Environmental Pollution 215 (2016) 66e76

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

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
b
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

a r t i c l e i n f o a b s t r a c t

Article history: This study aimed to investigate the oxidative stress process and bioaccumulation the racemic/(
)-/(þ)-
Received 1 December 2015 2,2ʹ,3,4ʹ,6-pentachlorobiphenyl were administered to adult zebrafish (Danio rerio) after prolonged
Received in revised form exposure of 56-days uptake and 49-days depuration experiments. Stereoselective accumulation was
18 April 2016
observed in adult samples after racemic exposure as revealed by decreased enantiomer fractions. The
Accepted 21 April 2016
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 stereo-
selectively induced oxidative stress owing to changes in reactive oxygen species, malondialdehyde
Keywords:
PCB91
contents, antioxidant enzyme activities and gene expressions in brain and liver tissues. In addition, the
Zebrafish stereoselective relationship between bioconcentration and oxidative stress were also presented in this
Oxidative stress study. Our findings might be helpful for elucidating the environmental risk of the two enantiomers of
Bioconcentration PCB91 that induce toxicity in aquatic organisms.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction two stable rotational enantiomers (Dai et al., 2014). The

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 toxico-
logical 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, Bei-
jing 100193, China.
** Corresponding author. 12 Zhongguancun South Street, Beijing 100081, China.
E-mail addresses: wangchengju@cau.edu.cn (C. Wang), qiujing@caas.cn (J. Qiu).
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

http://dx.doi.org/10.1016/j.envpol.2016.04.075
0269-7491/© 2016 Elsevier Ltd. All rights reserved.
 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 cyto-
toxic 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 (El-
Amrani 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 environ-
ment” 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 rela-
tionship between bioaccumulation and oxidative stress and the
differences induced by racemic/(þ)-/(
)- PCB91 were also deter-
mined. To our knowledge, this is the first study to systematically
evaluate the bioaccumulation and oxidative stress mechanism
induced by environmental concentrations of two different enan-
tiomers 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 re-
agents used in this study were of HPLC grade, and the other re-
agents 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 concen-
trations 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 trans-
ferred to clean aerated water, which was renewed every 24 h
during the depuration experiment. Five randomly selected in-
dividuals 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 expres-
sion 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 deter-
mined. 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 heart-
shaped flasks and concentrated to near-dryness by rotary evapo-
ration (Shanghai Ailang Instruments, Shanghai, China) at 35  C. The
68 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 isooc-
tane 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).
2.5. Enzyme activity, ROS and MDA contents
The collected brain/liver tissues were homogenized in 50 mM
cold potassium phosphate buffer (pH 7.4) containing 0.5 mM EDTA-
2Na. 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.
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 complemen-
tary 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 b-
actin 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 second-
eluting 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).
When SS was not reached, BCF was calculated as the first-order
kinetic model (Eq. (2))
dCB dCB
¼ k1  CW
k2  CB ðaccumulationÞ
dt dt
¼
k2  CB ðelimiationÞ (2)
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:
k1  
CB ¼  1
e
k2 t ðaccumulationÞ CB
k2
¼ CB;0  e
k2 t ðelimiationÞ (3)
When the SS is reached, Eq. (4) can be obtained as follows:
BCFk ¼ CB =CW ¼ k1 =k2 (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 corre-
lation 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 signifi-
cant differences between solvent or matrix-matched calibration
curves were observed, thus excluding the presence of critical ma-
trix 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
Table 1
The recovery, repeatability and reproducibility at three spiked levels for (þ)-/(
)- PCB91 in water and adult zebrafish.
Spiked level (þ)-PCB91
Recovery (%) Repeatability %RSD

1
Fish 2.5 mg kg 91.3 6.7
25 mg kg
1 96.7 11.9
250 mg kg
1 92.0 4.3
Water 0.02 mg L
1 98.7 5.8
0.2 mg L
1 100.4 10.0
2 mg L
1 101.2 8.0
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, sug-
gesting 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. 1-
c) 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 signifi-
cant concentrations. Chiral PCB91 has previously been shown to
remain non-racemic in aquatic organisms collected from PCB-
polluted 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 concen-
trations 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/Bruns-
wick 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 zebra-
fish (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
69
(
)-PCB91
Reproducibility %RSD Recovery (%) Repeatability %RSD Reproducibility %RSD
7.3 98.0 8.2 6.3
5.3 100.0 10.0 9.1
4.4 93.3 6.5 6.0
8.0 104.7 7.7 6.9
4.2 103.3 5.6 5.4
5.7 102.7 6.0 9.0
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 concen-
trations 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 elec-
tronic 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 oxida-
tion 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 pro-
tecting 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 ac-
tivity 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 3.0-
fold 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
70 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 U3d U7d U14d U21d U35d U42d

0.458 ± 0.009 0.440 ± 0.014 0.446 ± 0.012 0.443 ± 0.017 0.406 ± 0.023 0.436 ± 0.006 0.426 ± 0.012

Exposure time (depuration)

U49d U56d D0d D7d D14d D21d D49d

0.432 ± 0.010 0.425 ± 0.007 0.425 ± 0.007 0.312 ± 0.023 0.354 ± 0.019 0.231 ± 0.038 0.287 ± 0.015

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, antiox-
idant activities in the liver tissue of treated groups varied more than
those in the brain tissue. For example, antioxidant enzyme activ-
ities were significantly higher in the liver tissue than in the brain
 T. Chai et al. / Environmental Pollution 215 (2016) 66e76 71

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 k1 (uptake) (L mg h
1) k2 (uptake) (mg h
1) k2 (depuration) (mg h
1) Log BCFk

Racemate
(þ)-PCB91 145.86 0.09 0.14 3.20
(
)-PCB91 183.47 0.09 0.08 3.32
Isomers
(þ)-PCB91 91.60 0.01 0.18 3.92
(
)-PCB91 307.61 0.11 0.07 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

A previous study revealed that the induction of antioxidant

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 treat-
ments 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 re-
sults about the effects of PCBs on antioxidant enzymes in fish have
been obtained. Antioxidant enzyme activities declined after expo-
sure of zebrafish embryos to PCB126 (Liu et al., 2014). However,
antioxidant enzyme activities were not found to be sensitive in-
dicators of oxidative stress after exposure of lake trout to PCB126
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 RT-
qPCR (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
72 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 dose-
dependent and whether there was a correlation between them
was necessary. To our knowledge, this is the first study to sys-
tematically 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 correla-
tion 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 dose-
dependently 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 cor-
relation 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 Liver

Racemate (þ)-PCB91 (
)-PCB91 Racemate (þ)-PCB91 (
)-PCB91

ROS
0.005
0.355
0.552* 0.598** 0.271
0.505*

MDA e e e
0.871**
0.639**
0.822**
SOD
0.029
0.251
0.221
0.260
0.276
0.183
CAT 0.706**
0.044
0.377
0.311
0.250 0.413
Gpx
0.441
0.291
0.184
0.439
0.727**
0.839**
sod
0.240
0.391
0.579*
0.505* 0.563*
0.035
cat 0.492*
0.092 0.266
0.531* 0.499* 0.032
Gpx
0.182 0.157
0.512*
0.606** 0.655**
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 Liver

Racemate (þ)-PCB91 (
)-PCB91 Racemate (þ)-PCB91 (
)-PCB91

SOD 0.693** 0.773** 0.596**
0.029 0.191 0.030

CAT
0.367
0.587*
0.287
0.493*
0.549* 0.102
Gpx 0.571* 0.437 0.247
0.199
0.082
0.416
sod
0.323 0.794**
0.134
0507* 0.767** 0.272
cat
0.404 0.650*
0.706*
0.355 0.838** 0.236
Gpx
0.796** 0.208
0.211
0.535* 0.697**
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 Liver

Racemate (þ)-PCB91 (
)-PCB91 Racemate (þ)-PCB91 (
)-PCB91

SOD-CAT
0.486*
0.385
0.240 0.284 0.226 0.220

SOD-Gpx
0.550* 0.775** 0.783** 0.831** 0.758** 0.591**
CAT-Gpx
0.389 0.075 0.083
0.013 0.405
0.330
SOD-sod 0.229 0.384
0.134
0.319 0.103
0.284
CAT-cat 0.589*
0.532* 0.412 0.389
0481* 0.318
Gpx-Gpx
0.147
0.075
0.058
0.245
0.456
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 antioxi-
dant system in adult zebrafish treated with chiral PCB91 was
assessed by calculating the correlation coefficients (Table 5). Cere-
bral 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 antago-
nistic action of cerebral enzymes between SOD-CAT and SOD-Gpx
after racemic exposure was affirmed by the strong correlation co-
efficients. However, the enzymes showed coordinated action be-
tween SOD and CAT in the brain tissues of rats (Hassoun and
Periandri-Steinberg, 2010). The coordinated action of hepatic en-
zymes 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 tran-
scription 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 tis-
sue after racemic exposure. There was significant negative corre-
lation 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 expo-
sure to chiral PCB91 could cause stereoselective accumulation and
stereoselective induce of oxidative stress in adult zebrafish. Dose-
dependent 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 Foun-
dation of China (No. 21477161 and 21177156) and China Post-
doctoral Science Foundation (No. 2015M570178).
 T. Chai et al. / Environmental Pollution 215 (2016) 66e76 75

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