ToxSci Advance Access originally published online on February 27, 2008
Toxicological Sciences 2008 103(1):158-168; doi:10.1093/toxsci/kfn035
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Influence of TCDD on Zebrafish CYP1B1 Transcription during Development
,1
,1
* Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan ROC
Department of Medical Technology, Chung Hwa University of Medical Technology, Tainan, Taiwan ROC
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon
Center for Marine Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan ROC
1 To whom correspondence should be addressed at National Taiwan Ocean University—Bioscience & Technology, 2, Pei-Ning Road Keelung 202-24, Taiwan, ROC. Fax: 886224622320. E-mail: chhu{at}mail.ntou.edu.tw. Correspondence may also be addressed to Donald R. Buhler, Department of Environmental and Molecular Toxicology, Oregon State College, 1143 ALS, Corvallis, OR 97331. Fax: 737-1784. E-mail: Donald.Buhler{at}oregonstate.edu.
Received September 29, 2007; accepted February 6, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Cytochrome P450 1B1 (CYP1B1) is a heme-containing monooxygenase that metabolizes various polycyclic aromatic hydrocarbons and aryl amines, as well as retinoic acid and steroid hormones. Here we report the cloning of an ortholog of CYP1B1 from zebrafish and the demonstration that transcription of zebrafish CYP1B1 was modulated by two types of mechanisms during different developmental stage. First in late pharyngula stage before hatching, CYP1B1 was constitutively transcribed in retina, midbrain–hindbrain boundary and diencephalon regions through a close coordination between aryl hydrocarbon receptor 2 (AHR2)–dependent and AHR2-independent pathways. After hatching, the basal transcription was attenuated and it could not be elicited upon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. In contrast, TCDD exposure induced de novo CYP1B1 transcription in larval branchial arches and heart tissues via an AHR2-dependent pathway. Blocking AHR2 translation completely eliminated the TCDD-mediated CYP1B1 transcription. However, we did not detect any types of CYP1B1 transcription in liver and kidney tissues through the developmental stage. It suggests that the constitutive and TCDD-inducible types of CYP1B1 transcriptions are modulated by distinct pathways with different tissue specificities. Finally, we investigated the role of CYP1B1 in TCDD-mediated embryonic toxicity. Because knockdown of CYP1B1 did not prevent TCDD-induced pericardial edema and cranial defects, it suggests that CYP1B1 is not involved in the developmental toxicity of dioxin.
Key Words: CYP1B1; transcription; zebrafish; embryo; larva; AHR2; TCDD.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Cytochrome P450 (CYP) is a superfamily of heme-containing monooxygenases that catalyze the oxidative and reductive metabolism of many drugs, environmental chemicals, and endogenous compounds (Guengerich et al., 2003
). On the basis of sequence similarity, the CYPs can be classified into various families and subfamilies (Nelson et al., 1996). In mammals, the CYP1 family consists with several closely related members, including CYP1A1, CYP1A2, and CYP1B1 (Nelson, 1999; Nelson et al., 1996). CYP1A1 is the first identified CYP1 member, which metabolizes important polycyclic aromatic hydrocarbon (PAH) carcinogens such as benzo[a]pyrene and benzo[a]anthracene. CYP1A2 is responsible for the metabolizing caffeine and carcinogens such as arylamines and aflatoxins. CYP1B1 was first purified from mouse (Pottenger et al., 1991). Subsequently, the CYP1B1 gene was cloned from mouse, rat and human species (Bhattacharyya et al., 1995; Savas et al., 1994; Sutter et al., 1994). CYP1B1 metabolizes several PAHs and arylamines, retinoic acid, as well as estradiol-17β (Bowes et al., 1996; Chen et al., 2000; Lee et al., 2003; Otto et al., 1991, 1992; Shimada et al., 1996). A recent study demonstrated that CYP1B1 was also involved in retinoic acid synthesis through a RALDH (retinal dehydrogenase)-independent pathway and directed RA-mediated patterning during developmental stage (Chambers et al., 2007). In addition to the mammalian species, orthologous members of CYP1A, CYP1B, and additional closely related CYP1C genes have been characterized in various teleost species (Godard et al., 2005; Itakura et al., 2005; Jonsson et al., 2007b).
In mammals, the transcriptional activities of CYP1B1 genes are controlled by distinct mechanisms in various tissues. A constitutive type of CYP1B1 transcription was found in human fetal thymus and kidney and adult extrahepatic tissues such as lung, kidney, heart, spleen, thymus, prostate, and endocrine-regulated tissues including breast, uterus, ovary and testis (Choudhary et al., 2005; Hakkola et al., 1997; Shimada et al., 1996). A similar type of transcription was also seen in murine fetal eye, hindbrain, branchial arches, forelimb bud, and kidney (Stoilov et al., 2004). After weaning, the CYP1B1 was expressed in adrenocortical cells (Brake et al., 1999). A number of transcription factors, including Sp1, cyclic adenosine monophosphate–response element–binding protein (CREB), and estrogen receptor, have critical roles in these AHR-independent CYP1B1 transcription (Beischlag and Perdew, 2005; Sissung et al., 2006; Tsuchiya et al., 2003; Zheng et al., 2003). In addition, epigenetic regulation, post-transcriptional modifications, and degradation pathways of CYP1B1 gene have also been explored (Tokizane et al., 2005; Widschwendter et al., 2004). However, the regulatory mechanism of these basal transcriptions still needs to be characterized.
In addition to the constitutive type of transcription, CYP1B1 gene can be induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons as well as polycyclic aromatic hydrocarbons (PAHs) (Alexander et al., 1997) through activation of a cellular aryl hydrocarbon receptor (AHR) signaling pathway (Zhang et al., 1998, 2003). In addition to normal tissues, CYP1B1 is also expressed in a wide range of human cancers in an AHR-independent manner (Murray et al., 1997).
In teleost species, two types of CYP1B genes have been characterized in carp, in which CYP1B1 is constitutively expressed in adult gills and its transcription can be induced by 3-methylcholanthene (3-MC) in liver, intestine and gills. In comparison, CYP1B2 is not expressed in normal carp tissues, but it can be induced in gills by 3-MC treatment (El-kady et al., 2004a, b). In channel catfish, CYP1B can be induced by conventional AHR ligands in blood, liver, gonad, and gill (Willett et al., 2006). In zebrafish, the CYP1 family genes exhibit two types of transcription. In adult fish, the CYP1A is expressed in liver, whereas the CYP1B1, CYP1C1, and CYP1C2 are all transcribed in heart and eye (Jonsson et al., 2007b). During developmental stages, the maximal level of basal CYP1B1 and CYP1C transcriptions appears within 2–3 dpf embryos, whereas the CYP1A transcription increases significantly after hatching (Jonsson et al., 2007a). In addition to the basal type of transcription, 3,3'4,4'5-pentachlorobiphenyl exposure induced CYP1 genes through an AHR2-dependent pathway in a wide ranges of adult tissues and embryos and caused a number of embryo toxic effects (Dong et al., 2004; Jonsson et al., 2007a; Prasch et al., 2003). Morpholino oligonucleotide (MO) knockdown of zebrafish AHR2 prevented CYP1A1 induction and protected against embryotoxicity (Dong et al., 2004; Prasch et al., 2003). Nevertheless, the AHR2-MO had no effect on the basal level of CYP1A transcription in normal embryos, it suggests that AHR2 may not be involved in controlling constitutive type of CYP1A message (Prasch et al., 2003). Up till now, the spatial and temporal expression pattern of zebrafish CYP1B1 gene during developing stages has not been studied.
In addition to AHR2, a recent study revealed another AHR protein, AHR1B, in zebrafish that could respond to TCDD effectively and activate target genes under the control of AHR response element (Karchner et al., 2005).
In this study we cloned a full-length CYP1B-like complimentary DNA (cDNA) from zebrafish by reverse transcription–PCR (RT-PCR), which was named as CYP1B1 by the cytochrome P450 Nomenclature Committee. The spatial/temporal expression pattern and induction of this gene in the developing embryo was determined by in situ RNA hybridization. Blocking AHR2-related signaling pathway eliminated the induction of CYP1B1 transcription by TCDD, but did not diminish the constitutive type of CYP1B1 transcription. This suggests that the zebrafish CYP1B1 gene is modulated by the close coordination between AHR2-dependent and AHR2-independent pathways. Blocking CYP1B1 translation did not prevent the TCDD-induced pericardial edema and cranial defects, suggesting that CYP1B1 was not involved in the developmental toxicity of dioxin.
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MATERIALS AND METHODS |
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Zebrafish embryos and xenobiotics exposure.
Wild-type (AB strain) zebrafish embryos were maintained at 28.5°C and were staged by hours postfertilization (hpf) (Kimmel et al., 1995
). For dioxin treatment, the newly fertilized embryos were continuously incubated at 28.5°C with 1nM waterborne TCDD. The TCDD of > 99% purity from Chem Service (West Chester, PA) was dissolved in dimethyl sulfate so that final solvent concentration was 0.003% for TCDD treatment.
Rapid amplification of the cDNA ends–PCR for the isolation of CYP1B1 gene.
To obtain the full-length sequence of zebrafish CYP1B1 cDNA, rapid amplification of the 5' and 3' cDNA ends (RACE) method was performed using a SMART RACE cDNA Amplification Kit (Clontech, CA) (Chenchik et al., 1998; Zhu et al., 2001). The gene-specific primers (GSP1: 5'-GGTTGATCCCATTTTGTTGGGTCGTGATTC-3', GSP2: 5'-CTCTGTCTACAGCTCTCCAGTGGATCATCC-3') and nested primers (NGSP1: 5'-TGGGTCGTGATTCAAAGACCATTGGTTGAC-3', NGSP2: 5'-TGGATCATCCTGCTACTTGTCAGGTACCCAG-3') were synthesized according to the sequence of zebrafish expressed sequence tag (EST) clone (GenBank accession number AF235139), which is similar to the sequence of mammalian CYP1B1. After sequencing the RACE products, a pair of gene-specific primers was designed based on the extreme end sequences of the cDNA and used to amplify the full-length cDNA. The amplified cDNA fragment was sequenced to confirm the nucleotide sequence (GenBank Accession number AY727864).
In situ hybridization.
Whole-mount in situ hybridization was performed as described (Westerfield, 2000). For the CYP1B1 probe, a digoxigenin (DIG)–labeled complimentary RNA fragment containing the complete open reading frame sequence of CYP1B1 gene was synthesized with the DIG RNA Labeling Kit (Sp6/T7) from Roche (IL). Hybridization was detected by anti-DIG antibody coupled to alkaline phosphatase.
MO and blocking gene expression.
The ahr2 and CYP1B1 antisense MOs, obtained from Gene Tools (Corvallis, OR), were designed to target the 5'-untranslated region (UTR) across the AUG start codon of zebrafish AHR2 (GenBank AAF063446) (5'-TGTACCGATACCCGCCGACATGGTT-3') (Wang et al., 2004) and CYP1B1 (5'-CAGAGCCAGCAGGACATCCATCATG-3') cDNA, respectively. Prior to injection, the morpholino was diluted to 0.3mM in 1X Danieau's solution, as described by Nasevicius and Ekker (2000). To block the protein translation, 12 ng (1.4 pmol in 4.6 nl) of gene-specific MOs was injected into more than 30 fertilized egg at the one-cell stage.
To confirm of the morpholino specificity, recombinant reporter plasmids containing AHR- and CYP1B1-EGFP fusion protein genes were constructed. For the AHR2-EGFP, a 219-bp DNA fragment corresponding to the 5'-UTR and partial coding sequence (nucleotides –169 to +50, including the MO target regions) of the zebrafish AHR2 messenger RNAs (mRNAs) was generated by PCR (forward primer, 5'-ccggggaattcCTGCCACTGACAAGAATTAC-3'; reverse primer, 5'-ccgggaccggtACGGGCTTCTTCCGTTTCTT-3'; the linker sequences are in lowercase type). For the CYP1B1-EGFP, a 563-bp DNA fragment corresponding to the 5'-UTR and partial coding sequence (nucleotides –194 to +369, including the MO target regions) of the zebrafish CYP1B1 mRNAs was generated by PCR (forward primer, 5'-ccggggaattcACGCGGGGAGATAAATTCTTG-3'; reverse primer, 5'-ccgggaccggtGGTGTAGTTACCGAAAGCCAT-3'). The amplified DNA segments were subcloned in frame into pcDNA3-EGFP (kindly provided by Dr Chung BC at Academia Sinica) using the EcoRI and AgeI sites to fuse the AHR2 and CYP1B1 partial sequence N-terminally of EGFP to produce the pCMV-AHR2:EGFP and pCMV-CYP1B1:EGFP reporter constructs, respectively. The orientation and accuracy of sequence were verified by DNA sequencing. One hundred and fifty picograms of recombinant reporter plasmid was injected alone or coinjected with 12 ng of gene-specific MOs into the fertilized egg at the 1-cell stage. The fluorescence of recombinant EGFP protein was assessed at 14 and 96 hpf stages.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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CYP1B1 Cloning
A computer homology search for genes similar to CYP1B have identified a zebrafish EST clone (GenBank accession number AF235139), which is similar to the sequence of human CYP1B1 cDNA (GenBank accession number NM_000104, with 66% identity) and mouse CYP1B1 cDNA (GenBank accession number NM_009994, with 64% identity). The 5'- and 3'-end of this CYP1B-like mRNA were obtained by RACE-PCR approach. A full-length cDNA fragment was obtained by RT-PCR using the 5'- and 3'-end primers and its nucleotide sequence was determined without ambiguity on both strands by primer walking. The complete 3267 bp cDNA (GenBank accession number AY727864) contained an open reading frame of 1581 bp encoding for a 526-amino acid protein with a single ATG initiation cordon at nt 195 and TGA termination signal at nt 1773. Two polyadenylation signals, AATAAA, are located at nucleotides 2552–2557 and 3163–3168, respectively. It is interesting that the human CYP1B1 mRNA also contains multiple polyadenylation sites at its 3'-UTR (Sutter et al., 1994
). On the basis of its nucleotide and amino acid sequence, the identified cDNA was assigned as CYP1B1 by the Cytochrome P450 Nomenclature Committee (http://drnelson.utmem.edu/CytochromeP450.html).
The encoded amino acid sequence of CYP1B1 shared high degree of sequence identity with other teleost CYP1B isoforms, such as 84% identity to carp CYP1B2 (AAR87722
[GenBank]
) and carp CYP1B1 (BAB39160
[GenBank]
), 72% identity to catfish (AAY90143
[GenBank]
), 70% identity to Japanese eel CYP1B (AAR99332
[GenBank]
), and 64% identity to plaice CYP1B (AB51367). It also shares 57% identity with human CYP1B1 (NP_000095
[GenBank]
) and 55% identity with mouse CYP1B1 (NP_034124
[GenBank]
) (Fig. 1). The putative substrate recognition sites (SRS) of CYP1B1 were predicted according to the mode of human CYP1B1 (Fig. 1) (Lewis et al., 2003). Within the six putative SRS regions, zebrafish CYP1B1 shared highly conserved residues with other teleost CYP1B1 proteins at SRS1 (residues 103–124), SRS2 (residues 209–221), SRS4 (residues 303–316), SRS5 (residues 373–382), and SRS6 (residues 482–491). However, the sequence of SRS1, 2, and 6 in teleost CYP1B1 are distinct from human and mouse CYP1B1 and the sequence of SRS4 and 5 are common in teleost and mammalian CYP1B1 proteins. It should be noted that the sequence of putative SRS3 (residues 244–248) (Fig. 1) is quite divergent in all CYP1B1 proteins that we compared here.
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Spatial Expression Pattern of CYP1B1 in Fish Embryo
The spatial and temporal expression profile of zebrafish CYP1B1 gene in the developing embryos was examined by whole-mount RNA in situ hybridization analysis (Figs. 2A–L). It appeared that initially the zebrafish CYP1B1 gene was expressed specifically in ocular cells at the 24 hpf stage (Figs. 2A and 2G). After that, the basal CYP1B1 transcription in the ocular cells reached the highest level between the 30 and 48 hpf stage. In addition to the ocular cells, the CYP1B1 mRNA was also detectable in the diencephalon and midbrain–hindbrain boundary (MHB) regions (Figs. 2B, 2C, 2H, and 2I). After hatching, the constitutive type of CYP1B1 transcription in the ocular cells gradually attenuated and its mRNA was negligible after 72 hpf (Figs. 2D–F and 2J–L). Comparing with mouse CYP1B1 gene, zebrafish CYP1B1 did not express in branchial arches, fin bud (limb) and kidney during developmental stage. Nevertheless, the zebrafish, human and murine CYP1B1 genes all displayed a transcriptional activity in embryonic retina.
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TCDD-Elicited De Novo CYP1B1 Transcription
In mammals, CYP1B1 gene was activated by TCDD exposure via AHR::ARNT signaling pathway (Kerzee and Ramos, 2001; Pitt et al., 2001; Tsuchiya et al., 2003
; Vidal et al., 2005). However, it is unclear whether TCDD affects zebrafish CYP1B1 transcription during early development. Here we presented that the constitutive expression of CYP1B1 in the eye and diencephalon was not further enhanced by TCDD at 48 hpf (Figs. 2M–O and 2S–U). Instead, starting at the 48 hpf stage, a novel CYP1B1 transcription was induced in the branchial arch region by TCDD (Fig. 2U). Start from 60 hpf, the TCDD-mediated de novo CYP1B1 transcription was also induced in heart (Figs. 2V–X). We did not detect any signal by using sense-strand probe (Figs. 2Y, 2Z). In contrast to the mouse CYP1B1 gene, which was constitutively transcribed in fetal branchial arches, here we showed that activation of AHR2 by TCDD is required for zebrafish CYP1B1 transcription in larval branchial arches. We did not detect CYP1B1 transcription in zebrafish liver and nephric tissues during developing stages even after treating with TCDD. It is worth noting that TCDD did not induce CYP1B1 transcription in liver of mice or rats (Buters et al., 1999; Dalton et al., 2000; Ryu et al., 1996; Savas et al., 1994; Walker et al., 1999).
CYP1B1 Transcription is Modulated by both of AHR2-Dependent and AHR2-Independent Pathways
Because the constitutive-type and TCDD-elicited zebrafish CYP1B1 mRNAs were transcribed in different tissues, it suggests that this transcription was regulated by different pathways. We used antisense MOs against AHR2 mRNAs to suppress its protein translation and examined the effect on CYP1B1 transcription. The effectiveness and duration of AHR2 morpholino was first assessed by the fluorescence of recombinant AHR2-EGFP reporter (Fig. 3). It showed that the fluorescence of AHR2-EGFP reporter started to appear at 14 hpf stage and it could be extended up to 96 hpf (Figs. 3A, 3E). The AHR2 morpholino almost completely eliminated the fluorescence of AHR2-EGFP protein between 14 and 96 hpf stage (Figs. 3C, 3G). To confirm the efficiency of AHR2 morpholino, we have examined the CYP1A1 expression in AHR2 morpholino-injected embryos. It appeared that TCDD treatment induced strong CYP1A1 transcription in larval blood vessels, liver and intestine (Fig. 3J). However, the AHR2 morpholino completely eliminated the TCDD-mediated CYP1A1 induction (Fig. 3L).
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In Figure 4, we presented that the constitutive transcription of CYP1B1 in the eye and diencephalon was almost completely eliminated by AHR2 morpholino (Figs. 4C, 4G). TCDD treatment only modestly enhanced the residual transcriptional activity (Figs. 4D, 4H). It suggests that the AHR2 was the major factor that coordinated with AHR2-independent pathway to modulate the maximal CYP1B1 transcription. Because TCDD did not further enhance the constitutive type of CYP1B1 expression before hatching (Figs. 2O, 2U and Figs. 4B, 4F), it is likely that the AHR2 was maximally activated by endogenous developing process.
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After hatching, our results presented that AHR2 morpholino completely blocked the TCDD-induced CYP1B1 transcription in branchial arches and heart tissues at 96 and 120 hpf stage (Figs. 4L, 4P, 4T, 4X). It suggests that the TCDD-mediated CYP1B1 induction in branchial arches and heart tissues was exclusively modulated by AHR2-dependent pathway (Figs. 2P–R, 2V–X and Figs. 4J, N, R, V). Differing from the early stage of development, external activation of AHR2 by TCDD is required to drive CYP1B1 transcription after hatching (Figs. 2D–F, 2J–L and Figs. 4I, M, Q, U).
CYP1B1 Knockdown did not Eliminate the Developmental Toxicity of TCDD
It has been shown that in zebrafish embryos TCDD causes a number of toxic responses, including pericardial edema, slowed blood flow, craniofacial malformation, and defects in erythropoiesis through an AHR/ARNT-dependent pathway (Carney et al., 2006b; Prasch et al., 2003). Because TCDD can induce de novo CYP1B1 transcription in branchial arches and heart tissues, here we investigated the role of CYP1B1 in TCDD-mediated developmental toxicity by MO knockdown assay. The effectiveness and duration of CYP1B1 morpholino was first assessed by the fluorescence of recombinant CYP1B1-EGFP reporter protein (Figs. 5A–H). It appeared that the CYP1B1-EGFP protein could be detected between 14 and 96 hpf stage (Figs. 5A, 5E). The CYP1B1 morpholino almost completely eliminated the CYP1B1-EGFP expression between 14 and 96 hpf stage (Figs. 5C, G). It suggests that the CYP1B1 morpholino has high efficiency and high specificity to block CYP1B1 translation up to 96 hpf.
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Next, we examined the effect of CYP1B1 knockdown on TCDD-mediated embryonic toxicity. Figures 5I–L presented that although the CYP1B1 morpholino effectively blocked CYP1B1-GFP expression, it did not eliminate the TCDD-induced pericardial edema (Figs. 5K, L). On the other hand, we did not detect any phenotype in CYP1B1 morphants (Fig. 5J).
Previously it was found that TCDD affected zebrafish craniofacial chondrogenesis (Teraoka et al., 2002). TCDD shortened the length of Meckel's cartilage and caused a slightly wider angle against the ethmoid plate of the upper jaw. Also, the ceratobranchial cartilage lost their angles with the opposite ones in later stages (Figs. 5Q, R) (Teraoka et al., 2002). Here we presented that blocking CYP1B1 translation did not reduced these toxic effects in lower jaw retardation. (Figs. 5S, T). On the other hand, the cartilage development in CYP1B1 morphants seems normal (Figs. 5O, P). Accordingly, our results suggests that the embryonic toxicity of TCDD is not mediated by de novo synthesized CYP1B1.
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DISCUSSION |
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Sequence Alignment of Zebrafish CYP1B1
CYP1B1 is a highly conserved heme-thiolate monooxygenase that metabolizes a number of procarcinogens including PAHs and aryl amines (Shimada et al., 1996
). In addition, human CYP1B1 hydroxylates the endogenous substrate estrogen and retinoic acid (Chen et al., 2000; Lee et al., 2003). Recent studies revealed that CYP1B1 also plays important role in certain human cancer development (Sissung et al., 2006). In this study we cloned an ortholog of CYP1B1 from zebrafish, which encoded a protein sequence sharing 64–84% identity with other teleost CYP1B1s. This suggests that the sequence of CYP1B1 is highly conserved in vertebrate species. Nevertheless, most of putative SRS in teleost CYP1B1 proteins are different from those of mammalian CYP1B1 sequences, suggesting that teleost and mammalian CYP1B1 proteins may have distinct substrate selectivity to meet their specific physiological functions. This hypothesis is supported by previous phylogenetic analysis, which suggested that mammalian and teleost CYP1B evolved divergently from each other (Godard et al., 2005). However, it is worth noting that the six residues in the active site of human CYP1B1 that orient the steroidal substrate estradiol for 4-hydroxylation, including Ser127, Ala133, Phe134, Phe231, Tyr 507, and Thr510 (Lewis et al., 1999), are all conserved in teleost CYP1B1. This suggests that the enzymatic activity of estrogen hydroxylation is likely to be conserved in vertebrate CYP1B1. However, the enzymatic activity of teleost CYP1B1 still remains to be established.
Recently, a tertiary structure of human CYP1A2 has been identified with a highly conserved canonical P450 fold structure. Surrounded by multiple 
-helices and β-sheet structures, a compact, closed active site cavity is highly adapted for the positioning and oxidation of relatively large, planar substrates (Sansen et al., 2007). Sequence alignment revealed that 16 of 22 residues lining the active site cavity in the CYP1A2 structure are conserved in human and zebrafish CYP1B1 (Table 1). It suggests that the planar active site in CYP1A2 is likely to be conserved in CYP1B1 to adapt for the oxidation of polycyclic aromatic compounds (Sansen et al., 2007). We noted that within the six putative SRS, the SRS2 and 3 are the least conserved regions between CYP1A2 and CYP1B1 and the sequence of SRS3 is quite divergent in all CYP1B1 proteins. It is likely that the SRS2 and 3 do not contact the nonpolar and planar substrates directly.
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Two Types of CYP1B1 Transcription Controlled by Distinct Pathways
In this study we presented evidence that the transcriptional activity of zebrafish CYP1B1 is controlled by distinct regulatory mechanism in different stages of development. First during late pharylgula stages before hatching, CYP1B1 is transcribed constitutively in ocular cells, MHB and telencephalon regions through a close coordination between AHR2-dependent and AHR2-independent pathway. TCDD did not enhance this basal transcription significantly. After hatching, this constitutive type of transcription is attenuated. Instead, a novel AHR2-dependent CYP1B1 transcription can be induced by TCDD in branchial arches and heart tissues during zebrafish larval stages.
Constitutive CYP1B1 transcription before hatching.
Although both of zebrafish and mouse embryos exhibit constitutive type of CYP1B1 transcription, these two species display quite different tissue selectivity in their CYP1B1 transcription. As revealed by loss-of-function assay, we found that the constitutive type of CYP1B1 transcription was almost completely eliminated by AHR2 knockdown before hatching. Although TCDD treatment did not enhance the constitutive CYP1B1 transcription significantly, it did enhance the residual transcriptional activity modestly. It suggests that the AHR2 was the major factor that coordinated with an AHR2-independent pathway to modulate the maximal CYP1B1 transcription. It seems likely that the AHR2 was maximally activated by endogenous developmental processes. Recently, a novel AHR factor, AHR1B, was found in zebrafish, which could respond to external TCDD and activate target genes through the XRE sequence (Karchner et al., 2005). We cannot exclude the possibility that the TCDD-responsive, AHR2-independent pathway involved in constitutive CYP1B1 transcription is in fact the AHR1B-related pathway.
Because the constitutive type of CYP1B1 transcription in the eye and brain tissues attenuated and it was no longer transcribed even exposed to TCDD after hatching, we anticipate that the accessibility of CYP1B1 promoter by regulatory factors was blocked in ocular cells and brain tissues after hatching due to changes in chromatin architecture.
Inducible transcription of CYP1B1 via AHR2-dependent pathway after hatching.
Although both of zebrafish CYP1A1 and CYP1B1 genes can be induced by TCDD treatment during larva stages through the AHR-ARNT signaling pathway, these two genes exhibit quite different expression patterns in TCDD-treated larva. Here we showed that TCDD exposure induced a novel CYP1B1 transcription in branchial arches and heart. In contrast, TCDD-induced strong CYP1A1 transcription in a broad range of tissues, including blood vessels, liver, skin, pronephros, brain, integument tissues, and fins (Andreasen et al., 2002; Wang et al., 2004). Nevertheless, knockdown of AHR2 substantially eliminated both of TCDD-induced CYP1A1 (Wang et al., 2004) and CYP1B1 transcriptions. It suggests that although AHR2 and ARNT factors are present ubiquitously in larval tissues, the CYP1B1 promoter in most tissues is nonpermissible for the AHR2-ARNT complex binding. It seems very likely that additional tissue-specific factors are required to coordinate with the liganded-AHR2 complex to initiate the CYP1B1 expression in branchial arches and heart tissues.
Developmental Toxicity of TCDD is not Mediated by CYP1B1
TCDD exposure causes aberrant zebrafish larval development, including edema, anemia, hemorrhage, and ischemia through AHR2/ARNT1 pathway (Billiard et al., 2006; Carney et al., 2004, 2006a; Prasch et al., 2006). However, knockdown the downstream target CYP1A1 gene did not prevent the signs of TCDD-induced developmental toxicity, such as pericardial edema, slowed blood flow, craniofacial malformation, and defects in erythropoiesis, suggesting that the increased CYP1A1 expression is not the culprit in TCDD-mediated developmental toxicity (Carney et al., 2004). Our results show that although CYP1B1 responded to TCDD exposure with increased transcription in larval branchial arches and heart tissues through the AHR2/ARNT1 pathway, knockdown of CYP1B1 expression did not suppress the TCDD-induced pericardial edema and craniofacial malformation. It indicates that CYP1B1 does not mediate the developmental toxicity of TCDD in zebrafish larvae. There must be some other targets of the AHR2/ARNT pathway, which mediate the signs of developmental toxicity after TCDD exposure (Handley-Goldstone et al., 2005). In addition, blocking CYP1B1 translation did not give any observable phenotypes, it suggests that CYP1B1 does not have critical role in fish development. Similar conclusion was also obtained in CYP1B1 knockout mice that lacking CYP1B1 has no phenotype, indicating that CYP1B1 is not critical for mouse development (Buters et al., 1999; Gonzalez and Kimura, 2003). However, the CYP1B1 null mice have altered responses to the toxic and carcinogenic effects of chemicals as compared with wild-type mice (Buters et al., 2003).
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FUNDING |
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National Science Council grants (Taiwan, ROC) (91-2313-B-019-033, 92-2313-B-019-031, and 93-2313-B-019-005) to C.H.H.; Academia Sinica (0240022381) to C.H.H.; and National Institutes of Health (ES00210 and ES11587) to D.R.B.
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REFERENCES |
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