TBBPA Induces Developmental Toxicity, Oxidative Stress, and Apoptosis in Embryos and Zebrafish Larvae (Danio rerio) Shengmin Wu, Guixiang Ji, Jining Liu, Shenghu Zhang, Yang Gong, Lili Shi Nanjing Institute of Environmental Sciences/Key Laboratory of Pesticide Environmental Assessment and Pollution Control, Ministry of Environmental Protection, Nanjing 210042, China
Received 15 December 2014; revised 6 February 2015; accepted 15 February 2015 ABSTRACT: Tetrabromobisphenol A (TBBPA) is currently one of the most frequently used brominated flame retardants and can be considered as a high production volume chemical. In this study, zebrafish embryos and larvae served as a biological model to evaluate TBBPA-induced developmental toxicity, oxidative stress, oxidant-associated gene expression, and cell apoptosis. Abnormalities, including hyperemia and pericardial edema, were induced in zebrafish larvae. The results showed that toxicity endpoints such as hatching rate, survival rate, malformation rate, and growth rate had a significant dose–response relationship with TBBPA. Further studies revealed that TBBPA did not alter the enzyme activities of Copper/Zinc Superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathioneperoxidase (GPx) at 0.10 mg/L, but decreased activities following exposure to 0.40, 0.70, and 1.00 mg/L. Despite the significantly decreased gene expression of Cu/Zn-SOD, CAT, and GPx1a in the 1.00 mg/L treatment group, other treatments (0.10, 0.40, 0.70 mg/L) did not alter gene expression. Moreover, Acridine orange staining results showed that apoptotic cells mainly accumulated in the brain, heart, and tail, indicating possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. Histological analysis also showed evidence of obvious heart impairment in TBBPA-treated groups. This study provides new evidence on the developmental toxicity, oxidative stress, and apoptosis of embryos and zebrafish larvae, which is important for the evaluation of environmental toxicity and chemical risk. C 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. V
Keywords: TBBPA; zebrafish; developmental toxicity; oxidative stress; apoptosis
INTRODUCTION
Correspondence to: S. Wu; e-mail: [email protected] Contract grant sponsor: High-technology Research, Development Program of the Ministry of Science and Technology of China. Contract grant number: No. 2013AA06A308. Contract grant sponsor: Central Public-interest Scientific Institution Basal Research Fund. Contract grant sponsor: Project of Technology Development Research for Scientific Research Institutes. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22131
Currently, tetrabromobisphenol A (TBBPA) is the largest brominated flame retardant chemical in production, which is used to improve the fire safety of electrical and electronic equipment (BSEF, 2008; ECB, 2008). The abundant production and use of brominated flame retardants has raised concerns regarding their fate and effects on the environment. The widespread use of TBBPA and its contaminants is frequently detected in the environment (Yang et al., 2012; Huang et al., 2014). It is typically detected at concentrations of parts per million (ppm) in sediments and sewage sludge near brominate flame retardant production facilities, and in
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parts per billion (ppb) at other sites (Hakk and Letcher, 2003; Hale et al., 2006; Kuramochi et al., 2008). Toxicity tests have shown that TBBPA is toxic to organisms, especially to aquatic animals. It is acutely toxic at low concentrations as shown in fish toxicity studies. Bluegill sunfish, exposed to TBBPA in water, became agitated and exhibit abnormal swimming behavior, while rainbow trout exhibit irritation, twitching, erratic swimming, dark discoloration, and labored respiration. Furthermore, fathead minnow show reduced survival of young at hatch and reduced survival and growth after 30 days (de Wit, 2002).The experiments in zebrafish suggest that during development, TBBPA may be more toxic than either bisphonel (BPA) or TBBPA dimethyl ether (McCormick et al., 2010; Kang et al., 2007). Zebrafish eggs exposed to TBBPA develop malformations and pericardial fluid accumulation, and some embryos fail to hatch (Kuiper et al., 2007). Recently, a study revealed decreased reproductive success in zebrafish exposed to environmentally relevant TBBPA concentrations (Kuiper et al., 2008). TBBPA is known to act as an immunotoxicant, a thyroid hormone disruptor, and cause neurobehavioral effects (Nkajima et al., 2009; Chan et al., 2011; Viberg et al., 2011). However, few studies have focused on the relationship between TBBPA, oxidative stress, and apoptosis. Recently, several studies have indicated that TBBPA may induce oxidative stress through the generation of reactive oxygen species (ROS) and cause apoptosis (Zatecka et al., 2013; Ritola et al., 2002). The effects of TBBPA on tubifex (Monopylephorus limosus) worms has revealed that the activities of several enzymes, namely superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase, are inhibited (Yaning et al., 2008), while studies on Eisenia fetida have shown that TBBPA induces the generation of ROS (Xue et al., 2009). Moreover, studies on freshwater Carassius auratus have shown that SOD and glutathione reductase (GR) are highly sensitive to TBBPA (Yan et al., 2007). Fish embryos have a weak antioxidant defense capacity, making them more sensitive to the teratogenic effects of oxidative compounds. Therefore, they are one of the most suitable organisms to act as bioindicator of the aquatic environment (Embry et al., 2010). Zebrafish embryos and larvae have all the advantages of an in vitro model and great efforts have been made to use them as tools for risk assessments of toxic chemicals in ecotoxicology studies. In addition to the activities of antioxidants such as SOD, CAT, and glutathione peroxidase (GPx), changes in the gene expression of these enzymes have been suggested to be markers of toxicity (Boix et al., 2013). In this study, the toxic effects of TBBPA on zebrafish larvae and embryos were evaluated. In addition, the enzyme activities of SOD, CAT, and GPx, as well as the mRNA expression levels of genes encoding for these antioxidants was monitored. Moreover, apoptosis in embryos and larvae was monitored using acridine orange staining after TBBPA
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exposure. Biopsies were also performed to assess apoptosis in organisms.
MATERIALS AND METHODS Chemicals TBBPA was obtained from Sigma-Aldrich (>98% purity). Stock solutions were stored at 220 C and prepared with 100% (v/v) dimethyl sulfoxide (DMSO, Sigma-Aldrich), which was used as a solvent control.
Experimental Animals The AB strain of zebrafish was obtained from the China Zebrafish Resource Center. Adult male and female fish were housed separately at ambient temperature (27 6 0.5 C) in holding tanks supplied with a continuous flow of aerated water for 7 days with a light:dark cycle of 14:10 h, pH from 6.0 to 7.5, and oxygen levels exceeding 80% (v/v) of the air saturation value. Fertilized zebrafish eggs were obtained from spawning adults in groups of male and female zebrafish at a ratio of 2:1 in tanks overnight. Spawning was induced in the morning when the light was turned on.
Dilution Medium The dilution water used was reconstituted water (ISO-63411982): Stock solution I: Cacl2 solution was prepared by dissolving 111.0065 g CaCl2 into 5000 mL deionized water. Stock solution II: MgSO4 solution was prepared by dissolving 61.6262 g MgSO4.7H2O into 5000 mL deionized water. Stock solution III: NaHCO3 solution was prepared by dissolving 32.3790 g NaHCO3 into 5000 mL deionized water. Stock solution IV: KCl solution was prepared by dissolving 2.8738 g KCl into 5000 mL deionized water. All chemicals were of analytical grade. Three days before the test, 1 L of each of solutions I–IV was mixed and the total volume was made up to 100 L with deionized water. The dilution water was aerated until oxygen saturation was achieved, and it was then stored for approximately 2 days without further aeration before use. The pH was 7.80, water hardness was approximately 245 mg/L(as CaCO3), and Ca/Mg was 4:1.
Exposure and Experimental Design In this study, groups of zebrafish embryos were exposed to the test solutions of TBBPA with nominal concentrations of 0.10, 0.40, 0.70, and 1.00 mg/L. The concentrations were selected based on a range-finding test that identified the concentration that would induce obvious effects during exposure. Solvent control groups [0.005% (v/v) DMSO] were also included in the study. Test solution (50 mL) was filled
TBBPA INDUCES DEVELOPMENTAL TOXICITY, OXIDATIVE STRESS, AND APOPTOSIS
TABLE I. Forward (F) and reverse (R) primers used for real-time PCR Gene
Accession No
Primer (50 to 30 )
CAT
AF170069
Cu/Zn-SOD
Y12236
GPx1a
NM_001007281
b-actin
NM_131031
F-agggcaactgggatcttaca R-tttatgggaccagaccttgg F-ggccaaccgatagtgttaga R-ccagcgttgccagtttttag F-caccctctgtttgcgttcc R-ctctttaatatcagcatca F-aagtgcgacgtggaca R-gtttaggttggtcgttcgtttga
in each test vessel under 72 h renewal conditions. Three replicates were assigned for each treatment group and control group, while the initial number of eggs was 30 for each group. The test embryos within 4 h postfertilization (hpf) were examined under a dissecting microscope, and those that had developed normally and had reached the gastrula stage were selected for further testing. During the test, the following conditions were maintained: Temperature for zebrafish embryos: 25 6 1.0 C; Oxygen concentration: > 60% (v/v) of the air saturation value; Duration: 8 days.
Apical observations performed on each tested embryo include hatching rate, survival rate, as well as abnormal appearance. At the end of the test, total length measurements and individual weights (dry weights, 24 h at 60 C) of individuals were measured.
Biochemical Assays Zebrafish larvae at 48, 96, 144, and 192 hpf were homogenized, respectively, in Tris-HCl buffer (100 mM, pH 7.4). Homogenates were centrifuged at 12,000g for 15 min at 4 C, and the supernatants were collected for biochemical parameter analysis. The activities of Copper/Zinc Superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathioneper oxidase (GPx) were assayed using diagnostic reagent kits (Sangon Biotech, Shanghai, China) according to the specified instructions from the manufacturers. Cu/Zn-SOD activity was assayed based on a modified method (Marklund et al., 1974). CAT activity was determined according to Sinha’s method (Sinha, 1972). GPx activity was measured according to Hafeman et al. (1974), which was based on a modified method using 5,5’-dithiobis-2-nitrobenzoic acid.
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trophotometry at 260 nm, and RNA purity was verified by the 260/280 nm ratio of 1.8 or greater. The cDNA was synthesized using the reverse transcriptase kit (Toyobo, Japan) in accordance with the product’s protocol. Oligonucleotide primes (Table I) were used to detect the gene expression of b-actin, Cu/Zn-SOD, CAT, and GPx1a using the SYBR green system (Toyobo, Japan). Real-time polymerase chain reaction (quantitative PCR) was carried out with the CFX96 Real-Time PCR Detection System (BioRad, USA) in sterile 96-well PCR plates. The reaction mixtures included 12.5 lL of 23SYBR Premix Ex TaqTM, 0.5 lL of PCR Forward and Reverse primers, and 1 lL of cDNA. The PCR conditions were as follows: 95 C for 30 s, followed by 40 cycles of 95 C for 5 s, 60 C for 30 s.
Acridine Orange (AO) Staining to Detect Apoptosis According to the test method (Tucker and Lardelli, 2007), one- to four-cell stage embryos and larvae (n 5 10) after 96 h TBBPA exposure were collected and divided into two groups: 1.0 mg/L treatment group and control group. The embryos and larvae stained with Acridine Orange (AO) (2 lg/mL in Hank’s medium) were wrapped with foil to prevent fluorescence quenching for 30 min and washed repeatedly with Hank’s medium. Stained tissues were pictured as whole mounts under a fluorescence microscope (Leica excitation light, 473 nm).
Histological Processing Larvae treated at 96 hpf with 1.0 mg/L TBBPA and the control group were fixed with 4% (w/v) paraformaldehyde overnight at 4 C, dehydrated with ethanol, cleared in xylene, and embedded in paraffin. After routine tissue processing and paraffin embedding, whole-body serial sections were cut at approximately 5-lm intervals. Sections were stained with hematoxylin and eosin, and observed under the microscope.
Statistical Analysis Values are expressed as the mean 6 standard error of the mean for all experiments. CAT, Cu/Zn-SOD, and GPx data were analyzed by one-way analysis of variance. When statistically significant differences were found between treatment groups, Dunnett’s test was used to determine which treatments were significantly different from unexposed controls. All statistical analyses were conducted using SPSS 17.0 and the threshold level of significance was set at p < 0.05.
Gene Expression
RESULTS
Total RNA was extracted from 20 embryos or hatched larvae using Trizol reagent (Takara Biochemicals, Dalian, China) following the manufacturer’s instructions. Total RNA concentrations were quantified immediately by ultraviolet spec-
Embryonic Dose Response Studies Zebrafish embryos were exposed to TBBPA to determine which concentration would affect the hatching rate, survival
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Fig. 1. (A) Hatchability rate, (B) survival rate, (C) cumulative malformation, as well as larvae length in zebrafish embryos and larvae exposed to different TBBPA concentrations (0.0, 0.10, 0.40, 0.70, 1.00 mg/L) for (D) 8 days (D). Values are the mean6 standard error. Values significantly different from the control are indicated by an asterisk (one-way analysis of variance, followed by the Dunnett’s test (SPSS 17.0), *p 0.05, **p 0.01).
rate, growth rate, and induce malformation. The toxic end points were examined after 8 days of TBBPA exposure. The hatching rates were, in the control group 83.3 6 4.02%, and 84.5 6 4.60%, 82.2 6 4.70%, and 82.2 6 4.70% in the 0.10, 0.40, and 0.70 mg/L treatment groups, respectively. No significant differences were observed between all groups. In contrast, the hatching rate in the 1.00 mg/L treatment group was 78.9 6 2.40%, which was significantly different compared with the control [P < 0.05; Fig. 1(A)]. The survival rate of embryos/larvae after TBBPA exposure for 8 days was 63.3 6 5.30% in 0.7 mg/L treatments, and 27.8 6 18.3% in1.0 mg/L treatments, respectively, which were significantly decreased relative to 87.8 6 2.2% in the control treatments. The survival rate of embryos/larvae in the 0.10 and 0.40 mg/L treatment groups was 90.0 6 3.70% and 88.9 6 4.30%, respectively, which was not significantly different from the control group [Fig. 1(B)]. TBBPA exposure for 8 days resulted in teratogenesis involving hyperemia and pericardial edema (Fig. 2). TBBPA exposure induced malformation rates of 17.8 6 10.3%, 74.4 6 2.63%, and 93.3 6 3.59% for the 0.40, 0.70, and
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1.00 mg/L treatment groups, respectively [Fig. 1(C)], which were significantly different from the control group. The length of larvae after 8 days TBBPA exposure was measured to assess whether TBBPA could impact zebrafish embryo and larvae growth. The mean length in the control group was 4.29 6 2.54 mm. No significant differences were observed between the control and 0.10 and 0.40 mg/L treatment groups. However, the 0.70 and 1.00 mg/L treatment groups resulted in lengths of 3.94 6 4.37 mm and 3.67 6 7.20 mm, respectively, which were significantly different when compared with the control [Fig. 1(D)].
Cu/Zn-SOD, CAT, and GPx Activities As indices of antioxidant status, the activities of Cu/ZnSOD, CAT, and GPx were measured to evaluate the presence of oxidative stress in embryonic zebrafish following exposure to TBBPA (Fig. 3). There was no significant change in Cu/Zn-SOD activity in the 0.10 mg/L treatment group relative to the control. While a significant decrease in Cu/Zn-SOD activity was observed in the 0.40, 0.70, and 1.00 mg/L exposure groups when compared with the control
TBBPA INDUCES DEVELOPMENTAL TOXICITY, OXIDATIVE STRESS, AND APOPTOSIS
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Fig. 2. Morphological abnormalities including hyperemia, pericardial edema, and tail deformities were caused by 0.4 mg/L TBBPA and higher concentrations. The control group had a normal appearance (A) and (C). Representative malformations are indicated by arrows in (B) and (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
after 3, 5, and 8 dpf [Fig. 3(A)]. Similarly, a significant decrease in CAT activity was observed in all groups, except the 0.10 mg/L group, when compared with control [Fig. 3(B)]. There was no change in GPx activity in the 0.10 mg/L treatment group, however, there was a significant decrease in activity in the 0.40, 0.70, and 1.00 mg/L treatment groups after 3, 5, and 8 dpf when compared with the control [Fig. 3(C)].
Cu/Zn-SOD, CAT, and GPx1a Gene Expression The Cu/Zn-SOD, CAT, and GPx1a gene expression were suppressed in embryos and larvae exposed to 1.00 mg/L TBBPA at 1, 3, 5, and 8 dpf (Fig. 4). There was no significant change in GPx1a gene expression in the 0.10, 0.40, and 0.70 mg/L treatment groups compared with control at the four detection times. Although significant differences in gene expression were observed between the individual treatment groups and the control group for Cu/Zn-SOD and
CAT, there were no obvious changes overall for in the 0.10, 0.40, and 0.70 mg/L treatment groups.
Analysis of Apoptosis AO staining was performed to detect TBBPA-induced apoptosis in embryos and larvae. After 5 dpf of treatment, notable signs of apoptosis were found to have mainly accumulated in the brain, heart, and tail (Fig. 5), suggesting possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. A suite of abnormalities including hyperemia and pericardial edema were induced in zebrafish embryos and larvae after TBBPA exposure.
Histopathology Histological analysis was performed using larvae paraffin sections. The morphology of cardiac tissue was evaluated visually under the microscope (Fig. 6). In the 1.00 mg/L TBBPA treatment group, pathological patterns in the
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DISCUSSION This study adopted zebrafish embryos and larvae as a biological model to evaluate TBBPA-induced developmental toxicity, oxidative stress, oxidant-associated gene expression,
Fig. 3. The activities of Cu/Zn-superoxide dismutase (Cu/ Zn-SOD, A), catalase (CAT, B), and glutathione peroxidase (Gpx, C) in zebrafish embryos and larvae after TBBPA exposure at 0.10, 0.40, 0.70, and 1.00 mg/L. Results are the mean 6 standard error of triplicate samples. Values significantly different from the control are indicated by an asterisk (one-way analysis of variance, followed by the Dunnett’s test (SPSS 17.0), *p 0.05, **p 0.01). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
morphology of cardiac tissue were observed with a decrease in myocardial cells and heart linearization when compared with the control. The results showed that TBBPA exposure restrained the heart development and caused cardiac chambers elongated.
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Fig. 4. Expression of Cu/Zn-SOD, CAT, and GPx1a in zebrafish embryos and larvae after exposure to 0.10, 0.40, 0.70, and 1.00 mg/L of TBBPA. Results are the mean 6 standard error of triplicate samples. Values significantly different from the control are indicated by an asterisk (one-way analysis of variance, followed by the Dunnett’s test (SPSS 17.0), *p 0.05, **p 0.01). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
TBBPA INDUCES DEVELOPMENTAL TOXICITY, OXIDATIVE STRESS, AND APOPTOSIS
Fig. 5. Apoptosis induced by TBBPA in zebrafish embryos and larvae. Zebrafish embryos and larvae were exposed to 1.00 mg/L TBBPA for 5 dpf and stained with acridine orange. Apoptotic cells accumulated in the brain, cardiac tissue, and tail of TBBPA-treated groups when compared with the control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and cell apoptosis. Abnormalities including hyperemia and pericardial edema were induced in zebrafish embryos and larvae. The results showed that SOD, CAT, and Gpx activities were repressed following exposure to 0.40, 0.70, and 1.00 mg/L TBBPA. Despite the significant decrease in Cu/ Zn-SOD, CAT, and GPx1a gene expression in the 1.00 mg/ L treatment group, gene expression in the 0.10, 0.40, and 0.70 mg/L treatment groups remained unaltered relative to the control. These results indicate that TBBPA induces oxidative stress and apoptosis in zebrafish embryos and larvae.
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With increasing concentrations of TBBPA, embryo and larvae abnormalities increased and the survival rate decreased. TBBPA exposure caused lethal effects on zebrafish embryos and larvae. Furthermore, exposure of embryos to increasing concentrations of TBBPA during early development resulted in a significant increase in malformation rate, as well as a significant decrease in hatching, growth, and survival rate. In addition, the results also showed that TBBPA may interfere with embryonic heart development and it indicated that there was a lack of formed elements in the circulating blood. Oxidative stress is a complex phenomenon involved in physiological and pathological processes. During the first stages of the organogenesis period, embryos present with weak antioxidant defense capacity, making them more sensitive to the teratogenic effects of test substances. As oxidative stress is the first response to environmental stressors, the embryos may initially initiate antioxidant and detoxification responses to TBBPA exposure. Evidence indicates disequilibrium between the formation of ROS and the embryo antioxidant defense system, which can affect embryo development and function, with possible etiology consequences of different congenital anomalies. Therefore, antioxidant defense is important in modulating oxidative stress-mediated events. The involvement of antioxidative enzymes, such as SOD, CAT, and GPx, has been suggested to play an important role in protecting cells from oxidative stress. SOD is crucial in catalyzing superoxide radicals to H2O2 and O2. CAT catalyzes the transformation of H2O2 to water, and GPx transforms hydroperoxides to hydroxyl compounds using GSH as a substrate. This study showed that relatively low concentrations of TBBPA do not induce significant changes in antioxidant enzyme activities (SOD, CAT, GPx) to mitigate oxidative stress, whereas relatively
Fig. 6. Control larvae (A) and larvae exposed to 1.0 mg/L TBBPA (B). Myocardial cells in the 1.0 mg/L TBBPA group were fewer and thinner heart was observed compared with the control (arrows). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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high concentration of TBBPA decrease enzyme activities to counter the oxidative stress. This result indicates that there is a threshold value. When the test concentration is below the threshold value, there is no significant change in enzyme activity; while enzyme activity is significantly inhibited when test concentrations are above the threshold. This threshold effect may explain the difference in SOD, CAT, and GPx activities following exposure to varying concentrations of TBBPA. Recently, research has focused on the link between oxidative stress enzyme activities and the related gene expression. In this study, we showed that Cu/Zn-SOD, CAT, and GPx1a gene expression is suppressed in embryos and larvae exposed to 1.00 mg/L TBBPA at 1, 3, 5, and 8 dpf. These results were consistent with the antioxidant enzyme activities. However, there was no significant decrease in Cu/ZnSOD, CAT, and GPx1a gene expression in the 0.40 and 0.70 mg/L exposure groups relative to the control, which was not consistent with the related enzyme activities. This result may be attributed to gene expression characteristics. When zebrafish are exposed to certain concentrations of the herbicide atrazine, antioxidant enzyme activity is inconsistent with related gene expression changes (Jin et al., 2009). Similarly, studies have shown that SOD, CAT, and the activities of other antioxidant enzymes decreased when zebrafish were exposed to perfluorinated dodecanoic acid and copper metal, but the related genes did not change (Craig et al., 2007; Liu et al., 2008). These discrepancies may be because gene expression is a relatively quick process, while enzyme activity is lengthy and involves complex processes, such as translation and post-translational modifications, which are subject to environmental impact. However, to fully understand the mechanism of TBBPA oxidative damage, further studies must be performed. AO staining revealed that apoptosis mainly occurred in the brain, heart, and tail, which suggests that the blood circulation system and nervous system may be potential targets of TBBPA. At the same time, we observed hyperemia and pericardial edema in zebrafish embryos and larvae. These effects were possibly because of the high percentage of apoptotic cells in the related sites and may partly explain the observed heart malformation. Another explanation is that hyperemia and pericardial edema may be induced by circulating blood cells and circulatory system failure, subsequently resulting in a decrease in myocardial cells and heart thinner, as observed by pathological analysis. It can be hypothesized that TBBPA-induced apoptosis in zebrafish embryos and larvae occurs through the production of excess ROS. This leads to altered gene expression regulation of oxidative stress mediators, such as SOD, CAT, and GPx. These enzymes play an important role in mitigating oxidative stress. However, when relatively high concentrations of TBBPA are present, enzyme activities decrease, and apoptosis is exacerbated in the brain, heart, and tail, which may induce blood circulation and nervous system develop-
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mental abnormalities. In any case, to be confirmed, this hypothesis needs further investigation.
CONCLUSION In summary, TBBPA can cause developmental toxicity in zebrafish embryos and larvae as a result of its effects on the actions of oxidative stress, related gene expression, and cell apoptosis. We conclude that: (1) The abnormalities including hyperemia and pericardial edema could be induced by TBBPA in zebrafish embryos and larvae. (2) The results showed there was a significant dose–response relationship between toxicity endpoints (hatching rate, survival rate, malformation rate, and growth rate) and TBBPA concentration. (3) The study showed that 0.10 mg/L TBBPA did not induce significant changes in antioxidant enzyme activities (SOD, CAT, GPx), while 0.40, 0.70, and 1.00 mg/L TBBPA decreased enzyme activities. (4) The Cu/Zn-SOD, CAT, and GPx1a gene expression were suppressed in embryos and larvae exposed to 1.00 mg/L TBBPA at 1, 3, 5, and 8 dpf. (5) Histological analysis showed evidence of obvious heart impairment in the TBBPA exposure group when compared with the control (6) AO staining results showed that apoptotic cells mainly accumulated in the brain, heart, and tail, indicating possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. These study results will help our understanding of the mechanism of TBBPA-induced oxidative stress and apoptosis. This study also shows that zebrafish embryos can serve as an effective model for studying developmental toxicity and oxidative stress, which is of great importance in evaluating environmental toxicity and the risks associated with various chemicals. The authors would like to thank Project of Technology Development Research for Scientific research institutes for their assistance for the research. Disclosure: The authors declare that they have no competing interests.
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Kuramochi H, Kawamoto K, Miyazaki K, Nagahama K, Maeda K, Li XW, Shibata E, Nakamura T, Sakai S. 2008. Determination of physicochemical properties of tetrabromobisphenol A. Environ Toxicol Chem 27:2413–2418.
Chan WK, Chan KM. 2011. Disruption of the hypothalamicpituitary axis in zebrafish embryo-larvae following waterborne exposure to BDE-47, TBBPA and BPA. Aquat Toxicol108: 106–111.
Liu Y, Wang J, Wei Y, Zhang H, Xu M, Dai J. 2008. Induction of time-dependent oxidative stress and related transcriptional effects of perfluorododecanoic acid in zebrafish liver. Aquat Toxicol 89:242–250.
Xue Y, Gu X, Wang X, Sun C, Xu X, Sun J, Zhang B. 2009. The hydroxyl radical generation and oxidative stress for the earthworm Eisenia fetida exposed to tetrabromobisphenol A. Ecotoxocology18:693–699.
Environmental Toxicology DOI 10.1002/tox
Received 15 December 2014; revised 6 February 2015; accepted 15 February 2015 ABSTRACT: Tetrabromobisphenol A (TBBPA) is currently one of the most frequently used brominated flame retardants and can be considered as a high production volume chemical. In this study, zebrafish embryos and larvae served as a biological model to evaluate TBBPA-induced developmental toxicity, oxidative stress, oxidant-associated gene expression, and cell apoptosis. Abnormalities, including hyperemia and pericardial edema, were induced in zebrafish larvae. The results showed that toxicity endpoints such as hatching rate, survival rate, malformation rate, and growth rate had a significant dose–response relationship with TBBPA. Further studies revealed that TBBPA did not alter the enzyme activities of Copper/Zinc Superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathioneperoxidase (GPx) at 0.10 mg/L, but decreased activities following exposure to 0.40, 0.70, and 1.00 mg/L. Despite the significantly decreased gene expression of Cu/Zn-SOD, CAT, and GPx1a in the 1.00 mg/L treatment group, other treatments (0.10, 0.40, 0.70 mg/L) did not alter gene expression. Moreover, Acridine orange staining results showed that apoptotic cells mainly accumulated in the brain, heart, and tail, indicating possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. Histological analysis also showed evidence of obvious heart impairment in TBBPA-treated groups. This study provides new evidence on the developmental toxicity, oxidative stress, and apoptosis of embryos and zebrafish larvae, which is important for the evaluation of environmental toxicity and chemical risk. C 2015 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2015. V
Keywords: TBBPA; zebrafish; developmental toxicity; oxidative stress; apoptosis
INTRODUCTION
Correspondence to: S. Wu; e-mail: [email protected] Contract grant sponsor: High-technology Research, Development Program of the Ministry of Science and Technology of China. Contract grant number: No. 2013AA06A308. Contract grant sponsor: Central Public-interest Scientific Institution Basal Research Fund. Contract grant sponsor: Project of Technology Development Research for Scientific Research Institutes. Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.22131
Currently, tetrabromobisphenol A (TBBPA) is the largest brominated flame retardant chemical in production, which is used to improve the fire safety of electrical and electronic equipment (BSEF, 2008; ECB, 2008). The abundant production and use of brominated flame retardants has raised concerns regarding their fate and effects on the environment. The widespread use of TBBPA and its contaminants is frequently detected in the environment (Yang et al., 2012; Huang et al., 2014). It is typically detected at concentrations of parts per million (ppm) in sediments and sewage sludge near brominate flame retardant production facilities, and in
C 2015 Wiley Periodicals, Inc. V
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parts per billion (ppb) at other sites (Hakk and Letcher, 2003; Hale et al., 2006; Kuramochi et al., 2008). Toxicity tests have shown that TBBPA is toxic to organisms, especially to aquatic animals. It is acutely toxic at low concentrations as shown in fish toxicity studies. Bluegill sunfish, exposed to TBBPA in water, became agitated and exhibit abnormal swimming behavior, while rainbow trout exhibit irritation, twitching, erratic swimming, dark discoloration, and labored respiration. Furthermore, fathead minnow show reduced survival of young at hatch and reduced survival and growth after 30 days (de Wit, 2002).The experiments in zebrafish suggest that during development, TBBPA may be more toxic than either bisphonel (BPA) or TBBPA dimethyl ether (McCormick et al., 2010; Kang et al., 2007). Zebrafish eggs exposed to TBBPA develop malformations and pericardial fluid accumulation, and some embryos fail to hatch (Kuiper et al., 2007). Recently, a study revealed decreased reproductive success in zebrafish exposed to environmentally relevant TBBPA concentrations (Kuiper et al., 2008). TBBPA is known to act as an immunotoxicant, a thyroid hormone disruptor, and cause neurobehavioral effects (Nkajima et al., 2009; Chan et al., 2011; Viberg et al., 2011). However, few studies have focused on the relationship between TBBPA, oxidative stress, and apoptosis. Recently, several studies have indicated that TBBPA may induce oxidative stress through the generation of reactive oxygen species (ROS) and cause apoptosis (Zatecka et al., 2013; Ritola et al., 2002). The effects of TBBPA on tubifex (Monopylephorus limosus) worms has revealed that the activities of several enzymes, namely superoxide dismutase (SOD), catalase (CAT), and glutathione-S-transferase, are inhibited (Yaning et al., 2008), while studies on Eisenia fetida have shown that TBBPA induces the generation of ROS (Xue et al., 2009). Moreover, studies on freshwater Carassius auratus have shown that SOD and glutathione reductase (GR) are highly sensitive to TBBPA (Yan et al., 2007). Fish embryos have a weak antioxidant defense capacity, making them more sensitive to the teratogenic effects of oxidative compounds. Therefore, they are one of the most suitable organisms to act as bioindicator of the aquatic environment (Embry et al., 2010). Zebrafish embryos and larvae have all the advantages of an in vitro model and great efforts have been made to use them as tools for risk assessments of toxic chemicals in ecotoxicology studies. In addition to the activities of antioxidants such as SOD, CAT, and glutathione peroxidase (GPx), changes in the gene expression of these enzymes have been suggested to be markers of toxicity (Boix et al., 2013). In this study, the toxic effects of TBBPA on zebrafish larvae and embryos were evaluated. In addition, the enzyme activities of SOD, CAT, and GPx, as well as the mRNA expression levels of genes encoding for these antioxidants was monitored. Moreover, apoptosis in embryos and larvae was monitored using acridine orange staining after TBBPA
Environmental Toxicology DOI 10.1002/tox
exposure. Biopsies were also performed to assess apoptosis in organisms.
MATERIALS AND METHODS Chemicals TBBPA was obtained from Sigma-Aldrich (>98% purity). Stock solutions were stored at 220 C and prepared with 100% (v/v) dimethyl sulfoxide (DMSO, Sigma-Aldrich), which was used as a solvent control.
Experimental Animals The AB strain of zebrafish was obtained from the China Zebrafish Resource Center. Adult male and female fish were housed separately at ambient temperature (27 6 0.5 C) in holding tanks supplied with a continuous flow of aerated water for 7 days with a light:dark cycle of 14:10 h, pH from 6.0 to 7.5, and oxygen levels exceeding 80% (v/v) of the air saturation value. Fertilized zebrafish eggs were obtained from spawning adults in groups of male and female zebrafish at a ratio of 2:1 in tanks overnight. Spawning was induced in the morning when the light was turned on.
Dilution Medium The dilution water used was reconstituted water (ISO-63411982): Stock solution I: Cacl2 solution was prepared by dissolving 111.0065 g CaCl2 into 5000 mL deionized water. Stock solution II: MgSO4 solution was prepared by dissolving 61.6262 g MgSO4.7H2O into 5000 mL deionized water. Stock solution III: NaHCO3 solution was prepared by dissolving 32.3790 g NaHCO3 into 5000 mL deionized water. Stock solution IV: KCl solution was prepared by dissolving 2.8738 g KCl into 5000 mL deionized water. All chemicals were of analytical grade. Three days before the test, 1 L of each of solutions I–IV was mixed and the total volume was made up to 100 L with deionized water. The dilution water was aerated until oxygen saturation was achieved, and it was then stored for approximately 2 days without further aeration before use. The pH was 7.80, water hardness was approximately 245 mg/L(as CaCO3), and Ca/Mg was 4:1.
Exposure and Experimental Design In this study, groups of zebrafish embryos were exposed to the test solutions of TBBPA with nominal concentrations of 0.10, 0.40, 0.70, and 1.00 mg/L. The concentrations were selected based on a range-finding test that identified the concentration that would induce obvious effects during exposure. Solvent control groups [0.005% (v/v) DMSO] were also included in the study. Test solution (50 mL) was filled
TBBPA INDUCES DEVELOPMENTAL TOXICITY, OXIDATIVE STRESS, AND APOPTOSIS
TABLE I. Forward (F) and reverse (R) primers used for real-time PCR Gene
Accession No
Primer (50 to 30 )
CAT
AF170069
Cu/Zn-SOD
Y12236
GPx1a
NM_001007281
b-actin
NM_131031
F-agggcaactgggatcttaca R-tttatgggaccagaccttgg F-ggccaaccgatagtgttaga R-ccagcgttgccagtttttag F-caccctctgtttgcgttcc R-ctctttaatatcagcatca F-aagtgcgacgtggaca R-gtttaggttggtcgttcgtttga
in each test vessel under 72 h renewal conditions. Three replicates were assigned for each treatment group and control group, while the initial number of eggs was 30 for each group. The test embryos within 4 h postfertilization (hpf) were examined under a dissecting microscope, and those that had developed normally and had reached the gastrula stage were selected for further testing. During the test, the following conditions were maintained: Temperature for zebrafish embryos: 25 6 1.0 C; Oxygen concentration: > 60% (v/v) of the air saturation value; Duration: 8 days.
Apical observations performed on each tested embryo include hatching rate, survival rate, as well as abnormal appearance. At the end of the test, total length measurements and individual weights (dry weights, 24 h at 60 C) of individuals were measured.
Biochemical Assays Zebrafish larvae at 48, 96, 144, and 192 hpf were homogenized, respectively, in Tris-HCl buffer (100 mM, pH 7.4). Homogenates were centrifuged at 12,000g for 15 min at 4 C, and the supernatants were collected for biochemical parameter analysis. The activities of Copper/Zinc Superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathioneper oxidase (GPx) were assayed using diagnostic reagent kits (Sangon Biotech, Shanghai, China) according to the specified instructions from the manufacturers. Cu/Zn-SOD activity was assayed based on a modified method (Marklund et al., 1974). CAT activity was determined according to Sinha’s method (Sinha, 1972). GPx activity was measured according to Hafeman et al. (1974), which was based on a modified method using 5,5’-dithiobis-2-nitrobenzoic acid.
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trophotometry at 260 nm, and RNA purity was verified by the 260/280 nm ratio of 1.8 or greater. The cDNA was synthesized using the reverse transcriptase kit (Toyobo, Japan) in accordance with the product’s protocol. Oligonucleotide primes (Table I) were used to detect the gene expression of b-actin, Cu/Zn-SOD, CAT, and GPx1a using the SYBR green system (Toyobo, Japan). Real-time polymerase chain reaction (quantitative PCR) was carried out with the CFX96 Real-Time PCR Detection System (BioRad, USA) in sterile 96-well PCR plates. The reaction mixtures included 12.5 lL of 23SYBR Premix Ex TaqTM, 0.5 lL of PCR Forward and Reverse primers, and 1 lL of cDNA. The PCR conditions were as follows: 95 C for 30 s, followed by 40 cycles of 95 C for 5 s, 60 C for 30 s.
Acridine Orange (AO) Staining to Detect Apoptosis According to the test method (Tucker and Lardelli, 2007), one- to four-cell stage embryos and larvae (n 5 10) after 96 h TBBPA exposure were collected and divided into two groups: 1.0 mg/L treatment group and control group. The embryos and larvae stained with Acridine Orange (AO) (2 lg/mL in Hank’s medium) were wrapped with foil to prevent fluorescence quenching for 30 min and washed repeatedly with Hank’s medium. Stained tissues were pictured as whole mounts under a fluorescence microscope (Leica excitation light, 473 nm).
Histological Processing Larvae treated at 96 hpf with 1.0 mg/L TBBPA and the control group were fixed with 4% (w/v) paraformaldehyde overnight at 4 C, dehydrated with ethanol, cleared in xylene, and embedded in paraffin. After routine tissue processing and paraffin embedding, whole-body serial sections were cut at approximately 5-lm intervals. Sections were stained with hematoxylin and eosin, and observed under the microscope.
Statistical Analysis Values are expressed as the mean 6 standard error of the mean for all experiments. CAT, Cu/Zn-SOD, and GPx data were analyzed by one-way analysis of variance. When statistically significant differences were found between treatment groups, Dunnett’s test was used to determine which treatments were significantly different from unexposed controls. All statistical analyses were conducted using SPSS 17.0 and the threshold level of significance was set at p < 0.05.
Gene Expression
RESULTS
Total RNA was extracted from 20 embryos or hatched larvae using Trizol reagent (Takara Biochemicals, Dalian, China) following the manufacturer’s instructions. Total RNA concentrations were quantified immediately by ultraviolet spec-
Embryonic Dose Response Studies Zebrafish embryos were exposed to TBBPA to determine which concentration would affect the hatching rate, survival
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Fig. 1. (A) Hatchability rate, (B) survival rate, (C) cumulative malformation, as well as larvae length in zebrafish embryos and larvae exposed to different TBBPA concentrations (0.0, 0.10, 0.40, 0.70, 1.00 mg/L) for (D) 8 days (D). Values are the mean6 standard error. Values significantly different from the control are indicated by an asterisk (one-way analysis of variance, followed by the Dunnett’s test (SPSS 17.0), *p 0.05, **p 0.01).
rate, growth rate, and induce malformation. The toxic end points were examined after 8 days of TBBPA exposure. The hatching rates were, in the control group 83.3 6 4.02%, and 84.5 6 4.60%, 82.2 6 4.70%, and 82.2 6 4.70% in the 0.10, 0.40, and 0.70 mg/L treatment groups, respectively. No significant differences were observed between all groups. In contrast, the hatching rate in the 1.00 mg/L treatment group was 78.9 6 2.40%, which was significantly different compared with the control [P < 0.05; Fig. 1(A)]. The survival rate of embryos/larvae after TBBPA exposure for 8 days was 63.3 6 5.30% in 0.7 mg/L treatments, and 27.8 6 18.3% in1.0 mg/L treatments, respectively, which were significantly decreased relative to 87.8 6 2.2% in the control treatments. The survival rate of embryos/larvae in the 0.10 and 0.40 mg/L treatment groups was 90.0 6 3.70% and 88.9 6 4.30%, respectively, which was not significantly different from the control group [Fig. 1(B)]. TBBPA exposure for 8 days resulted in teratogenesis involving hyperemia and pericardial edema (Fig. 2). TBBPA exposure induced malformation rates of 17.8 6 10.3%, 74.4 6 2.63%, and 93.3 6 3.59% for the 0.40, 0.70, and
Environmental Toxicology DOI 10.1002/tox
1.00 mg/L treatment groups, respectively [Fig. 1(C)], which were significantly different from the control group. The length of larvae after 8 days TBBPA exposure was measured to assess whether TBBPA could impact zebrafish embryo and larvae growth. The mean length in the control group was 4.29 6 2.54 mm. No significant differences were observed between the control and 0.10 and 0.40 mg/L treatment groups. However, the 0.70 and 1.00 mg/L treatment groups resulted in lengths of 3.94 6 4.37 mm and 3.67 6 7.20 mm, respectively, which were significantly different when compared with the control [Fig. 1(D)].
Cu/Zn-SOD, CAT, and GPx Activities As indices of antioxidant status, the activities of Cu/ZnSOD, CAT, and GPx were measured to evaluate the presence of oxidative stress in embryonic zebrafish following exposure to TBBPA (Fig. 3). There was no significant change in Cu/Zn-SOD activity in the 0.10 mg/L treatment group relative to the control. While a significant decrease in Cu/Zn-SOD activity was observed in the 0.40, 0.70, and 1.00 mg/L exposure groups when compared with the control
TBBPA INDUCES DEVELOPMENTAL TOXICITY, OXIDATIVE STRESS, AND APOPTOSIS
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Fig. 2. Morphological abnormalities including hyperemia, pericardial edema, and tail deformities were caused by 0.4 mg/L TBBPA and higher concentrations. The control group had a normal appearance (A) and (C). Representative malformations are indicated by arrows in (B) and (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
after 3, 5, and 8 dpf [Fig. 3(A)]. Similarly, a significant decrease in CAT activity was observed in all groups, except the 0.10 mg/L group, when compared with control [Fig. 3(B)]. There was no change in GPx activity in the 0.10 mg/L treatment group, however, there was a significant decrease in activity in the 0.40, 0.70, and 1.00 mg/L treatment groups after 3, 5, and 8 dpf when compared with the control [Fig. 3(C)].
Cu/Zn-SOD, CAT, and GPx1a Gene Expression The Cu/Zn-SOD, CAT, and GPx1a gene expression were suppressed in embryos and larvae exposed to 1.00 mg/L TBBPA at 1, 3, 5, and 8 dpf (Fig. 4). There was no significant change in GPx1a gene expression in the 0.10, 0.40, and 0.70 mg/L treatment groups compared with control at the four detection times. Although significant differences in gene expression were observed between the individual treatment groups and the control group for Cu/Zn-SOD and
CAT, there were no obvious changes overall for in the 0.10, 0.40, and 0.70 mg/L treatment groups.
Analysis of Apoptosis AO staining was performed to detect TBBPA-induced apoptosis in embryos and larvae. After 5 dpf of treatment, notable signs of apoptosis were found to have mainly accumulated in the brain, heart, and tail (Fig. 5), suggesting possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. A suite of abnormalities including hyperemia and pericardial edema were induced in zebrafish embryos and larvae after TBBPA exposure.
Histopathology Histological analysis was performed using larvae paraffin sections. The morphology of cardiac tissue was evaluated visually under the microscope (Fig. 6). In the 1.00 mg/L TBBPA treatment group, pathological patterns in the
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DISCUSSION This study adopted zebrafish embryos and larvae as a biological model to evaluate TBBPA-induced developmental toxicity, oxidative stress, oxidant-associated gene expression,
Fig. 3. The activities of Cu/Zn-superoxide dismutase (Cu/ Zn-SOD, A), catalase (CAT, B), and glutathione peroxidase (Gpx, C) in zebrafish embryos and larvae after TBBPA exposure at 0.10, 0.40, 0.70, and 1.00 mg/L. Results are the mean 6 standard error of triplicate samples. Values significantly different from the control are indicated by an asterisk (one-way analysis of variance, followed by the Dunnett’s test (SPSS 17.0), *p 0.05, **p 0.01). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
morphology of cardiac tissue were observed with a decrease in myocardial cells and heart linearization when compared with the control. The results showed that TBBPA exposure restrained the heart development and caused cardiac chambers elongated.
Environmental Toxicology DOI 10.1002/tox
Fig. 4. Expression of Cu/Zn-SOD, CAT, and GPx1a in zebrafish embryos and larvae after exposure to 0.10, 0.40, 0.70, and 1.00 mg/L of TBBPA. Results are the mean 6 standard error of triplicate samples. Values significantly different from the control are indicated by an asterisk (one-way analysis of variance, followed by the Dunnett’s test (SPSS 17.0), *p 0.05, **p 0.01). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
TBBPA INDUCES DEVELOPMENTAL TOXICITY, OXIDATIVE STRESS, AND APOPTOSIS
Fig. 5. Apoptosis induced by TBBPA in zebrafish embryos and larvae. Zebrafish embryos and larvae were exposed to 1.00 mg/L TBBPA for 5 dpf and stained with acridine orange. Apoptotic cells accumulated in the brain, cardiac tissue, and tail of TBBPA-treated groups when compared with the control. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
and cell apoptosis. Abnormalities including hyperemia and pericardial edema were induced in zebrafish embryos and larvae. The results showed that SOD, CAT, and Gpx activities were repressed following exposure to 0.40, 0.70, and 1.00 mg/L TBBPA. Despite the significant decrease in Cu/ Zn-SOD, CAT, and GPx1a gene expression in the 1.00 mg/ L treatment group, gene expression in the 0.10, 0.40, and 0.70 mg/L treatment groups remained unaltered relative to the control. These results indicate that TBBPA induces oxidative stress and apoptosis in zebrafish embryos and larvae.
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With increasing concentrations of TBBPA, embryo and larvae abnormalities increased and the survival rate decreased. TBBPA exposure caused lethal effects on zebrafish embryos and larvae. Furthermore, exposure of embryos to increasing concentrations of TBBPA during early development resulted in a significant increase in malformation rate, as well as a significant decrease in hatching, growth, and survival rate. In addition, the results also showed that TBBPA may interfere with embryonic heart development and it indicated that there was a lack of formed elements in the circulating blood. Oxidative stress is a complex phenomenon involved in physiological and pathological processes. During the first stages of the organogenesis period, embryos present with weak antioxidant defense capacity, making them more sensitive to the teratogenic effects of test substances. As oxidative stress is the first response to environmental stressors, the embryos may initially initiate antioxidant and detoxification responses to TBBPA exposure. Evidence indicates disequilibrium between the formation of ROS and the embryo antioxidant defense system, which can affect embryo development and function, with possible etiology consequences of different congenital anomalies. Therefore, antioxidant defense is important in modulating oxidative stress-mediated events. The involvement of antioxidative enzymes, such as SOD, CAT, and GPx, has been suggested to play an important role in protecting cells from oxidative stress. SOD is crucial in catalyzing superoxide radicals to H2O2 and O2. CAT catalyzes the transformation of H2O2 to water, and GPx transforms hydroperoxides to hydroxyl compounds using GSH as a substrate. This study showed that relatively low concentrations of TBBPA do not induce significant changes in antioxidant enzyme activities (SOD, CAT, GPx) to mitigate oxidative stress, whereas relatively
Fig. 6. Control larvae (A) and larvae exposed to 1.0 mg/L TBBPA (B). Myocardial cells in the 1.0 mg/L TBBPA group were fewer and thinner heart was observed compared with the control (arrows). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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high concentration of TBBPA decrease enzyme activities to counter the oxidative stress. This result indicates that there is a threshold value. When the test concentration is below the threshold value, there is no significant change in enzyme activity; while enzyme activity is significantly inhibited when test concentrations are above the threshold. This threshold effect may explain the difference in SOD, CAT, and GPx activities following exposure to varying concentrations of TBBPA. Recently, research has focused on the link between oxidative stress enzyme activities and the related gene expression. In this study, we showed that Cu/Zn-SOD, CAT, and GPx1a gene expression is suppressed in embryos and larvae exposed to 1.00 mg/L TBBPA at 1, 3, 5, and 8 dpf. These results were consistent with the antioxidant enzyme activities. However, there was no significant decrease in Cu/ZnSOD, CAT, and GPx1a gene expression in the 0.40 and 0.70 mg/L exposure groups relative to the control, which was not consistent with the related enzyme activities. This result may be attributed to gene expression characteristics. When zebrafish are exposed to certain concentrations of the herbicide atrazine, antioxidant enzyme activity is inconsistent with related gene expression changes (Jin et al., 2009). Similarly, studies have shown that SOD, CAT, and the activities of other antioxidant enzymes decreased when zebrafish were exposed to perfluorinated dodecanoic acid and copper metal, but the related genes did not change (Craig et al., 2007; Liu et al., 2008). These discrepancies may be because gene expression is a relatively quick process, while enzyme activity is lengthy and involves complex processes, such as translation and post-translational modifications, which are subject to environmental impact. However, to fully understand the mechanism of TBBPA oxidative damage, further studies must be performed. AO staining revealed that apoptosis mainly occurred in the brain, heart, and tail, which suggests that the blood circulation system and nervous system may be potential targets of TBBPA. At the same time, we observed hyperemia and pericardial edema in zebrafish embryos and larvae. These effects were possibly because of the high percentage of apoptotic cells in the related sites and may partly explain the observed heart malformation. Another explanation is that hyperemia and pericardial edema may be induced by circulating blood cells and circulatory system failure, subsequently resulting in a decrease in myocardial cells and heart thinner, as observed by pathological analysis. It can be hypothesized that TBBPA-induced apoptosis in zebrafish embryos and larvae occurs through the production of excess ROS. This leads to altered gene expression regulation of oxidative stress mediators, such as SOD, CAT, and GPx. These enzymes play an important role in mitigating oxidative stress. However, when relatively high concentrations of TBBPA are present, enzyme activities decrease, and apoptosis is exacerbated in the brain, heart, and tail, which may induce blood circulation and nervous system develop-
Environmental Toxicology DOI 10.1002/tox
mental abnormalities. In any case, to be confirmed, this hypothesis needs further investigation.
CONCLUSION In summary, TBBPA can cause developmental toxicity in zebrafish embryos and larvae as a result of its effects on the actions of oxidative stress, related gene expression, and cell apoptosis. We conclude that: (1) The abnormalities including hyperemia and pericardial edema could be induced by TBBPA in zebrafish embryos and larvae. (2) The results showed there was a significant dose–response relationship between toxicity endpoints (hatching rate, survival rate, malformation rate, and growth rate) and TBBPA concentration. (3) The study showed that 0.10 mg/L TBBPA did not induce significant changes in antioxidant enzyme activities (SOD, CAT, GPx), while 0.40, 0.70, and 1.00 mg/L TBBPA decreased enzyme activities. (4) The Cu/Zn-SOD, CAT, and GPx1a gene expression were suppressed in embryos and larvae exposed to 1.00 mg/L TBBPA at 1, 3, 5, and 8 dpf. (5) Histological analysis showed evidence of obvious heart impairment in the TBBPA exposure group when compared with the control (6) AO staining results showed that apoptotic cells mainly accumulated in the brain, heart, and tail, indicating possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. These study results will help our understanding of the mechanism of TBBPA-induced oxidative stress and apoptosis. This study also shows that zebrafish embryos can serve as an effective model for studying developmental toxicity and oxidative stress, which is of great importance in evaluating environmental toxicity and the risks associated with various chemicals. The authors would like to thank Project of Technology Development Research for Scientific research institutes for their assistance for the research. Disclosure: The authors declare that they have no competing interests.
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TBBPA INDUCES DEVELOPMENTAL TOXICITY, OXIDATIVE STRESS, AND APOPTOSIS
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Environmental Toxicology DOI 10.1002/tox
