ToxSci Advance Access originally published online on April 2, 2008
Toxicological Sciences 2008 104(1):124-134; doi:10.1093/toxsci/kfn066
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An Aryl Hydrocarbon Receptor Repressor from Xenopus laevis: Function, Expression, and Role in Dioxin Responsiveness during Frog Development
Biology Department, Kenyon College, Gambier, Ohio 43022
1 To whom correspondence should be addressed at Biology Department, Kenyon College, 302A College Park St., Fischman Wing 202, Gambier, OH 43022. Fax: (740) 427-5741. E-mail: powellw{at}kenyon.edu.
Received February 15, 2008; accepted March 14, 2008
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Xenopus laevis and other frogs are extremely insensitive to the toxicity of xenobiotic ligands of the aryl hydrocarbon receptor (AHR), including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Premetamorphic life stages are especially insensitive, and they are reported to be refractory to induction of Cytochrome P4501As, which are readily induced in older animals. The AHR repressor (AHRR) is a member of the AHR gene family. AHRR expression is induced by TCDD; it then represses AHR in an apparent negative feedback loop. In this study, we sought to test the hypothesis that constitutive AHRR expression underlies the lack of TCDD responsiveness in frog early life stages. We determined the sequence of an AHRR complimentary DNA encoding an 85.3-kDa protein sharing 52–55% identity with the bHLH/PAS domains of other AHRRs. In transient transfection assays, X. laevis AHRR inhibited TCDD-induced reporter gene expression mediated by either X. laevis AHR paralog, AHR1
or AHR1β. AHRR messenger RNA was expressed at low levels in embryos (Nieuwkoop-Faber stage 33–38; approximately 52 h.p.f.) and was induced approximately twofold following TCDD exposure (42 ng/g wet weight). In contrast, AHRR exhibited higher constitutive expression and was induced more than threefold in tadpoles at stage 52–55 (prometamorphic; 
4 weeks postfertilization) and in isolated viscera of stage 62 tadpoles (in the metamorphic climax; 7 weeks postfertilization). Although the magnitude of induction was smaller, the temporal pattern of AHRR expression and inducibility resembled that of CYP1A6. Thus, attenuated transcriptional activation of AHR target genes and low TCDD toxicity in X. laevis embryos cannot be explained by constitutive, high-level expression of AHRR. Key Words: dioxin; cytochrome P450; nonmammalian species; aryl hydrocarbon receptor; embryo.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant and potent toxicant in most vertebrates. Exposure to TCDD and structurally related halogenated aromatic hydrocarbons (HAHs) is associated with a wide range of deleterious physiological and developmental effects (McLachlan, 2001
; Peterson et al., 1993; Safe, 1995). Compared with other vertebrates, frogs are up to 1000-fold less sensitive to TCDD-induced mortality (Beatty et al., 1976; Jung and Walker, 1997). Although some changes in sublethal endpoints have been associated with HAHs (Sakamoto et al., 1995, 1997; Fisher et al., 2003; Gutleb et al., 1999, 2007), these effects resulted from long-term, high-level exposures. Furthermore, they were delayed until metamorphosis (Gutleb et al., 1999, 2000, 2007) rather than being associated with development of the cardiovascular system, as is typical in fish (Belair et al., 2001; Elonen et al., 1998; Henry et al., 1997) and birds (Ivnitski et al., 2001; Walker and Catron, 2000).
Toxicity of dioxin-like HAH compounds is mediated by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor of the basic helix-loop-helix/Per-ARNT-Sim family of proteins (Gu et al., 2000). Following ligand binding in the cytoplasm, the AHR translocates to the nucleus, dissociates from a complex of chaperone proteins, and dimerizes with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein. This transcriptionally active heterodimer binds cis-acting DNA elements (xenobiotic response elements [XREs]) and alters the expression of target genes (reviewed in Hankinson, 1995, 2005; Schmidt and Bradfield, 1996). The AHR complex may also cause changes in gene expression patterns through complex interactions with other signaling pathways (reviewed in Puga et al., 2005). The best characterized targets of the AHR pathway are Cytochrome P4501A (CYP1A) genes, which are strongly induced (Whitlock, 1999).
Specific features of the expression or function of the AHR frequently underlie differences in TCDD sensitivity among vertebrate species and populations (reviewed in Hahn, 1998). Indeed, the relative insensitivity of Xenopus laevis (African clawed frog) to TCDD toxicity is associated with AHRs displaying low affinity for TCDD (Lavine et al., 2005). In addition to the taxon-specific AHR properties, however, several lines of evidence suggest that factors related to the TCDD response at specific developmental stages may also play a role in the overall lack of HAH toxicity in X. laevis and other frogs. The earliest developmental stages exhibit very low responsiveness to AHR agonists. Although there are anecdotal reports of CYP1A induction by 3-methylcholanthrene (3MC) in early X. laevis embryos (Ohi et al., 2003), published guidelines for the Frog Embryo Teratogenesis Assay–Xenopus (FETAX), which encompasses primary organogenesis events during the first 96 h of development, have long noted the apparent low expression levels of cytochromes P450 necessary for the biotransformation of polynuclear aromatic hydrocarbons into toxic intermediates (ASTM, 1998). For example, toxicity of the AHR agonist benzo[a]pyrene is not significant in the absence of exogenously added microsomes isolated from Aroclor 1254-treated rats (ASTM, 1998; Propst et al., 1997). Similarly, CYP1A7 messenger RNA (mRNA) is not induced by Aroclor 1254 (Jelaso et al., 2003). Responsiveness to AHR agonists seems to increase in later life stages. Metamorphic tadpoles of the green frog (Rana clamitans) and northern leopard frog (Rana pipiens) display a greater capacity for PCB metabolism and elimination than tadpoles that have not commenced metamorphosis (Leney et al., 2006b), and CYP1A genes are clearly induced in X. laevis adult liver following 3MC exposure (Fujita et al., 1999). The low responsiveness of early frog life stages stands in contrast to mouse embryos, in which TCDD-inducible CYP1A1 expression can be measured in blastocysts (Wu et al., 2002), and to zebrafish embryos, in which CYP1A induction by TCDD can be observed within 24 h of fertilization (Tanguay et al., 1999). The weak response to AHR agonists in early frog life stages cannot be explained by the relatively low affinity AHRs, because the same AHRs are expressed at reasonably high levels at both earlier CYP1A-refractory stages and later, more responsive stages (Lavine et al., 2005; Ohi et al., 2003).
The AHR repressor (AHRR) is a member of the AHR protein family that attenuates AHR-driven transcriptional activation (Mimura et al., 1999). Lacking ligand-binding capacity (Karchner et al., 2002), the AHRR nonetheless dimerizes with ARNT and binds XREs in a fashion very similar to the AHR itself (Karchner et al., 2002; Mimura et al., 1999). Induction of AHRR mRNA by AHR agonists suggests that the protein acts in a negative feedback loop to downregulate AHR activity (Karchner et al., 2002; Mimura et al., 1999).
In the studies reported here, we sought to test the hypothesis that constitutive, high-level expression of AHRR underlies the low responsiveness of frog embryos to TCDD by attenuating AHR-mediated transcriptional activation. Xenopus laevis is a compelling model species for these studies for several reasons. Well defined components of the AHR signaling pathway include two AHRs, AHR1 and AHR1β, which bind TCDD and induce XRE-driven reporter gene expression (Lavine et al., 2005); two ARNT paralogs, ARNT (Bollerot et al., 2001) and ARNT2 (Rowatt et al., 2003), which can interact with AHRs in vitro; and two CYP1As, CYP1A6 (MC1) and CYP1A7 (MC2), both induced in response to 3MC (Fujita et al., 1999) and TCDD (Lavine et al., 2005). Xenopus laevis is also a long standing model of vertebrate development in which the temporal sequence of events from fertilization to the completion of metamorphosis is documented in great detail (Nieuwkoop and Faber, 1994), facilitating comparisons between well defined stages. Finally, the widespread use of this species in FETAX (American, 1998) and related developmental toxicity tests (e.g., Gutleb et al., 2007) makes the understanding of molecular mechanisms of chemical toxicity and resistance important in determining how data from these tests can be extrapolated to other species, including humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Animals and TCDD exposures.
Xenopus laevis frogs were obtained from Nasco (Fort Atkinson, WI) and Xenopus Express (Plant City, FL). Pairs of frogs were injected with human chorionic gonadotropin and allowed to breed as described (Dawson et al., 1992
). Embryos were sorted for viability and maintained in FETAX buffer (American, 1998) until they reached the desired developmental stage (Nieuwkoop and Faber, 1994). Embryos (NF stage 33–38) were exposed to 1,6 [3H]-TCDD (Chemsyn, Lenexa, KS) dissolved in dimethyl sulfoxide (DMSO) or to vehicle only for approximately 24 h at 26°C (water-borne exposure in FETAX solution; Philips et al., 2006). For tadpoles, a drop of [3H]-TCDD in DMSO was applied to the ventral surface (NF stage 52–57 and 62–64) as described previously (Philips et al., 2006); animals were held in FETAX buffer for 24 h prior to sampling. TCDD body burden was determined via liquid scintillation counting of solubilized animals as described (Jung and Walker, 1997; Philips et al., 2006).
Complementary DNA cloning and plasmid construction.
Total RNA was isolated from embryos and tadpoles using STAT-60 (Tel-Test, Friendswood, TX). Partial complementary DNAs (cDNAs) encoding X. laevis AHRR were amplified by reverse transcription PCR (RT-PCR) using total RNA extracted from TCDD-exposed tadpoles (stage 62–64) using the GeneAmp Gold RNA PCR Reagent Kit (Applied Biosystems, Foster City, CA). Degenerate PCR primers were designed based on a putative AHRR sequence from Xenopus tropicalis identified in the U.S. Department of Energy Joint Genome Institute X. tropicalis v4.0 genome database. Primers F10 and B15 were synthesized by Operon (Huntsville, AL; sequences listed in Table S1). Cycling conditions: 95°/10 min; [94°/30 s; 60°/30 s; 72°/90 s] x five cycles; [94°/30 s; 58°/30 s; 72°/90 s] x five cycles; [94°/30 s; 55°/30 s; 72°/90 s] x 27 cycles; 72°/7 min.
The 5' and 3' end sequences were determined using the SMART RACE cDNA amplification kit (Clontech, Mountain View, CA). Gene specific primers for 5' RACE were AZad14, AZad17, and AZad13; for 3' RACE, the gene specific primers were AZad36, AZad36, and AZad49 (Table S1). PCR cycling conditions for 5' and 3' RACE were [94°/5 s; 72°/3 min] x five cycles; [94°/5 s; 70°/10 s; 72°/3 min] x three cycles; [94°/5 s; 67°/10 s; 72°/3 min] x 35 cycles. For nested PCR: [94°/5 s; 66°/10 s; 72°/3 min] x 25 cycles; 72°/4 min. Sequences containing the 3' end of the AHRR gene were readily obtained, but 5' RACE products were incomplete, and additional reactions were performed using primers 5race1, 5race14, 5race23, 5race34, AZ5R1, AZ5R3, AZ5R7 (Table S1). Finally, 5' RACE reactions were performed using the GeneRacer kit (Invitrogen, Carlsbad, CA), which employs high temperatures to disrupt secondary structure in target RNAs, and primer SS1 (Table S1). PCR conditions used were: 94°/2 min; [94°/30 s; 68°/1 min] x five cycles; [94°/30 s; 65°/1 min] x five cycles; [94°/30 s; 60°/30 s] x 30 cycles; 72°/10 min. The contiguous sequence was determined using 33 overlapping PCR products of various lengths. The open reading frame derived from these sequences was synthesized and cloned into the pCMVTNT expression vector by Epoch Biolabs (Sugar Land, TX).
Sequence alignment and phylogenetic analysis.
A full length consensus sequence was determined with AssemblyLign software (Accelrys, San Diego, CA) using at least three clones of each overlapping PCR product The translated AHRR amino acid sequence was aligned with amino acid sequences for other AHR family proteins using ClustalX (Thompson et al., 1997). The neighbor-joining algorithm (Saitou and Nei, 1987) was used to construct a phylogenetic tree based on the conserved bHLH/PAS domains of these proteins.
Genome walking.
Genomic DNA was purified from adult X. laevis liver tissue using the Easy-DNA kit (Invitrogen). The Genome Walking kit (Clontech) was employed to amplify genomic DNA sequences upstream of the 5' ends of AHRR and CYP1A6 cDNAs. Briefly, genomic DNA was digested with one of four different blunt-cutting restriction endonucleases and ligated to genome walker adaptors to create four DNA libraries. The upstream region was then amplified using gene specific primers designed near the 5' end of each known cDNA sequence (Table S2) in sequential PCR and nested PCR reactions with Advantage2 DNA polymerase (Clontech). Cycling conditions for primary PCR were: [94°/2 s, 72°/3 min] x five cycles; [94°/2 s, 67° 3 min] x 32 cycles; 67°/4 min. Nested PCR reactions used 4% of the primary PCR reaction as template and only 20 cycles at the 67° annealing temperature. This process provided fragments of varying lengths in each of the four DNA libraries. The longest PCR product was isolated and cloned into the p-GEMT Easy (Promega, Madison, WI) in order to obtain the most sequence information possible. Cloned fragments were subsequently subcloned into pGL3-Basic (Promega) to generate luciferase reporter genes driven by the endogenous promoter and upstream regions (pGL3/AHRR and pGL3/CYP1A6).
Quantitative RT-PCR.
AHRR and CYP1A6 mRNA expression were compared in TCDD-treated and DMSO-treated X. laevis at developmental stage 33–38, stage 52–55, and stage 62–64. β-Actin expression was used as an endogenous control. Total RNA samples from each stage were treated with DNase to completely remove genomic DNA (Turbo DNA-free; Ambion, Austin, TX). Each PCR reaction used SYBR Green PCR Master Mix (Applied Biosystems) and specific primers (Table S3) with cDNA synthesized from 10 ng total RNA using random hexamers and the TaqMan Reverse Transcriptase kit (Applied Biosystems). PCR conditions used were 95°/10 min; [95°/15 s; 60°/1 min] x 45 cycles in an ABI7500 Real Time PCR System. Dissociation curves were examined for each experiment. Relative expression was determined using the 
Ct method in SDS v1.4 software (Applied Biosystems). Statistical analyses were performed using Prism 4.0b (GraphPad, San Diego, CA) or Minitab v. 14 (Minitab, Inc., State College, PA).
Transactivation assays.
COS-7 cells (ATCC, Manassas, VA) were maintained at 37° with 5% CO2 in Dulbecco's modified Eagle's medium (Sigma) and 10% fetal bovine serum (Invitrogen). Repression of the TCDD-dependent transcriptional activity of X. laevis AHR1, AHR1β, and mouse AHR by AHRR were measured in reporter gene assays as described (Lavine et al., 2005). Cells were cotransfected with expression constructs for X. laevis ARNT1; AHRR; AHR1, AHR1β, or mouse AHR; the Renilla luciferase transfection control pRL-TK (Promega); and either the XRE containing firefly luciferase reporter pGudLuc6.1 (Long et al., 1998), pGL3/AHRR, or pGL3/CYP1A6. AHR1, AHR1β, and AHRR were in the pCMVTNT plasmid, whereas mouse AHR (gift from Dr C. Bradfield) and X. laevis ARNT (Open Biosystems, Huntsville, AL) were in pSPORT, all driven by the CMV promoter. 30,000 cells were plated in each well of a 48-well plate. After 24 h, 50 ng AHR1, AHR1β, or mouse AHR, 50 ng ARNT, 50 ng AHRR, 20 ng of reporter construct, and 3 ng pRL-TK were transfected into triplicate wells using Lipofectamine 2000 (Invitrogen). The total amount of transfected DNA was kept constant (300 ng) by addition of pCMVTNT plasmid containing no insert. Five hours following transfection, cells were treated with DMSO vehicle (0.5%) or TCDD (50nM). Cells were lysed 18 h after dosing. The Dual Luciferase Assay kit (Promega) and a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA) were used to measure luminescence. Luminescence values are given as the ratio of firefly luciferase units to Renilla luciferase units (relative luciferase units [RLUs]).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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CYP1A Expression and Inducibility during X. laevis Development
The presence of very low levels of microsomal P450-like activities in X. laevis embryos has long been noted in FETAX guidelines (ASTM, 1998
), and CYP1A7 is not induced by Aroclor1254 (Jelaso et al., 2002) during the first days of development, suggesting that AHR signaling is attenuated in premetamorphic frogs. On the other hand, Ohi et al. (2003) reported induction of CYP1A7 by 3MC in early developmental stages. We directly compared the induction of CYP1A6 and CYP1A7 by TCDD at distinct stages during X. laevis development. Initial experiments using semiquantitative RT-PCR detected neither constitutive expression of the CYP1As nor induction by TCDD during stage 47 (age 5.5 days; near the end of primary organogenesis). However, by stage 55 (age 32 days; prometamorphic), both CYP1As were clearly induced (Fig. 1A).
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Subsequent experiments examined CYP1A induction by more sensitive and quantitative means, in the measurement of both TCDD exposure levels and CYP1A transcript abundance. These studies used RNA isolated from six individual stage 52–55 tadpoles from three different clutches (one TCDD-treated tadpole and one vehicle-treated tadpole per clutch) or RNA isolated from pools of twenty five stage 36–37 embryos from three unique clutches (one pool of 25 TCDD-treated embryos plus one pool of 25 vehicle-treated embryos per clutch). Real time PCR reactions from each cDNA sample were performed in triplicate and results from all reactions from a given treatment group were averaged. In whole animals CYP1A6 mRNA was detected at stage 36/37 (age
2 days, 5 h) and was induced approximately 12-fold by TCDD (41–43 ng/g wet weight; Fig. 1B). In stage 55 animals, CYP1A6 mRNA was induced 19-fold, despite a lower body burden of TCDD (20–21 ng/g). CYP1A6 was readily induced in older animals, approximately 126-fold in the viscera isolated from stage 62–64 tadpoles (during the metamorphic climax) with a TCDD burden of only 3–8 ng/g (Fig. 1D). However, because RNA in these experiments was derived from isolated visceral organs rather than whole animals, the values cannot be directly compared with those from earlier life stages. Although the magnitude of CYP1A induction was roughly comparable during premetamorphic and prometamorphic development, constitutive CYP1A6 expression was approximately 60-fold higher in stage 52–55 tadpoles than in stage 36–37 embryos. TCDD-induced CYP1A6 mRNA from stage 52–55 tadpoles was over 1170-fold more abundant than in untreated stage 36–37 embryos (Fig. 1E). Taken together, these experiments demonstrate a markedly lower TCDD response in earlier life stages that corresponds to the lack of toxicity. We hypothesized that the dramatically reduced CYP1A6 expression and lack of TCDD toxicity during early X. laevis development result from repression of AHR signaling related to constitutive, high-level expression of the AHRR.
Cloning X. laevis AHRR cDNA
To isolate AHRR cDNA sequences from X. laevis, we used RT-PCR and RACE as described previously (Lavine et al., 2005). The resulting cDNA encodes a protein of 754 amino acids and 85.3 kDa (GenBank Accession Number EU156964). The amino acid sequence contains readily recognizable hallmark motifs, including bHLH and PAS A domains. Also typical of AHRR sequences, residues beyond the N-terminal half of the PAS domain are poorly conserved with AHRs and with other AHRRs (Fig. 2A). It shares 60 to 67% identity with known AHRRs in the N-terminal region and 26–34% overall. Phylogenetic analysis placed the X. laevis sequence unambiguously in the same clade with other AHRR sequences (Fig. 2B).
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AHRR Function: Repression of AHR1
and AHR1β ActivityTo determine the ability of the X. laevis AHRR to repress AHR1
or AHR1β, COS-7 cells were transfected with expression constructs for X. laevis AHR1 or AHR1β, ARNT, and AHRR. Cells were also cotransfected with pGudLuc6.1 (Long et al., 1998), containing a mouse CYP1A1 5' flanking region, the MMTV promoter, and the firefly luciferase cDNA. In cells cotransfected with 50 ng of AHRR expression plasmid, TCDD-dependent transactivation of luciferase expression by X. laevis AHR1, AHR1β, and mouse AHR was repressed by 95, 78, and 69% respectively (Fig. 3). Constitutive reporter expression, evident in cells expressing AHR1β or mouse AHR, was reduced to near background levels. As the amount of AHRR plasmid used in the cotransfection with AHR and ARNT increased, the repression of constitutive and TCDD-induced reporter expression strengthened in a dose-dependent manner for both AHR1 and AHR1β (Fig. 4). The threshold amount of AHRR plasmid for this effect was greater than 0.5 ng (Fig. 4). Because X. laevis AHRR could repress both AHR1 and AHR1β, and because AHR1β activity appeared to be more robust in the initial assays, only AHR1β was used in further experiments.
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Activity of both AHR and AHRR was strengthened by coexpression with exogenous ARNT. Addition of X. laevis ARNT plasmid in transient transfections substantially increased TCDD-induced, AHR1β–mediated transactivation, although exogenous ARNT was not required for AHR signaling (Fig. 5, lanes 2 and 6). This finding is consistent with observations of heterologously expressed AHR from other species and suggests that a background of functional endogenous ARNT activity is present in COS-7 cells (Ema et al., 1994
; Evans et al., 2005). In TCDD-treated cells, cotransfection with 10 ng AHRR expression plasmid without exogenous X. laevis ARNT resulted in a 15% decrease in AHR1β-mediated reporter gene expression in TCDD-treated cells (Fig. 5, lanes 2 and 3), compared with a 44% reduction when cells were also transfected with 50 ng exogenous ARNT (Fig. 5, lanes 6 and 7). More substantial decreases were observed with greater amounts of AHRR regardless of the addition of exogenous ARNT (Fig. 5, lanes 4, 5, 8, and 9). The ability of exogenous ARNT to augment the activity of small amounts of AHRR echoes previous observations of zebrafish AHRR function in transactivation assays (Evans et al., 2005). This may also reflect heightened ability of X. laevis AHR and AHRR to interact with ARNT derived from the same species.
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AHRR Expression and Inducibility
In other vertebrates, AHRRs are inducible by halogenated and nonhalogenated AHR ligands (Evans et al., 2005
; Karchner et al., 2002; Mimura et al., 1999). This induction is largely mediated by interactions of the AHR:ARNT complex with XREs, frequently located within 2 kb upstream of the core promoter (Karchner et al., 2002; Mimura et al., 1999). DNA sequence upstream of the X. laevis AHRR promoter is curiously devoid of XREs (Fig. 6A; GenBank Accession Number EU333283). One possible means of AHR-mediated transcriptional activation may involve a sequence at –1127 resembling the XREII sequence (Sogawa et al., 2004), an element reported to support AHR:ARNT transcriptional activation via protein-mediated contacts with DNA. To test the role of this possible XREII, we constructed a luciferase reporter containing the X. laevis AHRR promoter and 1931 bp upstream and assayed the ability of this construct to direct TCDD-induced luciferase expression in conjunction with AHR1β. Both constitutive and TCDD-induced reporter expression were minimal, although positive control constructs containing the mouse CYP1A1 enhancer or the X. laevis CYP1A6 promoter and enhancer (GenBank Accession Number EU333284) were clearly responsive to TCDD (Fig. 6B). This result suggests a different scheme for the regulation of AHRR expression, perhaps involving XREs more distant or downstream from the transcriptional start site. Notably, the first intron of the human AHRR gene (Cauchi et al., 2003) contains three XREs.
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We next sought to characterize AHRR inducibility by examining mRNA expression in X. laevis embryos and tadpoles. To test the hypothesis that relatively high level, constitutive expression of AHRR underlies low CYP1A6 expression in X. laevis early life stages, we used quantitative RT-PCR to measure AHRR mRNA expression in untreated and TCDD-exposed X. laevis at NF stages 33–38, 52–55, and 62–64. TCDD exposure caused approximately 1.8-fold induction of AHRR mRNA in stage 36–37 embryos (41–43 ng TCDD/g), and threefold induction of AHRR mRNA in stage 52–55 tadpoles (20–21 ng TCDD/g; Figs. 7A and 7B). Additionally, AHRR was induced approximately 3.2-fold by TCDD in isolated viscera of stage 62–64 tadpoles with a substantially lower TCDD body burden (3–8 ng/g; Fig. 7C). Constitutive expression of AHRR was approximately 10-fold greater in stage 52–55 tadpoles than in stage 36–37 embryos (Fig. 7D). In stage 52–55 tadpoles treated with TCDD, AHRR mRNA expression was approximately 30-fold greater than expression in untreated stage 36–37 embryos. These results suggest that like the CYP1As, AHRR expression is very low in the early life stages; in later stages, the constitutive expression level and the magnitude of induction by TCDD is greater. Notably, the relatively low level of constitutive AHRR expression in stage 36–37 embryos is inconsistent with our initial hypothesis and is likely inadequate to account for low levels of TCDD-induced CYP1A6 expression during early life stages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Induction of CYP1A is a sensitive biomarker of exposure to AHR ligands in multiple life stages of many vertebrate species. Previous data from the development of the FETAX procedure (ASTM, 1998
; Propst et al., 1997) and subsequent studies (e.g., Jelaso et al., 2003) suggest that during early developmental stages, CYP1A genes from X. laevis (CYP1A6 and CYP1A7) exhibit low expression levels, both constitutively and following xenobiotic exposure. The lack of CYP1A induction contrasts with other, more TCDD-sensitive species, in which CYP1A can be induced well before the completion of primary organogenesis, within 24 h.p.f. in zebrafish (Tanguay et al., 1999) and even in the mouse blastocyst (Wu et al., 2002). Data from the present study provide additional evidence that X. laevis CYP1As are expressed at very low levels during the premetamorphic life stages, at least until the end of primary organogenesis (stage 48). Although we were able to detect TCDD-induced increases in CYP1A6 mRNA expression during this time frame, substantially higher body burdens of TCDD resulted in slightly lower induction levels relative to later stages (Figs. 1B and 1C), and the overall expression level at stage 36/37 was one to two orders of magnitude lower than that observed even in untreated tadpoles at stage 55 (Fig. 1E).
AHRR and TCDD Sensitivity
We sought to test the hypothesis that constitutive, high-level expression of AHRR underlies the low expression of CYP1A6 during early frog development. Although studies in mouse, human, and fish systems demonstrate that AHRR mRNA is typically expressed as a response to TCDD or 3MC exposure, constitutive expression in different tissue types and cell lines is highly variable (Fujita et al., 2002; Karchner et al., 2002; Mimura et al., 1999). Tsuchiya et al. (2003) examined AHRR expression in several human cell lines derived from different tissue types. They found that HeLa cells expressed far more AHRR mRNA than other lines examined, a property that may underlie the lack of induction of CYP1A1, 1A2, 1B1, and AHRR by TCDD and 3MC in this cell line (Tsuchiya et al., 2003). Intrinsic AHRR activity may also affect TCDD responsiveness. Fujita et al. (2002) noted a significant correlation between a specific human AHRR polymorphism and the incidence of micropenis, a developmental pathology that possibly results from in utero exposure to low levels of TCDD (Gray et al., 1997). They hypothesized that reduced AHRR activity associated with a specific genotype may leave individuals more susceptible to AHR-mediated toxicity (Fujita et al., 2002).
Research in several other systems observed no link between AHRR and reduced sensitivity to AHR ligands. For example, AHRR expression patterns in TCDD-sensitive Long-Evans and TCDD-resistant Han/Wistar rats were very similar, suggesting that AHRR expression provides no mechanistic basis for the difference in TCDD toxicity (Korkalainen et al., 2004). Several other studies have examined a potential role for the AHRR in the heritable resistance to the toxic effects of AHR ligands exhibited by fish populations native to highly polluted industrial sites. Karchner et al. (2002) determined that the expression and tissue distribution of AHRR mRNA from TCDD- and PCB-resistant killfish (Fundulus heteroclitus) from New Bedford Harbor, MA resembled fish from a reference site, and Meyer et al. (2003) made similar observations of PAH-resistant killifish from Virginia's Elizabeth River. Basal AHRR expression in HAH-resistant Atlantic tomcod (Microgadus tomcod) from the highly polluted Hudson River (New York) was not altered and could not explain the CYP1A-refractory phenotype in this population (Roy et al., 2006). Rather, there was a positive correlation between CYP1A and AHRR expression in both sensitive and resistant populations (Roy et al., 2006). Our observations of AHRR expression in frogs reinforce the notion that AHRR is regulated in coordinate fashion with CYP1A. Fish populations, rat strains, and frog life stages that are refractory to CYP1A induction also exhibit low expression and inducibility of AHRR. Thus, although AHRR seems to play a role in differential TCDD responsiveness in human cell lines (Tsuchiya et al., 2003), in no case has constitutive, high-level AHRR expression or activity been demonstrated to attenuate TCDD responsiveness or toxicity in an actual organism. Alternative regulatory mechanisms, perhaps involving transcriptional cofactors that function with AHR (reviewed in Hankinson, 2005), may play an important role in developmental differences in TCDD toxicity and CYP1A induction.
Evolution and AHRR Gene Multiplicity
Phylogenetic analysis placed the X. laevis AHRR in the same clade with other AHRR sequences (Fig. 2B). This analysis is consistent with the model of evolutionary descent of AHR family proteins proposed by Karchner et al. (2002), which suggests that AHRR and AHR proteins diverged from a common ancestor following a gene duplication event in an early chordate ancestor. Subsequent duplications of genes encoding AHR family members occurred in common vertebrate ancestors as well as in specific groups (Hahn et al., 2006). Xenopus laevis is a pseudotetraploid species, having undergone a relatively recent genome duplication event (Hughes and Hughes, 1993). This genome duplication likely resulted in the creation of multiple AHRR paralogs, as observed for the AHRs in this species (Lavine et al., 2005). It is unclear if the predicted duplicate AHRR now exists as an actively transcribed gene. Our extensive efforts to clone AHRR sequences via RT-PCR with various primer pairs, both degenerate and specific to well conserved regions of the cDNA, always yielded a single, identical product. Evidence at other loci suggests that X. laevis can, at least in the functional sense, revert to the diploid state (Krotoski et al., 1985). Even if an additional AHRR paralog exists, it may well exhibit substantial redundancy with the protein already identified with regard to both expression and function, and likewise contribute nothing to the suppression of CYP1A expression in early developmental stages.
Beyond Transcriptional Regulation: Toxicokinetic Considerations
The lack of TCDD toxicity in frogs likely results from the contributions of several different molecular and physiological mechanisms. AHRs from X. laevis are 25- to 50-fold less responsive to TCDD in reporter gene assays than mouse AHRb–1 and exhibit correspondingly low TCDD affinity in saturation binding assays (Lavine et al., 2005). Additionally, tadpoles of green frogs, leopard frogs, American toads (Jung and Walker, 1997), and X. laevis (Philips et al., 2006) all exhibit remarkably rapid TCDD elimination rates, with half-lives varying from one to six days, much lower than values measured in the highly sensitive lake trout (Salvelinus namaycush), in which TCDD has a half-life of around five weeks (Walker et al., 1991). Significant TCDD clearance has a relatively late onset in X. laevis, however, beginning only after the first 96 h of development (Philips et al., 2006), subsequent to the completion of cardiovascular developmental events targeted by TCDD in fish. During the corresponding "window of cardiovascular and hematopoietic toxicity" defined in zebrafish (Belair et al., 2001), TCDD body burdens remain substantial. Thus, rapid elimination is unlikely to explain the low responsiveness to TCDD during early frog development (Philips et al., 2006).
Recent studies in ranid frogs highlight the potential role of bioavailability of lipophilic contaminants in the responsiveness of the organisms and their ability to perform metabolic transformation. In green frogs and leopard frogs, both elimination and biotransformation of PCBs increase during metamorphosis relative to both earlier larval and later adult stages (Leney et al., 2006a). Leney et al. (2006a) offer the hypothesis that increased PCB metabolism may result from the rapid consumption of lipid stores during metamorphosis, mobilizing the contaminants sequestered within and making them available to the biotransformation enzymes and/or the regulatory systems that induce enzyme expression. Our own previously published observations of TCDD clearance in X. laevis embryos and tadpoles prior to metamorphosis suggest that TCDD elimination rate increases with age, perhaps coordinate with the consumption of embryonic yolk and the onset of feeding (Philips et al., 2006). Increased mobility of AHR ligands due to depletion of lipid stores consumed during development might make the compounds available for interaction with AHR in various tissues, facilitating the apparent age-related increase in constitutive and induced expression of CYP1As as well as overall toxicity related to AHR-driven changes in gene expression. In addition to AHR signaling and related transcriptional regulatory mechanisms, physiological and metabolic changes during development may play important roles in determining dioxin sensitivity at different frog life stages.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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Supplementary data (Tables S1, S2, and S3) are available online at http://toxsci.oxfordjournals.org/.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAFUNDINGREFERENCES |
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National Institute of Environmental Health Sciences (R15 ES011130); an Education Grant from the Howard Hughes Medical Institute to Kenyon College; and the Kenyon College Summer Science Scholars Program.
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ACKNOWLEDGMENTS |
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We thank Christopher M. Gillen (Kenyon College) for his assistance in revising early drafts of the manuscript.
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