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Iwata, Hisato,Yamaguchi, Keisuke,Takeshita, Yoko,Kubota, Akira,Hirakawa, Shusaku,Isobe, Tomohiko,Hirano, Masashi,Kim, Eun-Young Elsevier 2015 Aquatic toxicology Vol.162 No.-
<P><B>Abstract</B></P> <P>This study aimed to elucidate the catalytic function of cytochrome P450 (CYP) 1 enzymes in aquatic mammals. Alkoxyresorufin <I>O</I>-dealkylation (AROD) activities including methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD), and benzyloxyresorufin <I>O</I>-dealkylation (BROD), and 2- and 4-hydroxylation activities of 17β-estradiol (E<SUB>2</SUB>) were measured by using yeast-expressed Baikal seal (<I>Pusa sibirica</I>) CYP1A1, 1A2, and 1B1 proteins. Heterologous protein expression of the Baikal seal CYP1s (bsCYP1s) in yeast microsomes was confirmed by reduced CO-difference spectra and immunoblotting. Heterologously expressed human CYP1 enzyme (hCYP1) activities were simultaneously measured and compared with those of bsCYP1 isozymes. Recombinant bsCYP1A1 protein showed the highest <I>V</I> <SUB>max</SUB> of EROD, followed by MROD, PROD, and BROD, similar to that of hCYP1A1. <I>V</I> <SUB>max</SUB>/<I>K</I> <SUB>m</SUB> ratios of all AROD activities catalyzed by bsCYP1A1 were lower than those catalyzed by hCYP1A1, suggesting less potential for AROD by bsCYP1A1. Enzymatic assays for bsCYP1A2 showed no or minimal AROD activities, while hCYP1A2 displayed MROD and EROD activities. bsCYP1B1 showed an AROD profile (EROD>BROD>MROD>>PROD) similar to that of hCYP1B1; however, <I>V</I> <SUB>max</SUB>/<I>K</I> <SUB>m</SUB> ratios of all AROD activities by bsCYP1B1 were higher. Yeast microsomes containing bsCYP1A1 and 1B1 and hCYP1A1, 1A2, and 1B1 metabolized E<SUB>2</SUB> to 2-OHE<SUB>2</SUB> and 4-OHE<SUB>2</SUB>, whereas bsCYP1A2 showed no such activity. Comparison of 4- and 2-hydroxylations of E<SUB>2</SUB> by CYP1As suggests that bsCYP1A1, hCYP1A1, and 1A2 preferentially catalyze 2- rather than 4-hydroxylation. As for CYP1B1, the <I>V</I> <SUB>max</SUB>/<I>K</I> <SUB>m</SUB> ratios suggest that both Baikal seal and human CYPs catalyze 4- rather than 2-hydroxylation. Interspecies comparison showed that bsCYP1B1 has higher metabolic potencies for both E<SUB>2</SUB> hydroxylations than does hCYP1B1, whereas the activity of bsCYP1A1 was lower than that of hCYP1A1. Messenger RNA expression levels of bsCYP1s in the liver of Baikal seals indicated that bsCYP1A1 and 1A2 enzymes contributed to 16.2% and 83.7% of total CYP1s, respectively; bsCYP1B1 accounted for only 0.06%. Addition of anti-human CYP1A1 antibody in seal liver microsomes suppressed EROD activity more than did anti-human CYP1A2 antibody. Therefore, EROD may be catalyzed by hepatic bsCYP1A1 but not bsCYP1A2, consistent with the results of yeast-expressed bsCYP1A1 and 1A2. <I>In silico</I> substrate-docking models of bsCYP1s suggested that the defect in bsCYP1A2 enzymatic activities may be accounted for by the Pro substitution of highly conserved Thr in the I-helix, which is involved in formation of a hydrogen bond with the hydroperoxy intermediate on the heme. This Thr-Pro substitution is evolutionarily conserved across aquatic mammals and could explain their lower metabolic potential for persistent organic pollutants.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Catalytic activities of Baikal seal CYP1A1 were lower than those of human CYP1A1. </LI> <LI> Baikal seal CYP1B1 showed higher catalytic activities than human CYP1B1. </LI> <LI> Catalytic activities by Baikal seal CYP1A2 showed no or a minimal detectable value. </LI> <LI> Pro317 substitution appears to render seal CYP1A2 incapable of its catalytic function. </LI> <LI> This substitution is evolutionarily conserved in aquatic mammals. </LI> </UL> </P>
Hirano, Masashi,Hwang, Ji-Hee,Park, Hae-Jeong,Bak, Su-Min,Iwata, Hisato,Kim, Eun-Young American Chemical Society 2015 Environmental science & technology Vol.49 No.6
<P>The aryl hydrocarbon receptor (AHR) mediates toxic responses to 2,3,7,8-tetrachlorodibenzo-<I>p</I>-dioxin (TCDD) and other dioxin-like compounds (DLCs). 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, we generated in silico homology models of the ligand-binding domain of avian AHRs based on <I>holo</I> human HIF-2α (PDB entry <ext-link ext-link-type='pdb' xlink:href='3H7W' xlink:type='simple'>3H7W</ext-link>). Molecular docking simulations of TCDD and other DLCs with avian AHR1s and AHR2s using ASEDock indicated that the interaction energy increased with the number of substituted chlorine atoms in congeners, supporting AHR transactivation potencies and World Health Organization TCDD toxic equivalency factors of congeners. The potential interaction energies of an endogenous AHR ligand, 6-formylindolo [3,2-<I>b</I>] carbazole (FICZ) to avian AHRs were lower than those of TCDD, which was supported by a greater potency of FICZ for in vitro AHR-mediated transactivation than TCDD. The molecular dynamics simulation revealed that mean square displacements in Ile324 and Ser380 of TCDD-bound AHR1 of 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 contribute to TCDD preference. For avian AHR2, the corresponding residues (Val/Ser or Val/Ala type) were not responsible for differential TCDD sensitivity. Application of the three-dimensional reference interaction site model showed that the stabilization of TCDD binding to avian AHRs may be due to the solvation effect depending on the characteristics of two amino acids corresponding to Ile324 and Ser380 in chicken AHR1. This study demonstrates that in silico simulations of AHRs and ligands could be used to predict isoform-, ligand-, and species-specific interactions.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/esthag/2015/esthag.2015.49.issue-6/es505733f/production/images/medium/es-2014-05733f_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/es505733f'>ACS Electronic Supporting Info</A></P>
Ishibashi, Hiroshi,Hirano, Masashi,Kim, Eun-Young,Iwata, Hisato American Chemical Society 2019 Environmental science & technology Vol.53 No.4
<P>In this study, we assessed the binding affinities of perfluoroalkyl substances (PFASs), including perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFSAs), to the ligand-binding domains (LBDs) of Baikal seal (<I>Pusa sibirica</I>; bs) and human (h) peroxisome proliferator-activated receptor alpha (PPARα). An in vitro competitive binding assay showed that six PFCAs and two PFSAs could bind to recombinant bs and hPPARα LBD proteins in a dose-dependent manner. The relative binding affinities (RBAs) of PFASs to bsPPARα were as follows: PFOS > PFDA > PFNA > PFUnDA > PFOA > PFHxS > PFHpA > PFHxA. The RBAs to bsPPARα showed a significant positive correlation with those to hPPARα. In silico PPARα homology modeling predicted that there were two ligand-binding pockets (LBPs) in the bsPPARα and hPPARα LBDs. Structure-activity relationship analyses suggested that the binding potencies of PFASs to PPARα might depend on LBP binding cavity volume, hydrogen bond interactions, the number of perfluorinated carbons, and the hydrophobicity of PFASs. Interspecies comparison of the in vitro binding affinities revealed that bsPPARα had higher preference for PFASs with long carbon chains than hPPARα. The in silico docking simulations suggested that the first LBP of bsPPARα had higher affinities than that of hPPARα; however, the second LBP of bsPPARα had lower affinities than that of hPPARα. To our knowledge, this is the first evidence showing interspecies differences in the binding of PFASs to PPARαs and their structure-activity relationships.</P> [FIG OMISSION]</BR>
Yoshinouchi, Yuka,Shimizu, Sachiko,Lee, Jin-Seon,Hirano, Masashi,Suzuki, Ken-ichi T.,Kim, Eun-Young,Iwata, Hisato Elsevier 2019 Ecotoxicology and environmental safety Vol.181 No.-
<P><B>Abstract</B></P> <P>To assess the effect of exposure to persistent organic pollutants (POPs) on the estrogen receptor (ER) signaling pathway in Baikal seals (<I>Pusa sibirica</I>), we investigated the molecular characterizations and functions of two Baikal seal ER (bsER) isoforms, bsERα and bsERβ. The bsERα and bsERβ cDNA clones isolated have an open reading frame of 595 and 530 amino acid residues, respectively. The tissue distribution analyses of bsER mRNAs showed that bsERα transcripts were primarily found in the ovary and uterus, and bsERβ in the muscle in wild Baikal seals. The immunofluorescence staining assay showed that 17β-estradiol (E<SUB>2</SUB>) treatment promoted the nuclear translocation of <I>in vitro</I>-expressed bsERα. Transient transfection of bsERα in U2OS cells enhanced the transcription of <I>pS2</I>, an ER target gene of E<SUB>2</SUB>. We then measured bsER-mediated transactivation potencies of POPs in an <I>in vitro</I> reporter gene assay system, in which a bsERα or bsERβ expression vector was transfected into COS-1 cells. For comparison, transactivation potencies of POPs on mouse ERs (mERα and mERβ) were also evaluated in the same manner. Results showed significant dose-dependent responses of bsERs and mERs when treated with <I>p,p’</I>-dichlorodiphenyltrichloroethane (<I>p,p’</I>-DDT), and <I>p,p’</I>-dichlorodiphenyldichloroethylene (<I>p,p’</I>-DDE). bsERs and mERs showed no response when exposed to polychlorinated biphenyls (PCBs) or 2,3,7,8-tetrachlorodibenzo-<I>p</I>-dioxin. Comparison of the dose-response curves of DDTs across species (bsERs vs. mERs) showed that bsERα had a response similar to mERα, but bsERβ was less sensitive than mERβ. Comparing the lowest observable effective concentrations of <I>p,p</I>′-DDT (2.8 μM) and <I>p,p</I>′-DDE (10 μM) for <I>in vitro</I> bsERα-mediated transactivation with their hepatic concentrations in wild Baikal seals indicated that some individuals accumulated these compounds at levels comparable to the effective concentrations, suggesting the potential disruption of the bsERα signaling pathway in the wild population by these compounds. Co-transfection experiments with bsER and the aryl hydrocarbon receptor (AHR) suggested that high accumulation of estrogenic compounds exerts a synergistic effect with dioxin-like congeners on ER signaling through AHR activation in the wild seal population.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Baikal seal estrogen receptor cDNA clones (bsERα and bsERβ) were isolated. </LI> <LI> <I>In vitro</I> bsER-expressed reporter gene assay system was constructed. </LI> <LI> Dose-dependent responses of bsERs to <I>p,p</I>′-DDT and <I>p,p</I>′-DDE were observed. </LI> <LI> Aryl hydrocarbon receptor activation by dioxins disrupts bsER signaling pathway. </LI> </UL> </P>
Dau, Pham Thi,Sakai, Hiroki,Hirano, Masashi,Ishibashi, Hiroshi,Tanaka, Yuki,Kameda, Kenji,Fujino, Takahiro,Kim, Eun-Young,Iwata, Hisato Academic Press 2013 Toxicological sciences Vol.131 No.1
<P>The constitutive androstane receptor (CAR) not only displays a high basal transcriptional activity but also acts as a ligand-dependent transcriptional factor. It is known that CAR exhibits different ligand profiles across species. However, the mechanisms underlying CAR activation by chemicals and the species-specific responses are not fully understood. The objectives of this study are to establish a high-throughput tool to screen CAR ligands and to clarify how CAR proteins from the Baikal seal (bsCAR) and the mouse (mCAR) interact with chemicals and steroid receptor coactivator 1 (SRC1). We developed the surface plasmon resonance (SPR) system to assess quantitatively the interaction of CAR with potential ligands and SRC1. The ligand-binding domain (LBD) of bsCAR and mCAR was synthesized in a wheat germ cell-free system. The purified CAR LBD was then immobilized on the sensor chip for the SPR assay, and the kinetics of direct interaction of CARs with ligand candidates was measured. Androstanol and androstenol, estrone, 17β-estradiol, TCPOBOP, and CITCO showed compound-specific but similar affinities for both CARs. The CAR-SRC1 interaction was ligand dependent but exhibited a different ligand profile between the seal and the mouse. The results of SRC1 interaction assay accounted for those of our previous in vitro CAR-mediated transactivation assay. In silico analyses also supported the results of CAR-SRC1 interaction; there is little structural difference in the ligand-binding pocket of bsCAR and mCAR, but there is a distinct discrimination in the helix 11 and 12 of these receptors, suggesting that the interaction of ligand-bound CAR and SRC1 is critical for determining species-specific and ligand-dependent transactivation over the basal activity. The SPR assays demonstrated a potential as a high-throughput screening tool of CAR ligands.</P>