Form 3

Annual Report
LaMer, Ehime University
Date (28, Feb, 2017)
To Director of LaMer
Principle Investigator:
Affiliation＿Kyung Hee University＿

Position ＿Associate 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

Searching of naturally occurring and anthropognic AHR ligands in wildlife
2. Members of project / meeting
NameAffiliation

PositionContribution partPIKim Eun-Young
Kyung Hee University

Associate

Data analysis

ProfessorMembers
Hwang Ji-Hee
Bak Su-Min

Kyung Hee UniversityGrad. Student

In vitro assay

Go Dong-HeeKyung Hee University

Grad. Student

In vitro assay

Kyung Hee University

Grad. Student

In silico analysis

CMESProfessor

Data analysis

LaMer Faculty
member in charge

Hisato Iwata3. Contents
We participated in the 19th Annual Meeting of Japan Society of Endocrine Disrupters
Research and made oral and poster presentations on December 8-9, 2016.
The followings are the titles and abstracts of presentations.

Structural characteristics of avian aryl hydrocarbon receptors to decipher dioxin
susceptibility and ecological factors related to their genotypes
Dioxin-related chemicals (DRCs) are highly accumulated in some population of birds. Oneof the molecular targets of DRCs is the aryl hydrocarbon receptor (AHR), aligand-dependent transcription factor that mediates toxic action of DRCs and physiologicalresponses including immune system. Avian species possess multiple AHR isoforms (AHR1,AHR1β, and AHR2) that exhibit species- and isoform-specific responses to ligands. To
account for the ligand preference in terms of the structural features of avian AHRs, wegenerated in silico homology models of the ligand-binding domain of avian AHRs based onHIF2 structural template (PDB code 3H7W). The molecular dynamics simulation showedthat the values of mean square displacements in Ile324 and Ser380 of TCDD-bound AHR1of the chicken, the most sensitive species to TCDD, were smaller than those in other avian
AHR1s, suggesting that the dynamic stability of these amino acid residues contributes toTCDD preference. Analysis of the 3D reference interaction site model showed that thestabilization of TCDD binding to avian AHRs may be due to the solvation effect dependingon the characteristics of two amino acids corresponding to Ile324 and Ser380 in chicken
AHR1. In addition to the in silico structural analysis, we investigated the ecological factors inavian species that may have driven natural selection pressure of AHR genotypes related todioxin sensitivity. Cluster analyses based on the association between ecological factors andAHR1 genotypes of 113 avian species were conducted. The results showed that 2 majorclusters and sub-clusters of the cluster 3 were associated with specific AHR1 genotypes
depending on ecological factors including the food and habitat. The majority of the species
with Ile_Ala type were the Passeriformes, which are omnivorous or herbivorous feeders in
the terrestrial environment. The species with Val_Ala type was primarily composed of
raptors and waterbirds, which have been exposed to naturally occurring dioxins like
1,3,7-tribromodibenzo-p-dioxin. These results suggest that ecological factors related to theexposure of natural dioxins contribute to natural selection of the avian AHR1 genotype,which consequently leads to different sensitivity to man-made dioxins.

Auto-induction mechanism of aryl hydrocarbon receptor 2 (AHR2) gene by AHR1 andAHR2 in the red seabreamThe toxic effects of dioxin-like compounds are mediated by the aryl hydrocarbon receptor(AHR). Our previous study identified two AHR isoforms (AHR1 and AHR2) from the redseabream (Pagrus major). Moreover, we found that AHR2 mRNA levels were remarkably
induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in the early life stage ofred seabream embryos, while AHR1 mRNA level was not altered. In this study, toinvestigate this isoform-specific regulatory mechanism of AHR transcripts, we cloned andcharacterized 5′-upstream regions of AHR1 and AHR2 genes from the red seabream. Bothof the 5′-upstream regions in AHR1 and AHR2 genes contained three potential
xenobiotic-responsive elements (XREs). In vitro assays using reporter plasmids containing
the 5′-upstream regions of each AHR gene demonstrated that only the 5′-upstream region ofAHR2 gene was activated by AHR1 and AHR2 proteins in a TCDD dose-dependent manner.
Electrophoretic mobility shift assay using mutations in each XRE of AHR2 gene showed thatXRE1, the closest XRE from the start codon in AHR2 gene, was mainly responsible for thebinding with TCDD-activated AHR protein. This suggests that TCDD-activated AHR1 andAHR2 proteins up-regulate the mRNA levels via the functional XRE1 in AHR2 gene. This
auto-induced AHR2 may amplify the signal transduction of its downstream targets including
CYP1A in the red seabream.

In silico/in vitro analyses of the interaction of avian AHR and natural ligands
The aryl hydrocarbon receptor (AHR) is a transcription factor activated by various
xenobiotics such as dioxin-like chemicals (DLCs) and polycyclic aromatic hydrocarbons.
Recent studies have shown that some endogenous and naturally occurring chemicals also
activate AHR as ligands. However, little is known about the potency of natural ligands totransactivate avian AHR. To screen potential natural ligands of avian AHR, we applied an invitro reporter gene assay system in which chicken CYP1A5-XREs reporter plasmid wastransfected into a chicken hepatocellular carcinoma cell line, LMH. The in vitro assays
demonstrated more than 2-fold induction of the reporter signal at the maximum
concentrationof

6-formylindolo[3,2-b]carbazole(FICZ),

indoxylsulfate,

1,3,7-tribromodibenzo-p-dioxin, and pyocyanin. FICZ showed the highest potency andefficacy among the tested chemicals, suggesting the preferable structure of FICZ for binding
with chicken AHR. Moreover, we carried out in silico analysis which predicts ligand-AHR
docking energy to develop high-throughput screening method for AHR ligand candidate
compounds. To select a proper AHR model for the in silico analysis, avian AHR1 PAS-B
homology models using the amino acid sequences of chicken AHR1, great cormorant AHR1,and black-footed albatross AHR1 were constructed from four crystal structures of HIF2α as
templates; PDB code 3H7W, 3H82, 4GHI, and 4ZQD. The relationships between

transactivation potencies (e.g. LOEL and IEF) of ligands (DLCs and natural ligands) via
avian AHR1s and ligand docking energies of the corresponding AHR1 homology models
were evaluated by coefficient of determination (R2) of logarithmic linear best-fit line. The
evaluation demonstrated that the relationships were largely changed, depending on theAHR1 homology models constructed from four HIF2α templates. The results suggest that
there is a limit of in silico ligand screening by using avian AHR1 homology models. Further
investigation of the selection of proper AHR homology models according to animal species
and structural characteristics of ligand candidate compounds is necessary to develop insilico high throughput AHR ligand screening method.

Molecular characterization of aryl hydrocarbon receptor (AHR) in Polar bears (Ursusmaritimus)
The aryl hydrocarbon receptor (AHR) is a key transcription factor which mediates the toxicityof dioxin-related chemicals (DRCs) by regulation of a battery of target genes including
cytochrome P450 1A1. The apex predator in the Arctic, the polar bear (Ursus maritimus), isa vulnerable species to exposure to DRCs via the food web. Thus the functional
characterization of polar bear AHR (pbAHR) contributes to assessment of the susceptibility
to and risk of DRCs. We cloned pbAHR which encodes 793 amino acids and investigated
the transactivation potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the in vitro
reporter-gene assay in which pbAHR expression plasmid was transfected into U-2 OS cells.
The in vitro assay showed that pbAHR had an insufficient induction of the reporter signal
even at the highest TCDD concentration. This result was due to a high basal level ofpbAHR-mediated transactivation in vehicle-treated cells. In contrast, C3H/lpr mouse AHR(C3H AHR) bore a low basal level in vehicle control and sufficient induction potencies in aTCDD dose dependent manner. To unravel the high basal level of pbAHR with no ligand,the expression levels of pbAHR and C3H AHR proteins expressed in U-2 OS cells werecompared by a western blot assay. The result indicated no difference in the expression
levels between the two AHR proteins; the difference in the protein expression level was not
the cause of high basal level of pbAHR. To verify the hypothesis that the unique structure ofpbAHR protein is responsible for the high basal level, we constructed two types of chimeric
AHR plasmids which switched the N-terminus and C-terminus of pbAHR with thecorresponding sites of C3H AHR and performed the in vitro assay using the chimeric AHR
plasmids. The results revealed that the chimeric AHR, which contains the N-terminal
domains of pbAHR for DNA binding, dimerization with ARNT, and ligand binding, had a highbasal level similar to wild type pbAHR. This suggests that the high basal level of pbAHR iscaused by the structural characteristics in the N-terminus.