Environmental Pollution 203 (2015) 40e49

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Cyhalofop-butyl has the potential to induce developmental toxicity, oxidative stress and apoptosis in early life stage of zebrafish (Danio rerio) Lizhen Zhu, Xiyan Mu, Kai Wang, Tingting Chai, Yang Yang, Lihong Qiu, Chengju Wang* College of Sciences, China Agricultural University, Beijing, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2014 Received in revised form 20 March 2015 Accepted 29 March 2015 Available online

Cyhalofop-butyl is a selective herbicide widely employed in paddy field, which can transfer into aquatic environments. However, details of the environmental risk and aquatic toxicity of cyhalofop-butyl have not been fully investigated. In this study, zebrafish (Danio rerio) embryos were exposed to a range of cyhalofop-butyl until 120 hour post-fertilization (hpf) to assess embryonic toxicity of the chemical. Our results demonstrated that cyhalofop-butyl was highly toxic to zebrafish embryos, with concentrationdependent negative effects in embryonic development. In addition, exposure to cyhalofop-butyl resulted in significant increases in reactive oxygen species (ROS) production and cell apoptosis in heart area. The mRNA levels of the genes related to oxidative stress and apoptosis were also altered significantly after cyhalofop-butyl exposure. Moreover, the activity of capspase-9 and caspase-3 were significantly increased. Therefore, we speculated that oxidative stress-induced apoptosis should be responsible for abnormal development during embryogenesis after cyhalofop-butyl exposure. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Cyhalofop-butyl Zebrafish embryo Developmental toxicity Oxidative stress Apoptosis

1. Introduction Cyhalofop-butyl(CB)2-[4-(4-cyano-2-fluorophenoxy)phenoxy] propanoic acid, butyl ester (R), is a member of the aryloxyphenoxypropionate(AOPP) group of herbicides introduced in the mid 1980s by DowElanco (Buehring et al., 2001). It is a postemergence herbicide, which has high selectivity between rice and target grasses such as Echinochloa crusgalli, Leptochloa chinensis and Alopecurus aequalis in rice paddy, due to different metabolism of the molecule (Ntanos and Koutrouba, 2000). Cyhalofop-butyl is formulated as an ester to translocate throughout the plant, moves in both xylem and phloem from the treated foliage to the root system, and accumulates in the meristematic region of the plant. Once in the plant, it rapidly hydrolyzes to the acid form, cyhalofopacid, the primary metabolite of cyhalofop-butyl and the herbicidal active form. In addition, cyhalofop acid inhibits ACCase, which is responsible for the biosynthesis of fatty acids (Ottis et al., 2005). Cyhalofop-butyl has been used as the main herbicide to combat annual rice grasses in many parts of the world for decades. It is applied as an emulsifiable concentrate (Clincher CA®) at a rate of

* Corresponding author. E-mail address: [email protected] (C. Wang). http://dx.doi.org/10.1016/j.envpol.2015.03.044 0269-7491/© 2015 Elsevier Ltd. All rights reserved.

210 g/ha (Lassiter et al., 2000). Unfortunately, the exposure is usually not limited to the location where it is employed, with relatively higher concentrations applied in environment. Due to used extensively in paddy field and transferred into aquatic environments easily, more consideration should be given to the potential hazards caused by cyhalofop-butyl to aquatic organisms. A few researchers have described the negative effects of cyhalofopbutyl induced in aquatic organisms. Wu et al. demonstrated that cyhalofop-butyl showed high acute toxicity to Rana limnocharis in lab (Wu et al., 2011). Huang et al. showed that cyhalofop-butyl had low toxicity to Bufo gargarizans tadpole, but it could cause malformed individuals (Huang et al., 2007). Bruni et al. recently indicated that Clincher containing cyhalofop-butyl was able to inhibit the growth of Marsilea quadrifolia, an endangered aquatic fern, even at very dilute concentrations (Bruni et al., 2013). However, limited studies have been conducted on the negative effects of cyhalofop-butyl exposure on fish, especially in the early life stages. Therefore, it is necessary to perform environmental toxicological studies of cyhalofop-butyl to reflect the environmental risk on fish. In recent years, numerous researches have employed zebrafish as a vertebrate model for acute and chronic tests, particularly in the fields of genetics and developmental biology (Eimon and Ashkenazi, 2010; Jin et al., 2011). Zebrafish embryos have numerous traits, including small in size, transparent, fertilized

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

externally, undergo rapid development, and easy to maintain and handle. Most organs of embryos become functional between 3 and 5 days post fertilization (dpf). In addition, fish embryonic development is more sensitive to environmental stress, compared with adults. More important, it has also been demonstrated that zebrafish share many common features with humans and many molecular pathways are evolutionarily conserved between zebrafish and humans (Goldsmith and Jobin, 2012; Yuan et al., 2014). Therefore, zebrafish embryos have been ideal models to evaluate the developmental toxicity of exposure to toxicants during early-life stages (Fraysse et al., 2006; Deng et al., 2009; Mu et al., 2013). Microscopically visible acute endpoints such as deformation, hatching rates, survival, can be used to assess the potential developmental toxicity, but not the mechanism of toxicant-induced effects. Alternatively, gene expression analysis, a highly sensitive and mechanism-based technology, is able to examine the effects at the molecular level, which can provide a better mechanistic understanding of toxicity (Voelker et al., 2007). In the present study, zebrafish embryos were used to investigate the developmental toxicity induced by cyhalofop-butyl exposure. The reactive oxygen species (ROS) level and the target organ of cyhalofop-butyl-induced apoptosis were also examined. In addition, the alteration of gene expression related to oxidative stress and apoptosis after cyhalofop-butyl exposure were analyzed to elucidate the potential mechanism induced by cyhalofop-butyl at the molecular level. Therefore, genes encoding antioxidant proteins including Cu/Znesuperoxide dismutase (Cu/ZneSOD), manganese superoxide dismutase (MneSOD), catalase (CAT), glutathione peroxidase (GPx), as well as the genes related to apoptosis pathway including p53, murine double minute 2 (Mdm2), B-cell lymphoma/ leukaemia-2 gene (Bcl-2), Bcl-2 associated X protein (Bax), Puma, apoptotic protease activating factor-1 (Apaf1), Caspase 3 (Cas3) and Caspase 9 (Cas9) were determined. To evaluate whether cyhalofopbutyl induces apoptosis via the caspase pathway, the activity of caspase-3 and caspase-9 were also examined. Overall, this information is intended to provide new insights into the toxicological mechanism of cyhalofop-butyl. 2. Materials and methods 2.1. Chemicals and reagents 98% Cyhalofop-butyl (CAS: 122008-85-9) was obtained from Anhui Futian Agrochemical CO., Ltd and the stock solution used for drug exposure was prepared with acetone AR. All of the other reagents utilized were of analytical grade. The reconstituted water was prepared in the lab with the formula of iso-7346-3, which contained 2 mmol/L Ca2þ, 0.5 mmol/L Mg2þ, 0.75 mmol/L Naþ, and 0.074 mmol/L Kþ (ISO, 1996).

41

2013). Four-hour post-fertilization (hpf) embryos were randomly distributed in 24-well culture plates for exposure to the test solutions with a cyhalofop-butyl concentration of 0.10, 0.20, 0.30, 0.40, 0.50, and 0.60 mg/L for 120 hpf. All of test solutions were made up using reconstituted water, designed on the basis of pre-experiment data. Reconstituted water served as the over-all control (0 mg/L), and solvent control which contained the same acetone content with the highest dosage solution was arranged. Three replicates for each concentration were used. Twenty wells were used in each plate and each well containing 2 ml of the respective treatment solutions and one viable embryo (blastula stage). The exposure solution was renewed every 24 h. During exposure, dead embryos/ larvae were immediately removed. The hatching and malformation of embryos were checked daily. Morphological development was observed by microscope. Abnormalities were recorded and the body length was measured by an Aigo GE-5 (made by Aigo Corp.). 2.3. Analytical analysis of exposure solutions Exposure solutions were analyzed at the beginning of exposure (T0) and before water renewal after 24 h (T24) of the experiments. The analysis of the concentrations of cyhalofop-butyl in water was performed with a high performance liquid chromatography (HPLC), using the equipment of Agilent 1200 system (Agilent, USA). Samples (20 mL) were extracted twice with acetonitrile (10 mL each time). Then, 1.5 mL of extract was pipetted into a centrifuge tube, which contained 100 mg anhydrous magnesium sulfate. 20 uL supernatant was injected after centrifugation for 3 min and filtered through a 0.22-um nylon filter. Chromatographic separation was performed with Aglitent Ezlipse XDB-C18 column (5.0 mm 150  4.6 mm, Agilent, USA). The mobile phase (acetonitrile: water ¼ 60: 40(V/V)) flow rate was 1 mL/min, the column temperature was 25  C, and UV detection was performed at 238 nm. The control solution was used as blanks for the method. 2.4. Acridine orange staining Embryo cell apoptosis was identified using AO staining (Chan and Cheng, 2003; Deng et al., 2009). After 96 h of exposure to the concentrations of cyhalofop-butyl, 10 larvae from each group (n ¼ 3) were washed twice in 30% Danieau's solution (58 mM of NaCl, 0.7 mM of KCl, 0.4 mM of MgSO4, 0.6 mM of Ca(NO3)2, and 5 mM of HEPES, pH 7.4), then transferred to 5 mg/ml of AO dissolved in 30% Danieau's solution for 20 min at room temperature. The larvae were then washed with 30% Danieau's solution three times for 5 min each. Before examination, the embryos were anesthetized with 0.03% MS-222 for 3 min. Apoptotic cells were identified with a fluorescence microscope (Olympus, Japan). 2.5. ROS measurement

2.2. Maintenance of zebrafish and embryo toxicity test Wild type AB-strain zebrafish (length 3.4 ± 0.5; weight 0.17 ± 0.05) were obtained from Beijing Hongdagaofeng Aquarium Department, kept in the flow-through feeding equipment (made by Esen Corp.) at 26  C with a photoperiod of 14/10 (light/dark) (Mu et al., 2013). The fish were fed twice daily with live brine shrimp. Male and female adult fish (male/female ratio was 1/2) were separated by isolation boards in spawning boxes overnight. In the following morning, spawning was triggered once the light was turned on and the isolation boards were removed. The embryos were siphoned from the spawning boxes, washed with reconstituted water. The embryonic acute toxicity test was conducted in accordance with a previously proposed method (Fraysse et al., 2006; Mu et al.,

The generation of ROS in the larvae exposed to cyhalofop-butyl was measured using dichlorofluorescein-diacetate (DCFH-DA). Briefly, 10 larvae were washed with cold-PBS (pH 7.4) twice and then homogenized in cold buffer (0.32 mM of sucrose, 20 mM of HEPES, 1 mM of MgCl2, and 0.5 mM of phenylmethyl sulfonylfluoride (PMSF), pH 7.4). The homogenate was centrifuged at 15,000  g at 4  C for 20 min. Twenty microliters of the homogenate supernatant was added to a 96-well plate and incubated at room temperature for 5 min, after which 100 ul of PBS (pH 7.4) and 8.3 ul of DCFH-DA stock solution (dissolved in DMSO, 10 mg ml1) were added to each well. The plate was incubated at 37  C for 30 min. The fluorescence intensity was measured using a microplate reader (TECAN, Infiinite F200, Switzerland) with excitation and emission at 485 and 530 nm, respectively. The ROS

42

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

Table 1 Sequences of primer pairs used in the real-time quantitative PCR reactions. Target gene

Primer sequences

Accession number

References

MneSod Cu/ZneSod Cat Gpx p53 Mdm2 Bax Bcl-2 Puma Apaf-1 Caspase-9 Caspase-3 b-Actin

Forward-CCGGACTATGTTAAGGCCATCT Reverse-ACACTCGGTTGCTCTCTTTTCTCT Forward-GTCGTCTGGCTTGTGGAGTG Reverse-TGTCAGCGGGCTAGTGCTT Forward-AGGGCAACTGGGATCTTACA Reverse-TTTATGGGACCAGACCTTGG Forward-AGATGTCATTCCTGCACACG Reverse-AAGGAGAAGCTTCCTCAGCC Forward-GGGCAATCAGCGAGCAAA Reverse-ACTGACCTTCCTGAGTCTCCA Forward-AAGCAGTGATCCTGAGAGTCC Reverse-ATCCGAAGACTCGCTGTTC Forward-GGCTATTTCAACCAGGGTTCC Reverse-TGCGAATCACCAATGCTGT Forward-AGGAAAATGGAGGTTGGGATG Reverse-TGTTAGGTATGAAAACGGGTGGA Forward-TGGAAAGCAGAGTGGACGAA Reverse-GATGGCAGGGCTGGATGA Forward-TTCTACAGTAAACGCCCACC Reverse-TATCTAGTATTTCCCCATATTCC Forward-AAATACATAGCAAGGCAACC Reverse-CACAGGGAATCAAGAAAGG Forward-CCGCTGCCCATCACTA Reverse-ATCCTTTCACGACCATCT Forward-CGAGCAGGAGATGGGAACC Reverse-CAACGGAAACGCTCATTGC

AY195857 Y12236 AF170069 AW232474 AF365873.1 AF010255.1 AF231015.1 NM_001030253.2 NM001045472 AF251502.1 NM_001007404.2 NM_131877.3 AF057040.1

Jin et al. (2010) Jin et al. (2010) Jin et al. (2010) Jin et al. (2010) Deng et al. (2009) Deng et al. (2009) Deng et al. (2009) Jin et al. (2010) Deng et al. (2009) Deng et al. (2009) Deng et al. (2009) Deng et al. (2009) Deng et al. (2009)

concentration was expressed in arbitrary units (DCF mg1 protein). 2.6. Gene expression analysis Zebrafish embryos were exposed to sub-lethal concentrations of 0.10, 0.20, and 0.30 mg/L concentrations. Total RNA was isolated from the 20 embryos or newly hatched zebrafish larvae exposed until 96 hpf using TRIzol (Tiangen Biotech, Beijing, China) reagent according to the manufacturer's protocol. The RNA quality in each sample was evaluated by the ratio of absorbance at (A260 nm)/ (A280 nm), using a UV1240 spectrophotometer (Perkin Elmer, USA) and the banding pattern on a 1% agarose formaldehyde gel. Firststrand complementary DNA (cDNA) was synthesized by reversetranscription (RT) reactions using a reverse transcriptase kit (Tiangen Biotech, Beijing, China) according to the manufacturer's protocols. Quantitative real-time PCR amplifications were carried out on ABI 7500 q-PCR system (Applied Biosystems, Foster City, CA) using the SYBR Green PCR Master Mix reagent kits (Tiangen Biotech, Beijing, China). The thermal cycle was as follows: denaturation for 10 min at 95  C, followed by 40 cycles at 95  C for 15 s, annealing at 60  C for 20 s and extension at 72  C for 32 s. The transcription of b-actin was used as a house-keeping gene. The gene expression levels were measured in triplicate for each treatment. The sequences of the primers used in this study were indicated in previous reports and listed in Table 1. The fold-change of the genes tested was calculated using the 2DDCt method. 2.7. Caspase activity measurement Caspase-3 and caspase-9 activity was measured by a colorimetric assay based on the extent to which acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVDepNA) and acetyl-Leu-Glu-His-Asp pnitroanilide (Ac-LEHDepNA), respectively, changed into a yellow formazan product (p-nitroaniline (pNA)) (Deng et al., 2009). Briefly, 20 larvae from each beaker (n ¼ 3) were washed with iced PBS (pH 7.4) and homogenized on ice for 20 min, after which they were centrifuged at 2000  g at 4  C for 5 min and the supernatant collected. The enzyme activity of the supernatant was determined

using a caspase assay kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. The caspase activity was expressed as the percentage enzyme activity compared to the control group. All of the experiments were carried out in triplicate. 2.8. Statistical analysis All statistical analyses were undertaken using SPSS16.0 (SPSS, Chicago, IL, USA). Differences were determined by one-way ANOVA, completed with Dunnett and Duncan post-hoc comparison. p < 0.05 was considered significant. Morphological and behavioral differences were compared using the images captured with an Aigo GE-5 (made by Aigo Corp.) 3. Results 3.1. Solvent effect Statistical analysis indicated that solvent control did not show any effect for all indicators in this study. Therefore, the solvent control could be used as control. 3.2. Analytical quantification of exposure solutions HPLC analysis of water samples, which were analytically quantified in triplicate, indicated that the exposure solutions ranged from approximately 82 to 103% of all the nominal concentrations (Table 2). All test solutions in this research were renewed daily. Therefore, the nominal dosage is able to represent the actual content in this research. 3.3. Embryonic development toxicity of cyhalofop-butyl The mortality of the embryos or larvae at four 72, 96 and 120 hpf) is shown in Fig. 1A. The that cyhalofop-butyl exposure caused mortality in a concentration-dependent manner. At

time points (48, results showed of the embryos cyhalofop-butyl

Table 2 Mean measured cyhalofop-butyl concentrations (mg/L ± SE) in the water during the experiment. Nominal concentration

Cyhalofop-butyl concentration (mg/L) ± SE 0.10

0.20

0.30

0.40

0.50

0.60

0.10 ± 0.01 0.08 ± 0.01

0.20 ± 0.01 0.17 ± 0.01

0.28 ± 0.01 0.26 ± 0.01

0.37 ± 0.02 0.35 ± 0.02

0.51 ± 0.01 0.44 ± 0.01

0.58 ± 0.02 0.52 ± 0.04

Measured concentration T0 T24 Bold signifies standard error, SE.

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

43

Fig. 1. Mortality (A) and cumulative malformation rate (B) in zebrafish embryos exposed to various cyhalofop-butyl concentrations until 96 h. The asterisks indicate significant differences from the control group (determined by Dunnett post-hoc comparison, *p < 0.05; **p < 0.01). Error bars represent the standard deviation.

concentrations of 0.30 mg/L and below, no significant difference in embryos mortality were observed, compared with control groups. Whereas notable difference was observed at 96 hpf at concentrations of 0.50 mg/L and 0.60 mg/L, with embryos suffered mortality as high as 43.3% and 60.1%, respectively, in which almost all of embryos exposed were dead at 120 hpf. The LC50 value of cyhalofop-butyl at 96 hpf was 0.57 mg/L. Exposure to cyhalofop-butyl resulted in a series of morphological deformations in the zebrafish embryos, including yolk sac, pericardial edema and yolk sac deformity (Fig. 2). The percentage of cumulative malformation following exposure to cyhalofop-butyl significantly increased with time and in a dose-dependent manner (Fig. 1B). Since 72 hpf, developmental abnormalities

increased in a concentration-dependent manner. Among these, the most pronounced morphological alterations were pericardial edema and yolk sac edema, when exposure to cyhalofop-butyl concentrations of 0.30 mg/L or greater. In addition, yolk sac deformity was observed in the 0.60 mg/L cyhalofop-butyl-treated group at 72 hpf. Even more strikingly, the cumulative malformation rate of the surviving fish at 0.50 and 0.60 mg/L cyhalofopbutyl-treated group increased to 96.8% and 100% at 96 hpf, respectively. Hatching is known to be a key step of zebrafish embryogenesis. As shown in Fig. 3A, embryos exposed to control began hatching out of the chorion at 48 hpf, while almost all the surviving embryos hatched at 96 hpf. Compared with the control, hatching was

Fig. 2. Embryos with morphological deformation after exposure to cyhalofop-butyl at 72 hpf. A. Embryo with pericardial edema (Pe). B. Embryo with yolk sac edema (Yse). C. Embryo with both pericardial edema (Pe) and yolk sac edema (Yse). D. Embryo with both yolk sac edema (Yse) and yolk sac deformation (Ysd). E. Embryo with pericardial edema (Pe), yolk sac edema (Yse) and yolk sac deformation (Ysd). F. Embryo in control group with normal pericardial, yolk sac at 72 hpf before hatching. Arrows mark the different position of the two abnormities.

44

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

Fig. 3. Hatching rates of zebrafish embryos for different periods. (A) The spontaneous movement at 24 hpf. (B) Heartbeat of zebrafish during each observation time. (C) Body length of the hatched larvae at 96 hpf. (D) The asterisks indicate significant differences from the control group (determined by Dunnett post-hoc comparison, *p < 0.05; **p < 0.01). Error bars represent the standard deviation.

delayed and hatching rates were significantly reduced after the embryos were exposed to cyhalofop-butyl concentrations of 0.10 mg/L or greater since 48 hpf. More striking hatching inhibitions were observed in the embryos exposed to 0.50 and 0.60 mg/L concentration, in which the surviving individuals even didn't hatch until 120 hpf. Compared with control groups, the spontaneous movements of embryos exposed to concentrations 0.10 mg/L were not significantly impacted by cyhalofop-butyl. However, as the concentration of cyhalofop-butyl increasing, the spontaneous movements of embryos continued to decrease (Fig. 3B). Heart rates were recorded in embryos or new hatched larvae at 48, 72, and 96 hpf, respectively. The heartbeat was inhibited by 0.20 mg/L or higher dosage cyhalofop-butyl at 48 hpf. At 96 hpf, significant heartbeat inhibition was found at concentrations of 0.50 and 0.60 mg/L (Fig. 3C). Furthermore, the body length of hatched larvae was also recorded until exposed to 96 hpf. Body length was not significantly affected by exposure to 0.10 mg/L cyhalofop-butyl, but a striking inhibition of the length growth induced by cyhalofop-butyl was observed in 0.20 mg/L and greater at 96 hpf (Fig. 3D).

3.4. Apoptosis analysis 3.4.1. AO staining Zebrafish larvae exposed to cyhalofop-butyl at 96 hpf were stained with Acridine Orange (AO). There were no obvious apoptotic cells observed in the control larvae, but considerable numbers of apoptotic cells were observed in all of the other cyhalofop-butyl-treated groups, mainly appeared around the heart area, in a dose-dependent manner (Fig. 4). 3.4.2. ROS measurement The ROS concentrations in the cyhalofop-butyl-treated embryos were significantly increased in the 0.30 and greater groups, while ROS levels remained unchanged in other exposure groups compared with the controls (Fig. 5). 3.4.3. Gene expression pattern of oxidative stress and apoptosis related gene Next, we examined the gene transcription related to the oxidative stress and apoptosis process in zebrafish embryos. The effective concentration at which deformation was initiated was

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

45

Fig. 4. The fish exposed to cyhalofop-butyl until 96 hpf were stained with acridine orange (AO). Apoptotic cells stained with AO appeared mainly in the heart region. (A) Control; (B) 0.30 mg/L; (C) 0.40 mg/L; (D) 0.50 mg/L.

0.30 mg/L, whereas the lowest concentration used in the study was 0.10 mg/L. Therefore, the embryos had been exposed to 0.10, 0.20, 0.30 cyhalofop-butyl concentrations for 96 h to assess the alteration of the genes. Fig. 6 shows that cyhalofop-butyl significantly upregulated the mRNA levels of Cat compared with the control group, with increases of about 2.18-fold, 2.06-fold and 2.71-fold, in the 0.10, 0.20, 0.30 mg/L cyhalofop-butyl treatment groups respectively. Similarly, Mn-Sod mRNA levels increased significantly in the 0.10, 0.20, 0.30 mg/L cyhalofop-butyl treatment groups, with increases of about 2.18-, 2.05- and 3.52-fold. The expression of Cu/ZneSod and Gpx were only significantly up-regulated in the 0.30 mg/L cyhalofop-butyl-treated groups. As shown in Fig. 7, a concentration-dependent up-regulation of the p53 gene expression was observed upon exposure to 0.10, 0.20, 0.30 mg/L cyhalofop-butyl, upregulated 2.36-, 2.54- and 2.67-fold relative to the control group. As for the expression of Bcl-2, no significant alteration occurred in any treatment groups. The mRNA expression of Bax gene, the Bcl-2-associated X protein, was significantly upregulated 2.10-fold in the 0.3 mg/L cyhalofop-butyltreated embryos relative to the control group. Thus, the Bcl-2/Bax ratio decreased after cyhalofop-butyl exposure, especially in the highest cyhalofop-butyl concentrations (0.30 mg/L). The quantity of

Fig. 5. ROS prodution in different concentrations cyhalofop-butyl-treated embryos at 96 hpf. The asterisks indicate significant differences from the control group (determined by Dunnett post-hoc comparison, *p < 0.05; **p < 0.01). Error bars represent the standard deviation.

the pro-apoptotic BH3-only gene, Puma, was significantly increased 3.92-, 4.30- and 3.61-fold in the 0.10, 0.20, 0.30 mg/L cyhalofopbutyl exposures, but not proportionally. The mRNA levels of Apaf1 in the all groups were not significantly higher than that in the control group (Fig. 7). Furthermore, the gene expression of caspase-3 and caspase-9 were examined to assess whether cyhalofop-butyl induces apoptosis via the caspase pathway. As shown in Fig. 7, the mRNA levels of caspase-3 increased significantly when exposed to all the three concentration of cyhalofop-butyl, while obvious induction was only observed in the mRNA levels of caspase-9 at highest treatment of cyhalofop-butyl compared with the control group. 3.4.4. Caspase activity measurement Caspase-3 activity of the embryos was significantly increased to 139.2% and 151.7% at the 0.50 and 0.60 mg/L cyhalofop-butyl exposure groups, respectively, compared with control groups. Similarly, an increased caspase-9 activity exhibited 129.3% and 169.9% at the 0.50 and 0.60 mg/L cyhalofop-butyl exposure groups, respectively (Fig. 8). 4. Discussion This study provided the first approach to evaluating the aquatic toxicity of cyhalofop-butyl in early life stage of zebrafish. From the data presented, it has been clearly indicated that cyhalofop-butyl strongly affects zebrafish embryos and induces teratogenicity to the embryos in a dose-dependent manner. The results also showed that the expression of genes encoding antioxidant proteins and genes related apoptosis were significantly changed, suggesting the important roles of oxidative stress and apoptosis in cyhalofopbutyl-induced developing toxicity. In the present study, the 96 h-LC50 value of cyhalofop-butyl to zebrafish embryos was 0.57 mg/L, suggesting that cyhalofop-butyl shows highly toxic to zebrafish embryos. Exposure to cyhalofopbutyl also caused types of developmental abnormalities, such as hatching inhibition, abnormal spontaneous movement, depressed heart rates, growth regression and morphological deformities during embryonic development. Among the malformations, the most pronounced morphological alterations were pericardial edema and yolk sac edema. The same malformation has been reported in zebrafish embryos exposed to environmental toxicants, such as BDE 47 (Lema et al., 2007), TCDD (Yamauchi et al., 2006), copper (Johnson et al., 2007), HBCD (Deng et al., 2009) and difenoconazole (Mu et al., 2013). Heart is the first functional organ developed in zebrafish (Glickman and Yelon, 2002). It should be noted that the heart beat

46

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

Fig. 6. Expression of the mRNA of MneSod (A), Cu/ZneSod (B), Cat (C) and Gpx (D) in zebrafish exposed to various concentrations of cyhalofop-butyl for 96 h. The asterisks indicate significant differences from the control group (determined by Dunnett post-hoc comparison, *p < 0.05; **p < 0.01). Error bars represent the standard deviation.

rates were inhibited in embryos, in addition, the malformation mainly occurred in the heart in our study. This effect is possibly due to the high percentage of apoptotic cells accumulating in the heart, according to the staining by AO. Thus, we may speculate that the developing heart may be an important potential target for cyhalofop-butyl toxicity in zebrafish, and apoptotic cells accumulating in the heart may result in heart malformations, depressing heart rates, eventually disturbing the early development of zebrafish. Similar results have also been reported in previous study. For example, Shi et al. demonstrated that PFOS induced malformation in heart and impacted heart beat rates, which might be mediated via apoptosis (Shi et al., 2008). Oxidative stress has become an important subject in aquatic toxicology (Livingstone, 2003). Exposure to chemical pollutants may interfere the balance between endogenous and exogenous reactive oxygen species (ROS), and the elevated levels of ROS can subsequently been eliminated by antioxidant enzymes including superoxidate dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), which convert superoxide anions (O 2 ) into H2O2 and then into H2O and O2 (Valavanidis et al., 2006; Zhang et al., 2009). In this study, it was observed that cyhalofop-butyl induced ROS generation in zebrafish embryos in the exposure group. At the same time, the mRNA levels of genes encoding antioxidant proteins had also significantly up-regulated in the zebrafish embryos after cyhalofop-butyl exposure. Thus, it is possible that oxidative stress had occurred in zebrafish after cyhalofop-butyl exposure and an increase in the activities of these enzymes contributed to the elimination of ROS from the cell induced by cyhalofop-butyl exposure. Activation of the p53 protein, which can lead to a variety of

outcomes, including cell cycle arrest and apoptosis, has been known as an effective indicator that the cell has entered an apoptotic state. The induction of p53 associated with environmental pollutants induced apoptosis in zebrafish has been intensely studied (Shi et al., 2008; Deng et al., 2009; C. Zeng et al., 2014). During apoptotic stimulation, a decrease of the ratio of Bcl-2/ Bax may lead to an induction of mitochondrial cytochrome c release (Hildeman et al., 2003). Typically, p53 translocates to the mitochondrial outer membrane following directly up-regulating the expression of the pro-apoptotic BH3-only gene, Puma, and down-regulating anti-apoptotic members of the Bcl-2 family (Eimon and Ashkenazi, 2010; Perfettini et al., 2004). In current study, the transcripts of p53 and Puma were up-regulated, while the ratio of Bcl-2/Bax was decreased upon the treatment with 0.30 mg/L cyhalofop-butyl, suggesting that the high concentration of cyhalofop-butyl might change the ratio between the Bcl-2 and Bax and lead to an induction of mitochondrial cytochrome c release and then activation of apoptosis through the p53 pathway. Caspase activity has been thought to be a crucial marker in determining the progress of cell apoptosis. It has been well known that caspase-3 plays an essential role in apoptosis, mainly by catalyzing the specific cleavage of many key cellular proteins (Liu et al., 2007; Zeng et al., 2014). It is well established that pathways to caspase-3 activation either depend on or independ on mitochondrial cytochrome c release and caspase-9 function (Porter and J€ anicke, 1999). Previous study also demonstrated that caspase-8 was involved in the extrinsic pathway while caspase-9 participated in the intrinsic pathway (Jin and El-Deiry, 2005). In this study, both the gene transcription level and activity of caspase-3 and caspase-9 were significantly increased, suggesting that caspase-3

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

may play a pivotal role in cyhalofop-butyl-induced apoptosis via caspase-9 in the early life stages of zebrafish. It has been considered that the production of oxidative stress should be responsible for inducing apoptosis in cells. Then,

47

oxidative stress and cell apoptosis can result in developmental malformations at early life stages (Yamashita, 2003). Several researches had demonstrated that the bidirectional interactions between oxidative stress and cell apoptosis could be induced by

Fig. 7. Expression of the mRNA of p53 (A), Mdm2 (B), Bax (C), Bcl-2 (D), Puma (E), Apaf-1 (F), Cas3 (G) and Cas9 (H) in zebrafish exposed to various concentrations of cyhalofop-butyl for 96 h. The asterisks indicate significant differences from the control group (determined by Dunnett post-hoc comparison, *p < 0.05; **p < 0.01). Error bars represent the standard deviation.

48

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49

Fig. 8. Caspase-3 (A) and caspase-9 (B) activity in zebrafish after exposure to various concentrations of cyhalofop-butyl until 96 h. The asterisks indicate significant differences from the control group (determined by Dunnett post-hoc comparison, *p < 0.05; **p < 0.01). Error bars represent the standard deviation.

environmental chemicals. For example, Buccellato et al. found that exposure to hyperoxia resulted in the activation of Bax at the mitochondrial membrane, cytochrome c release, and cell death in primary rat alveolar epithelial cells, which demonstrated that Bax activation at the mitochondrial membrane required the generation of ROS (Buccellato et al., 2004). Zhao et al. investigated that acetofenate-induced intracellular ROS generation and DNA damage resulted in apoptosis through the p53 signal pathway in mouse macrophage cells (Zhao et al., 2009). Similar results had also been found in fish. Jin et al. suggested that CYP has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in zebrafish (Jin et al., 2011). MCLR-induced apoptosis had been demonstrated correlating with generation of ROS (Zeng et al., 2014). Therefore, it is possible that the increased malformation in embryos/larvae in this study can be explained by ROS-induced apoptosis. Taking these results together, it can be hypothesized that cyhalofop-butyl increases ROS generation, which induces apoptosis in zebrafish embryos with the activation of p53. Then, p53 translocates to mitochondria and transcribes the genes that encode proapoptotic proteins. This leads to the up-regulation of the apoptosis mediator, Puma, which in turn triggers the transactivation of Bax. Then Bax induces a selective process of membrane permeabilization. While caspase-3, a key executor of apoptosis, is triggered by caspase-9. These enzymes promote the cleaving of death substrates, which leads to apoptosis and the malformation of the embryos. It is worth noting that, in the present study, 0.30 mg/L cyhalofop-butyl could inflict apparent effects on zebrafish embryos at 72 hpf, and 0.10 mg/L cyhalofop-butyl was sufficient to alter the expression levels of several genes related to oxidative stress and apoptosis of zebrafish embryos. A previous study, which had explored degradation behavior and environmental safety of cyhalofop-butyl in paddy of Changsha, China, indicated that the detected residual cyhalofop-butyl in paddy water one day after spraying reached 1.67 mg/L, with a half-life of about 3.18 days (Guo et al., 2008). Wu et al. indicated that the half-life of cyhalofop acid in paddy water were 1.01e1.53 days (Wu et al., 2014). According to the results, this detected residual was much higher than the acute effective concentration and three days' half life is sufficient to cause harm to fish infields. Therefore, the application of cyhalofop-butyl is a serious threat to aquatic organisms cultured

in paddy water. 5. Conclusions In conclusion, our study showed developmental toxicity in zebrafish embryos exposed to cyhalofop-butyl, indicated by hatching inhibition, abnormal spontaneous movement, slow heart rate, growth regression and morphological deformities. The generation of ROS and the appearance of cell apoptosis in heart area could reveal the mechanism of the developmental toxicity induced by cyhalofop-butyl. Moreover, we also demonstrated that the mRNA levels of genes related to oxidative stress and apoptosis were also altered significantly in cyhalofop-butyl treated zebrafish embryos. Thus, the information presented in this study is helpful to provide integral and systematical information in terms of the mechanisms of developmental toxicity and cyhalofop-butylinduced oxidative stress and apoptosis in fish. Furthermore, exploring the multi-directional interactions among the oxidative stress and cell apoptosis will provide us further insight into the comprehensive knowledge on the environmental. Acknowledgments This work was financially supported by National Twelfth FiveYear Plan for Science & Technology (Grant No.2011BAE06B09). References Bruni, I., Gentili, R., De Mattia, F., Cortis, P., Rossi, G., Labra, M., 2013. A multi-level analysis to evaluate the extinction risk of and conservation strategy for the aquatic fern Marsilea quadrifolia L. in Europe. Aquat. Bot. 111, 35e42. Buccellato, L.J., Tso, M., Akinci, O.I., Chandel, N.S., Budinger, G.R., 2004. Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells. Biochem. J. 279, 6753e6760. Buehring, N., Baldwin, F., Talbert, R., Scherder, E., Lovelace, M., 2001. Graminicides in programs for broad-spectrum weed control in rice. Res. Series-Arkansas Agric. Exp. Stn. 58e61. Chan, P.K., Cheng, S.H., 2003. Cadmium-induced ectopic apoptosis in zebrafish embryos. Archives Toxicol. 77, 69e79. Deng, J., Yu, L., Liu, C., Yu, K., Shi, X., Yeung, L.W., Lam, P.K., Wu, R.S., Zhou, B., 2009. Hexabromocyclododecane-induced developmental toxicity and apoptosis in zebrafish embryos. Aquat. Toxicol. 93, 29e36. Eimon, P., Ashkenazi, A., 2010. The zebrafish as a model organism for the study of apoptosis. Apoptosis 15, 331e349. Fraysse, B., Mons, R., Garric, J., 2006. Development of a zebrafish 4-day embryolarval bioassay to assess toxicity of chemicals. Ecotoxicol. Environ. Saf. 63,

L. Zhu et al. / Environmental Pollution 203 (2015) 40e49 253e267. Glickman, N., Yelon, D., 2002. Cardiac development in zebrafish: coordination of form and function. Cell Dev. Biol. 13, 507e513. Goldsmith, J.R., Jobin, C., 2012. zebrafish as a model system of human pathology. J. Biomed. Biotechnol. 817341. Think small. Guo, Z., Huang, F., Xu, Z., 2008. Residue dynamics of 10% fenoxaprop-p-ethyl cyhalofop-butyl EC in rice. J. Ecol. Rural Environ. 24, 51e54. Hildeman, D.A., Mitchell, T., Kappler, J., Marrack, P., 2003. T cell apoptosis and reactive oxygen species. J. Clin. Invest. 111, 575e581. Huang, F., Guo, Z., Xu, Z., Yang, R., 2007. Toxicity of cyhalofop-butyl and fenoxapropethyl to Tadpole. J. Agro-Environ. Sci. 26, 1063e1066. ISO, 1996. Water Qualitye-Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamiltone-Buchanan (Teleostei, Cyprinidae)]. Jin, Y., Zhang, X., Shu, L., Chen, L., Sun, L., Qian, H., Liu, W., Fu, Z., 2010. Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish (Danio rerio). Chemosphere 78, 846e852. Jin, Y., Zheng, S., Pu, Y., Shu, L., Sun, L., Liu, W., Fu, Z., 2011. Cypermethrin has the potential to induce hepatic oxidative stress, DNA damage and apoptosis in adult zebrafish (Danio rerio). Chemosphere 82, 398e404. Jin, Z., El-Deiry, W.S., 2005. Review overview of cell death signaling pathways. Cancer Biol. Ther. 4, 139e163. Johnson, A., Carew, E., Sloman, K.A., 2007. The effects of copper on the morphological and functional development of zebrafish embryos. Aquat. Toxicol. 84, 431e438. Lassiter, R., Simpson, D., Grant, D., Richburg, J., Langston, V., Mann, R., 2000. Efficacy and crop tolerance of cyhalofop post-applied in direct seeded rice. Proc. Annu. Meeting-Southern Weed Sci. Soc. 170e170. Lema, S.C., Schultz, I.R., Scholz, N.L., Incardona, J.P., Swanson, P., 2007. Neural defects and cardiac arrhythmia in fish larvae following embryonic exposure to 2,20 ,4,40 tetrabromodiphenyl ether (PBDE 47). Aquat. Toxicol. 82, 296e307. Liu, C., Yu, K., Shi, X., Wang, J., Lam, P.K., Wu, R.S., Zhou, B., 2007. Induction of oxidative stress and apoptosis by PFOS and PFOA in primary cultured hepatocytes of freshwater tilapia (Oreochromis niloticus). Aquat. Toxicol. 82, 135e143. Livingstone, D., 2003. Oxidative stress in aquatic organisms in relation to pollution and aquaculture. Rev. Med. Veterinaire 154, 427e430. Mu, X., Pang, S., Sun, X., Gao, J., Chen, J., Chen, X., Li, X., Wang, C., 2013. Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays. Environ. Pollut. 175, 147e157. Ntanos, D., Koutrouba, S., Mavrotas, c., 2000. Barnyardgrass (Echinochloa crus-galli) control in water-seeded rice (Oryza sativa) with cyhalofop-butyl. Weed Sci. Soc.

49

Am. 14, 383e388. Ottis, B.V., Mattice, J.D., Talbert, R.E., 2005. Determination of antagonism between cyhalofop-butyl and other rice (Oryza sativa) herbicides in barnyardgrass (Echinochloa crus-galli). J. Agric. Food Chem. 53, 4064e4068. Perfettini, J.-L., Kroemer, R.T., Kroemer, G., 2004. Fatal liaisons of p53 with Bax and Bak. Nat. Cell Biol. 6, 386e388. Porter, A.G., J€ anicke, R.U., 1999. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6, 99e104. Shi, X., Du, Y., Lam, P.K., Wu, R.S., Zhou, B., 2008. Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS. Toxicol. Appl. Pharmacol. 230, 23e32. Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M., 2006. Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants. Ecotoxicol. Environ. Saf. 64, 178e189. Voelker, G., Rohwer, S., Bowie, R.C., Outlaw, D.C., 2007. Molecular systematics of a speciose, cosmopolitan songbird genus: defining the limits of, and relationships among, the Turdus thrushes. Mol. Phylogenetics Evol. 42, 422e434. Wu, C., Zhao, X., Wu, S., Cheng, L., Wang, Y., Chang, T., Yu, R., Ping, L., 2011. Toxicity and risk of herbicide cyhalofop-butyl on Rana limnocharis. Acta Agric. Zhejiangensis 23, 771e775. Wu, J., Wang, K., Zhang, Y., Zhang, H., 2014. Determination and study on dissipation and residue determination of cyhalofop-butyl and its metabolite using HPLCMS/MS in a rice ecosystem. Environ. Monit. Assess. 186, 6959e6967. Yamashita, M., 2003. Apoptosis in zebrafish development. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 136, 731e742. Yamauchi, M., Kim, E.Y., Iwata, H., Shima, Y., Tanabe, S., 2006. Toxic effects of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryo: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquat. Toxicol. 80, 166e179. Yuan, G., Wang, Y., Yuan, X., Zhang, T., Zhao, J., Huang, L., Peng, S., 2014. T-2 toxin induces developmental toxicity and apoptosis in zebrafish embryos. J. Environ. Sci. 26, 917e925. Zeng, C., Sun, H., Xie, P., Wang, J., Zhang, G., Chen, N., Yan, W., Li, G., 2014. The role of apoptosis in MCLR-induced developmental toxicity in zebrafish embryos. Aquat. Toxicol. 149, 25e32. Zhang, X., Xie, P., Li, D., Tang, R., Lei, H., Zhao, Y., 2009. Time-dependent oxidative stress responses of crucian carp (Carassius auratus) to intraperitoneal injection of extracted microcystins. Bull. Environ. Contam. Toxicol. 82, 574e578. Zhao, M., Zhang, Y., Wang, C., Fu, Z., Liu, W., Gan, J., 2009. Induction of macrophage apoptosis by an organochlorine insecticide acetofenate. Chem. Res. Toxicol. 22, 504e510.