Potencies of Red Seabream AHR1- and AHR2-Mediated

Article pubs.acs.org/est

Potencies of Red Seabream AHR1- and AHR2-Mediated Transactivation by Dioxins: Implication of Both AHRs in Dioxin Toxicity Su-Min Bak,† Midori Iida,‡ Masashi Hirano,‡ Hisato Iwata,‡ and Eun-Young Kim*,†,§ †

Department of Life and Nanopharmaceutical Science and §Department of Biology, Kyung Hee University, Hoegi Dong, Dongdaemun-Gu, Seoul 130-701, Korea ‡ Laboratory of Environmental Toxicology, Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan S Supporting Information *

ABSTRACT: 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, 1,2,3,7,8PeCDD, 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. In silico 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.

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INTRODUCTION Dioxins and related compounds (DRCs) including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (Co-PCBs) are environmental contaminants that cause a broad spectrum of toxicities in both humans and wildlife. The toxic effects are mostly mediated by the aryl hydrocarbon receptor (AHR).1,2 DRC-bound AHR is translocated into the nucleus, and then forms a heterodimer with AHR nuclear translocator (ARNT). The AHR-ARNT complex activates the transcription of multiple target genes through the interaction with xenobiotic responsive element (XRE) located in the promoter region of these genes.3 Cytochrome P450 (CYP) 1A is one of the most sensitive target genes that is regulated by AHR activated with DRCs.4 To date, there is sufficient evidence indicating that AHR-mediated response is a common mechanism for DRC toxicities in vertebrates.5,6 DRCs are detected as complex mixtures of various congeners in the environment and animal tissues. For evaluating the potential risk of a mixture of DRCs, a concept that incorporates © 2013 American Chemical Society

CYP1A induction equivalency factor (IEF) or toxic equivalency factor (TEF) of each DRC congener has been established.7−11 The IEF and TEF are derived from the relative potency (REP) of each congener to a reference congener, 2,3,7,8-tetrachlorinated dibenzo-p-dioxin (TCDD) for AHR-mediated CYP1A induction and comprehensive toxic effects, respectively.7,10 The TEFs were initially provided for mammals by the WHO working group in 1994.7 Later, the TEFs were separately assigned for mammals, birds, and fish.8 The IEFs and TEFs are applied to estimate total 2,3,7,8-TCDD CYP1A-induction equivalents (IEQs) and toxic equivalents (TEQs), respectively, as the sum of the concentration of each congener multiplied by its corresponding IEF/TEF. Fish TEFs have been derived mostly from data on the early life stage mortality and CYP1A induction of the rainbow trout.8 Received: Revised: Accepted: Published: 2877

November 5, 2012 February 11, 2013 February 12, 2013 February 12, 2013 dx.doi.org/10.1021/es304423w | Environ. Sci. Technol. 2013, 47, 2877−2885

Environmental Science & Technology

Article

Isolation of rsCYP1A 5′-Flanking Region. To obtain the rsCYP1A 5′-flanking region, the genomic DNA of the red seabream was isolated from the liver of three adults using Wizard SV Genomic DNA Purification System (Promega). The putative promoter/enhancer of the rsCYP1A gene was cloned using the Universal Genome Walker Kit (Clontech). PCR amplification was performed under the following conditions: 30 s at 94 °C, 7 cycles of 2 s at 94 °C and 4 min at 72 °C, 32 cycles of 2 s at 94 °C and 4 min at 67 °C, and 4 min at 67 °C. Oligonucleotides used were as follows: rsCYP1A 50R, 5′CATCGGCCTTCATGACATTGTTGAACT-3′; and adaptor primer 1, 5′-GTAATACGACTCACTATAGGGC-3′. The PCR products were cloned into the pGEM-T Easy Vector (Promega) and sequenced using ABI 3130 Genetic Analyzer (Applied Biosystems). Mat Inspector (Genomatix) was used for searching the binding sites of transcription factors including AHR. Expression of rsAHR1 and rsAHR2 Proteins. pFLAGCMV4 (Sigma) which is a 6271 bp vector was used to obtain FLAG tag sequence. The FLAG sequence corresponding to a peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, was inserted into the N-terminus of rsAHR1 pcDNA3.1/Zeo+ vector by using PCR primers: F-NheI pflag, 5′-CGT TTA GTG AAC CGC TAG CAT TAA TTC-3′; and R-BstI pflag, 5′-CCG GGA TCC TCT TAAGTC GAC-3′. The same FLAG sequence was added into the N-terminus of rsAHR2 pcDNA3.1/Zeo+ vector by using PCR primers: F-NheI pflag and R-EcoRV pflag, 5′-GAC TGG TAC CGA TAT CAG ATC TAT CG-3′. The green African monkey kidney cell line, COS-7 cells were transiently transfected with 6 μg of the expression plasmid encoding FLAG-tagged rsAHR1 or rsAHR2 using LTX Reagent (Invitrogen). Similarly, the transfection of pFLAG-CMV4 with no rsAHR sequence as a negative control was carried out in parallel. Five hours after transfection, the cell media were exchanged. Following 18 h incubation, the cells were rinsed with cold PBS, harvested with 1 mL PBS using a cell scraper, and transferred to a microcentrifuge tube. After the collection of cells by centrifugation, the supernatant was removed and total protein was extracted in 90 μL of cold RIPA buffer (1% nonylphenoxy-polyethoxylethanol 40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM sodium orthovanadate, and protease inhibitor cocktail [Promega] in PBS). The extracts were centrifuged at 13 000 rpm for 10 min at 4 °C. Protein concentrations of supernatants were determined by a Bio-Rad (Bradford) Protein Assay Kit (Bio-Rad 500−0006) using a spectrophotometer (Shimadzu UV-1700). Standard curves were established by using serially diluted bovine serum albumin standard (Sigma P-0834). Cell lysates containing 35 μg protein were loaded on each lane after boiling at 95 °C. Proteins were separated by SDS−polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene fluoride membrane. Blots were blocked for 60 min in Tris-buffered saline with 0.1% Tween 20 (TBS-T) containing 5% nonfat dried milk, and were then incubated with an anti-FLAG monoclonal antibody (Sigma F3165) in TBS-T containing 5% nonfat dried milk overnight at 4 °C. Following the washing in TBS-T, a horseradish peroxidase-conjugated secondary antibody was incubated for 60 min in TBS-T containing 5% nonfat dried milk. Membranes were then washed in TBS-T. Emerged bands on the membrane were visualized using ECL Prime Western Blotting Detection System (Amersham Biosciences). Reporter Gene Assay. The cDNAs of rsAHRs isolated in our previous study11 were subcloned into the pcDNA3.1/Zeo+

Since the TEFs are based upon the studies on limited species, WHO working group manifested the need for separate sets of TEFs for more disparate fish species.8 To apply the TEF concept, the involvement of AHR as the mode of action of DRCs is a prerequisite. However, fish possess at least two types of paralogous AHR genes, designated as AHR1 and AHR2, although mammals have only one AHR. A battery of zebrafish studies revealed that 2,3,7,8-TCDD toxicities and CYP1A induction are mediated only by AHR2, not by AHR1a.12,13 On the other hand, both AHR1 and AHR2 from Atlantic killifish (Fundulus heteroclitus) (kfAHR1 and kfAHR2) showed specific binding to 2,3,7,8-TCDD, and 2,3,7,8-TCDD- and ARNTdependent interactions with XRE.5 This suggests that ligand dependent function of AHR may vary among different species. Hence, functional studies on each AHR isoform of a variety of fish are necessary for assessing the ecological risk of DRCs. As far as we know, no information is available on dose-dependent transactivation potencies of each AHR isoform by various DRCs in fish. In the red seabream (Pagrus major), cDNAs of two AHR isoforms denoted as rsAHR1 and rsAHR2 have been isolated.14 We have previously reported that the tissue expression profiles of rsAHR1 and rsAHR2 are isoform-specific; rsAHR1 mRNA is expressed primarily in the brain, heart, ovary, and spleen of adult fish, while rsAHR2 mRNA is observed in all tissues examined, indicating the distinct role of each rsAHR. Furthermore, CYP1A induction has been observed in red seabreams treated with 3-methylcholanthrene (3-MC) and 2,3,7,8-TCDD,15,16 indicating that the AHR-CYP1A signaling pathway may be conserved in this fish species. The objective of this study is thus to investigate potencies of rsAHR1- and rsAHR2-mediated transactivation by the exposure to individual DRCs by using a reporter gene assay where rsAHR1 or rsAHR2 expression vector was transiently transfected in COS-7 cells together with a reporter plasmid containing red seabream CYP1A (rsCYP1A) 5′-flanking region with multiple XREs. From the dose-dependent luciferase gene induction of major DRC congeners via rsAHR1 or rsAHR2, we estimated 50% effective concentrations (EC50) and REPs. Red seabream IEFs of DRC congeners for each rsAHR were determined from the respective REPs. Based on these results, we compared the sensitivity to DRC congeners and IEFs in this species with those in other species. To evaluate the binding potentials of tested DRCs to those of rsAHRs, threedimensional models of the ligand-binding domain (LBD) of rsAHR1 and rsAHR2 were built, and docking simulations of DRCs were performed. Furthermore, the mode of action of DRCs through rsAHRs in this species is also discussed in terms of comparative biology.

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MATERIALS AND METHODS DRC Congeners and 3-MC. Individual standard solutions of 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, PCB126, and 3-MC were purchased from Wellington Laboratories Inc. and AccuStandard Inc. (New Haven, CT, USA) and used as test compounds in this study. Information on the impurities of DRCs has already been reported elsewhere.17 For reporter gene assays, test solutions of individual congeners were prepared by serially diluting the stock solutions with the medium for cell culture. The examined DRC congeners were selected based on a previous paper that proposed TEFs of a variety of DRC congeners in fish.8 2878

dx.doi.org/10.1021/es304423w | Environ. Sci. Technol. 2013, 47, 2877−2885


Article

Figure 1. Nucleotide sequence of rsCYP1A gene 5′-flanking region. Nucleotides are numbered from the putative transcription start site, with negative numbers representing the 5′-flanking region. The start site of transcription was determined by comparing nucleotide sequences cloned in this study with those of 5′-UTR of rsCYP1A cDNA reported by Mizukami et al. (1994).15 The start sites of transcription and translation are marked by arrows. The putative XREs identified using MatInspector (www.genomatix.de) are boxed. Other pertinent sequences are labeled and indicated by underline: CAAT Box, CCAAT binding factors; E-Box, E-box binding factors; GATA-1, GATA binding factor-1; HNF-1, hepatic nuclear factor-1.

lysates were measured using a Multi-Mode Microplate Reader (BioTek Synergy2). The final luminescence values were expressed as the ratio of firefly luciferase unit to the Renilla luciferase unit. Estimation of REPs and IEFs. To estimate the REPs of tested congeners for each rsAHR, dose−response curves were plotted as a relative unit to the maximum response of 2,3,7,8TCDD (% 2,3,7,8-TCDD max) against logarithmically transformed doses (Figure S1). For each rsAHR construct, the dose−response curves of tested compounds except PCB126 were obtained from at least nine replicates of three independent experiments. As for PCB126, data from six replicates of two independent experiments were used to obtain the dose− response curve. EC50 values of tested compounds were obtained by using Prism 4.0 (Graph Pad) from the dose− response curves. Methods for the estimation of REPs based on a systematic framework have already been reported elsewhere.11,17,21 rsAHR1-IEFs and rsAHR2-IEFs of individual congeners were determined as an average of the minimum and maximum REPs of each DRC for the respective rsAHR (Figure S1). Homology Modeling and Docking Simulation. The homology modeling for the AHR LBD and the in silico analysis of the interaction potential between ligands and the AHR LBD were performed using the programs of Molecular Operating Environment (MOE)22 (Chemical Computing Group Inc., Montreal, QB, Canada). To build homology models of rsAHR1- and 2-LBD, and zebrafish AHR (zfAHR) 1a-, 1b-, and 2-LBD, we used the crystal structure of human hypoxia-

expression vector (Invitrogen). Firefly luciferase reporter plasmid was constructed by inserting the rsCYP1A 5′-flanking region into the pGL4.10 vector (Promega). COS-7 was maintained in Dulbecco’s modified Eagle’s medium (SigmaAldrich) supplemented with fetal bovine serum (10% final concentration) at 37 °C under 5% CO2. Since COS-7 cells express endogenous AHR protein only at a low level, this cell line has been widely used to characterize the function of AHR from a variety of species.5,11,13,17,18 Fifty thousand cells/well were seeded in 24 well plates. Transfections of vectors with Lipofectamine LTX (Invitrogen) were carried out in triplereplicated wells 18 h after the seeding of cells. Renilla luciferase vector (pGL4.74 [pRLuc/TK], Promega) was used as a transfection control. Total 300 ng of DNA (20 ng of rsCYP1A reporter plasmid, 50 ng of chicken ARNT1 expression plasmid,20 2.5 ng of each rsAHR1 or rsAHR2 expression plasmids, 5 ng of pGL4.74 control vector, and 222.5 ng pcDNA3.1/Zeo+ empty vector) was mixed with 1 μL of Lipofectamine LTX, and the mixture was then added to the cells. After 5 h incubation, the media were exchanged with dextran-coated charcoal (DCC)-stripped DMEM with DCCstripped 10% FBS for COS-7 cells. The cells were then treated with serially diluted concentrations of selected seven DRC congeners, 3-MC, or solvent control (0.001% DMSO) for 18 h. Cells were lysed 19 h after ligand treatment with 150 μL of Passive Lysis Buffer (Promega). Luciferase activities derived from the activation of rsCYP1A-XREs were determined using a Dual-Luciferase Reporter Gene Assay Kit (Promega) according to the manufacturer’s instruction. The luciferase activities in 2879



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inducible factor 2α (hHIF-2α) PAS-B (Protein Data Bank: 3F1P.A)23 as a template. The sequence of HIF-2α among proteins of which the crystalline structure is deposited in the Protein Data Bank produced the best alignments with rsAHR1 and rsAHR2 LBDs. Amino acid sequences of target AHR LBDs and hHIF-2α were aligned and carefully checked to avoid deletions or insertions in each sequence. The pairwise sequence identities between the target AHR LBDs and the template are presented in Table S1. The structures of AHR LBDs were optimized by AMBER99 force field with energy gradient of 0.0524 and adjusted through the energy minimization using the MMF94X force field.25 The ligand-binding site was determined using the MOE Alpha Site Finder. Molecular docking simulations were performed to simulate the binding of DRCs to AHR LBDs using ASEDock (Ryoka Systems Inc., Tokyo, Japan).26 Prior to the ASEDock, ligand structures were constructed using the ISIS/Draw (MDL Information Systems Inc.), and were rendered and minimized. A total of 250 conformations for each chemical were generated by Lowmode MD. The parameters used in step 1 were as follows: cutoff value, 4.5; RMS gradient, 10; and energy threshold, 500. The parameters used in step 2 were as follows: cutoff value, 8; RMS gradient, 0.1. The energy of ligand-AHR LBD complex was refined using MMFF94x of MOE under the limited conditions in which the side chains of amino acids were fixed.25 Each docking simulation was evaluated as U-dock score (kcal/mol) [U_ele (electric energy) + U_vdw (van der Waals energy) + U_solv (solvation energy) + U_strain (strain energy)].

Figure 2. Expression of rsAHR1 and rsAHR2 proteins in COS-7 cells. Expression of proteins from FLAG-tagged rsAHR1 and rsAHR2 plasmids transfected in COS-7 cells was examined by Western blot analysis. pFLAG CMV4 vector with no rsAHR sequence was used as a negative control for rsAHR protein expression.

and rsAHR2 induced the transactivation of the reporter plasmid containing a rsCYP1A 5′-flanking region in a dose-dependent manner (Figure 3). EC50 values of 2,3,7,8-TCDF, PCB126, and 3-MC were almost similar between rsAHR1 and rsAHR2 (Table 1). For 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 1,2,3,4,7,8HxCDD, 2,3,4,7,8-PeCDF, and 1,2,3,4,7,8-HxCDF, EC50 values of rsAHR1 were 3.8 to 7.0 times lower than those of rsAHR2. Comparison of EC50 values among tested compounds indicated that, for both rsAHRs, 2,3,4,7,8-PeCDF had a higher potential than 2,3,7,8-TCDD. 3-MC seems to be a high-potential ligand for both rsAHR isoforms. REPs and IEFs of DRC Congeners for rsAHR1 and rsAHR2. Results showed that, for both rsAHR1 and rsAHR2, maximal plateau responses were achieved for all the tested compounds (Figure 3). Hence, the REP value of each compound was calculated as the ratio of ECx of 2,3,7,8TCDD (TCDD-ECx) that induces X% 2,3,7,8-TCDD max relative to the concentration of the compound (REPx) that induces the response corresponding to 2,3,7,8-TCDD-ECx. For 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, 2,3,7,8-TCDF, 2,3,4,7,8PeCDF, and 1,2,3,4,7,8-HxCDF, REP values in rsAHR1- and rsAHR2-mediated responses were given in the range of 10− 90% 2,3,7,8-TCDD max (Table 2). No REP value was obtained for PCB126 in rsAHR1-mediated responses, because the responses were less than 10% 2,3,7,8-TCDD max (Figure 3). IEFs of tested congeners for rsAHR1- and rsAHR2-mediated responses are summarized in Table 3. rsAHR1 and rsAHR2 showed a similar profile of IEFs. The IEF results revealed that the most potent congener, 2,3,7,8-TCDF was more potent than 2,3,7,8-TCDD for both rsAHR-mediated transcriptional activation, showing IEF of >1.0. The IEF of 2,3,4,7,8-PeCDF was also more than that of 2,3,7,8-TCDD for rsAHR1 (1.5), and was close to 1.0 for rsAHR2 (0.93). 1,2,3,7,8-PeCDD, 1,2,3,4,7,8-HxCDD, and 1,2,3,4,7,8-HxCDF had lower IEFs (0.13−0.38). The IEF of PCB126 for rsAHR2 was much lower than those of other congeners (0.0085). In Silico Homology Modeling and Docking Simulations. To investigate the ligand binding potential as the function of rsAHR1 and rsAHR2 isoforms, we performed in silico docking simulations for seven DRCs and 3-MC. Homology models for zfAHR1a, zfAHR1b, zfAHR2, rsAHR1, and rsAHR2 were built based on the crystal structure of hHIF2α PAS-B. Sequences of AHR PAS domains against the template hHIF-2α, pairwise aligned with MOE, are shown in Figure S2. The root-mean-square distance (RMSD) values of the Cα atoms for superimposition of the homology models of

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RESULTS Sequence Analysis of rsCYP1A 5′-Flanking Region. We succeeded in isolating approximately 2.7 kb of rsCYP1A 5′flanking region from the red seabream (Figure 1). Sequence analysis by MatInspector indicated multiple potential DNA elements including five XREs, TATA box, CAAT box, E-box, hepatic nuclear factor-1 (HNF-1), and GATA binding factor-1 (GATA) (Figure 1). To predict the function of XREs, the matrix similarity (MS) score of each XRE was calculated using the position weight matrix.19 Two XREs, XRE3 (0.92) and XRE5 (0.92), with a greater MS score than the threshold (0.85) were identified. This result suggests that the rsCYP1A 5′flanking region isolated in this study has a potential for the transcriptional activation through rsAHRs. Expression of rsAHR1 and rsAHR2 Proteins. To confirm whether rsAHR1 and rsAHR2 proteins are expressed in COS-7 cells, FLAG-tagged rsAHR1 and rsAHR2 expression vectors were transfected into COS-7 cells, and the cell lysates were subjected to Western blotting with an anti-FLAG monoclonal antibody (Figure 2). Each protein derived from rsAHR1 or rsAHR2 construct was detected at the predicted molecular size (95 kDa for FLAG-tagged rsAHR1 and 110 kDa for FLAGtagged rsAHR2). In contrast, only a faint band from the negative control vector, pFLAG-CMV4 with no rsAHR sequence was seen around the predicted molecular size of rsAHRs. This result indicates that both rsAHR1 and rsAHR2 were translated from the respective expression plasmids in COS-7 cells. EC50 for rsAHR1 and rsAHR2 Transactivations by DRC Congeners and 3-MC. To investigate the transactivation potencies of both rsAHR isoforms by DRC congeners and 3MC, we constructed the reporter gene assay system using COS7 cells. For all the compounds examined in this study, rsAHR1 2880



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Figure 3. Responses of rsAHR1- and rsAHR2-mediated transactivation by the exposure to 7 selected DRC congeners and 3-MC. Responses of tested compounds via each rsAHR are presented as a value (%) relative to the maximum response obtained by 2,3,7,8-TCDD (% 2,3,7,8-TCDD max). Data of the responses are plotted as mean ± SEM. S.C.: solvent (DMSO) control. For more details on the dose−responses of each compound, refer to Figure S3 in Supporting Information.

positive values for all the tested AHRs. The docking pairs with zfAHR1a had extremely high U-dock values, when compared to those with other AHRs.

Table 1. EC50 Values of Seven Selected DRCs Congeners and 3-MC Based on rsAHRl- and rsAHR2-Mediated Transcriptional Activation in in Vitro Reporter Gene Assays

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EC50 (nM) Congener

rsAHRl

rsAHR2

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 PCB126 3-MC

0.073 0.17 0.17 0.19 0.042 0.098 0.35 0.094

0.51 0.66 0.67 0.28 0.16 0.48 0.38 0.050

DISCUSSION In the present study, an AHR-driven reporter gene assay system was constructed by transfecting the expression vector of rsAHR1 or rsAHR2 into the COS-7 cells together with a reporter vector containing 5′-flanking region of rsCYP1A gene. rsAHR1- and rsAHR2-mediated transactivation potencies of selected seven DRCs and 3-MC were investigated using the constructed system. Both rsAHR1 and rsAHR2 isoforms dosedependently enhanced the transcriptional activities of the reporter gene by the treatment with these compounds (Figure 3 and Figure S3). This suggests that both rsAHR isoforms play functional roles in the induction of rsCYP1A. EC50 values for seven DRC congeners were determined from the respective dose−response curves for rsAHR1 and rsAHR2 (Table 1). The comparison of EC50 values indicates that the transactivation potentials of some DRCs were different between rsAHR1 and rsAHR2. Lower EC50 of 2,3,4,7,8-PeCDF for both rsAHRs than those of 2,3,7,8-TCDD revealed that 2,3,4,7,8PeCDF is more potent for the transcriptional activation. This observation is similar to results in cells containing the AHR1 construct of some avian species.27 It has been suggested that, when compared to 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF contains an additional electronegative Cl atom that may contribute to

zfAHR1a-, 1b-, and 2-LBDs and rsAHR1- and 2-LBDs with the crystal structure of hHIF-2α were 0.90, 1.3, 0.84, 0.87 and 0.93 Å, respectively. The docking simulations of each AHR with DRCs and 3-MC were carried out using ASEDock. Results of the docking of rsAHRs and zfAHRs with 2,3,7,8-TCDD are shown in Figure 4. The docking potential as U-dock value which indicates the binding energy for each pair of AHRcompound is summarized in Table 4. The U-dock values (kcal/ mol) of 2,3,7,8-TCDD were 148 for zfAHR1a, 7.62 for zfAHR1b, 2.48 for zfAHR2, 42.5 for rsAHR1, and 10.6 for rsAHR2, showing positive values for all the cases. Likewise, the docking simulations of other tested compounds also gave

Table 2. Minimum and Maximum Values of REPs for DRC Congenersa rsAHRl

rsAHR2

congener

REPmin

REPmax

REPmin

REPmax

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 PCB126

0.28 (REP21) 0.53 (REP16) 1.7 (REP34) 1.0 (REP56) 0.35 (REP16) NA

0.060 (REP78) 0.051 (REP77) 3.3 (REP66) 1.9 (REP86) 0.19 (REP79) NA

0.73 (REP10) 0.26 (REP10) 1.1 (REP10) 1.5 (REP10) 0.38 (REP10) 0.013 (REP10)

0.020 (REP60) 0.0068 (REP29) 1.9 (REP90) 0.39 (REP67) 0.011 (REP34) 0.0039 (REP14)

a

Relative potency at x% response (REPx) was calculated by dividing 2,3,7,8-TCDDECx with ligand ECx. NA: No REP was available, because the maximum response was less than 10% of 2,3,7,8-TCDD response. 2881



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Table 3. Comparison of IEFs, REPs, and TEFs in Fisha luciferase induction in COS-7 cells

CYP1A induction

IEFs DRCs 2,3,7,8TCDD 1,2,3,7,8PeCDD 1,2,3,4,7,8HxCDD 2,3,7,8TCDF 2,3,4,7,8PeCDF l,2,3,4,7,8HxCDF PCB126

rsAHRl

rsAHR2

LC50

rtAHR2ab

ZF-L cell CYP1A mRNA inductionc

RLT-Wl cell CYP1A activity (in vitro)d

RBT liver CYP1A activity (in vivo)e

WHO TEFi

REPs RTG-2 cell CYP1A mRNA Inductionf

RBTELS mortalityg,h

fish

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

0.17

0.38

1.3

3.6

2.6

1.8

1.6

0.7

1.0

0.29

0.13

-

3.2

1.1

0.4

0.1

0.3

0.5

2.5

1.5

0.5

0.07

0.2

0.5

0.5

0.03

0.05

1.5

0.93

0.2

8.1

1.9

2.0

0.1

0.3

0.5

0.27

0.20

-

-

-

-

0.4

0.3

0.1

0.0085

0.2

0.01

0.02

0.005

0.06

0.005

0.005

NA

a

b

NA: No IEF was available, because the maximum response was less than 10% of 2,3,7,8-TCDD response. -: No data provided. IEFs in rtAHR2derived COS-7 cell reporter gene assay system.18 cREPs determined by the induction potencies of CYPlA mRNA in zebrafish liver cell line.33 dREPs determined by the oxygenase activity of CYPIA in rainbow trout liver cell line.34 eREPs determined by the oxygenase activity of CYP1A in rainbow trout liver.35 fREPs determined by the induction potencies of CYP1A mRNA in rainbow trout gonad ceil line.36 g,hREPs determined by LC50 of rainbow trout early life stage mortality.37,38 iFish TEFs proposed by the World Health Organization.8

same type of AHRs (Ile-322 and Ala-378 in rsAHR1, and Ile317 and Ala-373 in rsAHR2) as ring-necked pheasant AHR1 (Figure S4), both rsAHRs may share the same mechanism as the pheasant AHR1 regarding the higher potency of 2,3,4,7,8PeCDF than 2,3,7,8-TCDD. The different transactivation potencies between rsAHR1 and rsAHR2 may be explained by the structural differences in rsAHRs. rsAHR1 and rsAHR2 show only 32% amino acid identity in full length (Table S2). Both rsAHRs are highly conserved in the N-terminus (65% identity in bHLH, and 64% and 72% identities in PAS A and B domains, respectively).14 In particular, rsAHRs showed 100% identity within the ligand binding cavity (T283, H285, F289, Y316, I319, F345, and A375) that is critical for a high binding affinity of 2,3,7,8TCDD.28 These comparisons indicate that rsAHR1 and rsAHR2 have similar functions in DNA binding, ligand binding, and heterodimerization with ARNT. In contrast, the transactivation domain in the C-terminus of rsAHRs is poorly conserved, sharing a 10% identity, and only rsAHR1 has a Qrich region.14 These results suggest that the structural difference in the C-terminal region of rsAHR paralogues may be responsible for the difference in the transactivation of rsCYP1A XRE-driven reporter gene. Several studies on AHRs in some fish models have reported distinct roles of AHR isoforms in dioxin toxicities. In zebrafish, zfAHR2 binds to 2,3,7,8-TCDD with high affinity, and is able to activate the transcription of a reporter gene under the control of XREs.12 In contrast, zfAHR1a lacks the binding ability to 2,3,7,8-TCDD and the transactivation potency.12 zfAHR1b protein that exhibits a high binding affinity to 2,3,7,8TCDD can transactivate the reporter gene with an efficacy comparable to that of zfAHR2, but displays a higher EC50 than zfAHR2.13 On the other hand, studies on Atlantic killifish revealed that both kfAHR1 and kfAHR2 showed specific binding abilities to 2,3,7,8-TCDD and enhanced transactivation potencies in the presence of 2,3,7,8-TCDD, although no data of dose-dependent transactivation by 2,3,7,8-TCDD and other congeners has been available so far.5,31

Figure 4. Molecular docking simulations of 2,3,7,8-TCDD with zebrafish and red seabream AHR LBDs. 2,3,7,8-TCDD was docked in the LBD of (A) rsAHR1, (B) rsAHR2, (C) zfAHR1b, (D) zfAHR2, and (E) zfAHR1a. The structural model was generated using the program Molecular Operating Environment (MOE), based on the crystal structure (PDB number: 3F1P.A) of hHIF2α PAS domain. The amino acid residues in the ligand binding pocket are displayed as sticks; blue for rsAHR1, red for rsAHR2, purple for zfAHR1b, pink for zfAHR2, and green for zfAHR1a. The structure of 2,3,7,8-TCDD is colored with light green. Secondary structure elements are represented as green arrows (strands) and red cylinders (helices).

greater hydrogen bonding with Ile-324 and Ala-380 in ringnecked pheasant AHR1.27 Given that the red seabream has the 2882



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Table 4. U-Dock Values Obtained from in Silico Docking Simulation of Tested Chemicals with AHR LBDs by Using ASEDock U-dock (kcal/mol)

a

ligand

M.W.a

rsAHR1

rsAHR2

zfAHR1A

zfAHR1B

zfAHR2

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-PeCF 1,2,3,4,7,8-HxCDF PCB126 3-MC

322 356 391 306 340 375 326 268

42.5 61.6 77.8 32.7 53.8 97.6 42.5 104

10.6 23.6 41.9 15.6 31.9 57.1 46.9 62.8

148 118 213 174 157 214 134 198

7.62 13.0 20.9 16.0 19.9 26.7 19.8 23.4

2.48 9.91 31.9 6.08 23.4 28.2 18.2 25.3

Molecular weight.

U-dock values for all the tested compounds were much lower than those of zfAHR1a that has impairs 2,3,7,8-TCDD binding ability, but were rather close to those of zfAHR1b that has a specific binding affinity to 2,3,7,8-TCDD. These results imply that the docking simulation may have a potential to predict the ligand of rsAHRs, although data of the crystal structure of AHR are necessary to raise the precision of the homology model. To validate whether potency of in vivo rsCYP1A induction by DRCs can be predicted from that of in vitro rsAHR-mediated transactivation by DRCs, we compared the in vitro 2,3,7,8TCDD-EC50 (0.073 nM for rsAHR1 and 0.51 nM for rsAHR2, respectively) with in vivo 2,3,7,8-TCDD-EC50 of rsCYP1A mRNA expression reported in our earlier study.16 The EC50 values for CYP1A mRNA induction in 2,3,7,8-TCDD-treated red seabreams at 36−66 h postfertilization were in the range of 0.096−0.29 ng/g wet weight of whole body (0.30 to 0.91 nM). These EC50 values were closer to that of the in vitro EC50 for rsAHR2 than for rsAHR1. Considering that expression levels of rsAHR2 mRNA were 3−30 times higher than those of rsAHR1 in red seabream embryos,16 the similar EC50 between the in vivo test and in vitro rsAHR2-mediated response is a convincing result. This suggests that the in vitro assay may be a useful tool to predict the in vivo rsAHR-mediated effect of DRCs. A previous study has reported that the REPs of DRC congeners for a rainbow trout AHR (rtAHR2α) regarding luciferase induction in COS-7 cells were positively correlated with their REPs for CYP1A induction in RTG-2, a rainbow trout gonad cell line (Table 3).18 The rtAHR2α-derived REPs in COS-7 cells also exhibited a significant positive correlation with their REPs for the mortality in the early life stage of the rainbow trout (Table 3).18 The correlations suggest that transactivation potencies of DRCs through AHR protein transiently expressed in COS-7 represent the REPs of the cell line and in vivo toxicities of the species from which the AHR originates. On the other hand, there are still some uncertainties to extrapolating in vivo toxicities of DRCs from in vitro AHR assays. In zebrafish, the in vivo role of zfAHR2 in TCDD-induced developmental toxicity has been well characterized by knockdown of zfAHR2 using a morpholino antisense oligonucleotide,30 while the role of zfAHR1b in TCDD toxicity has not yet been demonstrated. Moreover, a recent study by Clark et al. (2010)32 showed that morpholino knockdown of kfAHR2 rescued Fundulus embryos from PCB126-induced cardiac deformities, but no rescue was observed by the knockdown of kfAHR1. Since most of the studies on AHR-based dioxin toxicities in fish species have focused on zebrafish, it has been believed that fish AHR2 isoform plays a critical role in the induction of toxicities and CYP1A by the exposure to dioxins, but AHR1 has much less contribution to dioxin toxicities.12,13,29,30 To our

When comparing the full-length amino acid sequences of rsAHR paralogs with zebrafish and killifish AHRs, rsAHR1 is the closest to zfAHR1b (58%) and rsAHR2 is to kfAHR2 (58%) and kfAHR1 is closer to zfAHR1b than zfAHR1a (Table S2). The relationships are supported by the phylogenetic tree of AHRs (Figure S5). This suggests that our rsAHR1 clone is an ortholog of zfAHR1b, but not of zfAHR1a. Given that both zfAHR1b and kfAHR2 have a binding affinity to 2,3,7,8-TCDD and transactivation potencies, it is likely that both rsAHR1 and rsAHR2 induced the transcriptional activity of rsCYP1A 5′flanking region by exposure to 2,3,7,8-TCDD and other congeners. These sequence comparison also supports that the red seabream possesses multiple functional AHR isoforms in terms of CYP1A induction. To characterize the IEFs of rsAHR1 and rsAHR2, we compared them with WHO fish TEFs (Table 3). The largest difference was observed for 2,3,7,8-TCDF, showing 50- and 30fold higher IEFs for rsAHR1 (2.5) and rsAHR2 (1.5) than the WHO fish TEF (0.05), respectively. IEFs of 2,3,4,7,8-PeCDF and 1,2,3,4,7,8-HxCDF were several-fold larger than the respective WHO TEFs (0.5 and 0.1) for both rsAHR. With respect to 1,2,3,7,8-PeCDD and 1,2,3,4,7,8-HxCDD, IEFs for rsAHR1 and rsAHR2 were several-fold lower than the respective WHO TEFs (1.0 and 0.5). The WHO fish TEFs are assigned based on REPs derived from in vivo rainbow trout egg injection studies, in vivo/in vitro CYP1A induction potencies and quantitative structure−activity relationship modeling. This approach for assigning fish TEFs may lead to the difference between IEFs and TEFs of tested congeners. Homology models for zfAHR1a, zfAHR1b, zfAHR2, rsAHR1, and rsAHR2 were built based on the crystal structure of hHIF-2α PAS-B, which shared low sequence identities with the target proteins (25.9% with zfAHR1a, 25.0% with zfAHR1b, 27.2% with zfAHR2, 25.0% with rsAHR1, and 25.9% with rsAHR2) as shown in Table S1. The low sequence identities may lower the accuracy of the homology models of these fish AHRs, although RMSD values were close to 1.0 in all the examined AHRs. In fact, U-dock scores estimated for tested fish AHRs showed positive values for all the ligands. For DRCs other than PCB126, the higher U-dock scores of rsAHR1 than rsAHR2 fail to explain the lower or comparable EC50 value for rsAHR1 than for rsAHR2. Similarly, it is difficult to account for the REP/IEF profile for each rsAHR by the magnitude of Udock scores; the docking depends on the molecular weight of ligands (TCDD/F < PeCDD/F < HxCDD/F) for each rsAHR, while REPs/IEFs do not (Table 4). However, in silico docking simulations for the binding of DRCs to rsAHRs and zfAHRs appeared to support that both rsAHRs enhanced the transcriptional activity in a ligand-dependent manner; both 2883



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knowledge, this is the first report on the ligand- and dosedependent transactivation potencies of AHR1B type isoform in fish species, suggesting a contribution of this isoform to toxicities of DRCs. The comparison of REPs/IEFs/TEFs in this and previous studies suggests that the ligand profile of the fish AHR is unexpectedly diversified across species. Hence, there are still uncertainties regarding the general application of WHO fish TEFs to non-model species. To address the uncertainties, information on REPs from more disparate fish species is necessary. Furthermore, it remains poorly understood how accurately the IEFs derived from our in vitro AHR-driven reporter gene assay can predict REPs of in vivo toxicities, and whether the IEFs are end point- and tissue-specific. Nevertheless, the present study gives insight into participation of multiple AHR isoforms in the toxicity of DRCs in fish. Moreover, our in vitro reporter gene assay could be a useful tool for screening AHR ligands and for determining the susceptibility to each ligand in the red seabream.

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(3) Mimura, J.; Fujii-Kuriyama, Y. Functional role of AhR in the expression of toxic effects by TCDD. Biochim. Biophys. Acta 2003, 1619, 263−268. (4) Fujii-Kuriyama, Y.; Mimura, J. Molecular mechanisms of AhR functions in the regulation of cytochrome P450 genes. Biochem. Biophys. Res. Commun. 2005, 338 (1), 311−7. (5) Karchner, S. I.; Franks, D. G.; Powell, W. H.; Hahn, M. E. Regulatory interactions among three members of the vertebrate aryl hydrocarbon receptor family: AHR repressor, AHR1, and AHR2. Biochem. J. 2002, 277, 6949−6959. (6) Hahn, M. E. The aryl hydrocarbon receptor: A comparative perspective. . Comp. Biochem. Physiol., Part C: Pharmacol., Toxicol. Endocrinol. 1998, 121 (1−3), 23−53. (7) Van den Berg, M.; De Jongh, J.; Poiger, H.; Olson, J. R. The toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and their relevance for toxicity. Crit. Rev. Toxicol. 1994, 24, 1−74. (8) Van den Berg, M.; Birnbaum, L.; Bosveld, A. T.; Brunstr€om, B.; Cook, P.; Feeley, M.; Giesy, J. P.; Hanberg, A.; Hasegawa, R.; Kennedy, S. W.; Kubiak, T.; Larsen, J. C.; Van Leeuwen, F. X.; Liem, A. K.; Nolt, C.; Peterson, R. E.; Poellinger, L.; Safe, S.; Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ. Health Perspect. 1998, 106 (12), 775−792. (9) Bols, N. C.; Whyte, J. J.; Clemons, J. H.; Tom, D. J.; Van den Heuvel, M. R.; Dixon, D. G. Use of liver cell lines to develop toxic equivalency factors and to derive toxic equivalency concentrations in environmental samples. In Ecotoxicology: Responses, Biomarkers and Risk Assessment (Zelikoff, J. T., Ed.) SOS Publications: Fair Haven, NJ, 1997; PP 329−350. (10) Haws, L. C.; Su, S. H.; Harris, M.; Devito, M. J.; Walker, N. J.; Farland, W. H.; Finley, B.; Birnbaum, L. S. Development of a refined database of mammalian relative potency estimates for dioxin-like compounds. Toxicol. Sci. 2006, 89 (1), 4−30. (11) Kim, E. Y.; Suda, T.; Tanabe, S.; Batoev, V. B.; Petrov, E. A.; Iwata, H. Evaluation of relative potencies for in vitro transactivation of the Baikal seal aryl hydrocarbon receptor by dioxin-like compounds. Environ. Sci. Technol. 2011, 45 (4), 1652−1658. (12) Andreasen, E. A.; Hahn, M. E.; Heideman, W.; Peterson, R. E.; Tanguay, R. L. The zebrafish (Danio rerio) aryl hydrocarbon receptor type 1 is a novel vertebrate receptor. Mol. Pharmacol. 2002, 62, 234− 249. (13) Karchner, S. I.; Franks, D. G.; Hahn, M. E. AHR1B, a new functional aryl hydrocarbon receptor in zebrafish: tandem arrangement of ahr1b and ahr2 genes. Biochem. J. 2005, 392, 153−161. (14) Yamauchi, M.; Kim, E. Y.; Iwata, H.; Tanabe, S. Molecular characterization of the aryl hydrocarbon receptors (AHR1 and AHR2) from red seabream (Pagrus major). Comp. Biochem. Physiol. Part C 2005, 141, 177−187. (15) Mizukami, Y.; Okauchi, M.; Arizono, K.; Ariyoshi, T.; Kito, H. Isolation and sequence of cDNA encoding a 3-methylcholanthreneinducible cytochrome P450 from wild red sea bream, Pagrus major. Mar. Biol. 1994, 120, 343−349. (16) Yamauchi, M.; Kim, E. Y.; Iwata, H.; Shima, Y.; Tanabe, S. Toxic effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryo: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquat. Toxicol. 2006, 80, 166−179. (17) Leena, Mol. T.; Kim, E. Y.; Ishibashi, H.; Iwata, H. In vitro transactivation potencies of black-footed albatross (Phoebastria nigripes) AHR1 and AHR2 by dioxins to predict CYP1A expression in the wild population. Environ. Sci. Technol. 2012, 46, 525−533. (18) Abnet, C. C.; Tanguay, R. L.; Heideman, W.; Peterson, R. E. Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: greater insight into species differences in toxic potency of polychlorinated dibenzo-pdioxin, dibenzofuran, and biphenyl congeners. Toxicol. Appl. Pharmacol. 1999, 159, 41−51.

ASSOCIATED CONTENT

S Supporting Information *

Detailed description of experimental procedures including “Schematic illustration showing the concept of REP and IEF” (Figure S1), “Multiple sequence alignment of secondary structure elements of AHR LBDs against hHIF-2α” (Figure S2), “Responses of rsAHR1- and rsAHR2-mediated transactivation by the exposure to PCDDs, PCDFs, PCB126 and 3MC” (Figure S3), “Multiple sequence alignment of AHR LBD amino acids in the region corresponding to residues 241−384 of the mouse AHR” (Figure S4), “Phylogenetic analysis of overall amino acid sequences of fish AHRs” (Figure S5), “Identities (%) of amino acid sequences of fish AHRs and hHIF-2a used for the construction of homology modeling” (Table S1) and “Identities (%) of full length and LBD amino acid sequences between rsAHRs and other AHRs” (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-2-961-2310. Fax: +82-2961-0244. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology to E. Y. K. (20100012150, 2012K2A2A4021504). This work was also supported by a grant from the Kyung Hee University in 2012 (20120465) and by Grant-in-Aid for Scientific Research (S) from the Japan Society for the Promotion of Science to H. I. (no. 21221004). The authors would like to thank Prof. An. Subramanian, Ehime University, for critical reading of this manuscript.

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REFERENCES

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Potencies of Red Seabream AHR1- and AHR2-Mediated

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