Form 3

Annual Report
LaMer, Ehime University
Date (25, 03, 2018)
To Director of LaMer
Principle Investigator: Kim Eun-Young
Affiliation＿Kyung Hee University＿＿＿＿Position ＿Professor＿
Name in print＿Kim Eun-Young＿＿＿＿＿＿＿

Include the report on the result of the project/meeting in a separate sheet.
1. Project / Meeting title
Screening of naturally occurring and anthropogenic ligands of wildlife AHRs and molecularmechanisms of ligand preference
2. Members of project / meeting
NameAffiliation

PositionContribution partPIKim

Kyung Hee University

Professor

Research management

Hwang Ji-HeeKyung Hee University

PhD student

In vitro analysis

Song U-Son

Kyung Hee University

MS student

In vitro analysis

Bak Jae-Gon

Kyung Hee University

MS student

In silico analysis

Koh Dong-Hee

Kyung Hee University

MS student

In silico analysis

Professor

Advice in data analysis

Eun-Young
Members

LaMer

Facultymember in charge

CMESIwata Hisato

Ehime University

In vitro and in silico AHR assays for assessing the risk of heavy oil-derived polycyclicaromatic hydrocarbons in the red seabream
Su-Min Bak1, Dong-Hee Koh2, Hisato Iwata1, Eun-Young Kim2
1Laboratory of Environmental Toxicology, Center for Marine Environmental Studies, Ehime

University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan
2Department of Life and Nanopharmaceutical Science and Department of Biology, Kyung

Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 130-701, Korea,

1. AIM
By the aftermath of the Great East Japan Earthquake on March 11, 2011, the Sanriku coastin the Tohoku Region was struck by a Tsunami. Furthermore, in Kesennuma, this Tsunami
destroyed oil storage tanks and resulted in the release of approximately 11.5 million liters ofheavy oil. The polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental
contaminants from fossil fuels and combustion of organic material. After the earthquake, thecontamination of the heavy oil-derived PAHs have been monitored using bivalves andsediment samples in Kesennuma Bay, and 27 PAHs were detected. Moreover, in the case ofaccidental oil spills, the marine ecosystem is exposed to the high-dose of a variety of PAHs.
However, the impact of PAHs pollution on marine organisms from the oil spill site has not yetbeen investigated.
Our previous study has constructed an in vitro reporter gene system with red seabream(Pagrus major) AHR1 and AHR2 (rsAHR1 and rsAHR2, respectively) for assessing thetransactivation potencies of dioxin-like congeners. TCDD induction equivalency factors(IEFs) for rsAHR1 and rsAHR2 were calculated on the basis of TCDD relative potency
derived from the dose-response of each congener. We also constructed in silico homology
models for rsAHR1 and rsAHR2 proteins and performed docking simulation for dioxin-likecongeners. The results indicated that both rsAHR have a binding affinity to dioxin-likecongeners and their interaction potency (U_dock values) were correlated with rsAHR
specific-IEFs obtained from in vitro study (r2=0.61 for rsAHR1, r2=0.52 for rsAHR2). Since
most of the studies on AHR-mediated toxicities of dioxin-like congeners in fish species havefocused on the zebrafish as a model species, it has been believed that fish AHR2 isoform
plays a critical role in the induction of toxicities and CYP1A by the exposure to dioxin-likecongeners, but AHR1 has much less contribution to their toxicities. However, our previous
data indicated that both AHR could contribute to the AHR mediated toxic effects, suggesting
the necessity of study on AHR from more diverse fish species.
The objective of this study is thus to estimate the risk of PAHs in Kesennuma fish based on

rsAHR assay systems using in vitro and in silico approaches. We investigated AHRtransactivation potencies of PAHs which were detected at high concentrations in the tissueof greenlings (Hexagrammos otakii) from Kesennuma. Based on the PAHs detected in thegreenling tissues, six PAHs were chosen. REPs of these PAHs compared to the potency ofbenzo[α]pyrene were calculated from the AHR transactivation potencies measured withrsAHR based-in vitro reporter gene assay established in our previous study. In addition,binding potencies of the six PAHs to each rsAHR were performed using in silico docking
simulations. Based on these results, we discussed the risk of heavy oil-derived PAHs inKesennuma fish.
2. PROCEDURE
2.1 Chemicals used for in vitro rsAHR reporter gene assay
Six PAHs including dibenzothiophene (99.6 % purity), phenanthrene (98.9 % purity),
acenaphthene(99.0%purity),

2,3,5-trimethylnaphthalene(98.6%purity),

1-methylphenanthrene (99.5 % purity) and benzo[α]pyrene (99.5 % purity) were purchasedfrom the Enzo Life Sciences, Inc. These chemicals were dissolved in dimethyl sulfoxide(DMSO) (Sigma), except the 1-methylphenanthrene dissolved in toluene and DMSO (1:9).
2.2 In vitro rsAHR reporter gene assay
The in vitro reporter gene assays were carried out according to methods previously reported.
Briefly, African green monkey kidney fibroblast cells (COS-7) were maintained in RPMI-1640
medium (Hyclone) supplemented with fetal bovine serum (10 % final concentration) at 37 °C
under 5 % CO2. Cells were seeded in 24-well plate at 5.0 × 104 cells per well. Transfectionsof vectors with Lipofectamine LTX (Invitrogen) were carried out in triplicate or quadruplicate
wells per dose 18 h after the seeding of cells. Total 300 ng of DNA, which contained 20 ng of
red seabream CYP1A-5XREs reporter vector, 50 ng of MRL/lpr mouse ARNT expression
vector, 3 ng of rsAHR1 or rsAHR2 expression vector, 0.2 ng of pGL4.75 [hRluc (Renillareniformis)/CMV] as a control vector, and 226.8 ng of 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 hrs incubation, the media were exchanged with dextran-coated charcoal(DCC)-stripped RPMI-1640 containing 10 % DCC-stripped FBS for reducing signals fromunknown endogenous ligands. The cells were then treated with serially diluted
concentrations of PAHs or solvent control (0.1% DMSO or 0.01% toluene/0.09% DMSO onlyfor 1-methylphenanthrene) for 18 h. Cells were lysed after the ligand treatment with 150 μLof passive lysis buffer (Promega). The activation of each reporter vector was determinedusing a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’sinstruction. The luciferase activities in lysates were measured using a multimode microplatereader (BioTek Synergy2). The fold changes of relative luciferase (Firefly/ Renilla) ratio werecalculated. Data are presented as the mean ± standard deviation (SD) from six to eighttechnical replicates from two individual experiments.
2.3 Evaluation of rsAHR transactivation potencies
Dose–response curves of PAHs for each rsAHR transactivation in in vitro assay were plotted
as a relative unit to the maximum response of benzo[α]pyrene (% benzo[α]pyrene max)
against logarithmically transformed doses. The 50 % effective concentration (EC50) values
and low observed effective concentration (LOEC) values for each rsAHR transactivation
were obtained by using GraphPad 5.0 (San Diego, CA). The estimation method of REPs
based on a systematic framework has already been reported. The B[α]P-REP were inducedby the average of benzo[α]pyrene-relative potency 20, 50 and 80 (B[α]P-REP20, -REP50and –REP80) values. Each B[α]P-REP was calculated as the concentration ratio: theconcentration at the 20, 50 or 80 % benzo[α]pyrene transactivation response and theconcentration at the showing corresponding response of PAH.
2.4 In silico rsAHR homology modeling and docking simulation
The in silico homology modeling for the ligand binding domain (LBD) of rsAHR1 and rsAHR2
and the docking simulation of PAHs with the rsAHR LBD models were conducted using theprograms of Molecular Operating Environment (MOE), ver. 2015.10 (Chemical Computing
Group Inc., Canada). The docking simulations were performed to estimate the potential
binding energy between PAHs and each rsAHR LBD pocket using alpha sphere andexcluded volume based ligand-protein docking (ASEDock) provided by Ryoka Systems Inc.,
Japan. To construct the LBD homology models of rsAHRs, we applied a crystal structure ofthe PAS-B domain sequence of human hypoxia-inducible factor 2α (huHIF2α) (PBD ID
3H7W.A) which is a closely allied paralog of the AHR LBD. Due to the uncertainties from lowidentities with the template (25.7% for rsAHR1-LBD and 26.6% for rsAHR2-LBD), fourrsAHR-LBD model structures were constructed for each rsAHR isoform by repeating thesame steps. The results were compared among the models. Details of the in silico AHR
homology modeling and ASEDock simulations methods were given in SupportingInformation.
3. RESULTS
3.1 Evaluation of transactivation potencies of PAHs via rsAHRs
To investigate the transactivation potencies of both rsAHR isoforms by six PAHs, in vitroreporter gene assay system was applied. PAHs except 1-methylphenanthrene induced bothrsAHR1 and rsAHR2 transactivation of the reporter plasmid containing rsCYP1A-5XREs in adose-dependent manner. Exposure to 1-methylphenanthrene indicated no induction for bothrsAHR isoforms. The efficacy of rsAHR2 for dibenzothiophene and acenaphthene washigherthanthatofrsAHR1

andbenzo[α]pyrene,phenanthrene,

and2,3,5-trimethylnaphthalene gave similar responses to both rsAHRs. The EC50 value wascalculated for benzo[α]pyrene, because a full sigmoidal dose-response curve was obtained
only for this chemical. EC50 value (0.016 nM) of benzo[α]pyrene for rsAHR2 transactivation
was 17.5-fold lower than that for rsAHR1 (0.28 nM).
TheLOECvaluesofphenanthrene,dibenzothiophene,acenaphthene,2,3,5-trimethylnaphthalene, and benzo[α]pyrene were 112, 271, 324, 294 and 0.40 nM forrsAHR1 and 56.1, 109, 324, 58.7 and 0.04 nM for rsAHR2, respectively. rsAHR2-derived
LOEC values were lower than rsAHR1-derived LOEC values, although the LOEC value ofacenaphthene for rsAHR2 was the same as that for rsAHR1. These results indicate that
rsAHR2 may be more sensitive to PAHs than rsAHR1. The LOEC value of benzo[α]pyrenefor rsAHR2 (0.04 nM) was lower than their hepatic concentrations on wet weight basis (0.16nM) detected in the greenlings from the heavy oil contaminated site, whereas LOEC valuesof other PAHs were greater than their respective hepatic concentrations.
B[α]P-REPvalues

ofphenanthrene,

dibenzothiophene,acenaphthene,

and2,3,5-trimethylnaphthalene, were calculated as an average value of benzo[α]pyrene relative
potency values; B[α]P-REP20, -REP50, and -REP80. When compare with thebenzo[α]pyrene, other PAHs showed lower potencies for the activation of rsAHRs (<1.0).
3.2 In silico rsAHR-LBD homology modeling and PAHs docking simulation
To develop the in silico analysis system for screening potential rsAHR ligands, we initiallyconstructed four rsAHR-LBD homology models per each rsAHR isoform. In comparison
between constructed models, the four homology models (rsAHR1- or rsAHR2-LBD #1, #2,#3 and #4) had a similar structure showing within <1.0 Å distances between their alphacarbon of amino acid, as root-mean-square distance (RMSD) values, but showed larger
distances in the loop structure with RMSD >1.0 Å. The results of phi (φ)-psi (ψ) dihedral
angles for each amino acid residue showed no outliers in the constructed rsAHR models.
Two rsAHR2-LBD homology models contained non-modifiable and unusual pC-N-CA angle
between peptide bond and alpha carbon-nitrogen bond.
The docking simulations of each rsAHR with PAHs were carried out using ASEDock. In
rsAHR1-LBD models (including #1, #2, #3 and #4 models), the U-dock values ranged from-7.42 (kal/mol) for acenaphthene to -24.6 (kcal/mol) for benzo[α]pyrene. In rsAHR2-LBD

models (including #1, #2, #3 and #4 models), the U-dock value range was from -15.6(kal/mol) for acenaphthene to -27.0 (kcal/mol) for benzo[α]pyrene. 1-Methylphenanthrenethat had no activation potency of rsAHR1 and rsAHR2 showed low U_dock values that weresimilar to that of phenanthrene.
4. PUBLICATION/CONFERENCE PRESENTATION
1. Su-Min Bak, Midori Iida, Anatoly A. Soshilov, Michael S. Denison, Hisato Iwata,
Eun-Young Kim, Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene byTCDD-activated AHR1 and AHR2 in the red seabream, 19th International Symposium onPollutant Responses in Marine Organisms, Jun 30-July 2, Abstract O-042, Matsuyama,Japan, 2017
2. Su-Min Bak, Haruhiko Nakata, Hisato Iwata, Eun-Young Kim, Assessment of heavy
oil-derived PAHs for red seabream AHR activation by in silico and in vitro approaches, 19thInternational Symposium on Pollutant Responses in Marine Organisms, Jun 30-July 2,Abstract P-010, Matsuyama, Japan, 2017