Regulation of zebrafish CYP3A65 transcription by AHR2

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Toxicology and Applied Pharmacology xxx (2013) xxx–xxx

YTAAP-12732; No. of pages: 11; 4C:

Contents lists available at SciVerse ScienceDirect

Toxicology and Applied Pharmacology

j ourna l homepage: www.e lsev ie r .com/ locate /ytaap

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Regulation of zebrafish CYP3A65 transcription by AHR2

Chin-Teng Chang a, Hsin-Yu Chung a, Hsiao-Ting Su a, Hua-Pin Tseng a, Wen-Shyong Tzou a,b, Chin-Hwa Hu a,b,⁎a Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwanb Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan

⁎ Corresponding author at: Institute of Bioscience and BOcean University, No.2, Pei-Ning Road, Keelung, 20224, T

E-mail address: [email protected] (C.-H. Hu).

0041-008X/$ – see front matter © 2013 Published by Elhttp://dx.doi.org/10.1016/j.taap.2013.04.010

Please cite this article as: Chang, C.-T., et al.dx.doi.org/10.1016/j.taap.2013.04.010

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Article history:Received 17 December 2012Revised 17 March 2013Accepted 13 April 2013Available online xxxx

Keywords:ZebrafishCYP3A65AHR2LiverIntestineDioxinKynurenine

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ROCYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug me-

tabolism. Inmammals, CYP3A genes are induced by various xenobiotics through processesmediated by PXR.Wepreviously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinaltissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin(TCDD). However, the underlyingmechanism of TCDD-mediated CYP3A65 transcription is unclear. Herewe gen-erated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5′flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP inlarval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP ex-pression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expressioncan be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE ele-ments in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced tran-scription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalianCYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatorymechanism for tel-eost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter.

© 2013 Published by Elsevier Inc.

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UNCO

RREIntroduction

Cytochrome P450 (CYP) is a superfamily of heme-containingmonooxygenases that catalyze the oxidation of a variety of metabolicintermediates and xenobiotic substances. Based on sequence similar-ities, the CYP superfamily can be sub-divided into hundreds of fami-lies and subfamilies; among these, the CYP1-3 families consist ofmajor enzymes involved in xenobiotic metabolism. Many of thesexenobiotic-metabolizing CYP genes may be induced by their metabol-ic substrates, or a variety of xenobiotics, through the action of nuclearreceptor transcription factors, which include pregnane X receptor(PXR), constitutive androstane receptor (CAR), and aryl hydrocar-bon receptor (AhR) (Guengerich, 2005; Tompkins and Wallace,2007; Waxman, 1999). In mammals, CAR and PXR play majorroles in xenobiotic-mediated CYP2 and CYP3 induction, respec-tively (Bertilsson et al., 1998; Kliewer et al., 1998; Lehmann etal., 1998;Moore et al., 2000;Waxman, 1999), whereas AhR mediatesCYP1 activation (Whitlock, 1999). Similar nuclear receptors andtheir targeted CYP genes are widely distributed in many species.

CYP3A family members are the most abundant CYPs in humanliver and small intestinal tissues, and have pivotal functions in drugmetabolism (Bork et al., 1989; McKinnon et al., 1995). It has been

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sevier Inc.

, Regulation of zebrafish CYP3

estimated that over 75% of currently used drugs are metabolized byCYP3As (Guengerich, 2008). Orthologous CYP3A genes are alsofound in other non-mammalian organisms, including chickens,amphibians, and fish. However, other CYP3 subfamilies, includingCYP3B, CYP3C, and CYP3D, are found exclusively in teleost species(Nelson, 2009). Through PXR action, mammalian CYP3A genes are in-ducible by a wide range of compounds, including steroids, antifungaldrugs, and antibiotics, as well as by various xenobiotics, such aspregnenolone 16α-carbonitrile (PCN), dexamethasone, mifepris-tone (RU486), spironolactone, and rifampicin, with differentialspecies- and gene-specific selectivity [for review, see Goodwin etal., 2002].

We have previously identified CYP3A65, a CYP3A ortholog that is con-stitutively expressed in liver and intestinal tissues, in zebrafish (Tseng etal., 2005). Unlike mammalian CYP3A genes, CYP3A65 expression isstrongly induced by tetrachlorodibenzo-p-dioxin (TCDD) during the lar-val stage. We observed that repression of AHR2 translation abrogatedbasal-type and TCDD-stimulated CYP3A65 transcription, suggesting thatthe AHR2-related signaling pathway has a pivotal function in CYP3A65transcription. However, the underlying mechanism has not been eluci-dated. Here, we generated two CYP3A65 promoter-driven transgenicfish lines, Tg(CYP3A65L:EGFP) and Tg(CYP3A65S:EGFP), both of whichexpressed EGFP in a pattern that recapitulated that of endogenousCYP3A65. Moreover, EGFP expression in these two lines was induced byTCDD treatment. Multiple copies of consensus AHR-response elements(AHREs) in the CYP3A65 promoter were found to be critical for

A65 transcription by AHR2, Toxicol. Appl. Pharmacol. (2013), http://

http://dx.doi.org/10.1016/j.taap.2013.04.010

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2 C.-T. Chang et al. / Toxicology and Applied Pharmacology xxx (2013) xxx–xxx

basal-type and TCDD-inducible reporter gene transcription. Accordingly,the AHR2 DNA- and ligand-binding domains are essential for CYP3A65gene transcription. To our knowledge, this is the first report demonstrat-ing that AHR2 plays a critical role in supporting CYP3A transcription.

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Results

AHR2 and PXR are required for constitutive CYP3A65 transcription

We previously demonstrated constitutive CYP3A65 transcrip-tion in zebrafish liver and intestinal tissues (Tseng et al., 2005).CYP3A65 transcription could be elicited by various PXR- andAHR-specific xenobiotics, including dexamethasone, rifampicin, and2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To determine the impor-tance of individual AHR2 and PXR factors in CYP3A65 transcription, weseparately knocked down AHR2 and PXR expression with morpholinooligonucleotides, and examined their influence on CYP3A65 transcrip-tion. We observed that reducing translation of either PXR or AHR2 dra-matically reduced endogenous transcription of CYP3A65 (Fig. 1),suggesting that both factors are required to support basal CYP3A65expression.

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Fig. 1. CYP3A65 transcription requires AHR2 and PXR function. (A–D) Lateral views ofCYP3A65 expression in wild-type (A), ahr2MO (B), pxrMO (C) and control MO (D) larvaeat 96 hpf. CYP3A65 expression in the liver and intestinal tissues was abolished followingtreatment with either ahr2 MO or pxr MO, suggesting that AHR2 and PXR are requiredfor basal transcription levels of CYP3A65. Abbreviations: lv, liver; in, intestine.

Please cite this article as: Chang, C.-T., et al., Regulation of zebrafish CYP3dx.doi.org/10.1016/j.taap.2013.04.010

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Recapitulation of the CYP3A65 transcriptional profile in transgenic lines

Although most identified CYP3A genes are responsive to PXR acti-vators, CYP3A65 is, to our knowledge, the first isolated CYP3A gene tobe induced by AHR activators. To elucidate the regulatory mechanismbehind TCDD-mediated induction of the CYP3A gene, we isolated a5.4-kb flanking sequence upstream of the CYP3A65 translationalstart site (−5445 to +39) from zebrafish genomic DNA. Analysis ofthis region using Promo 3.0 (Farre et al., 2003; Messeguer et al.,2002) revealed multiple copies of consensus motifs for several tran-scription factors, including AHR/AHR2, PXR, and Cdx1 (SupplementalFig. 1), suggesting that the CYP3A65 core promoter resides in thisfragment. To analyze the activity of this putative CYP3A65 core pro-moter, the 5.4-kb fragment and a series of 5′-truncation fragments,including the 4.9 kb (−4898 to +39), 2.1 kb (−2066 to +39),1.4 kb (−1371 to +39) and 0.7 kb (−693 to +39) sequences up-stream of the translational start site, were cloned into the Tol2 ex-pression vector upstream of EGFP (Kawakami et al., 2004). One-cellembryos were co-injected with Tol2 mRNA and one of the constructs,and the percentage of larvae displaying fluorescent EGFP expressionin gastrointestinal (GI) tissues at the 5-dpf stage was used to evaluateconstruct promoter activity. We observed that when the injected con-structs contained an insert shorter than 2-kb, i.e., pCYP3A65-0.7 kb:EGFP and pCYP3A65-1.4 kb:EGFP, only a small percentage of larvae(less than 20%) exhibited EGFP expression in the liver and intestinal tis-sues (Figs. 2A, and D–E), the locations where endogenous CYP3A65mRNA is transcribed (Tseng et al., 2005). In contrast, when the insertswere larger than 2-kb, i.e., pCYP3A65-2.1 kb:EGFP, pCYP3A65-4.9 kb:EGFP, and pCYP3A65-5.3 kb:EGFP, a higher percentage (40–50%) ofthe injected larvae displayed EGFP expression in liver and intestinal tis-sues (Figs. 2D–E). Nevertheless, approximately 30–40% of the injectedembryos failed to express EGFP (Figs. 2C–E), regardless of the constructinjected. The failure rate was higher (over 60%) when embryos wereinjected with pCYP3A65-1.4 kb:EGFP (Figs. 2D–E). These results sug-gest that the 2.1 kb fragment upstream of CYP3A65 is sufficient todrive transient EGFP expression in the liver and intestinal tissues.

We next characterized the transcriptional activity of the CYP3A65promoter in stable transgenic lines. To generate stable transgeniclines, pCYP3A65-2.1 kb:EGFP or pCYP3A65-5.3 kb:EGFP-injected lar-vae displaying transient expression in the liver and intestinal tissueswere raised to adulthood as F0, and were then out-crossed with awild-type strain. EGFP-positive F1 embryos were raised to generatethe stable transgenic lines Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) that contained either the 2.1-kb (short) or the 5.3-kb (long)CYP3A65 upstream flanking sequence insert, respectively. The trans-genic EGFP gene was transmitted according to Mendelian inheritance,because 50% of the F3 fish exhibited a GI-specific EGFP expressionpattern when the transgenic F2 fish were crossed with wild-type fish.

During developing stages, the Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) lines exhibited weak EGFP fluorescence in the liver bud and fore-gut regions at 2 dpf (Figs. 3G and L), which became stronger by 3 dpf(Figs. 3H and M). From 4 dpf, intense EGFP expression was observedin the liver and intestinal tissues in both lines (Figs. 3I–J and N–O). Incontrast, whole-mount in situ hybridization revealed that basal tran-scription of endogenous CYP3A65 mRNA was just barely detectable at3 dpf (Fig. 3C). Transcription increased dramatically at 4 and 5 dpf inthe liver and intestinal tissues (Figs. 3D–E). It is noteworthy that trans-genic EGFP and endogenous CYP3A65mRNA exhibited an almost identi-cal expression pattern in 4 dpf Tg(CYP3A65S:EGFP) larvae (Fig. 3D, P).The expanded EGFP expression domains observed in 4 and 5 dpf larvae(Figs. 3I–J and N–O) were caused by out-of-focus emission of fluores-cent protein.

In summary, we generated two stable transgenic lines, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which recapitulate an endogenousCYP3A65 expression pattern. However, Tg(CYP3A65S:EGFP) exhibitedslightly stronger EGFP expression in the liver, and Tg(CYP3A65L:EGFP)




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Fig. 2. Characterization and transcriptional activity analysis of the CYP3A65 promoter. (A–C) Representative images of 5 dpf larvae displaying different types of transient EGFP ex-pression patterns, including GI-specific (A), non-specific (expressed in non-GI tissues) (B) and no expression (C). Abbreviations: lv, liver; in, intestine. (D) Schematic of thepCYP3A65-5.3 kb:EGFP, pCYP3A65-4.9 kb:EGFP, pCYP3A65-2.1 kb:EGFP, pCYP3A65-1.4 kb:EGFP, and pCYP3A65-0.7 kb:EGFP constructs (left), and a summary of their EGFP ex-pression patterns in a transient expression assay (right). Consensus AHR2 motifs are indicated by the gray blocks. For each construct, the number of injections and the percentageof injected larvae displaying EGFP expression in GI tissues (GI-specific) and non-GI tissues (non-specific), and those lacking EGFP expression (no expression) are indicated on theright. (E) Cumulative percentage of injected 5 dpf larvae displaying GI-specific, non-specific and deficient EGFP expression. Approximately 40–50% of the larvae injected withpCYP3A652.1-kb:EGFP, pCYP3A654.9-kb:EGFP or pCYP3A655.3-kb:EGFP constructs displayed a GI-specific EGFP expression pattern.

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REexhibited stronger EGFP expression in the intestine at 4 dpf. Using fluo-

rescentmicroscopy,we could detect tissue-specificCYP3A65promoter ac-tivity as early as 2 dpf, which is earlier than the stage at which we coulddetect endogenous CYP3A65 transcription using in situ hybridization.This difference is likely due to the higher sensitivity of fluorescent EGFPdetection compared with conventional RNA hybridization analysis.
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UNCOThe CYP3A65 promoter can be activated by TCDD and an endogenous

AHR ligand through an AHR2-dependent pathway

To elucidate the CYP3A65 transcription mechanism, newly fertilizedTg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) embryos were incubatedwith 1 nM TCDD, and their EGFP expression profiles were assessed byfluorescence microscopy. We found that TCDD exposure did not in-crease EGFP expression in the Tg(CYP3A65S:EGFP) and Tg(CYP3A65S:EGFP) lines before 3 dpf (Figs. 4A, B, G, and H). At 4 and 5 dpf, intenseEGFP expression was detected in both transgenic lines (Figs. 4C–F andI–L). Similar to our previous observations for endogenousCYP3A65 tran-scription, AHR2 knockdown abrogated basal and TCDD-induced EGFPexpression in both lines (Figs. 5G–L and O). It appears that theTg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) lines precisely recapitulatethe AHR2-dependent sensitivity of CYP3A65 to TCDD. It is of note thatEGFP expression in the Tg(CYP3A65S:EGFP) line was also enhanced byan endogenous AHR ligand, kynurenine (Kyn) (Figs. 5M–N), which isan intermediate metabolite generated by tryptophan degradation viatryptophan-2,3-dioxygenase (TDO) (DiNatale et al., 2010; Opitz et al.,2011). In human HepG2 40/6 cells, 100 nM kynurenine or highercould induce significant CYP1A1 transcriptional activity through an


AHR-dependent pathway. This finding further implies that CYP3A65transcription is mediated by an AHR-related signaling pathway.

The AHRE is indispensable for basal and TCDD-induced Tg(CYP3A65:EGFP) transcription

As illustrated in Fig. 2, there are multiple consensus AHR-response el-ements (AHREs) in the CYP3A65 upstream promoter sequence, which arerecognized by the aryl hydrocarbon receptor (AhR)–ARNT complex. Toexamine the significance of these AHRE motifs in CYP3A65 transcription,we mutated the AHREs in the upstream fragment to generate two noveltransgenic lines, Tg(CYP3A65L-mAHRE:EGFP) and Tg(CYP3A65S-mAHRE:EGFP), and assessed their effects on transgenic reporter expression. Wefound that mutating the AHRE motifs in the CYP3A65 promoter dramati-cally decreased constitutive EGFP expression (Figs. 6D–E). Moreover,TCDD exposure was no longer able to induce EGFP transcription(Figs. 6F–G), suggesting that the consensus AHRE motifs play a criticalrole in mediating basal and TCDD-mediated CYP3A65 transcription.

The AHR2 DNA- and ligand-binding domains are required for CYP3A65transcription

Although most nuclear receptors function by transducing the tran-scriptional information of their cognate response element, some nuclearreceptors regulate transcription via direct protein–protein interactions,which are independent of binding to their cognate DNA response ele-ments. For example, AHR functions as a coactivator, and enhances E2F1or ER-alpha target genes at non-AHRE sites independently of binding toDNA (Ohtake et al., 2003; Watabe et al., 2010). To clarify whether AHR2




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Fig. 3. The transgenic lines Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) recapitulate the endogenous CYP3A65 expression profile during the larval stage. (A–E) Lateral views of en-dogenous CYP3A65 expression in wild-type embryo/larvae at 1 dpf (A), 2 dpf (B), 3 dpf (C) 4 dpf (D), and 5 dpf (E) stages. (F–O) Fluorescent EGFP expression in Tg(CYP3A65L:EGFP) (F–J) and Tg(CYP3A65S:EGFP) (K–O) embryos/larvae at 1 dpf (F, K), 2 dpf (G, L), 3 dpf (H, M) 4 dpf (I, N), and 5 dpf (J, O). The presumptive liver and foregut tissues in2–3 dpf transgenic embryos are circled with dots. (P) Lateral view of EGFP expression in Tg(CYP3A65S:EGFP) larvae at 4 dpf.


Umediates CYP3A65 transcription through its DNA binding activity, we cre-ated a truncated form of AHR2 (AHR2dDB) that lacks a functionalDNA-binding domain (Tanguay et al., 1999). Tg(CYP3A65L:EGFP) embryosat the 1-cell stage were co-injected with in vitro-transcribed AHR2dDBcRNA and AHR2 morpholinos, to assess whether AHR2dDB could rescueendogenous CYP3A65 transcription and EGFP expression. We found thatwithout a functional DNA-bindingdomain, AHR2dDBwasunable to effec-tively rescue endogenous CYP3A65 transcription or EGFP expression inAHR2 morphants of this transgenic line (Figs. 7G–H and K). Conversely,wild-type AHR2 cRNA could restore endogenous CYP3A65 transcriptionand EGFP expression to 50–60% of the original level (Figs. 7E–F and K).This finding suggests that AHR2 requires a functional DNA binding do-main to mediate CYP3A65 transcription.


It was previously demonstrated that AHR regulates TGF-β1 expres-sion and cell cycle progression in a ligand-independent manner(Chang et al., 2007).We found that the endogenous CYP3A65 and trans-genic reporter genes could be transcribed without agonist treatmentduring development, which prompted us to examinewhether AHR2 re-quired its ligand to mediate CYP3A65 transcription. A previous studydemonstrated that theHis296 andGln388 residues in AHR2 play criticalligand-binding roles. Substituting these residues modifies the electro-static environment of the ligand-binding pocket, resulting in decreasedligand binding affinity (Bisson et al., 2009). We generated an AHR2cRNA (AHR2mtLB) encoding a mutant protein in which the Gln388andHis296 residues are substitutedwith histidine and tyrosine, respec-tively (i.e., Q388H and H296Y), to abolish its ligand binding affinity. We




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Fig. 4. Transgenic Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) larvae are sensitive to TCDD exposure. (A–F) Fluorescent EGFP expression in control (A, C, and E) and TCDD-treated(B, D, and F) Tg(CYP3A65S:EGFP) embryos/larvae at 3 dpf (A and B), 4 dpf (C and D), and 5 dpf (E and F). (G–L) Fluorescent EGFP expression in control (G, I, and K) andTCDD-treated (H, J, and L) Tg(CYP3A65L:EGFP) embryos/larvae at 3 dpf (G and H), 4 dpf (I and J), and 5 dpf (K and L). TCDD enhanced CYP3A65 promoter-driven EGFP expressionat the 4- and 5-dpf stages, but not at the 3-dpf stage.


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demonstrated that AHR2mtLB cRNA was unable to restore CYP3A65promoter-driven EGFP expression in AHR2 morphants of eithertransgenic line (Figs. 7I–K), suggesting that AHR2 requires endoge-nous ligand binding to fulfill its transcriptional functions.

Clustering of AHRE binding sites in teleost CYP3 genes

Unlike mammalian CYP3A genes, we found that the zebrafishCYP3A65 gene is controlled by the PXR and AHR2 nuclear receptors.The multiple AHRE motifs in the 5′-flanking sequence play criticalroles in AHR2-mediated transcription. This raises the interesting ques-tion of whether multiple AHRE motif copies are conserved in otherCYP3 genes. We collected the upstream 5 kb sequences of CYP3 genesfrom 19 model terrestrial organisms and teleost species (Table 2). Wenoted that 78% of the teleost CYP3 genes contained 3 or more AHRE se-quences in their promoter sequences. Conversely, only 9% of the terres-trial CYP3 genes harbor 3 or more AHRE sequences in their promoters(Table 2). Nevertheless, most CYP1 genes in all species contain multiple(3 or more) AHRE sequences in their promoters. This result suggeststhat AHR/AHR2-mediated CYP3 transcription is a common regulatorymechanism in teleost species.

Discussion

Although both PXR and AHR2 are expressed during early develop-mental stages (Bertrand et al., 2007; Karchner et al., 2005), their down-stream target, CYP3A65, is transcribed only in mature liver andintestinal tissues after hatching. It is likely that PXR and AHR2 require ac-tivation by endogenous ligands inmature liver and intestinal tissues dur-ing larval stages tomediate CYP3A65 transcription.We demonstrate herethat the AHR2 ligand-binding domain is indispensable for driving basalCYP3A65 transcription, suggesting that AHR2 ligand activation is a criticalprocess for transcription. Although the native embryonic PXR and AHR2ligands have not been fully elucidated, recent studies suggest that PXR


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and AHR2may be activated by lipid and amino acid intermediatemetab-olites, respectively. First, it was demonstrated that zebrafish PXR couldbe activated by cyprinol sulfate (Krasowski et al., 2005), which is thedominant biliary emulsifier (Farber et al., 2001) synthesized in theliver, and is released into the intestine to emulsify dietary fats (Russell,2003).Wehave also demonstrated that AHR2 is activated by kynurenine,a metabolite intermediate of tryptophan degradation that activatesmammalian AHR activity. A previous study revealed that zebrafishAHR2 functions as a mammalian AHR in response to TCDD(Andreasen et al., 2002). It is noteworthy that expression of manyof the genes required for cholesterol and tryptophan metabolismcommences from the early stages of development (Anderson etal., 2011; Rauch et al., 2003; Thisse et al., 2001), suggesting that in-termediate metabolites used as native PXR and AHR2 activators areproduced prior to the onset of exogenous feeding to mediateCYP3A65 transcription during larval stages.

CYP3A65 is a PXR-, AHR2-dependent gene that can effectively re-spond to PXR and AHR2 activation. Depletion of either PXR or AHR2eliminates CYP3A65 transcription completely, raising the possibilitythat interactions between these two nuclear factors are necessaryfor CYP3A65 expression. Recent studies have revealed several instanceof crosstalk betweenAHR, PXR and other nuclear receptors or transcrip-tion factors (Pascussi et al., 2008), in which the agonist-activated AHR–ARNT heterodimer directly associates with the estrogen receptorsER-alpha and ER-beta, thereby resulting in the activation of a numberof estrogen-responsive genes in the absence of estrogen (Ohtake et al.,2003).

The presence of multiple AHREs in the promoters of teleost CYP3family members suggests that AHR/AHR2-mediated CYP3 transcriptionis a common regulatorymechanism in teleost species. Itmay also reflectthe different environments that terrestrial and aquatic organisms en-counter. In this study, we established the Tg(CYP3A65L:EGFP) andTg(CYP3A65S:EGFP) transgenic lines, which express EGFP in a mannersimilar to that of endogenous CYP3A65. Therefore, these transgenic




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lines may be of potential use as biomarkers to screen for environmentalcontaminations in vivo that enhance or inhibit CYP3A65 expression viaPXR and AHR2.

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Although we demonstrate here that transcription of zebrafishCYP3A65 can be greatly induced by TCDD or other AHR agonists throughan AHR2-dependent pathway, it remains unclear whether this is

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Fig. 6. AHREs are required for fluorescent GPF expression in Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) larvae. (A) Schematic map of pCYP3A65L-mAHRE:EGFP andpCYP3A65S-mAHRE:EGFP constructs. In both constructs, the consensus AHRE motifs (GCGTG) were mutated to AAATA (indicated by red blocks). (B–G) Fluorescent EGFP expressionin control (B–E) and TCDD-treated (F–G) Tg(CYP3A65L:EGFP) (B), Tg(CYP3A65S:EGFP) (C), Tg(CYP3A65L-mAHRE:EGFP) (D and F), and Tg(CYP3A65S-mAHRE:EGFP) (E and G) 5-dpflarvae. Removing the AHRE motifs from the CYP3A65 promoter abolished its constitutive transcriptional activity and responsiveness to TCDD treatment. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)


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accompanied by a corresponding increase in CYP3A65 protein, or an en-hancement of its enzymatic activity.
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Morpholino and cRNA injection. Wild-type zebrafish were obtainedfrom the Taiwan Zebrafish Core Facility in Taipei. The fish weremaintained and housed as previously described (Westerfield, 2000).All embryos were grown in 0.003% 1-phenyl-2-thiourea after 14 hpostfertilization (hpf) at 28.5 °C to block pigmentation andmediate visu-alization. Morpholinos (Gene Tools, Philomath, Oregon) were designedagainst Danio rerio ahr2 (GenBank™ accession AAF063446) and pxr/nr1i2 (GenBank™ accession DQ69792). The ahr2 translation-blockingmorpholino (ATG-MO) (5′-TGTACCGATACCCGCCGACATGGTT-3′) wasdesigned to target the ahr2 start codon (underlined) as previously de-scribed (Tseng et al., 2005). Likewise, the pxr translation-blockingmorpholino (5′-TCATATAAGCGGGACATTGACGTAC-3′) was designed totarget the pxr start codon (underlined). To block AHR2 or PXR translation,16 ng (1.4 pmol) of ahr2 morpholinos or 24 ng (2.1 pmol) of pxrmorpholinos were injected into 1–2 cell embryos, respectively. A

Fig. 5. EGFP expression in Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) larvae requires AHR2scription (A, D, G, and J) and fluorescent EGFP expression (B–C, E–F, H–I, and K–L) in wild-typlarvae at 4-dpf. Larvae were treated with TCDD and/or injected with ahr2 morpholinos (MOBasal and TCDD-induced CYP3A65 transcription and EGFP expression were eliminated in ahwith 10–nM (M) or 100–nM (N) kynurenine, at 5 dpf. The CYP3A65 promoter is induced by tby the AHR pathway. (O) Quantitative RT-PCR of the CYP3A65 and EGFP genes in control, a


morpholino oligonucleotidewith irrelevant sequenceswas used as a stan-dard control (Gene Tools, LLC, Oregon).

For capped RNA synthesis, ahr2 full-length cDNA was cloned intothe pT7TS vector. After linearization, the plasmid was transcribed invitro using T7 RNA polymerase and the mMESSAGE mMACHINE kitfrom Ambion (Austin, TX). For the rescue assay, 50 pg of purifiedwild-type or mutant ahr2 cRNA was co-injected with ahr2 ATG-MOinto 1–2 cell-stage embryos.

The mutant forms of AHR2, including AHR2ΔDB (internal deletionof amino acid residues 36–76, lacking the DNA-binding domain)(Tanguay et al., 1999) and AHR2mtLB (mutated ligand-binding do-main with H296Y and Q388H amino acid substitutions) (Bisson etal., 2009) were generated by site-directed mutagenesis using overlapextension PCR (Ho et al., 1989).

Whole-mount in situ hybridization. The CYP3A65 (bases 11–2037)(Tseng et al., 2005) template was amplified from cDNA and clonedinto the pGEM-T-easy® vector (Promega). To generate probes, a line-arized plasmid was used as an in vitro transcription template for theDIG RNA labeling kit (Roche Applied Science, Indianapolis, IN, USA)according to the manufacturer's instructions. Whole-mount in situ

, and can be induced by the AHR agonist kynurenine. (A–L) Endogenous CYP3A65 tran-e (A, D, G, J), Tg(CYP3A65S:EGFP) (B, E, H, and K), and Tg(CYP3A65L:EGFP) (C, F, I, and L)): control (A–C), TCDD-treated (D-F), ahr2 MO (G–I), TCDD-treated in ahr2 MO (J–L).

r2 MO larvae. (M–N) Fluorescent EGFP expression in Tg(CYP3A65L:EGFP) larvae treatedreatment with a human AHR agonist, suggesting that CYP3A65 transcription is mediatedhr2 MO, and TCDD-treated Tg(CYP3A65S:EGFP) larvae at 5 dpf.


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Fig. 7. TheAHR2DNA- and ligand-binding domains are required for CYP3A65 transcription. (A-J) Endogenous CYP3A65 transcription (A, C, E, G, and I) andfluorescent EGFP expression (B, D, F, H,and J) in Tg(CYP3A65L:EGFP) larvae at 5 dpf. Embryoswere injectedwith the following: control (no injection) (A and B), ahr2morpholino (MO) (C–J); ahr2MOandwild-type ahr2 cRNA (E andF), ahr2MO and ahr2dDB cRNA (G and H), ahr2MO and ahr2mtLB cRNA (I and J). AHR2 cRNA lacking a functional DNA or ligand-binding domain was unable to restore CYP3A65 expression inahr2morphants, suggesting that CYP3A65 transcription ismediated by ligand-bound AHR2 through a direct protein-DNA interaction. (K) Quantitative RT-PCR of the CYP3A65 and EGFP genes inthe indicated Tg(CYP3A65L:EGFP) larvae at 5 dpf.

Table 1t1:1

t1:2 Primers used to generate 5′-deletions of the CYP3A65 promoter.

t1:3 Forward primers:

t1:4 5.4 kb (Forward) 5′-ccgggGAATTCACCCAGTAACGCCACCGCA-3′t1:5 4.9 kb (Forward) 5′-ccgggGAATTCCCCATCTCTGGAAAATATCC-3′t1:6 2.1 kb (Forward) 5′-ccgggGAATTCTTGGCTTCATCACTCTGTCT-3′t1:7 1.4 kb (Forward) 5′-ccgggGAATTCTTCTGTGTGGAGTTTGCATG-3′t1:8 0.7 kb (Forward) 5′-ccgggGAATTCGAGCAGTGAAAACACACTTC-3′t1:9 Reverse primer: 5′-ccgggGGATCCGTTTCTGCCGAGAAGAACAT-3′

t1:10 The spacer (ccggg) and restriction sites (EcoRI, GAATTC; BamHI, GGATCC) in eacht1:11 primer are italicized and underlined, respectively.



hybridization was performed as previously described (Westerfield,2000). Nitro blue tetrazolium/bromo-4-chloro-3-indolyl phosphate(Roche) was used as a substrate for color development.

Generation of transgenic lines. DNA fragments containing 0.7, 1.4, 2.1,4.9, and 5.3 kb 5′-flanking sequences from the CYP3A65 transcriptionalstart site (Gene ID 553969) were amplified from genomic DNA using aseries of primers (Table 1). After sequence verification, these DNAfragments were cloned into the Tol2 transposon vector, pT2KXIGΔin(Urasaki et al., 2006), to generate the pT2-CYP3A65-0.7 K-EGFP, pT2-


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Table 2t2:1

t2:2 Number of AHREs in the 5.0 kb-flanking sequences of terrestrial and teleost CYP1 and CYP3 promoters.

t2:3 Terrestrial species Terrestrial species

t2:4 Homo sapiens (human) CYP3A4 1 Homo sapiens (human) CYP1A1 14t2:5 CYP3A5 1 CYP1B1 9t2:6 CYP3A7 0 CYP1A2 6t2:7 CYP3A43 2 Pongo pygmaeus abelii (orangutan) CYP1A2 2t2:8 Pan troglodytes (chimpanzee) CYP3A4 1 CYP1B1 0t2:9 CYP3A5 2 Callithrix jacchus (marmoset) CYP1A2 1t2:10 CYP3A7 0 Mus musculus (mouse) CYP1A1 14t2:11 Macaca mulatta (rhesus) CYP3A5 2 CYP1A2 3t2:12 CYP3A7 0 CYP1B1 14t2:13 CYP3A43 3 Rattus norvegicus (rat) CYP1A2 1t2:14 CYP3A64 2 CYP1A1 11t2:15 Callithrix jacchus (marmoset) CYP3A4 2 Cavia porcellus (guinea pig) CYP1A1 5t2:16 CYP3A5 1 CYP1A2 2t2:17 CYP3A90 4 Felis catus (cat) CYP1A1 16t2:18 Mus musculus (mouse) CYP3A11 1 CYP1A2 1t2:19 CYP3A13 0 Canis familiaris (dog) CYP1A2 12t2:20 CYP3A16 0 CYP1B1 10t2:21 CYP3A25 0 Ovis aries (sheep) CYP1A1 6t2:22 CYP3A41a 0 Bos taurus (cow) CYP1A2 1t2:23 CYP3A41b 0 CYP1B1 9t2:24 CYP3A44 0 Gallus gallus (chicken) CYP1A1 14t2:25 CYP3A57 0 CYP1A4 14t2:26 CYP3A59 0 Xenopus tropicalis (frog) CYP1A1 11t2:27 Rattus norvegicus (rat) CYP3A3 0 CYP1C1 2t2:28 CYP3A11 1 CYP1D1 1t2:29 CYP3A13 1 Teleost speciest2:30 Cavia porcellus (guinea pig) CYP3A14 0 Danio rerio (zebrafish) CYP1A1 17t2:31 CYP3A15 1 CYP1B1 3t2:32 CYP3A17 0 CYP1C1 3t2:33 Oryctolagus cuniculus (rabbit) CYP3A6 2 CYP1C2 0t2:34 Canis familiaris (dog) CYP3A12 2 CYP1D1 6t2:35 CYP3A26 3t2:36 Equus caballus (horse) CYP3A89 1t2:37 CYP3A93 2t2:38 CYP3A94 1t2:39 CYP3A95 1t2:40 CYP3A96 2t2:41 CYP3A97 1t2:42 Ovis aries (sheep) CYP3A24 0t2:43 Bos taurus (cow) CYP3A5 1t2:44 CYP3A4 1t2:45 CYP3A5 0t2:46 Gallus gallus (chicken) CYP3A7 2t2:47 Xenopus tropicalis (frog) CYP3A4 4t2:48 CYP3A5 1t2:49 Teleost speciest2:50 Danio rerio (zebrafish) CYP3A65 6t2:51 CYP3C1 1t2:52 CYP3C112 3t2:53 CYP3C4 1t2:54 Tetraodon nigroviridis (tetraodon) CYP3A48 3t2:55 CYP3B1 6t2:56 CYP3B2 5t2:57 Takifugu rubripes (fugu) CYP3A48 1t2:58 CYP3B1 4t2:59 CYP3B2 8t2:60 CYP3D1 11t2:61 Gasterosteus aculeatus (stickleback) CYP3A48 3t2:62 CYP3A117 3t2:63 CYP3A118 3t2:64 CYP3A119 3t2:65 CYP3B7 3t2:66 CYP3D1 3t2:67 Oryzias latipes (medaka) CYP3A38 3t2:68 CYP3A40 1t2:69 CYP3B3 3t2:70 CYP3B4 6t2:71 CYP3B5 1t2:72 CYP3B6 6

t2:73 Promoters containing three or more AHRE-binding sites are highlighted.


CYP3A65-1.4 K-EGFP, pT2-CYP3A65-2.1 K-EGFP, pT2-CYP3A65-4.9 K-EGFP and pT2-CYP3A65-5.3 K-EGFP plasmids. To observe the transientexpression activity of these constructs, 25 ng of the plasmids were


co-injected with 25 ng of capped Tol2 transposase mRNA into 1-cell em-bryos, and EGFPfluorescencewas examined at 96 hpf (Kawakami, 2005).The pCS-TP plasmid encoding the transposase (Kawakami et al., 2004)




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was linearized and used as a template for in vitro transcription with themMessage mMachine kit (Ambion, Foster City, CA, USA), in accordancewith the manufacturer's instructions.

To generate stable transgenic lines, multiple pT2-CYP3A65-2.1 K-EGFP and pT2-CYP3A65-5.3 K-EGFP-injected larvae displayingtransient expression in the liver and intestinal tissues were raised toadulthood (F0) and crossed with wild-type fish. F1 embryos exhibitingEGFP fluorescencewere raised to adulthood and re-screened to establishTg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP) stable transgenic lines. Togenerate mutant constructs, the consensus AHRE motifs (GCGTG) inpT2-CYP3A65-2.1 K-EGFP and pT2-CYP3A65-5.3 K-EGFP were changedto AAATA by site-directed mutagenesis using overlap extension PCR(Ho et al., 1989). The verified constructs were used to generate theTg(CYP3A65S-mAHRE:EGFP) and Tg(CYP3A65L-mAHRE:EGFP) stable lines.

RT-qPCR. Total RNA was extracted from embryos using TRIzol(Invitrogen), treated with DNase I (Roche), and then reverse transcribedwith Superscript-II (Invitrogen). Quantitative (q)PCR was performedusing the Power SYBR Q-PCR Master Mix (BioNoVas) in a Bio-Rad iQ5Gradient Real-Time PCR System (Bio-Rad). qPCR conditions were:95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for1 min. The following primers were designed using Primer Express Soft-ware, version 3.0 (Applied Biosystems, Carlsbad, California, U.S.A.):

CYP3A65 forward, 5′-CTTCGGCACCATGCTGAGAT-3′;CYP3A65 reverse, 5′-AGATACCCCAGATCCGTCCATA-3′;egfp forward, 5′-CAACAGCCACAACGTCTATATCAT-3′;egfp reverse, 5′-ATGTTGTGGCGGATCTTGAAG-3′;beta-actin forward, 5′-CCCCGAGAGGACAACAATGTA-3′;beta-actin reverse, 5′-TGAGGAGGGCAAAGTGGTAAA-3′.

Duplicate mean values were calculated according to the Ct quanti-fication method using beta-actin transcript levels as a reference fornormalization. Relative quantification was determined using theΔΔCt method, where relative expression = 2−ΔΔCt.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.taap.2013.04.010.

Conflict of interest

The authors have no conflicts of interest to declare.

Acknowledgments

This work was supported by grants from the National Science Councilof Taiwan (NSC96-2312-B-019-001-MY2, NSC97-2627-B-019-001, andNSC98-2627-B-019-001), the Ministry of Education, and the Center forthe Excellence of Marine Bioenvironmental and Biotechnology (NTOU),which were awarded to C.H.H., and the National Science Council ofTaiwan (NSC96-2113-M-019-002-MY2), which was awarded to W.S.T.

We thank S. P. L. Hwang (Taiwan Zebrafish Core Facility at Taipei(ZCAS), NSC100-2321-B-001-030) and M. S. Yu (Taiwan ZebrafishCore Facility at Zhunan (ZeTH), NSC 100-2321-B-400-003) for pro-viding zebrafish.

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Regulation of zebrafish CYP3A65 transcription by AHR2