４ 研究内容

In vitro screening of natural and dietary ligands as potential modulators against
multiple avian AHR isoforms
Gee-Yea Lim, Jong-In CHOI, Eun-Young KIM
Department of Biology and Department of Biomedical and Pharmaceutical Sciences,
Kyung Hee University[Aim]
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that plays
an important role in assessing the toxicity of environmental pollutants, such as dioxinlike compounds (DLCs). While early studies of AHR focused on its role in regulating theexpression of drug-metabolizing enzymes and mediating the toxicity of DLCs, recent
reports suggest that natural ligands, including indole-3-carbinol (I3C), 3,3'diindolymethane (DIM), resveratrol, daidzein, and quercetin, also play a role in various
in vivo regulations. These natural ligands have been found to have health benefits or risks
due to their antioxidant, growth inhibitory, estrogenic, or antiestrogenic properties.
Indoles such as I3C and DIM have been shown to have immunomodulatory properties bybalancing the activity of regulatory T cells (Treg) and T helper 17 cells (Th17). Whilemost studies of AHR in avian species have focused on DLCs, few studies have examined
the transcriptional response of natural ligands. In this study, we examined the in vitro
response of chicken and cormorant AHR isoforms to natural ligands. This study will help
expand the use of natural ligands as AHR modulators.
[Procedure]In this study, an in vitro reporter gene assay was performed. The cells, COS-7 cells (5 ×104 cells/well) or LMH cells (6 × 104 cells/well), were seeded on 24-well plates. A totalof 300 ng of plasmid DNA was transfected into the cells. Expression vectors containing
ckAHR1, ckAHR2, ccAHR1, ckARNT1, ccARNT1 were previously constructed byinserting the respective full-length cDNA into the pcDNA3.1 Zeo (+) plasmid. For COS7 cells, 3 ng of AHR isoform, 20 ng of CYP1A5, 50 ng of ARNT, 226.8 ng of pcDNA3.1
Zeo (+) and 0.2 ng of CMV Renilla were used. For the LMH cells, 20 ng of ckCYP1A5and 0.2 ng of CMV Renilla were used. The ligands used in the transfected cells are FICZ,I3C, DIM, quercetin, resveratrol, baicalin, genistein and daidzein. DMSO was used assolvent control. As for the sample obtained by cell lysis, the firefly luciferase activity wasmeasured using the dual luciferase reporter assay system, and the overall transcriptional

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activity of AHR was measured with the relative luciferase unit (RLU) value. Prism 5 wasused to calculate the 50% effective concentration (EC50) for AHR transcriptional activity.
The Dunnett's test was used for statistical processing to assess significance.
[Results and Discussion]
The sensitivity of chicken AHR1 and AHR2 (ckAHR1, ckAHR2) and LMH cells to FICZ
and natural ligands (I3C, daidzein, baicalin, etc.) was investigated by measuringtranscriptional activity and calculating EC50 values. FICZ elicited similar EC50 values of0.041 nM, 0.028 nM, and 0.049 nM for ckAHR1, ckAHR2, and LMH cells, respectively.
Comparison of EC50 values for ckAHR1 and ckAHR2 revealed that I3C and daidzein
were more sensitive in ckAHR1, whereas DIM and quercetin were more sensitive inckAHR2. Both ckAHR1 and ckAHR2 showed similar EC50 values of 6.5 uM and 4.7 uM,
respectively, in the case of baicalin. The EC50 values of natural ligands in LMH cells werecomparable to those of ckAHR1 compared to ckAHR2. This may be due to the higher
endogenous expression level of ckAHR1 in LMH cells compared to ckAHR2. Cormorant
AHR1 (ccAHR1) had a similar EC50 for FICZ to ckAHR1, suggesting similar sensitivity
across species and AHR isoforms. In a comparison between chickens and cormorants,
ckAHR1 was slightly more sensitive than ccAHR1 for all natural ligands except
resveratrol, which showed no significant response in both chickens and cormorantscompared to controls. These results suggest that transcriptional activity by native ligands
in avian AHR is species- and ligand-dependent.
[Perspectives in future]
This study clarified transcriptional responses of eight natural ligands in chickens andcormorants with multiple AHR isoforms. Avian AHR isoforms exhibited transcriptional
activation ability for most natural ligands, but reactivity was species-specific, withchicken AHR1 being slightly more sensitive than cormorant AHR1. The sensitivity tonatural ligands differed between isoforms of AHR1 and AHR2, suggesting species- andligand-specific regulation of these responses.

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