Ecotoxicology and Environmental Safety 181 (2019) 214–223

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

In vitro and in silico AHR assays for assessing the risk of heavy oil-derived T
polycyclic aromatic hydrocarbons in fish
Su-Min Baka, Haruhiko Nakatab, Dong-Hee Kohc, Jean Yooa, Hisato Iwataa, Eun-Young Kimc,d,∗
a
Laboratory of Environmental Toxicology, Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama, 790-8577, Japan
b
Faculty of the Advanced Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Chuo-ku, Kumamoto, 860-8555, Japan
c
Department of Life and Nanopharmaceutical Science, Kyung Hee University,26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of Korea
d
Department of Biology, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: In the aftermath of the Great East Japan Earthquake of March 11, 2011, marine fish in Kesennuma Bay, Japan,
Polycyclic aromatic hydrocarbon have been contaminated with heavy oil containing polycyclic aromatic hydrocarbons (PAHs). To estimate the
Aryl hydrocarbon receptor risk of six PAHs (benzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene,
In vitro reporter gene assay and 1-methylphenanthrene), which have been detected at high levels in the tissues of fish from Kesennuma Bay,
In silico docking simulation
we attempted to evaluate the effects of these PAHs on the fish aryl hydrocarbon receptor (AHR) signaling
Greenling
Heavy oil
pathway. We initially measured PAH concentrations and cytochrome P4501A catalytic activities (EROD:
ethoxyresorufin-O-deethylase and MROD: methoxyresorufin-O-demethylase) as markers of AHR activation in
greenlings (Hexagrammos otakii) collected from Kesennuma Bay in 2014. The results showed that alkylated PAH
concentrations and EROD/MROD activities were higher in sites close to the oil-spilled sites than in the control
site, suggesting AHR activation by spilled alkylated PAHs. We then investigated AHR-mediated responses to
these PAHs in the in vitro reporter gene assay system where red seabream (Pagrus major) AHR1 (rsAHR1) or
rsAHR2 expression plasmids were transiently transfected into COS-7 cells. The in vitro assay showed rsAHR
isoform-, PAH-, and dose-dependent transactivation potencies. The relative effective concentrations of benzo[α]
pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene, acenaphthene, and 1-methylphenan-
threne that induce 20% of the maximum benzo[α]pyrene response (REC20-BaP) for rsAHR1 activation were
0.052, 38, 79, 88, 270 nM, and no response, respectively, and those for rsAHR2 activation were 0.0049, 32, 53,
88, 60 nM, and no response, respectively. The results showed that the REC20-BaP values of benzo[α]pyrene for
both the rsAHR1 and rsAHR2 isoforms were lower than the concentrations (0.041–0.20 nM) detected in the
muscle tissue of fish from Kesennuma Bay, while the REC20-BaP values of other PAHs were higher than their tissue
concentrations. In silico rsAHR homology modeling and subsequent ligand docking simulation analyses indicated
that the rsAHR activation potencies of PAHs could be predicted from a rsAHR2 model. This study shows that in
vitro and in silico rsAHR analyses may be a useful tool for assessing the risks to fish contaminated with PAHs.

1. Introduction
In the aftermath of the Great East Japan Earthquake of March 11,
2011, the Sanriku Coast in the Tohoku Region was struck by a tsunami.
In Kesennuma Bay, this tsunami destroyed oil storage tanks and
resulted in the release of approximately 11.5 million liters of heavy oil.
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environ-
mental contaminants derived from three sources: petrogenic PAHs from
spilled oil, pyrogenic PAHs from the burning of organic matter, and
biogenic PAHs from the transformation of natural organic precursors

Abbreviations: PAH, polycyclic aromatic hydrocarbon; AHR, aryl hydrocarbon receptor; EROD, ethoxyresorufin-O-deethylase; MROD, methoxyresorufin-O-de-
methylase; AOP, adverse outcome pathway; MIE, molecular initiating event; CYP1A, cytochrome P450 1A; EPA, Environmental Protection Agency; REP, relative
potency; IEF, induction equivalency factor; 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; IEQ, induction equivalent; IDL, instrumental detection limit; IQL,
instrumental quantification limit; MQL, method quantification limit; BaP, benzo[α]pyrene; LBD, ligand binding domain; LOEC, lowest observable effect con-
centration
∗
Corresponding author. Department of Life and Nanopharmaceutical Science, Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 02447,
Republic of Korea
E-mail addresses: bak.sumin.vk@ehime-u.ac.jp (S.-M. Bak), nakatah@kumamoto-u.ac.jp (H. Nakata), totoro9209@khu.ac.kr (D.-H. Koh),
jeanyoo@korea.kr (J. Yoo), iwata.hisato.mz@ehime-u.ac.jp (H. Iwata), eykim08@khu.ac.kr (E.-Y. Kim).

https://doi.org/10.1016/j.ecoenv.2019.06.008
Received 28 February 2019; Received in revised form 31 May 2019; Accepted 2 June 2019
Available online 10 June 2019
0147-6513/ © 2019 Elsevier Inc. All rights reserved.
S.-M. Bak, et al.
(Neff, 1997; 2002). After the earthquake, contamination by the heavy
oil-derived PAHs was monitored using mussel and sediment samples in
the east coast Tohoku district (Mizukawa et al., 2017; Onozato et al.,
2016). In Kesennuma Bay, 27 PAHs were consequently detected in
mussels (Mizukawa et al., 2017). Higher levels of alkylated PAHs
(methylated phenanthrene, pyrene, and chrysene) were detected in the
mussels (Mizukawa et al., 2017), supporting earlier reports that alky-
lated PAHs are more abundant than non-alkylated PAHs in petrogenic
PAH-contaminated sediments (Neff, 2002; Sporstol et al., 1983). It has
been established that the marine ecosystem in Kesennuma Bay has been
exposed to a variety of PAHs. However, the impact of these PAHs on
marine organisms at the oil-spilled site has not yet been investigated.
Exposure to mixed PAHs elicits developmental defects such as
pericardial and yolk sac edema, reduced craniofacial structures, and
body axis defects in the embryos of pink salmon (Oncorhynchus gor-
buscha), pacific herring (Clupea pallasi), killifish (Fundulus heteroclitus),
Australian rainbowfish (Melanotaenia fluviatilis), zebrafish (Danio rerio),
and red seabream (Pagrus major) (Carls et al., 1999; Couillard, 2002;
Hannah et al., 1982; Heintz et al., 1999; Huang et al., 2012; Incardona
et al., 2005, 2006; Marty et al., 1997; Pollino and Holdway, 2002; Zhao
et al., 2017) . However, identifying the major toxic inducers among the
PAHs and assessing the risks of mixed PAHs are still difficult because
the toxicities of individual PAHs in fish are not well understood.
Recent trends in toxicology and risk assessment require less costly
and more rapid alternative approaches focusing on non-apical end-
points. As an alternative approach, the adverse outcome pathway
(AOP) concept has emerged (Ankley et al., 2010). AOPs link a mole-
cular initiating event (MIE) to an adverse outcome via key events, in a
manner specified by the key event relationships. The AOPs of PAH
exposure in fish species are not yet fully understood due to limited
information on MIEs, which warrant further study.
One well-known PAHs-MIE is their interaction with the aryl hy-
drocarbon receptor (AHR). PAHs have been recognized as AHR agonists
in teleost fish based on their ability to bind to AHRs and to induce an
AHR target gene, cytochrome P450 1A (CYP1A) (Billiard et al., 2002;
Bols et al., 1999; Hahn and Stegeman, 1994). Accompanied by exposure
to AHR agonists such as PAHs, CYP1A expression levels and its catalytic
activities (e.g., EROD: ethoxyresorufin-O-deethylase and MROD:
methoxyresorufin-O-demethylase) were correlated with developmental
defects in fish embryos (Billiard et al., 2004; Yamauchi et al., 2005).
Thus, the toxicity of PAHs has been most often linked to the AHR sig-
naling pathway. The AHR is a transcription factor containing basic
helix-loop-helix-PAS domains and is involved in many biological and
physiological processes, including development, cellular differentiation
and proliferation, xenobiotic metabolism, and immune defense (Murray
et al., 2014). Thus, measuring the AHR transactivation potency of PAHs
as a MIE is one approach that is critical for the hazard and risk as-
sessment of oil-derived PAHs (Barron et al., 2004; Pieterse et al., 2013).
The US Environmental Protection Agency (EPA) scientific advisory
board acknowledged that the relative potency (REP) approach re-
mained the most pragmatic method for the cancer risk assessment of a
mixture of PAHs (US EPA, 2011). Estimating individual PAHs' potencies
for AHR activation facilitates the risk assessment of total PAHs. Several
studies have developed REPs for 30 PAHs based on their AHR binding
affinity and AHR transactivation assays in mammalian systems (Nisbet
and LaGoy, 1992; Pieterse et al., 2013). As for the fish system, a total of
34 PAHs were investigated using piscine hepatocytes, in vivo admini-
strated embryos, and cell lines (PLHC-1 and RTL-W1) (Barron et al.,
2004). Whereas in vitro mammalian AHR reporter gene assays have
been applied to PAHs, an in vitro fish AHR reporter gene system has not
been applied for estimating the REPs of PAHs.
Our previous study developed an in vitro reporter gene system with
red seabream (Pagrus major) AHR1 and AHR2 (rsAHR1 and rsAHR2,
respectively) for assessing the transactivation potencies of dioxin-like
congeners (Bak et al., 2013). The CYP1A induction equivalency factors
(IEFs) for rsAHR1 and rsAHR2 were calculated on the basis of potency
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Ecotoxicology and Environmental Safety 181 (2019) 214–223
relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) derived
from the dose-response of each congener. We also constructed in silico
homology models for rsAHR1 and rsAHR2 proteins and performed
docking simulations for dioxin-like congeners. The results indicated
that both rsAHRs have a binding affinity to these congeners and their
interaction potencies (U_dock values) were correlated with rsAHR
specific-IEFs obtained from the in vitro rsAHR-driven reporter gene
study (r2 = 0.61 for rsAHR1, r2 = 0.52 for rsAHR2). Since most studies
on the AHR-mediated toxicities of dioxin-like congeners in fish species
have focused on zebrafish as a model species, it is believed that the fish
AHR2 isoform plays a critical role in the induction of toxicities and
CYP1A through exposure to dioxin-like congeners, but that AHR1
contributes much less to toxicities (Andreasen et al., 2002; Karchner
et al., 2005). However, our previous data indicated that both AHRs
could contribute to the toxic effects of dioxin-like congeners, suggesting
more attention should be paid to AHRs from diverse fish species other
than the zebrafish (Bak et al., 2017).
The objective of this study was thus to estimate the risk of heavy oil-
derived PAHs in fish from Kesennuma Bay. We initially measured PAH
concentrations and hepatic EROD and MROD activities as markers of
AHR activation in greenlings (Hexagrammos otakii) collected from
Kesennuma Bay in 2014. We then investigated the AHR transactivation
potencies of six PAHs which were detected in the tissue of greenlings
from Kesennuma Bay, using the in vitro rsAHR-driven reporter gene
assay established in our previous study (Bak et al., 2013). The relative
effective concentrations of these PAHs that induce 20% of the max-
imum benzo[α]pyrene (BaP) response (REC20-BaP) or induce 20% of the
maximum 2,3,7,8-TCDD response (REC20-TCDD) and their respective
REPs, which were compared to the potency of BaP or 2,3,7,8-TCDD,
were obtained from the in vitro rsAHR responses to the PAHs. We then
calculated the total BaP-based induction equivalents (IEQs) of the PAHs
in the tissues of greenlings and compared the geographical trends of the
IEQs with those of EROD and MROD activities in the greenlings. In
addition, the binding potencies of the six PAHs to each rsAHR were
estimated by in silico docking simulations. The relationship between the
in vitro rsAHR transactivation and in silico binding potencies was ex-
amined and the applicability of an in silico approach for screening PAHs
as rsAHR ligands was validated.
2. Materials and methods
2.1. Fish collection
In November 2014, six greenlings were captured from the inner part
of Kesennuma Bay (Fig. 1), which is the closest to the heavy oil-spilled
site (site A), 16 from Oshima Nada, which is located near site A (site B),
and 15 from the east coast of the Karakuwa Peninsula, which is outside
Kesennuma Bay and used as a control site (site C). The liver and muscle
tissues of greenlings were removed from the bodies at a laboratory
temporally set up on the coast of the Bay and immediately stored in a
freezer. For the liver samples, we measured EROD and MROD as cata-
lytic markers of CYP1A. To measure the concentrations of non-alkylated
and alkylated PAHs accumulated in the greenlings, we used muscle
tissues. To compare the PAH data from 2014 with those collected in
earlier years, PAH data of fish samples collected in 2011 and 2012 were
sourced from Nakata et al. (unpublished data). The number of samples
used for each measurement is summarized in Table S1.
2.2. Determination of PAH concentrations in fish from Kesennuma Bay
A total 22 non-alkylated PAHs and 25 alkylated PAHs and its 7
homologues were analyzed following a method previously published
(Nakata et al., 2014; Wang et al., 2003) with some modifications. Ap-
proximately 1–5 g of muscle tissue was homogenized with anhydrous
Na2SO4, and then extracted with a mixture of dichloromethane:hexane
(1:1) in a Soxhlet apparatus. The aliquot of the extract was
S.-M. Bak, et al.
Fig. 1. Location of sampling sites around Kesennuma Bay, Japan. A: The inner part of Kesennuma Bay was the closest to a heavy oil-spilled site. B: Oshima Nada
received the outflow of oil-contaminated water. C: The east coast of the Karakuwa Peninsula was set as a reference control site.
concentrated and analyzed for extractable lipid. Deuterated PAH sur-
rogate standards (acenaphthene-d10, phenanthrene-d10, chrysene-d12,
and perylene-d14, Supelco, Bellefonte, USA) were added to the re-
maining sample extract. The sample extract was cleaned using gel
permeation chromatography followed by silica gel column chromato-
graphy. The eluate was concentrated, and PAHs were determined by
gas chromatography (Agilent 7890A, Agilent Technologies, USA) cou-
pled with mass spectrometry (Agilent 5975C). The GC column used was
a BPX-5 fused silica capillary column (60 m × 0.25 mm id., S&G Sci-
entific Inc., Australia). The oven temperature was programed to in-
crease from 80 to 160 °C at 20 °C/min, then to 310 °C at 3 °C/min, and
hold for 20 min. As for the alkylated PAH homologues, such as me-
thylnaphthalenes (C1 to C4-N), methylfluorenes (C1 to C3-F), methyl-
dibenzothiophenes (C1 to C4-DBT), methylphenanthrene/anthracenes
(C1 to C4-PHE/AN), methylfluoranthene/pyrenes (C1 to C3-FLTH/PY),
methylbenzo[α]anthracene/chrysene (C1 to C3-BaA/CHRY), and me-
thylbenzopyrene/perylenes (C1 to C2-BP/PERY) were classified and
quantitated using a straight baseline integration method (Wang et al.,
2003). The monitored ions for individual PAH compounds and their
alkylated homologues were shown in Table S2.
The recoveries of deuterated PAHs ranged from 68 to 125% in the
present study. A standard reference material (SRM) of marine sediment
certified by international atomic energy agency-383, IAEA-383
(Villeneuve et al., 1998) was analyzed to ensure the accuracy and
precision of PAH concentrations determined in this study. As shown in
Table S2, concentrations of individual PAHs determined in this study
agreed well with recommended values of SRM.
The instrumental detection limits (IDLs) and quantification limits
(IQLs) of the PAHs were calculated using the following formulas
(Ministry of Environment, Japan, 2009):
IDLs = t ( n− 1, 0.05) × 2 × SD
IQLs = 10 × SD
where t (n−1, 0.05) gives the t value appropriate for the 95% con-
fidence level with n−1 degrees of freedom and the number of mea-
surements. The method quantification limits (MQLs) of the non-alky-
lated PAHs ranged from 0.002 to 0.006 wet weight (ng/g ww).
As for the alkylated PAHs groups, no quantification limit was given
for grouped PAHs, because they were quantitated using a straight
baseline integration method, and not as alkylated compounds.
Among the PAHs analyzed, five PAHs (dibenzothiophene, phenan-
threne, acenaphthene, 2,3,5-trimethylnaphthalene, and 1-methylphe-
nanthrene) were detected at higher concentrations in the muscle tissues
of 2011 greenlings. These PAHs, together with BaP as a well-known
AHR agonist, were selected for further in vitro and in silico analyses.
BaP, phenanthrene, and acenaphthene belong to the EPA's 16 priority
PAHs and 2,3,5-trimethylnaphthalene and 1-methylphenanthrene are
alkylated PAHs.
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Ecotoxicology and Environmental Safety 181 (2019) 214–223
2.3. In vitro rsAHR reporter gene assay
Standard chemicals for the six PAHs including dibenzothiophene
(99.6% purity), phenanthrene (98.9% purity), acenaphthene (99.0%
purity), 2,3,5-trimethylnaphthalene (98.6% purity), 1-methylphenan-
threne (99.5% purity), and BaP (99.5% purity) were purchased from
Enzo Life Sciences, Inc. (Fig. S1). These chemicals were dissolved in
dimethyl sulfoxide (DMSO) purchased from Sigma, except 1-methyl-
phenanthrene which was dissolved in toluene and DMSO (1:9).
The in vitro reporter gene assays were carried out according to a
method previously reported (Bak et al., 2013). Briefly, African green
monkey kidney fibroblast cells (COS-7) were maintained in RPMI-1640
medium (Hyclone) supplemented with fetal bovine serum (FBS, 10%
final concentration) at 37 °C under 5% CO2. Cells were seeded in a 24-
well plate at 5.0 × 104 cells per well. Transfections of vectors with Li-
pofectamine LTX (Invitrogen) were carried out in triplicate or quad-
ruplicate after an 18-h seeding of cells. A total of 300 ng DNA, which
contained 20 ng red seabream CYP1A-5XREs reporter vector (Bak et al.,
2013), 50 ng MRL/lpr mouse ARNT expression vector (Cho et al., 2015),
3 ng rsAHR1 or rsAHR2 expression vector (Bak et al., 2013), 0.2 ng
pGL4.75 (hRluc (Renilla reniformis)/CMV) as a control vector, and
226.8 ng pcDNA3.1/Zeo (+) empty expression vector, was mixed with
1 μL of LTX and the mixture was then added to the COS-7 cells. After 5 h
incubation, the media were exchanged with dextran-coated charcoal
(DCC)-stripped RPMI-1640 containing 10% DCC-stripped FBS for re-
ducing signals from unknown endogenous ligands. The cells were then
treated with serially diluted concentrations of PAHs or control solvent
(0.1% DMSO or 0.01% toluene/0.09% DMSO only for 1-methylphe-
nanthrene). After ligand treatment for 18 h, cells were lysed with
150 μL of passive lysis buffer (Promega). The activation of each reporter
vector was determined using a Dual-Luciferase Reporter Assay System
(Promega) according to the manufacturer's instructions. The luciferase
activities in lysates were measured using a multimode microplate
reader (BioTek Synergy2). The fold changes of the luciferase activity
(firefly/Renilla) ratio in PAH-treated wells compared with those in
control solvent wells were calculated. Data are presented as the
mean ± standard deviation (SD) from six to eight technical replicates
in two independent experiments.
To evaluate the species sensitivity of the rsAHR transactivation
potencies of PAHs, we conducted the in vitro assay with zebrafish AHR2
(zfAHR2) for BaP, phenanthrene, and acenaphthene.
2.4. Estimation of REPs and RECs
Dose–response curves of the PAHs for each rsAHR transactivation in
the in vitro assay were plotted as relative units to the maximum re-
sponse of BaP against logarithmically transformed doses. The 50% ef-
fective concentration (EC50) values of PAHs for each rsAHR
S.-M. Bak, et al.
transactivation were obtained using GraphPad 5.0 (San Diego, CA). The
REC20-BaP of each PAH was defined as the lowest observable effective
concentration (LOEC). To compare the potencies of these PAHs with
those of dioxin-like compounds (DLCs), REC20-TCDD values were also
calculated for six PAHs and seven DLCs; 2,3,7,8-TCDD, 1,2,3,7,8-pen-
tachlorodibenzo-p-dioxin (1,2,3,7,8-PeCDD), 1,2,3,4,7,8-hexa-
chlorodibenzo-p-dioxin (1,2,3,4,7,8-HxCDD), 2,3,7,8-tetra-
chlorodibenzofuran (2,3,7,8-TCDF), 2,3,4,7,8-pentachlorodibenzofuran
(2,3,4,7,8-PeCDF), 1,2,3,4,7,8-hexachlorodibenzofuran (1,2,3,4,7,8-
HxCDF), and 3,3ʹ,4,4ʹ,5-pentachlorinated biphenyl (PCB126). The raw
data of each rsAHR transactivation in the in vitro assay from the seven
DLCs were sourced from our previous paper (Bak et al., 2013).
REPs were estimated on the basis of a systematic framework which
was previously proposed (Kim et al., 2011; Thuruthippallil et al., 2013;
Villeneuve et al., 2000). BaP-relative potency 20, 50, and 80 (BaP-
REP20, -REP50, and -REP80) values were calculated as concentration
ratios; the concentration that induces 20, 50, and 80% of the maximum
BaP response divided by the concentration that induces the corre-
sponding response of each PAH, respectively. The BaP-REP of each PAH
was expressed as the average of the BaP-REP20, -REP50, and -REP80
values. The BaP-IEQs for the PAHs in greenling samples were calculated
by the sum of the rsAHR2-derived BaP-REP for a given PAH multiplied
by the concentration of the respective PAH. To calculate the IEQ values,
0.001 ng/g ww was assigned for PAHs with non-detected (N.D.) con-
centrations and 0.004 ng/g ww, a median value of MQL
(0.002–0.006 ng/g ww), was assigned for PAHs with non-quantified
(N.Q.) concentrations.
2.5. In silico rsAHR homology modeling and docking simulations
The in silico homology modeling for the ligand binding domain
(LBD) of rsAHR1 and rsAHR2 and the docking simulations of PAHs with
the rsAHR LBD models were conducted using the Molecular Operating
Environment, ver. 2015.10 (Chemical Computing Group Inc., Canada).
The docking simulations were performed to estimate the potential
binding energy of PAHs with each rsAHR LBD pocket using alpha
sphere and excluded volume-based ligand-protein docking (ASEDock)
provided by Ryoka Systems Inc., Japan (Goto et al., 2008). To construct
homology models of rsAHR LBDs, we applied the crystal structure (PDB
ID 3H7W.A) of the PAS-B domain sequence of human hypoxia-inducible
factor 2α, which is a closely allied protein to the rsAHR. Because of
uncertainties due to low identities with the template (25.7% for the
rsAHR1 LBD and 26.6% for the rsAHR2 LBD) (Fig. S2), four rsAHR LBD
model structures were constructed for each rsAHR isoform by the steps
described in the workflow of Fig. S3. The results of the docking simu-
lations of PAHs were shown as the binding potential energy, the U-dock
value (kcal/mol). To establish the relationship between the in silico
potential binding energy and the in vitro rsAHR transactivation potency,
we additionally estimated the potential binding energies for the DLCs.
Detailed methods for the in silico rsAHR homology modeling and ASE-
Dock simulations are given in the Supporting Information.
2.6. CYP spectral and catalytic activity assays
Liver microsomal fractions of the greenlings collected from
Kesennuma Bay in 2014 were prepared following the method described
by Guengerich et al. (1982). Approximately 1 g of liver tissue sample
was homogenized in 1 mL of cold homogenization buffer (50 M Tris-
HCl, 0.15 M KCl, pH 7.4–7.5) with a teflon-glass homogenizer (10
passes) and the homogenate was centrifuged for 10 min at 750×g. The
supernatant was centrifuged at 12,000×g for 10 min, and the gained
supernatant fraction was further centrifuged at 105,000×g for 98 min.
The fraction containing cytosol was removed, and the microsomal
pellets were resuspended in one volume of TEDG buffer (50 mM Tris-
HCl, 1 mM EDTA, 1 mM dithiothreitol, 20% vol/vol glycerol, pH
7.4–7.5). The microsomal fractions were frozen immediately in liquid
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Ecotoxicology and Environmental Safety 181 (2019) 214–223
nitrogen, and stored at −80 °C until use.
The protein concentrations in the microsomal fractions were de-
termined using the bicinchoninic acid method. BCA Protein Assay
Reagent (Pierce, Rockford, IL) and bovine serum albumin as a standard
were used for the protein assay. The absorbance at 560 nm was mea-
sured using a multiwell plate reader (SpectraFluor Plus, Tecan Austria
GmbH, Groedig, Austria). The hepatic microsomal CYP content of CO-
treated samples was determined from the dithionite difference spectra
at 450 nm with a DU800 spectrophotometer (Beckman Coulter, Inc.).
EROD and MROD activities were determined as previously de-
scribed with slight modification (Kubota et al., 2005, 2006). The en-
zymatic activities were performed by adding 0.002 mM ethoxyresorufin
for EROD and 0.005 mM methoxyresorufin for MROD with 0.33 mM
NADPH to the liver microsomes. The resorufin formed by the CYP1A
enzymatic activity was excited at 535 nm wavelengths, and detected at
595 nm wavelengths using a multiwall plate reader (SpectraFluor Plus).
2.7. Statistical analysis
Statistical analyses were performed using SPSS version 21.0 (SPSS
Inc., Chicago, IL, USA). Significant differences in PAH concentrations
and MROD/EROD activities among fish collection sites were analyzed
using ANOVA, followed by Scheffe or Games-Howell post hoc tests
subsequent to a Shapiro-Wilk normality test. Differences with p < 0.05
were regarded as statistically significant. To estimate the correlation
between in vitro REC20-TCDD values and in silico potential binding energy
(U_dock value, kcal/mol), Pearson correlation analyses were performed
using GraphPad 5.0 (San Diego, CA, USA).
3. Results
3.1. PAH levels in fish from Kesennuma Bay
The muscle samples of greenlings collected from the inner part of
Kesennuma Bay (site A), Oshima Nada (site B), and the Karakuwa
Peninsula (site C) in November 2014 were analyzed for a total of 22
non-alkylated PAHs, including the EPA's 16 priority PAHs and 25 al-
kylated PAHs and its 7 homologues (Fig. 2 and Table S3). The PAHs
concentrations were compared with data of PAHs in greenlings col-
lected from the same sites in 2011 and 2012 (Nakata et al., unpublished
data). In 2014 greenling muscles, fifteen non-alkylated PAHs, including
acenaphthylene, acenaphthene, fluorene, dibenzothiophene, phenan-
threne, anthracene, fluoranthene, pyrene, benz[a]anthracene, chry-
sene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[e]pyrene,
BaP, indeno[1,2,3-cd]pyrene, and benzo[ghi]perylene, and three alky-
lated PAHs, including 2,3,5-trimethylnaphthalene, 9,9-dimethyl-
fluorene (9,9-DMFL), and 1-methylpyrene (1-MPY), were detected at
levels higher than the MQLs.
Comparisons of concentrations of PAHs in 2011 and 2014 showed
that both ∑non-alkylated and ∑alkylated PAHs showed a decreasing
trend, except for the non-alkylated PAHs at site C (Fig. 2). For non-
alkylated PAHs, the 2011 and 2012 greenling muscles at site A had
significantly higher concentrations than the 2014 samples. For alky-
lated PAHs, the concentrations at site B decreased significantly over the
sampling years: 2011 > 2012 > 2014. Comparisons of concentrations
of PAHs among sampling sites showed that both ∑non-alkylated and
∑alkylated PAHs showed a decreasing trend from site A to site C, except
for the non-alkylated PAHs in 2014, which showed higher concentra-
tions at site C due to phenanthrene, anthracene, fluorene, and pyrene
detected at concentrations over their MQL values (Fig. 2). For ∑alky-
lated PAHs, a significant geographical trend was found in the 2012
samples: site A > B > C.
3.2. rsAHR transactivation potencies of PAHs
To investigate the transactivation potencies of six PAHs for both
S.-M. Bak, et al. Ecotoxicology and Environmental Safety 181 (2019) 214–223

Fig. 2. Concentrations of non-alkylated and al-

kylated PAHs in greenling muscle tissues. A: Inner
part of Kesennuma Bay. B: Oshima Nada. C: The east
coast of the Karakuwa Peninsula. The statistical dif-
ferences in sites A and B compared with site C were
examined by one way-ANOVA and Games-Howell
post hoc tests following a Shapiro-Wilk test. The
significant site-differences are expressed as an as-
terisk within the same year and the significant year-
differences are expressed as different alphabetical
letters, a, b, and c (p < 0.05). The data of con-
centrations of PAHs from sites B and C in 2014 and
from site C in 2011 were excepted from statistical
analysis because of the non-normal distribution and
insufficient data number (n = 2), respectively, and
represented as not available (N.A.). Concentration
data in 2011 and 2012 are cited from Nakata et al.
(unpublished data).

Fig. 3. Dose-response curves for in vitro rsAHR transactivation by PAHs. The responses of tested compounds are presented as a percent (%) relative to the
maximum response obtained from benzo[α]pyrene exposure. Data are plotted as mean ± SEM. S.C.: solvent control.

rsAHR isoforms, an in vitro reporter gene assay system was applied. All Table 1
the PAHs except 1-methylphenanthrene induced both rsAHR1-and REC20-B[α]P and REP values obtained by in vitro reporter gene assays.
rsAHR2-mediated responses in a dose-dependent manner (Fig. 3 and Ligand REC20-BaP (nM)a BaP-REPa
Fig. S4). Exposure to 1-methylphenanthrene induced no response for
either rsAHR isoform. The transactivation efficacies of dibenzothio- rsAHR1 rsAHR2 zfAHR2 rsAHR1 rsAHR2
phene and acenaphthene for rsAHR2 were higher than those for
Benzo[α]pyrene 0.052 0.0049 0.12 1 1
rsAHR1, whereas BaP, phenanthrene, and 2,3,5-trimethylnaphthalene Phenanthrene 79 53 99 0.0025 0.00022
gave similar efficacies for both rsAHRs. EC50 was calculated only for Dibenzothiophene 38 32 – 0.0033 0.00060
BaP because a full sigmoidal dose-response curve was obtained only for Acenaphthene 270 60 300 0.00026 0.00041
this chemical. The EC50 (0.016 nM) of BaP for rsAHR2 transactivation 2,3,5-Trimethylnaphthalene 88 88 – 0.0014 0.00008
1-Methylphenanthrene N.A. N.A. – N.A. N.A.
was 17.5-fold lower than that for rsAHR1 (0.28 nM).
The REC20-BaP values of phenanthrene, dibenzothiophene, ace- ‘-’ not examined.
naphthene, 2,3,5-trimethylnaphthalene, and BaP were 79, 38, 270, 88, N.A. denotes not available because no AHR activation was obtained by the
and 0.052 nM for rsAHR1 and 53, 32, 60, 88, and 0.0049 nM for chemical treatment.
rsAHR2, respectively (Table 1). The rsAHR2-derived REC20-BaP values a
REC20-BaP and B[α]P-REP values for each PAH were calculated by the
of BaP and acenaphthene were lower than their respective rsAHR1- methods indicated in the Materials and Methods (Supplementary Table S4).
derived values, whereas phenanthrene, dibenzothiophene, and 2,3,5-
trimethylnaphthalene had similar REC20-BaP values for both rsAHR1 and similar to the average concentration in 2014. The REC20-BaP values of
rsAHR2. the other PAHs were over 100-fold greater than their respective muscle
The REC20-BaP and EC50 values of BaP for rsAHR2 (0.0049 and concentrations.
0.016 nM, respectively) were lower than the average concentrations on The BaP-REPs of phenanthrene, dibenzothiophene, acenaphthene,
a wet weight basis (0.15 ± 0.05, 0.07 ± 0.004, and and 2,3,5-trimethylnaphthalene were calculated as an average of their
0.048 ± 0.006 nM in 2011, 2012, and 2014, respectively) detected in respective BaP-REP20, -REP50, and -REP80 (Fig. 3, Table 1, and Table
the muscles of greenlings caught from the heavily oil-contaminated bay S4). Comparison of BaP-REPs among the examined PAHs showed that
(site A), whereas the REC20-BaP value of BaP for rsAHR1 (0.052 nM) was BaP had over 500-fold higher potencies than other PAHs. The REC20-BaP
also lower than the average concentrations in 2011 and 2012 and values of BaP, phenanthrene, and acenaphetene for zfAHR2, which is a

218
S.-M. Bak, et al. Ecotoxicology and Environmental Safety 181 (2019) 214–223

well-known major AHR isoform in the zebrafish (Andreasen et al.,

2002; Karchner et al., 2005) were 0.12, 99, and 300 nM, respectively
(Fig. S4 and Table 1). The EC50 was calculated only for BaP because a
full sigmoidal dose-response curve was obtained only for this chemical.
The EC50 value (0.42 nM) of BaP for zfAHR2 transactivation was 27-
fold higher than that for rsAHR2 (0.016 nM) and similar to that for
rsAHR1 (0.28 nM).
In our previous study, the TCDD-REP values of seven DLCs were
estimated using the same in vitro rsAHR1/2-derived reporter gene assay
system (Bak et al., 2013). To compare the rsAHR transactivation po-
tencies between PAHs and DLCs, the REC20-TCDD values of the PAHs
were calculated (Table S5). For rsAHR1, the REC20-TCDD (0.052 nM) and
EC50 (0.28 nM) values of BaP were higher than those of TCDD (REC20-
TCDD: 0.0064 nM and EC50: 0.073 nM). In contrast, for rsAHR2, the
REC20-TCDD (0.0049 nM) and EC50 (0.016 nM) values of BaP were lower
than those of TCDD (REC20-TCDD: 0.079 nM and EC50: 0.52 nM).
Fig. 5. EROD and MROD activities in the liver of greenlings collected in
2014. The statistical differences in these enzymatic activities in sites A and B
3.3. In silico rsAHR-LBD homology modeling and PAH docking simulations compared with site C were examined by one way-ANOVA and Scheffe post hoc
tests. Significant differences between sampling sites are expressed as the dif-
To develop the in silico analysis system for screening potential ferent alphabetical letters, a and b (p < 0.05).
rsAHR-active PAHs, we initially constructed homology models for each
rsAHR isoform following the workflow shown in Fig. S3. The results of
with the rsAHR1-specific log-transformed REC20-TCDD values; Pearson
the phi (φ)-psi (ψ) dihedral angles for each amino acid residue showed
R2 = 0.13 (p = 0.24). In contrast, the U_dock values from the rsAHR2-
no outliers in the constructed rsAHR1-and rsAHR2-LBD models (Fig.
LBD homology model showed a significant correlation with REC20-TCDD
S6).
values; Pearson R2 = 0.87 (p < 0.0001).
The docking simulations of each rsAHR with the six PAHs and seven
DLCs were carried out using ASEDock (Table S5). The lowest U-dock
(kcal/mol) values were −23.4 kcal/mol of 2,3,7,8-TCDF for the 3.5. EROD and MROD activity in fish from Kesennuma Bay
rsAHR1-LBD model and −26.9 kcal/mol of 2,3,4,7,8-PeCDF for the
rsAHR2-LBD model. The U-dock values of the six PAHs from the The data on EROD and MROD in greenlings were available only for
rsAHR1-LBD docking simulation ranged from −7.4 kal/mol for ace- the liver samples collected in 2014 (Fig. 5 and Table S6). EROD dis-
naphthene to −10.7 kcal/mol for BaP. For rsAHR2-LBD, the U-dock played greater activity than MROD. Both EROD and MROD activities
values ranged from −16.4 kal/mol for acenaphthene to −26.5 kcal/ indicated a geographical trend (A > B > C) among sites; fish from site
mol for BaP. 1-Methylphenanthrene, with no activation potency for A had significantly higher activities than those from site B (p < 0.05)
either rsAHR1 or rsAHR2, showed low U_dock values that were similar and C (p < 0.005), whereas no significant differences in these activ-
to those of non-methylated phenanthrene (data not shown). ities were detected between sites B and C.

3.4. Relationships between in vitro and in silico measurements 3.6. BaP-IEQs in fish

To evaluate the results from the in silico docking simulations, we
examined whether the rsAHR-specific REC20-TCDD values of the PAHs
and DLCs obtained by the in vitro reporter gene assays could be pre-
dicted from the potential binding energies (U_dock values) derived from
the in silico docking simulations (Fig. 4 and Table S5). The U_dock va-
lues from the rsAHR1-LBD homology model showed a poor correlation
The total IEQs of the six PAHs were calculated by the summation of
the rsAHR2-derived BaP-REP values multiplied by their respective PAH
concentrations in greenling muscles (Table S7 and Fig. S7). BaP was a
dominant contributor to the total IEQ value in all sites due it giving the
highest BaP-REP. The total IEQs from the 2014 muscle samples were
similar between sites. This is because of the small variation in the

Fig. 4. Relationships between U_dock values and REC20-TCDD values for rsAHR1-and rsAHR2. Binding potential energy, U-dock values, were calculated from
docking simulations of six PAHs and seven DLCs to a rsAHR1-LBD (A) or rsAHR2-LBD homology model (B). REC20-TCDD values were calculated based on normal-
ization to TCDD responses. The dose-responses of the seven DLCs are cited from Bak et al. (2013) and REC20-TCDD values were recalculated based on these dose-
response curves. The results of Pearson correlation analysis are expressed as R2 with p values.

219
S.-M. Bak, et al.
distribution of BaP concentrations in these sites and the low contribu-
tions of dibenzothiophene, phenanthrene, acenaphthene, 2,3,5-tri-
methylnaphthalene, and 1-methylphenanthrene to the total IEQs.
4. Discussion
In the present study, we detected PAHs in the muscles of greenlings
from Kesennuma Bay. Mizukawa et al. (2017) collected blue mussels
(Mytilus galloprovincialis) from the oil-spilled area (near site A) in Sep-
tember 2011 after the earthquake in Kesennuma Bay and reported total
concentrations of 2700 ng/g dry weight (240 ng/g ww) of 27 PAHs in
the mussels. Fourteen PAHs were analyzed in common between the
mussels and our fish muscles in Kesennuma Bay; phenanthrene, an-
thracene, 1-methylphenanthrene, fluorene, pyrene, benzo[α]anthra-
cene, chrysene, benzo[b]fluoranthene, benzo[e]pyrene, BaP, perylene,
indeno[1,2,3-cd]pyrene, benzo[ghi]perylene, and coronene. Compar-
ison of the total concentrations of the 14 PAHs showed that the levels in
the mussel tissues (61 ng/g ww) were higher than those in the greenling
muscles from site A (2.68 ng/g ww), site B (2.26 ng/g ww), and site C
(1.89 ng/g ww) in 2011. The concentrations of the 14 PAHs (29 ng/g
ww) in mussels collected from Kesennuma Bay in 2014 were still higher
than those of greenling muscles in the same year (site A: 0.34 ng/g ww
and site B: 0.21 ng/g ww). This suggests that fish have been exposed to
high levels of PAHs, but accumulated less because of their high meta-
bolic capacity for PAHs. This could also be partially attributed to fish
being mobile and mussels being bound to one location (Livingstone,
1998; Varanasi et al., 1989). The EU established a guideline (maximum
level; 12 ng/g ww) of four PAHs (benzo[α]pyrene, benz[α]anthracene,
benzo[b]fluoranthene, and chrysene) in fish as foodstuff (European
Commission, 2011). The total concentrations of these four PAHs in the
muscles of greenlings caught in 2011 and 2014 (0.060–0.13 ng/g ww)
were much lower than this maximum EU guideline.
The geographical distributions of non-alkylated and alkylated PAHs
were A > B > C in 2011, 2012, and 2014, except for the non-alky-
lated PAH concentration in the 2014 samples. Contamination with al-
kylated PAHs greatly decreased in site B over the sampling years. The
average alkylated PAHs/non-alkylated PAHs ratio as an index of spilled
heavy oil was higher in site A (0.70, 0.91, and 0.85 for 2011, 2012, and
2014, respectively) and site B (0.61, 0.51, and 0.65 for 2011, 2012, and
2014, respectively) than in the control site C (0.21, 0.20 and 0.014 for
2011, 2012, and 2014, respectively). These results suggest that the al-
kylated PAHs in Kesennuma Bay originated from the spilled heavy oil
and indications of the spilled heavy oil were still detectable in sites A
and B even in 2014.
To assess the effects of potential AHR ligands in non-model fish, we
previously constructed a rsAHR-driven reporter gene assay system by
transiently transfecting the expression vector of rsAHR1 or rsAHR2 into
COS-7 cells with a reporter vector rsCYP1A-5XREs. Using this con-
structed in vitro system, the rsAHR1-and rsAHR2-mediated transacti-
vation potencies of the six PAHs were estimated. Both rsAHR1 and
rsAHR2 dose-dependently induced the transactivation of the rsCYP1A
promoter following treatment with each PAH except for 1-methylphe-
nanthrene. Although it is known that unsubstituted PAHs with two and
three aromatic hydrocarbon rings are generally inactive to AHRs in fish,
avian, and mammalian species (Barron et al., 2004), our results in-
dicated that when fish are exposed to high levels of PAHs, the PAHs
could be potent AHR ligands and activate AHR-mediated signaling
pathways. Higher EROD and MROD activities in the liver of fish col-
lected at site A, close to the heavy oil-spilled site, also support that fish
AHRs are activated by PAHs, in particular alkylated PAHs.
BaP is a well-known AHR agonist in fish such as the rainbow trout,
salmon, zebrafish, and red seabream (Barron et al., 2004; Bo et al.,
2014; Hestermann et al., 2000; Incardona et al., 2005). In the present
study, BaP caused dose-dependent transactivation of both rsAHR1 and
rsAHR2. The EC50 (0.28 nM) of BaP for rsAHR1 transactivation was
17.5-fold higher than that (0.016 nM) for rsAHR2. This suggests that
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Ecotoxicology and Environmental Safety 181 (2019) 214–223
rsAHR2 may be more sensitive to BaP than rsAHR1. Compared with the
zfAHR2-EC50 (0.42 nM) measured in the same method, rsAHR2 was
more sensitive to BaP than zfAHR2 (Fig. S6). The EC50 of BaP for
rsAHR1 (0.28 nM) was approximately 4-fold higher than that of 2,3,7,8-
TCDD (0.073 nM) and the EC50 of BaP for rsAHR2 (0.016 nM) was
approximately 32-fold lower than that of 2,3,7,8-TCDD (0.51 nM) (Bak
et al., 2013). The EC50 of BaP for EROD induction in the rainbow trout
liver cell line, RTL-W1, was 28.5 nM which was also higher than those
in our rsAHR1/2-based reporter gene system (Bols et al., 1999). Com-
parison with the mouse hepatoma cell line-based chemically activated
luciferase expression system (Han et al., 2004) indicated that the LOEC
of BaP (10 nM) was higher than the REC20-BaP values (0.052 nM for
rsAHR1 and 0.0049 nM for rsAHR2) in our rsAHR reporter gene system.
Our in vitro rsAHR1-and rsAHR2-derived REC20-BaP values were lower
than the in ovo LOEC (0.18 μg/L; 0.71 nM) reported for morphological
deformities in red seabream embryos (Zhao et al., 2017). The con-
centrations (0.01–0.051 ng/g ww; 0.041–0.20 nM) detected in the
muscles of oil-contaminated greenlings during 2011, 2012, and 2014
were higher than or similar to the REC20-BaP and EC50 values in this in
vitro study. This comparison implies that contamination with BaP could
induce the activation of the AHR signaling pathway in Kesennuma fish.
Phenanthrene is a weak AHR agonist and has been widely detected
in oil-spilled sites (Incardona et al., 2005,2006). Although phenan-
threne has been reported to be a non-inducer of EROD activity in
rainbow trout RTL-W1 cell lines, hybrid tilapia (Oreochromis niloticus
female × O. aureus male) and yellowfin seabream (Acanthopagrus latus)
showed dose-dependent EROD induction following phenanthrene ex-
posure (Bols et al., 1999; Shirmohammadi et al., 2018; Wenju et al.,
2009). Our in vitro assay showed clear dose-dependent transactivation
potencies of phenanthrene for rsAHR1 and rsAHR2. Both rsAHR1 and
rsAHR2 showed similar REC20-BaP values, 38 and 32 nM, respectively.
These REC20-BaP values were 3-fold lower than the REC20-BaP values
(99 nM) for zfAHR2. Comparison of in ovo and in vitro studies indicated
that in vitro rsAHR1-and rsAHR2-derived REC20-BaP values were ap-
proximately 100-fold lower than the in ovo LOEC (600 μg/L; 3.4 μM) for
red seabream embryo deformities (Zhao et al., 2017). Studies on phe-
nanthrene have reported that toxic effects are mediated by both AHR
dependent and independent pathways (Willett et al., 2001). Exposure to
phenanthrene caused cardiac toxicity in zebrafish embryos, but this
effect was not rescued by AHR2-molpholino injections, suggesting the
involvement of AHR independent pathways (Willett et al., 2001). The
large differences in the in vitro REC20-BaP values and in ovo LOECs may
thus be explained by the different sensitivities of in vitro and in ovo
experimental systems and also by the contributions of AHR in-
dependent pathways.
Exposure to dibenzothiophene showed clear dose-dependent re-
sponses through both rsAHR1 and rsAHR2. Dibenzothiophene has been
recognized as a noncompetitive inhibitor of CYP1A at mRNA and pro-
tein expression levels in fish such as the zebrafish, killifish, and rainbow
trout (Wassenberg et al., 2005). It remains unclear whether our result is
specific for the red seabream. On the other hand, zebrafish exposed to a
high concentration (100 mg/L) of dibenzothiophene exhibited cardiac
abnormality, tail curvature, yolk and pericardial edema, and reduced
growth, which are induced by AHR activation (Buccafusco et al., 1981).
In accordance with this result, our in vitro rsAHR assay suggests that a
high concentration of dibenzothiophene may be responsible for these
toxic effects via the AHR signaling pathway.
Acenaphthene is classified as a non-carcinogenic EPA priority pol-
lutant. Although it has been reported that acenaphthene was a no- or
low-potent inducer of CYP1A (Barron et al., 2004), this PAH transac-
tivated both rsAHR1 and rsAHR2 in a dose-dependent manner in the
present study. Comparison of rsAHR responses showed that the REC20-
BaP of rsAHR2 (60 nM) was approximately 4.5-fold lower than that of
rsAHR1 (272 nM), suggesting that rsAHR2 is more responsive to ace-
naphthene than rsAHR1. The 96-h LC50 values for acenaphthene were
reported to be 608 μg/L (corresponding to 3.94 μM) in the fathead
S.-M. Bak, et al.
minnow (Pimephales promelas) and to be 1700 μg/L (corresponding to
11.0 μM) in the bluegill (Lepomis macrochirus) (Cairns and Nebeker,
1982; Meier et al., 2000; US EPA., 1980). Compared with these 96-h
LC50 values of acenaphthene, our rsAHR1 and rsAHR2-derived REC20-
BaP values (270 nM for rsAHR1 and 60 nM for rsAHR2) were 10–100
times lower. However, the toxic effects of acenaphthene have not been
well investigated because this compound has been regarded as a low
risk due to its being non-carcinogenetic for vertebrates and due to its
non-persistent properties (< 1-day half-life in the bluegill). Our study
suggests that acenaphthene is a weak AHR agonist in fish.
Exposure to 2,3,5-trimethylnaphthalene induced the dose-depen-
dent activation of rsAHR1 and rsAHR2; 88 nM for both rsAHR1-and
rsAHR2-REC20-BaP values. Hong et al. (2015) applied the H4IIE-Luc
bioassay for estimating AHR-mediated transactivation potency and re-
ported no response to 2,3,5-trimethylnaphthalene. In addition, no in-
formation is available for the toxicological properties of this compound
in fish. To the best of our knowledge, this is the first report on the
activation of the AHR signaling pathway by this compound in fish.
1-Methylphenanthrene is known as a weak AHR agonist. The dose
response curves obtained by a yeast bioassay expressing human AHR
exhibited that 1-methylphenanthrene was five times more potent than
phenanthrene (Sun et al., 2014). An EROD assay using primary rat
hepatocytes showed a low induction potency by 1-methylphenanthrene
(Petrulis et al., 2001). For fish, numerous developmental abnormalities,
such as pericardial and yolk sac edema, dorsal curvature, and tail
malformations were reported in 1-methylphenanthrene-treated zebra-
fish larvae (Wolińska et al., 2011). However, in the present study, 1-
methylphenanthrene treatment failed to induce rsAHR1-and rsAHR2-
driven transactivation. These results suggest that there may be differ-
ences in responses to this PAH across species.
The REC20-BaP values of BaP for both rsAHR1- (0.052 nM) and
rsAHR2- (0.0049 nM) mediated transactivations were less than their
actual muscle concentrations on a wet weight basis (a range of
0.010–0.051 ng/g which corresponds to 0.041–0.20 nM) in greenling
muscles collected in 2011–2014, but the REC20-BaP values of other PAHs
were over their respective concentrations (Table 1 and Table S3). These
results suggested that fish in Kesennuma Bay may have suffered from
toxic effects of BaP. On the other hand, our PAH monitoring showed
that the concentrations and IEQs of BaP and other PAHs, which were
examined in the in vitro rsAHR assays, in the muscle of greenlings from
the heavy oil-contaminated sites A and B were similar to their values in
the control site C (Fig. 2). The geographical distribution of these PAHs
did not coincide with that of EROD/MROD activities which showed an
A > B > C pattern. Rather than these limited PAHs, the EROD/MROD
activities may be accounted for by the distribution of alkylated PAHs
(Figs. 2 and 5). The rsAHR2 may be a major AHR isoform in the toxicity
of PAHs because of its lower EC50 and REC20-BaP values than rsAHR1
and because of an auto-induction mechanism of the rsAHR2 gene by the
rsAHR ligand (Bak et al., 2017). Considering the results in this and
previous studies, exposure to a mixture of alkylated PAHs originated
from spilled heavy oil may have elicited adverse effects through the
AHR2-signaling pathway in the greenlings that inhabit sites A and B,
close to the heavy oil-spilled site. Since the concentration of PAHs in
Kesennuma Bay has gradually decreased from 2011 to 2014 (Mizukawa
et al., 2017; Onozato et al., 2016), the effect mediated by AHR signaling
in fish as sensitive as the red seabream seems to be limited in terms of
temporal and geographical trends.
In silico ligand screening methods have been constructed for a
variety of nuclear receptors, not only from model animals but also from
non-model animals. For AHR, amphibian, avian, and mammalian AHR-
based in silico analyses have illustrated the applicability of these
methods as a screening system (Fraccalvieri et al., 2013; Hirano et al.,
2015; Larsson et al., 2018; Lo Piparo et al., 2006; Shoots et al., 2015;
Wu et al., 2009; Yuan et al., 2013; Xing et al., 2012). However, there
are few reports on in silico analysis using fish AHR homology models
except for zebrafish AHR models constructed for predicting AHR
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Ecotoxicology and Environmental Safety 181 (2019) 214–223
ligands (Bisson et al., 2009). Our previous publication reported that the
rsAHR transactivation potencies of DLCs were correlated with their
U_dock values for rsAHRs, depending on their Cl numbers and sub-
stituted positions (Bak et al., 2013). In addition, the present study de-
monstrated that the U_dock values of PAHs from only the rsAHR2-LBD
homology model showed a significant correlation with the rsAHR2-
derived REC20-TCDD values, but that no correlation was obtained for
rsAHR1 (Fig. 4). This suggests that the rsAHR2-LBD model is more
predictable for examining the in vitro transactivation potencies of PAHs
than the rsAHR1-LBD model. The rsAHR2 model may thus be a useful
tool for assessing the transactivation potencies of not only DLCs but also
PAHs.
5. Conclusions
The present study clearly demonstrated that fish in Kesennuma Bay
have been exposed to high levels of non-alkylated and alkylated PAHs
originating from heavy oil spilled by the Great East Japan Earthquake
of March 11, 2011. Although contamination levels of PAHs have gra-
dually been decreasing, indications of the spilled heavy oil were still
found in 2014, with higher alkylated PAH concentrations and EROD/
MROD activities in fish from the bay area than those in fish from the
control site. This suggests that fish have suffered contamination by
PAHs, in particular alkylated homologues, in the spilled heavy oil in
Kesennuma Bay. Our in vitro rsAHR transactivation assay supported that
PAHs were able to activate fish AHR in a chemical- and concentration-
dependent manner. This study also suggests that AHR isoform- and
species-specific transactivation potencies could be a critical tox-
icological issue for the further refinement of the risk assessment of
PAHs in fish. This study provides an application of in vitro and in silico
fish AHR ligand screening systems, in particular for PAHs. Further
evaluation of the contribution of alkylated PAHs to AHR activation is
necessary to better assess the risk of heavy oil contamination.
Acknowledgements
This research was supported by the National Research Foundation
of Korea (NRF) grant funded by the Ministry of Education, Science and
Technology, Korea [2016K2A9A2A08003746 and
2016R1A2B4007714]; the Ministry of Environment, Korea through the
‘Environmental Health R&D Program’ [2017001370001]; and the
Grants-in-Aid for Scientific Research from the Japan Society for the
Promotion of Science (JSPS) (S) [No. 26220103] and (B) [No.
15H02852]. This study was also supported by the Ministry of
Education, Culture, Sports, Science and Technology, Japan through a
project on Joint Usage/Research Center–Leading Academia in Marine
and Environment Pollution Research (LaMer).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ecoenv.2019.06.008.
References
Andreasen, E.A., Hahn, M.E., Heideman, W., Peterson, R.E., Tanguay, R.L., 2002. The
zebrafish (Danio rerio) aryl hydrocarbon receptor type 1 is a novel vertebrate re-
ceptor. Mol. Pharmacol. 62 (2), 234–249. https://doi.org/10.1124/mol.62.2.234.
Ankley, G.T., Bennett, R.S., Erickson, R.J., Hoff, D.J., Hornung, M.W., Johnson, R.D., ...
Serrrano, J.A., 2010. Adverse outcome pathways: a conceptual framework to support
ecotoxicology research and risk assessment. Environ. Toxicol. Chem. 29 (3),
730–741. https://doi.org/10.1002/etc.34.
Bak, S.M., Iida, M., Hirano, M., Iwata, H., Kim, E.Y., 2013. Potencies of red seabream
AHR1-and AHR2-mediated transactivation by dioxins: implication of both AHRs in
dioxin toxicity. Environ. Sci. Technol. 47 (6), 2877–2885. https://doi.org/10.1021/
es304423w.
Bak, S.M., Iida, M., Soshilov, A.A., Denison, M.S., Iwata, H., Kim, E.Y., 2017. Auto-in-
duction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by TCDD-activated
AHR1 and AHR2 in the red seabream (Pagrus major). Arch. Toxicol. 91, 301. https://
S.-M. Bak, et al.
doi.org/10.1007/s00204-016-1732-9.
Barron, M.G., Heintz, R., Rice, S.D., 2004. Relative potency of PAHs and heterocycles as
aryl hydrocarbon receptor agonists in fish. Mar. Environ. Res. 58 (2), 95–100.
https://doi.org/10.1016/j.marenvres.2004.03.001.
Billiard, S.M., Bols, N.C., Hodson, P.V., 2004. In vitro and in vivo comparisons of fish-
specific CYP1A induction relative potency factors for selected polycyclic aromatic
hydrocarbons. Ecotoxicol. Environ. Saf. 59 (3), 292–299. https://doi.org/10.1016/j.
ecoenv.2004.06.009.
Billiard, S.M., Hahn, M.E., Franks, D.G., Peterson, R.E., Bols, N.C., Hodson, P.V., 2002.
Binding of polycyclic aromatic hydrocarbons (PAHs) to teleost aryl hydrocarbon
receptors (AHRs). Comp. Biochem. Physiol. B Biochem. Mol. Biol. 133 (1), 55–68.
https://doi.org/10.1016/S1096-4959(02)00105-7.
Bisson, W.H., Koch, D.C., O'Donnell, E.F., Khalil, S.M., Kerkvliet, N.I., Tanguay, R.L., ...
Kolluri, S.K., 2009. Modeling of the Aryl Hydrocarbon Receptor (AhR) ligand binding
domain and its utility in virtual ligand screening to predict new AhR ligands. J. Med.
Chem. 52 (18), 5635–5641. https://doi.org/10.1021/jm900199u.
Bo, J., Gopalakrishnan, S., Chen, F.Y., Wang, K.J., 2014. Benzo[α]pyrene modulates the
biotransformation, DNA damage and cortisol level of red sea bream challenged with
lipopolysaccharide. Mar. Pollut. Bull. 85 (2), 463–470. https://doi.org/10.1002/tox.
21777.
Bols, N.C., Schirmer, K., Joyce, E.M., Dixon, D.G., Greenberg, B.M., Whyte, J.J., 1999.
Ability of polycyclic aromatic hydrocarbons to induce 7-ethoxyresorufin-o-deethylase
activity in a trout liver cell line. Ecotoxicol. Environ. Saf. 44 (1), 118–128. https://
doi.org/10.1006/eesa.1999.1808.
Buccafusco, R.J., Ells, S.J., LeBlanc, G.A., 1981. Acute toxicity of priority pollutants to
bluegill (Lepomis macrochirus). Bull. Environ. Contam. Toxicol. 26 (1), 446–452.
https://doi.org/10.1007/BF01622118.
Cairns, M.A., Nebeker, A.V., 1982. Toxicity of acenaphthene and isophorone to early life
stages of fathead minnows. Arch. Environ. Contam. Toxicol. 11 (6), 703–707. https://
doi.org/10.1007/BF01059158.
Carls, M.G., Rice, S.D., Hose, J.E., 1999. Sensitivity of fish embryos to weathered crude
oil: Part I. Low-level exposure during incubation causes malformations, genetic da-
mage, and mortality in larval pacific herring (Clupea pallasi). Environ. Toxicol.
Chem. 18 (3), 481–493. https://doi.org/10.1002/etc.5620180317.
Cho, S.W., Suzuki, K.I., Miura, Y., Miyazaki, T., Nose, M., Iwata, H., Kim, E.Y., 2015.
Novel role of hnRNP-A2/B1 in modulating aryl hydrocarbon receptor ligand sensi-
tivity. Arch. Toxicol. 89, 2027. https://doi.org/10.1007/s00204-014-1352-1.
Couillard, C.M., 2002. A microscale test to measure petroleum oil toxicity to mummichog
embryos. Environ. Toxicol. 17 (3), 195–202. https://doi.org/10.1002/tox.10049.
European Commission, 2011. Commission Regulation (EU) No 835/2011 of 19 August
2011 amending Regulation (EC) No 1881/2006 as regards maximum levels for
polycyclic aromatic hydrocarbons in foodstuffs. Off. J. Eur. Union 20 (8), L215–L216.
Fraccalvieri, D., Soshilov, A.A., Karchner, S.I., Franks, D.G., Pandini, A., Bonati, L., ...
Denison, M.S., 2013. Comparative analysis of homology models of the Ah receptor
ligand binding domain: verification of structure–function predictions by site-directed
mutagenesis of a nonfunctional receptor. Biochem 52 (4), 714–725. https://doi.org/
10.1021/bi301457f.
Goto, J., Kataoka, R., Muta, H., Hirayama, N., 2008. ASEDock-docking based on alpha
spheres and excluded volumes. J. Chem. Inf. Model. 48 (3), 583–590. https://doi.
org/10.1021/ci700352q.
Guengerich, F.P., Dannan, G.A., Wright, S.T., Martin, M.V., Kaminsky, L.S., 1982.
Purification and characterization of liver microsomal cytochromes P-450: electro-
phoretic, spectral, catalytic, and immunochemical properties and inducibility of eight
isozymes isolated from rats treated with phenobarbital or. beta.-naphthoflavone.
Biochem 21 (23), 6019–6030. https://doi.org/10.3109/00498258209038945.
Hahn, M.E., Stegeman, J.J., 1994. Regulation of cytochrome P4501A1 in teleosts: sus-
tained induction of CYP1A1 mRNA, protein, and catalytic activity by 2, 3, 7, 8-tet-
rachlorodibenzofuran in the marine fish Stenotomus chrysops. Toxicol. Appl.
Pharmacol. 127 (2), 187–198. https://doi.org/10.1006/taap.1994.1153.
Han, D., Nagy, S.R., Denison, M.S., 2004. Comparison of recombinant cell bioassays for
the detection of Ah receptor agonists. Biofactors 20 (1), 11–22. https://doi.org/10.
1002/biof.5520200102.
Hannah, J.B., Hose, J.E., Landolt, M.L., Miller, B.S., Felton, S.P., Iwaoka, W.T., 1982.
Benzo (a) pyrene-induced morphologic and developmental abnormalities in rainbow
trout. Arch. Environ. Contam. Toxicol. 11 (6), 727–734. https://doi.org/10.1007/
BF01059161.
Heintz, R.A., Short, J.W., Rice, S.D., 1999. Sensitivity of fish embryos to weathered crude
oil: Part II. Increased mortality of pink salmon (Oncorhynchus gorbuscha) embryos
incubating downstream from weathered Exxon Valdez crude oil. Environ. Toxicol.
Chem. 18 (3), 494–503. https://doi.org/10.1002/etc.5620180318.
Hestermann, E.V., Stegeman, J.J., Hahn, M.E., 2000. Relative contributions of affinity and
intrinsic efficacy to aryl hydrocarbon receptor ligand potency. Toxicol. Appl.
Pharmacol. 168 (2), 160–172. https://doi.org/10.1006/taap.2000.9026.
Hirano, M., Hwang, J.H., Park, H.J., Bak, S.M., Iwata, H., Kim, E.Y., 2015. In silico
analysis of the interaction of avian aryl hydrocarbon receptors and dioxins to deci-
pher isoform-, ligand-, and species-specific activations. Environ. Sci. Technol. 49 (6),
3795–3804. https://doi.org/10.1021/es505733f.
Hong, S., Lee, S., Choi, K., Kim, G.B., Ha, S.Y., Kwon, B.O., Giesy, J.P., 2015. Effect-
directed analysis and mixture effects of AhR-active PAHs in crude oil and coastal
sediments contaminated by the Hebei Spirit oil spill. Environ. Pollut. 199, 110–118.
https://doi.org/10.1016/j.envpol.2015.01.009.
Huang, L., Wang, C., Zhang, Y., Li, J., Zhong, Y., Zhou, Y., Zuo, Z., 2012. Benzo [a] pyrene
exposure influences the cardiac development and the expression of cardiovascular
relative genes in zebrafish (Danio rerio) embryos. Chemosphere 87 (4), 369–375.
https://doi.org/10.1016/j.chemosphere.2011.12.026.
Incardona, J.P., Carls, M.G., Teraoka, H., Sloan, C.A., Collier, T.K., Scholz, N.L., 2005.
222
Ecotoxicology and Environmental Safety 181 (2019) 214–223
Aryl hydrocarbon receptor–independent toxicity of weathered crude oil during fish
development. Environ. Health Perspect. 113 (12), 1755–1762. https://doi.org/10.
1289/ehp.8230.
Incardona, J.P., Day, H.L., Collier, T.K., Scholz, N.L., 2006. Developmental toxicity of 4-
ring polycyclic aromatic hydrocarbons in zebrafish is differentially dependent on AH
receptor isoforms and hepatic cytochrome P4501A metabolism. Toxicol. Appl.
Pharmacol. 217 (3), 308–321. https://doi.org/10.1016/j.taap.2006.09.018.
Karchner, S.I., Franks, D.G., Hahn, M.E., 2005. AHR1B, a new functional aryl hydro-
carbon receptor in zebrafish: tandem arrangement of ahr1b and ahr2 genes. Biochem.
J. 392 (1), 153–161. https://doi.org/10.1042/BJ20050713.
Kim, E.Y., Suda, T., Tanabe, S., Batoev, V.B., Petrov, E.A., Iwata, H., 2011. Evaluation of
relative potencies for in vitro transactivation of the Baikal seal aryl hydrocarbon
receptor by dioxin-like compounds. Environ. Sci. Technol. 45 (4), 1652–1658.
https://doi.org/10.1021/es102991s.
Kubota, A., Iwata, H., Goldstone, H.M., Kim, E.Y., Stegeman, J.J., Tanabe, S., 2006.
Cytochrome P450 1A4 and 1A5 in common cormorant (Phalacrocorax carbo): evo-
lutionary relationships and functional implications associated with dioxin and related
compounds. Toxicol. Sci. 92 (2), 394–408. https://doi.org/10.1093/toxsci/kfl001.
Kubota, A., Iwata, H., Tanabe, S., Yoneda, K., Tobata, S., 2005. Hepatic CYP1A induction
by dioxin-like compounds, and congener-specific metabolism and sequestration in
wild common cormorants from Lake Biwa, Japan. Environ. Sci. Technol. 39 (10),
3611–3619. https://doi.org/10.1021/es048771g.
Larsson, M., Fraccalvieri, D., Andersson, C.D., Bonati, L., Linusson, A., Andersson, P.L.,
2018. Identification of potential aryl hydrocarbon receptor ligands by virtual
screening of industrial chemicals. Environ. Sci. Pollut. Res. Int. 25 (3), 2436–2449.
https://doi.org/10.1007/s11356-017-0437-9.
Livingstone, D.R., 1998. The fate of organic xenobiotics in aquatic ecosystems: quanti-
tative and qualitative differences in biotransformation by invertebrates and fish.
Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 120 (1), 43–49. https://doi.org/10.
1016/S1095-6433(98)10008-9.
Lo Piparo, E., Koehler, K., Chana, A., Benfenati, E., 2006. Virtual screening for aryl hy-
drocarbon receptor binding prediction. J. Med. Chem. 49 (19), 5702–5709. https://
doi.org/10.1021/jm060526f.
Marty, G.D., Hinton, D.E., Short, J.W., Heintz, R.A., Rice, S.D., Dambach, D.M., Stegeman,
J.J., 1997. Ascites, premature emergence, increased gonadal cell apoptosis, and cy-
tochrome P4501A induction in pink salmon larvae continuously exposed to oil-con-
taminated gravel during development. Can. J. Zool. 75 (6), 989–1007. https://doi.
org/10.1139/z97-120.
Meier, P.G., Choi, K., Sweet, L.I., 2000. Acute and chronic life cycle toxicity of ace-
naphthene and 2, 4, 6-trichlorophenol to the midge Paratanytarsus parthenogen-
eticus (Diptera: Chironomidae). Aquat Toxicol. 51 (1), 31–44. https://doi.org/10.
1016/S0166-445X(00)00104-1.
Mizukawa, K., Hirai, Y., Sakakibara, H., Endo, S., Okuda, K., Takada, H., Ogawa, H.,
2017. Spatial distribution and temporal trend of anthropogenic organic compounds
derived from the 2011 east Japan earthquake. Arch. Environ. Contam. Toxicol. 1–11.
https://doi.org/10.1007/s00244-017-0389-6.
Murray, I.A., Patterson, A.D., Perdew, G.H., 2014. Ah receptor ligands in cancer: friend
and foe. Nat. Rev. Cancer 14 (12), 801. https://doi.org/10.1038/nrc3846.
Ministry of Environment, 2009. Introduction to Environmental Monitoring Procedure of
Hazardous Chemicals (Kagakubussitsu Kankyoujittai Tyousajissi No Tebiki). Japan.
http://www.env.go.jp/chemi/anzen/chosa/tebiki-h20.pdf.
Nakata, H., Uehara, K., Goto, Y., Fukumura, M., Shimasaki, H., Takikawa, K., Miyawaki,
T., 2014. Polycyclic aromatic hydrocarbons in oysters and sediments from the
Yatsushiro Sea, Japan: comparison of potential risks among PAHs, dioxins and di-
oxin-like compounds in benthic organisms. Ecotoxicol. Environ. Saf. 99, 61–68.
https://doi.org/10.1016/j.ecoenv.2013.10.005.
Neff, J.M., 1997. Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Applied
Science Publishers Ltd., London.
Neff, J.M., 2002. Bioaccumulation in Marine Organisms: Effect of Contaminants from Oil
Well Produced Water. Elsevier.
Nisbet, I.C., LaGoy, P.K., 1992. Toxic equivalency factors (TEFs) for polycyclic aromatic
hydrocarbons (PAHs). Regul. Toxicol. Pharmacol. 16 (3), 290–300. https://doi.org/
10.1016/0273-2300(92)90009-X.
Onozato, M., Nishigaki, A., Okoshi, K., 2016. Polycyclic aromatic hydrocarbons in sedi-
ments and bivalves on the pacific coast of Japan: influence of tsunami and fire. PLoS
One 11 (5), e0156447. https://doi.org/10.1371/journal.pone.0156447.
Petrulis, J.R., Chen, G., Benn, S., LaMarre, J., Bunce, N.J., 2001. Application of the
ethoxyresorufin‐O‐deethylase (EROD) assay to mixtures of halogenated aromatic
compounds. Environ. Toxicol. 16 (2), 177–184. https://doi.org/10.1002/tox.1022.
Pieterse, B., Felzel, E., Winter, R., Van Der Burg, B., Brouwer, A., 2013. PAH-CALUX, an
optimized bioassay for AhR-mediated hazard identification of polycyclic aromatic
hydrocarbons (PAHs) as individual compounds and in complex mixtures. Environ.
Sci. Technol. 47 (20), 11651–11659. https://doi.org/10.1021/es403810w.
Pollino, C.A., Holdway, D.A., 2002. Toxicity testing of crude oil and related compounds
using early life stages of the crimson-spotted rainbowfish (Melanotaenia fluviatilis).
Ecotoxicol. Environ. Saf. 52 (3), 180–189. https://doi.org/10.1006/eesa.2002.2190.
Shirmohammadi, M., Salamat, N., Ronagh, M.T., Movahedinia, A., Hamidian, G., 2018.
Using cell apoptosis, micronuclei and immune alternations as biomarkers of phe-
nanthrene exposure in yellowfin seabream (Acanthopagrus latus). Fish Shellfish
Immunol. 72, 37–47. https://doi.org/10.1016/j.fsi.2017.10.039.
Shoots, J., Fraccalvieri, D., Franks, D.G., Denison, M.S., Hahn, M.E., Bonati, L., Powell,
W.H., 2015. An aryl hydrocarbon receptor from the salamander ambystoma mex-
icanum exhibits low sensitivity to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin. Environ. Sci.
Technol. 49 (11), 6993–7001. https://doi.org/10.1021/acs.est.5b01299.
Sporstol, S., Gjos, N., Lichtenthaler, R.G., Gustavsen, K.O., Urdal, K., Oreld, F., Skei, J.,
1983. Source identification of aromatic hydrocarbons in sediments using GC/MS.
S.-M. Bak, et al.
Environ. Sci. Technol. 17 (5), 282–286.
Sun, Y., Miller, C.A., Wiese, T.E., Blake, D.A., 2014. Methylated phenanthrenes are more
potent than phenanthrene in a bioassay of human aryl hydrocarbon receptor (AhR)
signaling. Environ. Toxicol. Chem. 33 (10), 2363–2367. https://doi.org/10.1002/etc.
2687.
Thuruthippallil, L.M., Kubota, A., Kim, E.Y., Iwata, H., 2013. Alternative in vitro ap-
proach for assessing AHR-mediated CYP1A induction by dioxins in Wild Cormorant
(Phalacrocorax carbo) population. Environ. Sci. Technol. 47 (12), 6656–6663.
https://doi.org/10.1021/es401155g.
U.S. Environmental Protection Agency (US EPA), 2011. SAB Review of EPA's
"Development of a Relative Potency Factor (RPF) Approach for Polycyclic Aromatic
Hydrocarbon (PAH) Mixtures (February 2010 Draft). Letter to the Honorable Lisa P.
Jackcon, EPA-SAB-11–004. https://yosemite.epa.gov/sab/sabproduct.nsf/0/
F24FBBBACA6EEABA852578570040C547/$File/EPA-SAB-11-004-unsigned.pdf.
U.S. Environmental Protection Agency (US EPA), 1980. Ambient water quality criteria for
acenaphthene, EPA-440/5-80-015. U.S. Environ. Prot. Agency, Office of Water
Regulations and Standards, Washington, DC.
Varanasi, U., Stein, J.E., Nishimoto, M., 1989. Biotransformation and Disposition of
Polycyclic Aromatic Hydrocarbons(PAH) in Fish. Metabolism of Polycyclic Aromatic
Hydrocarbons in the Aquatic Environment, vol. 1989. CRC Press, Inc., Boca Raton
Florida, pp. 93–149 20 fig, 15 tab, 171 ref. NOAA Contract Y 01-CP-40507.
Villeneuve, D.L., Blankenship, A.L., Giesy, J.P., 2000. Derivation and application of re-
lative potency estimates based on in vitro bioassay results. Environ. Toxicol. Chem.
19 (11), 2835–2843. https://doi.org/10.1002/etc.5620191131.
Villeneuve, J.P., Carvalho, F., Cattini, C., 1998. World-wide and Regional
Intercomparison for the Determination of Organochlorine Compounds, Petroleum
Hydrocarbons, and Sterols in the Sediment Sample IAEA-383. IAEA Report No.65.
Wang, Z., Hollebone, B.P., Fingas, M., Fieldhouse, B., Sigouin, L., Landriault, M., Smith,
P., Noonan, J., Thouin, G., Weaver, J.W., 2003. Characteristics of Spilled Oils, Fuels,
and Petroleum Products: 1. Composition and Properties of Selected Oils. EPA, pp. 600
R-03,072.
Wassenberg, D.M., Nerlinger, A.L., Battle, L.P., Di Giulio, R.T., 2005. Effects of the
polycyclic aromatic hydrocarbon heterocycles, carbazole and dibenzothiophene, on
223
Ecotoxicology and Environmental Safety 181 (2019) 214–223
in vivo and in vitro cypia activity and polycyclic aromatic hydrocarbon‐derived
embryonic deformities. Environ. Toxicol. Chem. 24 (10), 2526–2532. https://doi.
org/10.1897/04-440R1.1.
Wenju, X.U., Yuanyou, L.I., Qingyang, W.U., Shuqi, W.A.N.G., Zheng, H., Wenhua, L.I.U.,
2009. Effects of phenanthrene on hepatic enzymatic activities in tilapia (Oreochromis
niloticus♀× O. aureus♂). J. Environ. Sci. 21 (6), 854–857. https://doi.org/10.
1016/S1001-0742(08)62352-9.
Willett, K.L., Wassenberg, D., Lienesch, L., Reichert, W., Di Giulio, R.T., 2001. In vivo and
in vitro inhibition of CYP1A-dependent activity in Fundulus heteroclitus by the
polynuclear aromatic hydrocarbon fluoranthene. Toxicol. Appl. Pharmacol. 177 (3),
264–271. https://doi.org/10.1006/taap.2001.9296.
Wolińska, L., Brzuzan, P., Woźny, M., Góra, M., Łuczyński, M.K., Podlasz, P., ... Piasecka,
A., 2011. Preliminary study on adverse effects of phenanthrene and its methyl and
phenyl derivatives in larval zebrafish, Danio rerio. Environ. Biotechnol. 7, 26–33.
Wu, B., Zhang, Y., Kong, J., Zhang, X., Cheng, S., 2009. In silico predication of nuclear
hormone receptors for organic pollutants by homology modeling and molecular
docking. Toxicol. Lett. 191 (1), 69–73. https://doi.org/10.1016/j.toxlet.2009.08.
005.
Xing, Y., Nukaya, M., Satyshur, K.A., Jiang, L., Stanevich, V., Korkmaz, E.N., ... Bradfield,
C.A., 2012. Identification of the Ah-receptor structural determinants for ligand pre-
ferences. Toxicol. Sci. 129 (1), 86–97. https://doi.org/10.1093/toxsci/kfs194.
Yamauchi, M., Kim, E.Y., Iwata, H., Tanabe, S., 2005. Molecular characterization of the
aryl hydrocarbon receptors (AHR1 and AHR2) from red seabream (Pagrus major).
Comp. Biochem. Phys. C. 141 (2), 177–187. https://doi.org/10.1016/j.cca.2005.06.
003.
Yuan, J., Pu, Y., Yin, L., 2013. Docking-based three-dimensional quantitative structure-
activity relationship (3D-QSAR) predicts binding affinities to aryl hydrocarbon re-
ceptor for polychlorinated dibenzodioxins, dibenzofurans, and biphenyls. Environ.
Toxicol. Chem. 32 (7), 1453–1458. https://doi.org/10.1002/etc.2191.
Zhao, Y., Wang, X., Lin, X., Zhao, S., Lin, J., 2017. Comparative developmental toxicity of
eight typical organic pollutants to red sea bream (Pagrosomus major) embryos and
larvae. Environ. Sci. Pollut. Res. Int. 24 (10), 9067–9078. https://doi.org/10.1007/
s11356-016-6282-4.