Enzymatic characterization of <i>in vitro</i>-expressed Baikal seal cytochrome P450 (CYP) 1A1, 1A2, and 1B1: Implication of low metabolic potential of CYP1A2 uniquely evolved in aquatic mammals

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>

Chicken embryo toxic effects of 1,3,7-TriBDD, a naturally produced dioxin

Jae Gon Park(박재곤),Hisato Iwata(이와타 히사토),Nguyen Minh Tue(투 엔구옌 민),Tatsuya Kunisue(쿠니스 타스야),Eun-Young Kim(김은영) 환경독성보건학회 2021 한국독성학회 심포지움 및 학술발표회 Vol.2021 No.5

Dioxins are known to induce a variety of toxic effects and biological alterations as chemical pollutants. Until now, most of dioxins have been reported that they are derived from human activities such as industrial, combustion, and incineration sources. However, recent researches have shown that naturally produced dioxins were detected. 1,3,7-tribromodibenzo-p-dioxin (1,3,7-TriBDD) is one of the natural occurred dioxins by algal species and abundant polybrominated dibenzo-p-dioxins in marine ecosystem. 1,3,7-TriBDD is assimilated in mussels, and ingested by water birds with tons of mussels. Thus, it is possible that 1,3,7-TriBDD could effect on bird species. But, despite this possibility, the effects of biological exposure on vertebrates such as birds are not well known. Therefore, we performed in ovo exposure test and hepatic transcriptomic analysis with chicken embryos to understand effect of 1,3,7-TriBDD exposure. Chicken embryos treated with 27μM and 137μM of 1,3,7-TriBDD and sacrificed after 21 day incubation. Treated chicken embryos showed decreased glucose level in 1,3,7-TriBDD treated embryos. In transcriptomic analysis, 733 and 596 genes were identified as differentially expressed genes (DEGs) from 6520 sequences in 27 and 137μM of 1,3,7-TriBDD-treated groups, respectively. Pathway analysis with DEGs (differentially expressed genes) suggested that 1,3,7-TriBDD possibly induces cancerous effects and alteration of metabolism. And these results were similar with results of TCDD, the representative dioxin, treated chicken embryos. Collectively, this study suggests the necessity to further investigate the effects of 1,3,7-TriBDD exposure in animals, considering a possibility of increased production of 1,3,7-TriBDD due to climate change such as global warming.

In Vitro Assessment of Activation of Baikal Seal (Pusa sibirica) Peroxisome Proliferator-Activated Receptor α by Polybrominated Diphenyl Ethers

Ishibashi, Hiroshi,Kim, Eun-Young,Arizono, Koji,Iwata, Hisato ACS AMERICAN CHEMICAL SOCIETY 2018 Environmental science & technology Vol.52 No.20

In Vitro and In Silico Evaluations of Binding Affinities of Perfluoroalkyl Substances to Baikal Seal and Human Peroxisome Proliferator-Activated Receptor α

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>

Potencies of Red Seabream AHR1- and AHR2-Mediated Transactivation by Dioxins: Implication of Both AHRs in Dioxin Toxicity

Bak, Su-Min,Iida, Midori,Hirano, Masashi,Iwata, Hisato,Kim, Eun-Young American Chemical Society 2013 Environmental science & technology Vol.47 No.6

<P>To evaluate species- and isoform-specific responses to dioxins and related compounds (DRCs) via aryl hydrocarbon receptor (AHR) in the red seabream (Pagrus major), we constructed a reporter gene assay system. Each expression plasmid of red seabream AHR1 (rsAHR1) and AHR2 (rsAHR2) together with a reporter plasmid containing red seabream CYP1A 5′-flanking region were transfected into COS-7 cells. The cells were treated with graded concentrations of seven DRC congeners including 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 2,3,7,8-TCDF, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, and PCB126. Both rsAHR1 and rsAHR2 exhibited dose-dependent responses for all the tested congeners. The rsAHR isoform-specific TCDD induction equivalency factors (rsAHR1- and rsAHR2-IEFs) were calculated on the basis of 2,3,7,8-TCDD relative potency derived from the dose–response of each congener. The rsAHR1-IEFs of PeCDD, HxCDD, TCDF, PeCDF, and HxCDF were estimated as 0.17, 0.29, 2.5, 1.5, and 0.27, respectively. For PCB126, no rsAHR1-IEF was given because of less than 10% 2,3,7,8-TCDD maximum response. The rsAHR2-IEFs of PeCDD, HxCDD, TCDF, PeCDF, HxCDF, and PCB126 were estimated as 0.38, 0.13, 1.5, 0.93, 0.20, and 0.0085, respectively. The rsAHR1/2-IEF profiles were different from WHO toxic equivalency factors for fish. <I>In silico</I> docking simulations supported that both rsAHRs have potentials to bind to these congeners. These results suggest that dioxin toxicities may be mediated by both rsAHRs in red seabreams.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/esthag/2013/esthag.2013.47.issue-6/es304423w/production/images/medium/es-2012-04423w_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/es304423w'>ACS Electronic Supporting Info</A></P>

The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (<i>Corvus macrorhynchos</i>)

Kim, Eun-Young,Inoue, Naomi,Koh, Dong-Hee,Iwata, Hisato Elsevier 2019 Ecotoxicology and environmental safety Vol.171 No.-

<P><B>Abstract</B></P> <P>To understand the role of aryl hydrocarbon receptor (AHR) isoforms in avian species, we investigated the functional characteristics of two AHR isoforms (designated as <I>jc</I>AHR1 and <I>jc</I>AHR2) of the jungle crow (<I>Corvus macrorhynchos</I>). Two amino acid residues corresponding to Ile<SUP>324</SUP> and Ser<SUP>380</SUP> (high sensitive type) in chicken AHR1 that are known to determine dioxin sensitivity were Ile<SUP>325</SUP> and Ala<SUP>381</SUP> (moderate sensitive type) in <I>jc</I>AHR1 and Val<SUP>306</SUP> and Ala<SUP>362</SUP> (low sensitive type) in <I>jc</I>AHR2. The quantitative comparison of the two <I>jc</I>AHR mRNA expression levels in a Tokyo jungle crow population showed that <I>jc</I>AHR2 accounted for 92.4% in the liver, while <I>jc</I>AHR1 accounted for only 7.6%. Both <I>in vitro</I>-expressed <I>jc</I>AHR1 and <I>jc</I>AHR2 proteins exhibited a specific binding to [<SUP>3</SUP>H]-labeled 2,3,7,8-tetrachlorodibenzo-<I>p</I>-dioxin (TCDD). Transactivation potencies for <I>jc</I>AHR1 and <I>jc</I>AHR2 in <I>in vitro</I> reporter gene assays were measured in <I>jc</I>AHR-expressed cells exposed to 16 dioxins and related compounds (DRCs). Both <I>jc</I>AHR1 and <I>jc</I>AHR2 were activated in a congener- and an isoform-specific manner. EC<SUB>50</SUB> value of TCDD for <I>jc</I>AHR2 (0.61 nM) was six-fold higher than that for <I>jc</I>AHR1 (0.098 nM), but <I>jc</I>AHR2 had higher transactivation efficacy than <I>jc</I>AHR1 in terms of the magnitude of response. The high transactivation efficacy of <I>jc</I>AHR2 in DRCs is in contrast to that of AHR2s in other avian species with low transactivation efficacy. Molecular docking simulations of TCDD with <I>in silico jc</I>AHR1 and <I>jc</I>AHR2 homology models showed that the two sensitivity-decisive amino acids indirectly controlled TCDD-binding modes through their surrounding amino acids. Deletion assays of <I>jc</I>AHR2 revealed that 736–805 amino acid residues in the C-terminal region were critical for its transactivation. We suggest that <I>jc</I>AHR2 plays a critical role in regulating the AHR signaling pathway, at least in its highly expressed organs.</P> <P><B>Highlights</B></P> <P> <UL> <LI> To understand the role of aryl hydrocarbon receptor (AHR) isoforms in avian species, we investigated the functional characteristics of two AHR isoforms (designated as <I>jc</I>AHR1 and <I>jc</I>AHR2) of the jungle crow (<I>Corvus macrorhynchos</I>). </LI> <LI> The quantitative comparison of the two <I>jc</I>AHR mRNA expression levels in a Tokyo jungle crow population showed that <I>jc</I>AHR2 accounted for 92.4% in the liver, while <I>jc</I>AHR1 accounted for only 7.6%. </LI> <LI> Apart from previous studies indicating that AHR1 is a major player that determines dioxin susceptibility in avian species, we clarify here that AHR2 in the jungle crow plays a critical role in AHR signaling, at least in the liver, due to its higher mRNA expression levels and abilities of TCDD binding and transactivation. </LI> </UL> </P>

자가면역질병과 관련있는 MRL과 C3H 마우스 안에서의 다이옥신수용체의 활성도 비교

See-Eun Cho,Yoshiaki Miura,Ken-ichi Suzuki,Tatsuhiko Miyazaki,MasatoNose,Hisato Iwata,Eun-Young Kim 한국환경독성학회 2012 한국독성학회 심포지움 및 학술발표회 Vol.2012 No.5

Alternative In Vitro Approach for Assessing AHR-Mediated CYP1A Induction by Dioxins in Wild Cormorant (<i>Phalacrocorax carbo</i>) Population

Thuruthippallil, Leena Mol,Kubota, Akira,Kim, Eun-Young,Iwata, Hisato American Chemical Society 2013 Environmental science & technology Vol.47 No.12

<P>Our line of papers revealed that the common (great) cormorant (<I>Phalacrocorax carbo</I>) possesses two isoforms of the aryl hydrocarbon receptor (<I>cc</I>AHR1 and <I>cc</I>AHR2). This paper addresses in vitro tests of the <I>cc</I>AHR signaling pathways to solve two questions: (1) whether there are functional differences in the two <I>cc</I>AHR isoforms, and (2) whether a molecular perturbation, cytochrome P450 1A (<I>cc</I>CYP1A) induction, in the population-level can be predicted from the in vitro tests. The transactivation potencies mediated by <I>cc</I>AHR1 and <I>cc</I>AHR2 were measured in COS-7 cells treated with 15 selected dioxins and related compounds (DRCs), where <I>cc</I>AHR1 or <I>cc</I>AHR2 expression plasmid and <I>cc</I>CYP1A5 promoter/enhancer-linked luciferase reporter plasmid were transfected. For congeners that exhibited dose-dependent luciferase activities, 2,3,7,8-tetrachlorodibenzo-<I>p</I>-dioxin (TCDD) relative potencies (REPs) and induction equivalency factors (IEFs) were estimated. <I>cc</I>AHR1-IEF profile was similar to WHO avian TCDD toxic equivalency factor (TEF) profile except for dioxin-like polychlorinated biphenyls that showed lower IEFs in <I>cc</I>AHR1-driven reporter assay. <I>cc</I>AHR2-IEF profile was different from WHO TEFs and <I>cc</I>AHR1-IEFs. Notably, 2,3,4,7,8-PeCDF was more potent than TCDD for <I>cc</I>AHR2-mediated response. Using <I>cc</I>AHR1- and <I>cc</I>AHR2-IEFs and hepatic DRC concentrations in the Lake Biwa cormorant population, total TCDD induction equivalents (IEQs) were calculated for each <I>cc</I>AHR-mediated response. Nonlinear regression analyses provided significant sigmoidal relationships of <I>cc</I>AHR1- and <I>cc</I>AHR2-derived IEQs with hepatic <I>cc</I>CYP1A5 mRNA levels, supporting the results of in vitro <I>cc</I>AHR-mediated TCDD dose–response curves. Collectively, our in vitro AHR reporter assay potentially could be an alternative to molecular epidemiology of the species of concern regarding CYP1A induction by AHR ligands.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/esthag/2013/esthag.2013.47.issue-12/es401155g/production/images/medium/es-2013-01155g_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/es401155g'>ACS Electronic Supporting Info</A></P>

Molecular and Functional Characterization of a Novel Aryl Hydrocarbon Receptor Isoform, AHR1β, in the Chicken (<i>Gallus gallus</i>)

Lee, Jin-Seon,Iwabuchi, Kohei,Nomaru, Koji,Nagahama, Nobumasa,Kim, Eun-Young,Iwata, Hisato Oxford University Press 2013 TOXICOLOGICAL SCIENCES Vol.136 No.2

<P>Dioxins including 2,3,7,8-tetrachlorodibenzo-<I>p</I>-dioxin (TCDD) cause toxic effects through activation of the aryl hydrocarbon receptor (AHR)–mediated signaling pathway. Our previous studies have investigated the function of 2 AHR isoforms (AHR1 and AHR2) in avian species and identified a third AHR in the chicken (<I>Gallus gallus</I>) genome. Knowledge of multiple avian AHRs is indispensable to understand molecular mechanisms of AHR-mediated toxic effects and establish risk assessment framework for environmental AHR ligands in avian species. In this study, we successfully isolated a third novel AHR1-like cDNA from chicken and designated it as chicken AHR1 beta (<I>ck</I>AHR1β). The mRNA expression of <I>ck</I>AHR1β was primarily detected in the liver, and the hepatic protein expression was confirmed by Western blotting. Although mRNA expression of <I>ck</I>AHR1β was not altered by <I>in ovo</I> TCDD exposure, <I>ck</I>AHR1β exhibited specific binding to [<SUP>3</SUP>H]TCDD, TCDD-dependent nuclear translocation, and interaction with xenobiotic responsive elements (XREs) and AHR nuclear translocators (ARNTs). <I>In vitro</I> XRE-driven reporter gene assays revealed <I>ck</I>AHR1β-mediated transactivation of TCDD in a dose-dependent manner, showing a 10-fold reduced sensitivity (high EC<SUB>50</SUB>) compared with that mediated by <I>ck</I>AHR1. The mutation of Val<SUP>371</SUP> to Ser<SUP>371</SUP> in the ligand-binding domain of <I>ck</I>AHR1β shifted the TCDD-EC<SUB>50</SUB> toward the value observed in <I>ck</I>AHR1, indicating the critical roles of the amino acid in sensitivity. Furthermore, <I>ck</I>AHR1β-mediated transactivation of TCDD was enhanced by 17β-estradiol (E2)-activated chicken estrogen receptor α (<I>ck</I>ERα), suggesting a positive cross talk between <I>ck</I>ERα and <I>ck</I>AHR1β signaling pathway. Both TCDD-induced and its enhanced activities by E2 were suppressed by the <I>ck</I>AHR repressor in a manner similar to <I>ck</I>AHR1. Collectively, our findings discover the role of <I>ck</I>AHR1β in dioxin toxicity and give an insight into the evolutionary history of the AHR signaling pathway.</P>

In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population.

Mol, Thuruthippallil Leena,Kim, Eun-Young,Ishibashi, Hiroshi,Iwata, Hisato American Chemical Society 2012 Environmental science & technology Vol.46 No.1

<P>Our previous studies have detected high levels of dioxins and related compounds (DRCs) including polychlorinated dibenzo-p-dioxins (PCDDs), furans (PCDFs), and coplanar PCBs (Co-PCBs) in the black-footed albatross (BFA), Phoebastria nigripes, from the North Pacific region. We have also cloned two aryl hydrocarbon receptors, AHR1 and AHR2, of the BFA. To evaluate the sensitivity to DRCs in the BFA and to assess the status of cytochrome P450 1A (CYP1A) induction in the wild population, this study investigated the mRNA expression levels of BFA AHR1 and AHR2 and also the transactivation potencies of each AHR by 15 selected DRC congeners. Quantitative real-time PCR of BFA AHR mRNAs showed that hepatic AHR1 is more highly expressed than AHR2. Transactivation by graded concentrations of individual DRCs was measured in COS-7 cells, where BFA AHR1 or AHR2 was transiently transfected. For congeners that exhibited AHR-mediated dose-dependent activities, 50% effective concentration (EC(50)) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) relative potencies (REPs) were estimated. Based on the estimates of the REPs, TCDD induction equivalency factors (IEFs) were determined. For BFA AHR1, PeCDF was equipotent to TCDD, but other congeners exhibited lower IEFs. For BFA AHR2, PCDD/F congeners except OCDD/F showed IEFs 1.0. Using BFA AHR1- or AHR2-IEFs and hepatic concentrations of DRCs in North Pacific BFAs, TCDD induction equivalents (IEQs) were calculated. We further constructed nonlinear regression models on the relationships between BFA AHR1- or AHR2-IEF derived total IEQ or WHO-TEF derived total TEQ and ethoxyresorufin-O-deethylase activity (EROD) in the liver of wild BFAs. The results indicated that the relationships of BFA AHR1- and AHR2-based IEQs and EROD were predictable from BFA AHR1- and AHR2-mediated transactivation by TCDD, respectively. Collectively, these results suggest that the in vitro assay incorporating the AHR of species of concern would be a useful tool to predict the sensitivity to DRCs in the species and CYP1A induction in the wild population.</P>