An assessment of risks of dioxins for aryl hydrocarbon receptor-mediated effects
in polar bear (Ursus maritimus) by in vitro and in silico approaches
Ji-Hee Hwang†, Kurunthachalam Kannan ‡, Thomas J. Evans§, Hisato Iwataǁ,Eun-Young Kim*††

Department of Life and Nanopharmaceutical Science and Department of Biology,
Kyung Hee University, Seoul, Korea‡
School of Public Health, State University of New York at Albany (SUNY), New York,USA§
United States Fish and Wildlife Service, Washington DC, USAǁ
Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama,Japan
Aim and Procedure
Polar bear (Ursus maritimus) populations accumulate dioxins and related compounds(DRCs) at levels that are of health concern. The toxicities of DRCs are primarily
mediated via aryl hydrocarbon receptor (AHR) signaling pathway. To estimate the riskof DRCs in polar bears, we initially cloned and sequenced pbAHR complementary
DNA (cDNA). We then constructed an in vitro reporter gene assay system using apbAHR expression plasmid and measured the transactivation potencies of DRCs via
pbAHR, of which the protein was transiently expressed in U2OS cells. In addition, webuilt chimeric AHR constructs from pbAHR and C3H/lpr mouse AHR (mAHR) andfurther chimeric AHR constructs with site-directed mutations, and investigated thefunctional roles of the Per-Arnt-Sim (PAS)-B domain and several amino acid residues
in the ligand binding domain (LBD) of pbAHR. To probe the role of specific aminoacid residues in pbAHR, protein ligand interaction fingerprint (PLIF) analyses based onin silico ligand-receptor docking simulations were performed. Finally, we assessed therisks of DRCs for pbAHR-mediated toxicity from the results of in vitro and in silico
analyses.
ResultIn vitro assays showed that the pbAHR was as sensitive to DRCs as C3H/lpr mouse
AHR, which is known to be highly sensitive to DRCs. Comparison of pbAHR
transactivation potencies indicated that TCDF, 2,3,4,7,8-PeCDF, and BaP exhibitedhigh induction equivalency factors (IEFs). Considering the concentrations of DRCs inpolar bears, PCB126 was found to be the most active inducer of pbAHR. The in vitrotransactivation potencies of ligands of pbAHR showed a significant relationship with insilico ligand docking energies in a pbAHR homology model. The protein ligand
interaction fingerprint (PLIF) analysis showed different interaction patterns according toligands. Several amino acids which are highly conserved among mammals, may be
involved in species-specific responses via backbone interactions with neighboring
amino acid residues which are specific to pbAHR.

Publication/conference presentation
Ji-Hee Hwang, Kurunthachalam Kannan, Thomas J. Evans, Hisato Iwata, Eun-Young
Kim, Assessment of Risks of Dioxins for Aryl Hydrocarbon Receptor-Mediated Effects
in Polar Bear (Ursus maritimus) by in Vitro and in Silico Approaches. Environmental
Science & Technology, in press.
Perspectives in future
A high correlation between the results of in silico pbAHR docking simulations and invitro pbAHR transactivation potencies offers a prospect of high-throughput screening ofAHR active compounds in polar bears. Our established approaches thus enable the risk
assessment of environmental contaminants in endangered species like polar bears forwhich in vivo toxicity test are ethically, logistically and technically difficult.

In vitro and in silico AHR assays for assessing the risk of heavy oil-derived
polycyclic aromatic hydrocarbons in fish
Su-Min Baka, Haruhiko Nakatab, Dong-Hee Kohc, Jean Yooa, Hisato Iwataa,
Eun-Young Kimc,d,*aLaboratory of Environmental Toxicology, Center for Marine Environmental Studies,
Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japanb
Faculty of the Advanced Graduate School of Science and Technology, Kumamoto
University. 2-39-1 Kurokami, Chuo-ku, Kumamoto 860-8555, Japanc
Department of Life and Nanopharmaceutical Science and dDepartment of Biology,
Kyung Hee University, 26, Kyungheedae-ro, Dongdaemun-gu, Seoul, 130-701, Korea
Aim and Procedure
In the aftermath of the Great East Japan Earthquake of March 11, 2011, marine fish inKesennuma Bay, Japan, have been contaminated with heavy oil containing polycyclicaromatic hydrocarbons (PAHs). The objective of this study was thus to estimate the riskof heavy oil-derived PAHs in fish from Kesennuma Bay. We initially measured PAH
concentrations and hepatic EROD and MROD activities as markers of AHR activationin greenlings (Hexagrammos otakii) collected from Kesennuma Bay in 2014. We theninvestigated the AHR transactivation potencies of six PAHs which were detected in thetissue 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 maximum 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 thencalculated 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 relationshipbetween the in vitro rsAHR transactivation and in silico binding potencies wasexamined and the applicability of an in silico approach for screening PAHs as rsAHRligands was validated.
ResultThe 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 tothese PAHs in the in vitro reporter gene assay system where red seabream (Pagrusmajor) AHR1 (rsAHR1) or rsAHR2 expression plasmids were transiently transfected
into COS-7 cells. The in vitro assay showed rsAHR isoform-, PAH-, anddose-dependent transactivation potencies. The relative effective concentrations ofbenzo[α]pyrene, dibenzothiophene, phenanthrene, 2,3,5-trimethylnaphthalene,acenaphthene, and 1-methylphenanthrene that induce 20% of the maximum
benzo[α]pyrene response (REC20-BaP) for rsAHR1 activation were 0.052, 38, 70, 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 valuesof benzo[α]pyrene for both the rsAHR1 and rsAHR2 isoforms were lower than theconcentrations (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.
Publication/conference presentation
Su-Min Bak, Haruhiko Nakata, Dong-Hee Koh, Jean Yoo, Hisato Iwata, Eun-YoungKim, In vitro and in silico AHR assays for assessing the risk of heavy oil-derived
polycyclic aromatic hydrocarbons in fish, Ecotoxicology and Environmental Safety, 181,214-223, 2019.
Perspectives in future
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 thatAHR isoform- and species-specific transactivation potencies could be a critical
toxicological 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 oilcontamination.