdehyde N-acetylcysteine 126 3,30 ,4,40 ,5-pentachlorobiphenyl reactive oxygen species superoxide dismutase 2,3,7,8-tetrachlorodibenzo-p-dioxin
Environmental Toxicology DOI 10.1002/tox
that PCB126 induced developmental toxicity is associated with oxidative stress. However, it is not known whether exposure of zebrafish embryos to PCB126 affects the antioxidant enzymes activities and related gene expression. The antioxidant enzymatic protection against ROS include superoxide dismutase (Sod), catalase (Cat), glutathione-Stransferase (Gst), and glutathione peroxidase (Gpx) enzymes (Lassen et al., 2008). Acute doses of TCDD or PCBs cause lipid peroxidation and significant oxidative damage to peritoneal and adhesion fibroblasts in the human (Saed et al., 2010) testicular and Sertoli cell (Krishnamoorthy et al., 2005). Twaroski et al. (2001) demonstrated that PCB77 decreased hepatic Gpx activity and Gpx1 messenger RNA (mRNA) gene expression in rats. However, Kern et al. (2002) showed that although TCDD induced oxidative stress enzymes in both adipocyte and liver of rats, the mRNA expression in these tissues was unaffected. Many studies have demonstrated that developmental and reproductive toxicities of TCDD and PCBs in mammal are associated with oxidative stress followed by changing the antioxidant enzymes activity and related gene expression, but the mechanism underlining cellular oxidative damage on developmental toxicity of DIPCBs in zebrafish is not clear. Monitoring and assessing the effects of aquatic contaminants is critical for protection of human health and the environment. Zebrafish is an established vertebrate model for pharmacology, ontogenetic development, and toxicology studies because of its small size, low cost, diverse adaptability, short breeding cycles, high fecundity, and transparent embryos (Dai et al., 2014). Zebrafish embryos are more sensitive than mammals to certain dioxin-like compounds, and early organogenesis is easy to observe (McGrath and Li, 2008; Yang et al., 2009). To better understand the mechanism of developmental toxicity induced by PCB126 including oxidative stress in zebrafish embryos, three paradigms, toxicological phenotypes (mortality and malformation rates), biochemical parameters [(CuZn-Sod, Cat, Gpx enzyme activities, and malondialdehyde (MDA) measurement], and molecular changes (Sod1, Cat, and Gpx mRNA expression) were studied from 3- to 168-h postfertilization (hpf), following exposure to PCB126.
MATERIALS AND METHODS Fish Maintenance and Embryos Collection Juvenile AB strain zebrafish (Danio rerio) were purchased from China Zebrafish Resources Center and cultured in an aquarium until sexual maturation. Fish were kept at 28 C in a 14:10 h light:dark cycle and maintained in a recirculation system to supply sufficient oxygen. Zebrafish were fed a tropical fish commercial flake food twice a day. Embryos were obtained from healthy adult fish with a ratio of 1:2 for
PCB126 INDUCED OXIDATIVE STRESS IN ZEBRAFISH EMBRYO
3
TABLE I. Sequences of primer pairs used in the real-time quantitative PCR Target Gene Sod1 Cat Gpx b-Actin
Primer Sequences F: 50 -GGCCAACCGATAGTGTTAGA-30 R: 50 -CCAGCGTTGCCAGTTTTTAG-30 F: 50 -CCAGCCACGCTTCCTTGAGT-30 R: 50 -TCTCTCGGCTTCATTTAGCACCT-30 F: 50 -GCCCGCATTCAGATTCCTCA-30 R: 50 -AACGCACCACTTGGCCCTC-30 F: 50 -GATGCGGAAACTGGCAAAGG-30 R: 50 -GAGGAGGGCAAAGTGGTAAACG-30
female to male within 2 h of spawning, and cultured using a standard procedure described by Westerfield (1995).
PCB Exposure for Deformity Screening 3,30 ,4,40 ,5-Pentachlorobiphenyl (PCB126, purity > 99%), obtained from Dr. Ehrenstorfer (GmbH Germany) was dissolved in dimethylsulfoxide (DMSO). Six hundred healthy developing embryos at 3 hpf were selected (using a stereo microscope) and randomly divided into five groups (n 5 120 per treatment). The embryos were exposed to PCB126 concentrations of 0, 16, 32, 64, and 128 lg/L in 0.01% DMSO (v/ v) in 6 mL solution (containing 38.7 mM NaCl, 1.0 mM KCl, 1.7 mM HEPES-NaOH, pH 7.2, 2.4 mM CaCl2; Dong et al., 2002), and incubated in 6-well clear polystyrene multiwell culture plates (Corning, NY) at 28 C. In deformity screening, each dose exposure was performed in triplicate and each replicate contained 40 embryos per well. During the study, dead embryos were removed immediately and 50% of the test medium changed each day until the end of the experiment at 168 hpf. Developmental toxicity was assessed using two end points, malformation rates and mortality. Cumulative mortality was recorded every 12 h from 12 to 168 hpf. The dead identified as embryo condensation, missing heartbeat, or failure to develop somites, were removed immediately from the multiwell plates. The number of developmental malformations including pericardial and yolk sac edema, spinal curvature, and head edema, were recorded every 12 h and imaged using a Nikon microscope (Tokyo, Japan) from 12 to 144 hpf.
Determination of CuZn-Sod, Cat, Gpx Activity, and MDA Content One thousand and five hundred healthy embryos were exposed to five concentrations of PCB126 (0, 16, 32, 64, and 128 lg/L) as above in triplicate (n 5 300 per treatment). At four time points, 24, 72, 96, and 132 hpf, 15–20 embryos/larvae from each treatment in triplicate (n 5 45–60 per timepoint) were collected and homogenized with 10 volumes of cold buffer [consisting of 10 mmol/L sucrose, 10 mmol/L Tris–HCl, and 0.1 mmol/L EDTA-2Na (pH 7.4)]. The homogenate was centrifuged at 2000 3 g for 10 min at 4 C,
Product Length (bp)
Accession Number
205
Y12236.1
127
AF170069.1
95
BC046044.1
116
AF057040
and the supernatant used for determination of CuZn-Sod, Cat, Gpx activities, and MDA and protein content, using assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on the method described by Liu et al. (2008). Of the 300 embryos, only 180–240 embryos (45–60 3 4 time points) were used in the analysis. The excess was to allow for the deaths that occurred during the experiment. MDA was quantified by the thiobarbituric acid (TBA) method according to the manufacturer’s instructions, and the red color produced by MDA reacting with TBA was measured spectrophotometrically at 532 nm.
Gene Expression Studies RNA Extraction and Reverse Transcription Nine hundred embryos (used in excess to allow for any deaths during the experiment) were used in triplicate with 180 per treatment (0, 16, 32, 64, and 128 lg/L). At four time points, 24, 72, 96, and 132 hpf, 10 embryos/larvae of each treatment in triplicate (n 5 30) were collected. Total RNA was extracted by Trizol Reagent (Invitrogen, Carlsbad, CA). The quality of RNA was evaluated based on the integrity of 28s and 18s by 2% agarose gel electrophoresis. First-strand complementary DNA (cDNA) was synthesized from 3 lg of total RNA using a reverse transcriptase kit of TaKaRa Biochemicals (Dalian, China).
Quantitative Real-Time Polymerase Chain Reaction The quantitative real-time polymerase chain reaction (qPCR) was performed using the SYBR green Premix Ex TaqTM kit (TaKaRa, Dalian, China), and the reaction measured using a Chromo4TM detector (Bio-rad, Hercules, CA). The gene-specific primers were designed with the Primer Premier 5.0 software. Primers sequences, amplicon sizes, and annealing temperatures are shown in Table I. The housekeeping gene b-actin of zebrafish was used as an internal standard to eliminate variations in mRNA and cDNA quantity and quality. 20 lL qPCR reaction was performed with 10 lL 2 3 SYBR green Premix, 0.2 lM of each target genespecific primer, and 1lL cDNA template. The housekeeping gene and three target genes were evaluated by three-step
Environmental Toxicology DOI 10.1002/tox
4
LIU ET AL.
activities and related gene mRNA expression were carried out with SPSS 13.0 (SPSS, Chicago, IL). Statistical differences were determined by one-way ANOVA followed by a post hoc Tukey’s HSD test. All data were expressed as mean 6 standard error of the mean (SEM). Values were considered statistically significant when P < 0.05 and highly significant when P < 0.01.
RESULTS Developmental Toxicity
Fig. 1. Dose-dependent increase in mortality of embryos at different PCB126 concentrations from 24- to 168-h postfertilization (hpf). Values are shown as mean 6 SEM of three replicates each with 40 embryos. Asterisk(s) represent statistically significant differences from the control at P < 0.05 (*) and P < 0.01 (**).
qPCR with the same thermal cycling conditions: denaturation for 45 s at 95 C, 40 cycles of 15 s at 95 C, annealing step of 15 s at 61 C, an elongation step of 30 s at 72 C, and a melting curve analysis performed to demonstrate the specificity of the PCR product as a single peak. The relative quantification of specific gene expression normalized for the bactin expression in each sample was expressed as a percentage of the control value as described by Livak and Schmittgen (2001).
Statistical Analysis Mortality and malformation rates data were analyzed using Excel (Microsoft 2003). Analysis of antioxidant enzyme
Fig. 2. Dose-dependent increase in malformation rates (% affected) of embryos exposed to different PCB126 concentrations from 48- to 144-h postfertilization (hpf). Values are shown as mean 6 SEM of three replicates each with 40 embryos. Asterisk(s) represent statistically significant differences from the control at P < 0.05 (*) and P < 0.01 (**).
Environmental Toxicology DOI 10.1002/tox
A large suite of developmental abnormalities and mortality induced by PCB126 were observed for 168 hpf. Although there was no obvious difference in mortality between PCB126treated groups and control group from 12 to 132 hpf, malformations increased significantly (P < 0.05) in the treated groups from 144 hpf onward (Fig. 1). As expected, the highest mortality was in the embryos exposed to 128 lg/L of PCB126 and was 100% at 168 hpf. Malformation rates were most pronounced at 72, 96, and 132 hpf exposure to 64 and 128 lg/L PCB126 and significantly increased (P < 0.01) from 132 hpf in all PCB126 treatments groups (Fig. 2), up to 100% at 132 hpf in 128 lg/L of PCB126-treated group. In general, both malformation rates and mortality increased in a dose- and timedependent manner from 60 hpf onward. Minor changes were detected in the highest treatment group until 60 h exposure to PCB126 but from 72 hpf onward, more and marked multiple deformities including pericardial edema and spinal curvature appeared and were most evident at 96 and 132 hpf (Fig. 3).
Antioxidant Enzyme Activity and MDA Content In general, compared to the control group, CuZn-Sod, Cat, and Gpx activities decreased in all groups during the experiment (Fig. 4). CuZn-Sod activity in PCB126-treated groups at 24 hpf was similar to the controls but was significantly lower (P < 0.01) in 128 lg/L PCB126-treated group at 72, 96, and 132 hpf [Fig. 4(A)]. CuZn-Sod activity decreased significantly (P < 0.01) by 29, 20, 37, and 28% on exposure to 16, 32, 64, and 128 lg/L PCB126, respectively, at 96 and132 hpf. Similarly, Cat activity also significantly decreased (P < 0.05) at 72 hpf in the 64 and 128 lg/L PCB126 groups [Fig. 4(B)]. At 96 and 132 hpf, Cat activity significantly decreased (P < 0.01) in all PCB126-treated groups and a marked decline to
