Stereoselective induction by 2,2ʹ,3,4ʹ,6-pentachlorobiphenyl in adult zebrafish (Danio rerio): Implication of chirality in oxidative stress and bioaccumulation

Environmental Pollution 215 (2016) 66e76

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Stereoselective induction by 2,2ʹ,3,4ʹ,6-pentachlorobiphenyl in adult zebrafish (Danio rerio): Implication of chirality in oxidative stress and bioaccumulation* Tingting Chai a, b, Feng Cui a, Xiyan Mu a, Yang Yang a, Suzhen Qi a, Lizhen Zhu a, Chengju Wang a, *, Jing Qiu b, ** a

College of Science, China Agricultural University, Beijing 100193, China Institute of Quality Standards & Testing Technology for Agro-Products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Key Laboratory of Agri-food Quality and Safety, Ministry of Agriculture, Beijing 100081, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 December 2015 Received in revised form 18 April 2016 Accepted 21 April 2016

This study aimed to investigate the oxidative stress process and bioaccumulation the racemic/(
)-/(þ)2,2ʹ,3,4ʹ,6-pentachlorobiphenyl were administered to adult zebrafish (Danio rerio) after prolonged exposure of 56-days uptake and 49-days depuration experiments. Stereoselective accumulation was observed in adult samples after racemic exposure as revealed by decreased enantiomer fractions. The two enantiomers of PCB91 accumulated at different rates with logBCFk values close to 3.7, suggesting that they were highly hazardous and persistent pollutants. Exposure to racemic/(
)-/(þ)- PCB91 stereoselectively induced oxidative stress owing to changes in reactive oxygen species, malondialdehyde contents, antioxidant enzyme activities and gene expressions in brain and liver tissues. In addition, the stereoselective relationship between bioconcentration and oxidative stress were also presented in this study. Our findings might be helpful for elucidating the environmental risk of the two enantiomers of PCB91 that induce toxicity in aquatic organisms. © 2016 Elsevier Ltd. All rights reserved.

Keywords: PCB91 Zebrafish Oxidative stress Bioconcentration

1. Introduction Polychlorinated biphenyls (PCBs) are inadvertently generated during certain industrial processes such as the production of paint pigments (Anezaki et al., 2014) and adhesives (Anezaki and Nakano, 2015), although their industrial application was discontinued in the late 1970s. Laboratory and epidemiological studies have shown that exposure to PCBs has various toxicological effects such as to the endocrine (Hall et al., 2003) and immune systems (Frouin et al., 2010). Recent studies have shown that most water sources have been polluted by different levels of PCBs (Adeogun et al., 2013; Deshpande et al., 2013). Thus, investigating the long-term toxicological effects of PCBs on aquatic ecosystems is necessary. A group of 19 PCB congeners is known to contain a chiral axis and exist as

*

This paper has been recommended for acceptance by Harmon Sarah Michele * Corresponding author. No. 2 Yuan mingyuan West Road, Haidian District, Beijing 100193, China. ** Corresponding author. 12 Zhongguancun South Street, Beijing 100081, China. E-mail addresses: [email protected] (C. Wang), [email protected] (J. Qiu). http://dx.doi.org/10.1016/j.envpol.2016.04.075 0269-7491/© 2016 Elsevier Ltd. All rights reserved.

two stable rotational enantiomers (Dai et al., 2014). The manufacturing process of PCBs results in their racemate release into the environment, but atropisomeric biological processes occur in organisms, such as biotransformation (Kania-Korwel and Lehmler, 2015). To our knowledge, most studies on chiral PCB91 have focused on the atropisomeric oxidation by mice (Wu et al., 2013) and rats (Warner et al., 2009), stereoselective enrichment in predatory fish species (Ross et al., 2011), and stereoselective biotransformation processes in a stream food web (Dang et al., 2010b). However, whether the two enantiomers of chiral PCB91 could stereoselectively produce toxicological effects in fish is not yet known. Fish, as bio-indicators of environmental pollution, play an essential role in the assessment of potential risk associated with contaminants in aquatic ecosystems since they are directly exposed to chemicals via surface run-off or indirectly through the ecosystem food chain (Sharma and Ansari, 2014). Zebrafish (Danio rerio), a typical small tropical aquarium fish, has a long history of general use in toxicological experiments (Mu et al., 2015b). It also serves as an informative model for understanding human biology (Basu and

T. Chai et al. / Environmental Pollution 215 (2016) 66e76

Sachidanandan, 2013). Many studies have reported that exposure to environmental chemicals induces oxidative stress in zebrafish (Jin et al., 2011; Mu et al., 2014). Oxidative stress results from the imbalance between reactive oxygen species (ROS) production and antioxidant defense mechanisms and induces DNA and membrane destabilization, leading to different pathologic processes (Sakuragui et al., 2013). Exposure to PCBs is associated with cytotoxic effects in organisms, by producing ROS at levels that exceed antioxidant cell defenses, thereby inducing oxidative damage (Halliwell and Gutteridge, 2007). The lipid peroxidation process, one of ROS targets, is used as a biomarker for oxidative damage. The principle consequences of peroxidation include decreased fluidity and increased permeability, causing leakage in some molecules and inhibition of membrane-bound enzymes (Barni et al., 2014). Fish, like many other vertebrate species, activate defense mechanisms against oxidative stress by producing antioxidant enzymes, including the following radical-scavenging enzymes: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) (Sakuragui et al., 2013). However, whether exposure of adult zebrafish to chiral PCB91 stereoselectively affects the oxidative stress process is not yet known. In addition to toxicity, the bioaccumulation potential of a chemical is also an important indicator for assessing environmental damage (Advait and Shanta, 2013). The differential toxicological effects of chiral PCB enantiomers might be attributed to their stereoselective bioaccumulation in vivo. Bioaccumulation is defined as the accumulation of chemicals in an organism via any mechanism such as breathing, ingestion, or direct contact. It is expressed by bioconcentration factor (BCF) for aquatic species (ElAmrani et al., 2012). A chemical compound with a BCF value higher than 100 on basis of wet weight could damage the health of an organism and then is classified as “dangerous to the environment” by the European Union (Advait and Shanta, 2013). But to identify bioaccumulative substances, regulatory authorities rely on the chemicals' Kow (n-octanol/water partition coefficient), which is a measurement of hydrophobicity (Kelly et al., 2007). Previous studies have shown that racemic PCB91 has lgKow of 5, indicating that it is particularly susceptible to bioaccumulation in fish (Walters et al., 2011). However, there is no detailed information on chiral PCBs in aquatic species. This study aimed to better understand the oxidative stress process and bioaccumulation after adult zebrafish were exposed to the racemate and two enantiomers of chiral PCB91 for a prolonged exposure. For the bioaccumulation study, the enantiomer fraction (EF) and BCF values were calculated. For toxicological analysis, the related parameters indicating oxidative stress, including ROS, malondialdehyde (MDA), and antioxidant enzymes activities in the brain and liver tissues, were investigated. The transcription of antioxidant genes was also determined to elucidate the mechanism of oxidative stress induced by racemic/(þ)-/(
)- PCB91. The relationship between bioaccumulation and oxidative stress and the differences induced by racemic/(þ)-/(
)- PCB91 were also determined. To our knowledge, this is the first study to systematically evaluate the bioaccumulation and oxidative stress mechanism induced by environmental concentrations of two different enantiomers of chiral PCB91 in adult zebrafish. Our findings might provide new insights into the stereoselective effects of chiral PCB91 on oxidative stress in fish. 2. Experimental section 2.1. Zebrafish maintenance Juvenile AB strain zebrafish (Danio rerio) were cultured in a fish facility (ESEN-ZF-SS; Esen, Beijing) at 26  C with a photoperiod of

67

14 h:10 h (light:dark). The total daily feed intake was 2% dried brine shrimp (Artemia, Futian Brand, Japan) of adult zebrafish weight. 2.2. Chemicals and reagents Racemic PCB91 (99.9%) was provided by Dr. Ehrenstorfer GmbH (Germany). (þ)-PCB91 and (
)-PCB91 were obtained by separating the racemate by using high-performance liquid chromatography (HPLC); the separation conditions were the same as those reported previously (Xu et al., 2015). The quantities and purities (99.0%) of the isomers were determined using gas chromatography-mass spectrometry (Dai et al., 2012). The racemic/(þ)-/(
)- PCB91 was dissolved in acetone for following experiments. All organic reagents used in this study were of HPLC grade, and the other reagents were of analytical grade. 2.3. Processing experiments This study conformed to the Chinese legislation and was approved by the independent animal ethics committee of the China Agricultural University. The experiments were performed using a semi-static system at 26  C. Adult zebrafish were exposed to racemate, (þ)-PCB91, and (
)-PCB91 at 1 mg L
1 plus one solvent control were performed in triplicate. All experiments were performed in 60-L glass aquariums containing 50 L of test solution; each aquarium contained 150 adult zebrafish (age, six months). Half of the water containing the test chemicals was renewed for each exposure at 24-h intervals during the uptake experiment. The used water (500 mL samples) was collected at each renewal period to determine the actual concentrations in exposure water after the exposure period. In all, 15 zebrafish, i.e., five from each aquarium, were selected from each exposure group and control group at 0 (6 h), 3, 7, 14, 21, 35, 42, 49 and 56 days from the start of the experiment to detect PCB91 concentrations in the adult samples. Further, 10 adult zebrafish at 7, 21, 42, and 49 days from the start of the experiment were randomly selected from each aquarium (5 for gene expression and 5 for enzyme activity). After 56-day exposure, the remaining zebrafish were transferred to clean aerated water, which was renewed every 24 h during the depuration experiment. Five randomly selected individuals were removed from each aquarium at 0 (0 h), 7, 14, 21, 42, and 49 days from the transfer time for the determination of PCB91 concentration. 10 adult zebrafish at 21 and 42 days from the transfer time were also randomly selected from each tank (5 for gene expression and 5 for enzyme activity). All selected adult zebrafish for gene expression and enzyme activity analyses were anesthetized with MS-222 on ice to death, and their liver and brain tissues were dissected. For gene expression analysis, the tissues were maintained overnight in RNA storage solvent at 4  C, and then stored at
80  C for RNA extraction without the RNA storage solvent. The obtained tissues were immediately placed on ice for determining enzyme activity. During the experiments, the exposure surroundings, including temperature, humidity, and light cycle, were maintained the same as those in the culture environment. 2.4. Analysis of PCB91 The levels of PCB91 in adult zebrafish and water were determined. Water samples (100 mL) were subjected to liquid-liquid extractions with n-hexane in a separatory funnel along with violently shaking. The n-hexane layer was transferred to heartshaped flasks and concentrated to near-dryness by rotary evaporation (Shanghai Ailang Instruments, Shanghai, China) at 35  C. The

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T. Chai et al. / Environmental Pollution 215 (2016) 66e76

concentrated solutions were then blown to dryness by nitrogen evaporation and the residue was again dissolved in 1 mL of isooctane for GC-MS analysis. Six adult zebrafish with whole body were weighed for follow-up progress conducted in a previous study (Ottonello et al., 2014) and were then processed for GC-MS analysis. An Agilent 7890A/5975C GC-MS system equipped with a Chirasil-Dex capillary column (25 m  0.25 mm; I.D. 0.25 mm df) from Agilent was used for concentration determinations, and the oven temperature was programmed as follows: 60  C for 2 min, 60e150  C at 10  C min
1 (held for 5 min), 150e180  C at 1  C min
1 (held for 22 min). SIM ions were m/z 326 (quantification ion), 328, and 324 (Dai et al., 2014).

When SS was not reached, BCF was calculated as the first-order kinetic model (Eq. (2))

dCB ¼ k1  CW
k2  CB ðaccumulationÞ dt ¼
k2  CB ðelimiationÞ

dCB dt

Supposing that the concentration of compound in fish is zero when t ¼ 0, and that in exposure media is constant, then Eq. (3) is obtained as follows:

CB ¼

 k1   1
e
k2 t ðaccumulationÞ k2

CB

2.5. Enzyme activity, ROS and MDA contents

¼ CB;0  e
k2 t ðelimiationÞ

The collected brain/liver tissues were homogenized in 50 mM cold potassium phosphate buffer (pH 7.4) containing 0.5 mM EDTA2Na. The homogenate was centrifuged at 12,000 rpm/min for 30 min at 4  C, and the supernatant was used for biochemical parameter analysis. The SOD activity was determined by measuring the inhibition of the photochemical reduction of pyrogallol (Wang et al., 2015). CAT and Gpx activities were measured as described by Mu et al. (Mu et al., 2014). MDA and ROS contents were determined using the respective assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturers' instructions.

When the SS is reached, Eq. (4) can be obtained as follows:

2.6. Gene expression studies Total RNA was extracted from liver/brain tissue using an RNAprep Pure Tissue Kit (Tiangen Biotech, China). The quality of the isolated RNA was evaluated based on the quality of the 28s and 18s rRNAs by 2% agarose gel electrophoresis and the purity of the RNA preparations was assessed based on the ratio of OD260/OD280. The concentration of the RNA was determined by OD260 using a UV1240 spectrophotometer (Perkin Elmer, USA). First-strand complementary DNA (cDNA) was synthesized from 0.5 mg of total RNA using a FastQuant RT Kit (Tiangen Biotech). Quantitative real-time polymerase chain reaction (qPCR) was performed using a SuperReal PreMix Plus Kit (Tiangen Biotech) and an ABI 7500 qPCR system (Applied Biosystems, USA). Primers are designed according to previous studies (Mu et al., 2015a). The housekeeping gene b-actin was used as an internal standard to eliminate variations in mRNA and cDNA quantity and quality. Three-step qPCR was used to quantify the relative levels of housekeeping and target genes: 95  C for 15 min; 40 cycles of 95  C for 10 s, 60  C for 20 s, and 72  C for 32 s. A melting curve analysis was also included to demonstrate the specificity of PCR product as a single peak. Relative quantification of target genes normalized to bactin levels was performed by the 2
DDCt method. 2.7. Statistical analysis EF was evaluated to express enantiomeric compositions (Chai et al., 2014) (Eq. (1)) for PCB91 as follows:

EF ¼ ð þ Þ=ð þ Þ þ ð
Þ

(1)

where (þ) and (
) are the concentrations of the first- and secondeluting isomers, respectively. The EF value was in the range of 0e1, and a value of 0.5 represents racemate. BCF was calculated according to the OECD305 guideline and defined for aquatic species as the ratio between the concentration in fish (CB) and that in the surrounding media (CW) at equilibrium (steady state, SS).

(2)

BCFk ¼ CB =CW ¼ k1 =k2

(3)

(4)

where CB is expressed in mg/kg, t is the exposure time (d), CW is expressed in mg/L, k1 is the first-order accumulation (L/kg wet weight per day), and k2 is the first-order elimination (per day). The SigmaPlot 12.0 software was used for the analysis of the kinetic model. Statistical analyses were performed using SPSS16.0 software. Differences were determined using one-way analysis of variance (ANOVA), followed by a posthoc Dunnett test. All data are expressed as mean ± standard error of the mean. Values with P < 0.05 were considered statistically significant. Pearson's correlation coefficients were calculated for pairs of biomarkers by using SPSS16.0 software.

3. Results and discussion 3.1. Method validation To assess the specificity of the method, blank extracts of matrix (fish/water) were processed independently. The signal produced were observed at the retention time ±0.5% of (þ)/(
)- PCB91 and then compared with that of a spiked sample, showing that the matrix was free from interferences. Five point solvent or matrix-matched calibration curve (5.0mg/ Le500 mg/L for racemate) were created. The EF values of racemic PCB91 were 0.525 ± 0.017. Satisfactory linearity was obtained for (þ)-PCB91 (R2 ¼ 0.9987) and (
)-PCB91 (R2 ¼ 0.9998). No significant differences between solvent or matrix-matched calibration curves were observed, thus excluding the presence of critical matrix effects. The sensitivity was estimated using the matrix-dependent LODs (limits of detection) and LOQs (limits of quantitation). The LODs and LOQs were defined as the concentrations that generated S/N of 3 and 10, respectively. The LODs and LOQs both for (þ)- and (
)PCB91 in fish were 1 mg/kg and 2.5 mg/kg, respectively. The LODs and LOQs both for (þ)- and (
)- PCB91 in water were 0.2 mg/kg and 0.5 mg/kg, respectively. The precision and accuracy were evaluated at three spiked concentration levels (2.5, 25 and 250 mg/kg) for each isomer in fish and that (0.02, 0.2 and 2 mg/L) for each isomer in water. Satisfactory recovery of the isomers in range of 91.3%e100.0% for fish and 98.7%e104.7% for water were obtained in Table 1. Relative standard deviations (RSD) for repeatability and reproducibility, were lower than 11.9% and 9.1% for fish, 10.0% and 9.0%, respectively.

T. Chai et al. / Environmental Pollution 215 (2016) 66e76

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Table 1 The recovery, repeatability and reproducibility at three spiked levels for (þ)-/(
)- PCB91 in water and adult zebrafish. Spiked level

Fish

Water


1

2.5 mg kg 25 mg kg
1 250 mg kg
1 0.02 mg L
1 0.2 mg L
1 2 mg L
1

(þ)-PCB91

(
)-PCB91

Recovery (%)

Repeatability %RSD

Reproducibility %RSD

Recovery (%)

Repeatability %RSD

Reproducibility %RSD

91.3 96.7 92.0 98.7 100.4 101.2

6.7 11.9 4.3 5.8 10.0 8.0

7.3 5.3 4.4 8.0 4.2 5.7

98.0 100.0 93.3 104.7 103.3 102.7

8.2 10.0 6.5 7.7 5.6 6.0

6.3 9.1 6.0 6.9 5.4 9.0

3.2. Stereoselective bioconcentration Variations in the normal concentrations of the three forms of chiral PCB91 in water were always below 20% during one day, suggesting that their concentrations remained constant in the exposed aquatic media. In addition, the EFs of the collected water samples were also calculated and in range of 0.489e0.497, suggesting that no isomerization was observed for either of the two isomers in water. The uptake and depuration profiles for racemic/(
)-/(þ)- PCB91 in adult zebrafish are shown in Fig. 1. No target compounds were detected in the control group. However, all three forms of chiral PCB91 accumulated in adult zebrafish (Fig. 1). EFs decreased with prolonged exposure time (Table 2), suggesting that stereoselective accumulation occurred during racemic exposure (Fig. 1-a). The accumulation of (þ)-PCB91 was considerably higher in the (þ)-PCB91eexposed group (Fig. 1-b) than in the (
)-PCB91- (Fig. 1c) and racemic-exposed groups. The racemic and (
)-PCB91 levels almost reached SS during the uptake experiment, whereas the concentration of (þ)-PCB91 remained high until depuration. After racemic/(þ)-/(
)- PCB91 exposure, the analyte concentration in adult zebrafish reduced after depuration, but still reaching significant concentrations. Chiral PCB91 has previously been shown to remain non-racemic in aquatic organisms collected from PCBpolluted sites. Invertebrates such as yellowfin shiner (Dang et al., 2010a) showed the presence of non-racemic EFs after PCB91 and non-racemic enantiomer enrichment in the Arctic char, indicating stereoselective bioaccumulation in piscivorous fishes (Lu et al., 2014), similar to those found in our study. Previous studies had also investigated the correlations between EFs and PCB concentrations to find the mechanism of stereoselective bioaccumulation. A linear correlation between total concentration and EFs of PCB91 were found in bottlenose dolphin blubber from the Turtle/Brunswick estuary (Ross et al., 2011). However, no correlation was found between total concentration and EFs of PCB91 in our study, which was consistent with our previous study on PCB149 in adult zebrafish (Chai et al., 2016). Our results imply that CYP metabolism other than stereochemical process affects PCB91 bioaccumulation in adult zebrafish (Hoekstra et al., 2002). BCF has been increasingly used to understand the impacts of chemicals on organisms, such as toxicokinetic and toxicodynamic modeling (Ashauer et al., 2006). The toxicokinetic values are shown in Table 3. The k1 and k2 values were obtained by fitting data to a non-linear regression curve, and BCFk values were obtained from k1 and k2. In Canada, chemicals with BCF values exceeding 5000 (logBCFk, 3.7) are considered to be indicative of bioaccumulation, and such chemicals are recommended for “virtual elimination” (Canada, 1995). In our study, the logBCFk values for the isomers were close to 3.7. The logBCFk values for common carp (Crucian carp) exposed to 1 mg L
1 dicofol (METI-NITE, 2002) and zebrafish eleutheroembryos exposed to 1 mg L
1 chlorpyrifos (El-Amrani et al., 2012) were 3.91 and 3.55, respectively. These values are in the range as those found in our study at the same exposure

concentration. In addition, a previous study showed no significant difference between the BCF values for low and high concentrations for most of general chemical substances (Burden et al., 2014). However, in our study, there was a slight difference in the log BCFk values between racemic exposure (3.20) and (þ)-PCB91 exposure (3.92) groups, which could be attributed to the fact that the (þ)-PCB91 concentration had not reached SS in adult zebrafish after exposure. 3.3. Effects on ROS and MDA contents Changes in ROS concentrations in the brain and liver tissues are shown in Fig. 2. The ROS concentration increased at the early exposure time, i.e., 7 d (U7d) and 21 d (U21d), but decreased with prolonged exposure in the brain tissue, whereas it continued to increase with exposure time in the liver tissue. Abnormal concentrations of ROS were observed in the brain and liver tissues even during depuration. PCB-induced oxidative stress has been reported to lead to the generation of ROS owing to the impairment of electronic flow during the redox process (Schlezinger and Struntz, 2006). Abnormal ROS generation could induce significant cellular damage, such as damage to membrane lipids, nucleic acids, and proteins (Taju et al., 2014). For example, PCB126 and PCB169 were shown to stimulate the release of ROS in scup liver microsomes (Schlezinger et al., 2006) and in intact porcine endothelial cells (Hennig et al., 2002). PCB126 significantly increased ROS activity in killifish embryos collected from contaminated sites (Arzuaga and Elskus, 2010). MDA is the main by-product of lipid peroxidation. Lipid oxidation could lead to the production of many secondary metabolites that can exacerbate oxidative damage. In our study, no significant changes were noted in the MDA content in the brain of adult zebrafish; however, in the liver (Fig. 3), MDA contents decreased with prolonged exposure during the uptake experiments, but increased during the depuration. 3.4. Effects on antioxidant enzyme activities The fold changes of antioxidant enzymes SOD, CAT, and Gpx in the brain and liver tissues compared to those in the control groups during the uptake and depuration experiments are shown in Fig. 4. Antioxidant enzymes are considered as unique systems for protecting organisms against the damage caused by activated ROS. O2
can be converted to H2O2 by SOD, and then to oxygen and water by CAT and Gpx (Jin et al., 2011). The SOD activities initially increased in the brain tissue at U7d and U21d after exposure to racemic, (þ)-, and (
)- PCB91. CAT activity decreased significantly at U7d but showed no obvious changes compared to the control except at U49d after racemic exposure. Irregular changes in Gpx activities were observed in the brain tissue. It significantly increased by about 1.8-, 2.0-, and 3.0fold compared to that of the control at U21d, but significantly decreased by about 0.2-, 0.1-, and 0.03-fold compared to that of the

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T. Chai et al. / Environmental Pollution 215 (2016) 66e76

Fig. 1. Profiles of accumulation and elimination in adult zebrafish exposed to the three forms of chiral PCB91. a: exposed to 1 mg L
1 racemic PCB91, b: exposed to 1 mg L
1 (þ)-PCB91 isomer, c: exposed to1 mg$L
1 (
)-PCB91 isomer. Solid circles (C) and triangles (:) correspond to the determined values, and error bars indicate standard deviations. Solid (d) and dash (e e) lines correspond to the expected values based on the model calculations.

Table 2 The enantiomer faction (EF) values of determined concentrations when adult zebrafish were exposed to racemic PCB91. Exposure time (uptake) U0d 0.458 ± 0.009

U3d 0.440 ± 0.014

U7d 0.446 ± 0.012

U49d 0.432 ± 0.010

U56d 0.425 ± 0.007

D0d 0.425 ± 0.007

U14d 0.443 ± 0.017

U21d 0.406 ± 0.023

U35d 0.436 ± 0.006

U42d 0.426 ± 0.012

D14d 0.354 ± 0.019

D21d 0.231 ± 0.038

D49d 0.287 ± 0.015

Exposure time (depuration) D7d 0.312 ± 0.023

control at U35d, after exposure to racemic, (þ)-, and (
)- PCB91, respectively. An initial increase in SOD activities in the brain tissue could be attributed to the increase in ROS concentration, and then it might have been inactivated by H2O2 (Buha et al., 2015). No obvious changes in CAT activity were noted in the brain tissue; this result was consistent with that reported previously for rats injected with

PCB in the cerebral areas (Marin-Prida et al., 2013). PCB91 induced SOD and Gpx but had no obvious effect on CAT, suggesting that H2O2 in the brain was mainly removed by Gpx. In general, antioxidant activities in the liver tissue of treated groups varied more than those in the brain tissue. For example, antioxidant enzyme activities were significantly higher in the liver tissue than in the brain

T. Chai et al. / Environmental Pollution 215 (2016) 66e76

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Table 3 Toxicokinetic parameters and bioconcentration factor (BCF) values obtained for chiral PCB91 after uptake and depuration of adult zebrafish exposed to 1 mg L
1 PCB91. Forms Racemate (þ)-PCB91 (
)-PCB91 Isomers (þ)-PCB91 (
)-PCB91

k1 (uptake) (L mg h
1)

k2 (uptake) (mg h
1)

k2 (depuration) (mg h
1)

Log BCFk

145.86 183.47

0.09 0.09

0.14 0.08

3.20 3.32

91.60 307.61

0.01 0.11

0.18 0.07

3.92 3.45

Fig. 2. The relative reactive oxygen species (ROS) contents in treated adult groups compared to that in the control group. a: brain tissues, b: liver tissues. Asterisks denote significant differences between treatments and control (determined by Dunnett posthoc comparison, *P < 0.05 and **P < 0.01). Error bars indicate standard deviations. Bars within the same exposure time (a, b, and c) that do not have the same letter are significantly different by Tukey's HSD posthoc test.

(Palace et al., 2009). 3.5. Effects on antioxidant gene expression

Fig. 3. The relative MDA contents in the liver tissue of treated adult groups compared to that in the control group. Asterisks denote significant differences between treatments and control (determined by Dunnett posthoc comparison, *P < 0.05 and **P < 0.01). Error bars indicate standard deviations. Bars within the same exposure time (a, b, and c) that do not have the same letter are significantly different by Tukey's HSD posthoc test.

tissue after depuration for 21 d (D21d). After depuration, the SOD enzyme activity was about 2.7-, 2.2-, and 2.2-fold higher and Gpx enzyme activity about 1.4-, 1.6-, and 1.6-fold higher than those of the control for racemic, (
)-, and (þ)- PCB91, respectively. Liver is known to be a primary detoxification organ for chemical metabolism; the different responses of antioxidant enzymes in different tissues might be attributed to the different antioxidant capacities of the tissues (Hegseth et al., 2011; Periandri-Steinberg, 2010; Pogrmic-Majkic et al., 2012). Effects of PCBs on oxidative stress-related enzymes have been investigated in various tissues of mammalian species (Gao et al., 2011). However, no conclusive results about the effects of PCBs on antioxidant enzymes in fish have been obtained. Antioxidant enzyme activities declined after exposure of zebrafish embryos to PCB126 (Liu et al., 2014). However, antioxidant enzyme activities were not found to be sensitive indicators of oxidative stress after exposure of lake trout to PCB126

A previous study revealed that the induction of antioxidant genes was an important adaption to oxidative stress induced by environmental chemicals (Song et al., 2012). Thus, expression of antioxidant genes was assessed in this study as a biomarker of oxidative stress to gain insight into the mechanism of antioxidant systems. The effects of racemic/(
)-/(þ)- PCB91 exposure in the brain and liver tissues on the mRNA levels of various genes (sod, cat, and Gpx) encoding antioxidant proteins were determined using RTqPCR (Fig. 5). In this study, prolonged exposure changed the expression levels of hepatic antioxidant genes of the treated groups more than that in the brain tissue. The mRNA levels were up-regulated at U7d, but down-regulated at U21d and U35d in the liver tissue after racemic/ (
)-/(þ)- PCB91 exposure. The hepatic kinetics of sod and Gpx expression were almost similar; they were significantly induced during uptake and depuration experiments. Cerebral cat gene was not significantly induced except for at U7d. The toxic effects of PCBs have been attributed to genotoxic effects involving the induction of polyploidy, telomere shortening, and micronuclei formation (Zhu et al., 2013). The mRNA expression levels of antioxidant genes in rare minnow larvae were remarkably higher after exposure to high concentrations of Aroclor1254 (Wu et al., 2014). Exposure of zebrafish embryos to PCB126 was shown to lead to the induction of sod gene expression (Na et al., 2009). However, the mechanisms underlying these effects are not completely known. 3.6. Stereoselective effects on oxidative stress Chiral PCB91 stereoselectively induced hepatic and cerebral oxidative stress in adult zebrafish after racemic/(
)-/(þ)- PCB91 exposure. Stereoselective changes of cerebral and hepatic ROS contents could be observed even at certain sampling points such as at D42d. According to the MDA contents in the liver, lipid

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T. Chai et al. / Environmental Pollution 215 (2016) 66e76

Fig. 4. The relative enzyme activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (Gpx) in the treated adult groups compared to those in the control group. a: brain tissues, b: liver tissues. Asterisks denote significant differences between treatments and control (determined by Dunnett posthoc comparison, *P < 0.05 and **P < 0.01). Error bars indicate standard deviations. Bars within the same exposure time (a, b, c, and d) that do not have the same letter are significantly different by Tukey's HSD posthoc test.

peroxidation was stereoselective during the uptake and depuration experiments. Antioxidant enzymes activities and gene expressions were stereoselectively induced by oxidative stress. SOD activity increased significantly in the brain tissue after exposure to racemic and (
)- PCB91, but not after exposure to (þ)- PCB91 at D21d. CAT activity significantly decreased in the liver tissue after exposure to racemic and (þ)- PCB91, but significantly increased after (
)- PCB91 exposure at U7d. Although no significant changes in sod expression in the brain tissue were noted at U35d after racemic exposure compared to that in the control group, significant changes were observed compared to the isomer-treated groups. Expression of cat in the liver tissue significantly increased by about 4.3-fold after racemic exposure, but significantly decreased by about 0.3-fold and 0.7-fold after (
)- and (þ)- PCB91 exposure, respectively, compared to those of the control group at D42d. In recent years, differences in oxidative stress response between different enantiomers of chiral chemicals have been investigated in mammals. A chiral synthetic pyrethroid, 1S-cis-bifenthrin induced considerably more hepatic oxidative stress than 1R-cis-bifenthrin, suggesting that oxidative stress in mice was induced in an enantiomer-specific manner (Jin et al., 2013b). Enantioselectivity in oxidative stress damage was observed when PC12 cells were exposed to chiral o,pʹ-DDD (Wang et al., 2013) and profenofos (Lu

and Yu, 2014). However, studies on the stereoselective induction of oxidative stress in fish species are rare. 1S-cis-bifenthrin was reported to have higher risk to induce oxidative stress in embryonic zebrafish by activating the mRNA expression of antioxidant genes (Jin et al., 2013a). Our study findings on stereoselective oxidative stress in the brain and liver tissues of adult zebrafish induced by chiral PCB91 suggest that ecotoxicological effects and health risks of chiral contaminants in aquatic system need to be evaluated.

3.7. Relationship between bioconcentration and oxidative stress Since PCBs adversely affected the oxidative status of the liver and brain tissues, determining whether these effects were dosedependent and whether there was a correlation between them was necessary. To our knowledge, this is the first study to systematically address the possible effects of PCBs on the parameters of oxidative stress. Dose-dependent changes in oxidative stress parameters induced by PCBs have been reported. Dose-dependent production of different biomarkers such as ROS and MDA was found in the brain tissues of rats exposed to subchronic doses of PCB126 (Hassoun et al., 2000). Low, but not high dose of PCB126, could increase the hepatic antioxidant enzyme activities in marine fish

T. Chai et al. / Environmental Pollution 215 (2016) 66e76

73

Fig. 5. The relative gene expression of sod, cat, and Gpx in the treated adult groups compared to those of the control group. a: brain tissues, b: liver tissues. Asterisks denote significant differences between treatments and control (determined by Dunnett post hoc comparison, *P < 0.05 and **P < 0.01). Error bars indicate standard deviations. Bars within the same exposure time (a, b, c, and d) that do not have the same letter are significantly different by Tukey's HSD posthoc test.

scup (Stenotomus chrysops) (Schlezinger and Stegeman, 2001). Exposure to subacute doses of Aroclor1254 induced oxidative stress in the liver of rats with dose-dependent changes in the investigated parameters such as antioxidant enzyme activities (Buha et al., 2015). The dose-dependent effects of PCBs on the parameters of oxidative stress are shown in Table 4. Significant positive correlation between bioconcentration and CAT activity and cat expression in the brain tissue suggested that increased PCB91 concentration activated CAT activity and enhanced the expression of cat. Cerebral

ROS contents and sod and Gpx expression decreased dosedependently in zebrafish exposed to (
)-PCB91. Hepatic MDA contents showed significant dose-dependent changes after racemic/(
)-/(þ)- PCB91 exposure. Significant negative correlation existed between the mRNA level of antioxidant enzymes and PCB91 concentration after racemic exposure, but significant positive correlation was found with PCB91 concentration after (þ)- PCB91 exposure. Reduced ROS production might result from the altered mRNA

Table 4 The correlation coefficients between bioconcentration and other factors. Biomarkers

Correlations coefficients Brain

ROS MDA SOD CAT Gpx sod cat Gpx

Liver

Racemate

(þ)-PCB91

(
)-PCB91

Racemate

(þ)-PCB91

(
)-PCB91


0.005 e
0.029 0.706**
0.441
0.240 0.492*
0.182


0.355 e
0.251
0.044
0.291
0.391
0.092 0.157


0.552* e
0.221
0.377
0.184
0.579* 0.266
0.512*

0.598**
0.871**
0.260
0.311
0.439
0.505*
0.531*
0.606**

0.271
0.639**
0.276
0.250
0.727** 0.563* 0.499* 0.655**


0.505*
0.822**
0.183 0.413
0.839**
0.035 0.032
0.354

*P < 0.05 according to Spearman's test; **P < 0.01 according to Spearman's test.

74

T. Chai et al. / Environmental Pollution 215 (2016) 66e76

Table 5 The correlation coefficients between reactive oxygen species (ROS) contents and other factors. Biomarkers

Correlations coefficients Brain

SOD CAT Gpx sod cat Gpx

Liver

Racemate

(þ)-PCB91

(
)-PCB91

Racemate

(þ)-PCB91

(
)-PCB91

0.693**
0.367 0.571*
0.323
0.404
0.796**

0.773**
0.587* 0.437 0.794** 0.650* 0.208

0.596**
0.287 0.247
0.134
0.706*
0.211


0.029
0.493*
0.199
0507*
0.355
0.535*

0.191
0.549*
0.082 0.767** 0.838** 0.697**

0.030 0.102
0.416 0.272 0.236
0.058

*P < 0.05 according to Spearman's test; **P < 0.01 according to Spearman's test.

Table 6 The calculated correlations coefficients between antioxidant enzyme activities and gene expression. Biomarkers

Correlations coefficients Brain

SOD-CAT SOD-Gpx CAT-Gpx SOD-sod CAT-cat Gpx-Gpx

Liver

Racemate

(þ)-PCB91

(
)-PCB91

Racemate

(þ)-PCB91

(
)-PCB91


0.486*
0.550*
0.389 0.229 0.589*
0.147


0.385 0.775** 0.075 0.384
0.532*
0.075


0.240 0.783** 0.083
0.134 0.412
0.058

0.284 0.831**
0.013
0.319 0.389
0.245

0.226 0.758** 0.405 0.103
0481*
0.456

0.220 0.591**
0.330
0.284 0.318
0.025

*P < 0.05 according to Spearman's test; **P < 0.01 according to Spearman's test.

levels and/or activities of antioxidant enzymes (Arzuaga and Elskus, 2010). Whether ROS was the key factor that affected the antioxidant system in adult zebrafish treated with chiral PCB91 was assessed by calculating the correlation coefficients (Table 5). Cerebral ROS contents were remarkably positively correlated with SOD activity after racemic/(
)-/(þ)- PCB91 exposure, indicating that increase of ROS contents activated the SOD enzyme to convert O2
to H2O2. High positive correlation was observed between cerebral ROS contents and Gpx activity after racemic exposure, suggesting that the increase of H2O2 activated Gpx enzyme to produce oxygen and water. However, high negative correlation was found between ROS contents and CAT activity in the brain tissue after (þ)-PCB91 exposure and in the liver tissue after racemic and (þ)-PCB91 exposure, suggesting that CAT activity was inactivated by ROS contents. High positive correlation between ROS contents and gene expression of antioxidant enzymes in the liver tissue after (þ)-PCB91 exposure suggested that alteration of ROS was mainly attributed to the altered mRNA levels of antioxidant enzymes. However, no obvious correlation was found between ROS contents and the antioxidant system. In addition, gene expression and antioxidant enzyme activity were adversely affected after PCB91 exposure. ROS contents inhibited the expression of Gpx but enhanced that of Gpx in the brain tissue of adult zebrafish exposed to racemate. This adverse effect was also observed between cat expression and CAT activity in the brain tissue of adult zebrafish exposed to (þ)-PCB91. The interaction of antioxidant enzymes and the role of gene expression to enzymes activities were determined by calculating the correlations coefficients of biomarkers (Table 6). The antagonistic action of cerebral enzymes between SOD-CAT and SOD-Gpx after racemic exposure was affirmed by the strong correlation coefficients. However, the enzymes showed coordinated action between SOD and CAT in the brain tissues of rats (Hassoun and Periandri-Steinberg, 2010). The coordinated action of hepatic enzymes between SOD and Gpx after racemic/(
)-/(þ)- PCB91 exposure could be attributed to the strong correlation coefficients. In addition, PCBs affect the related gene expression and produce

toxic effects on antioxidant enzyme activities (Buha et al., 2015). However, the mechanism underlying this relationship was unclear. Antioxidant enzyme activities were not adequately controlled at the transcription level in zebrafish after acute copper exposure (Craig et al., 2007). No obvious correlation between gene transcription and enzyme activities was found in the (
)-PCB91-treated groups in our study as well. This could be attributed to the fact that gene transcription is transient, whereas enzymes activities are complex and long-lasting (Regoli et al., 2011; Yang et al., 2008). However, in our study, significant positive correlation was noted between mRNA expression of cat and CAT activity in the brain tissue after racemic exposure. There was significant negative correlation between mRNA expression of cat and CAT activity in the brain and liver tissues after (þ)-PCB91 exposure. These conflicting results between mRNA expression and antioxidant enzyme activities were also observed in zebrafish after atrazine exposure (Jin et al., 2010). In summary, our results demonstrated that waterborne exposure to chiral PCB91 could cause stereoselective accumulation and stereoselective induce of oxidative stress in adult zebrafish. Dosedependent changes in oxidative stress were also observed and ROS as a key factor affected the antioxidant system. The certain interaction of antioxidant enzymes and gene expression existed. Thus, results in our study could help to better understand the stereoselective toxicological mechanism of chiral PCB91 in adult zebrafish and properly assess PCB risk of aquatic containment.

Competing interest The authors declare no competing financial interest.

Acknowledgments This work was funded by the National Natural Science Foundation of China (No. 21477161 and 21177156) and China Postdoctoral Science Foundation (No. 2015M570178).

T. Chai et al. / Environmental Pollution 215 (2016) 66e76

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