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, OHIO 43022. Fax: 7404275741. E-mail: powellw{at}kenyon.edu.
Received May 17, 2005; accepted June 2, 2005
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ABSTRACT |
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Key Words: aryl hydrocarbon receptors; TCDD; Xenopus laevis; FETAX.
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INTRODUCTION |
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Compared to other vertebrate groups, HAH effects in amphibians are poorly characterized. However, several studies suggest that ranid frogs exhibit substantial insensitivity to TCDD toxicity during both early development (Beatty et al., 1976; Jung and Walker, 1997
) and adult life stages (Beatty et al., 1976
). Jung and Walker (1997)
estimated that embryos and tadpoles of green frogs (Rana clamitans), leopard frogs (Rana pipiens), and American toads (Bufo americanus) are 100 to 1000-fold less sensitive to TCDD-induced lethality than most fish species. Like ranid frogs, embryos of Xenopus laevis (African clawed frog; family Pipidae) suffer little mortality following acute exposure to TCDD (Dell'Orto et al., 1998
; Jung and Walker, 1997
) or polychlorinated biphenyl (PCB) mixtures (Gutleb et al., 1999
, 2000
). Some studies report measurable, HAH-induced changes in sublethal endpoints, including increased edema (Sakamoto et al., 1995
), anemia, and erythrocyte apoptosis (Sakamoto et al., 1997
), as well as delayed increases in mortality (Gutleb et al., 1999
), reduced rate of metamorphosis, and increased incidence of tail deformities (Fisher et al., 2003
). However, the most frequent and severe effects resulted only from long-term, high-level exposures, beginning at least 2 weeks after fertilization, consistent with reduced overall sensitivity to HAH exposure relative to fishes.
Most (if not all) biological effects of dioxin-like HAH compounds are mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor from the basic helix-loop-helix/PAS family of proteins (Gu et al., 2000). Following ligand binding in the cytosol, the AhR protein translocates to the nucleus, dissociates from a complex of chaperone proteins, and forms a heterodimer with the ARNT (aryl hydrocarbon receptor nuclear translocator) protein (Hoffman et al., 1991
). This transcriptionally active complex binds cis-acting DNA elements (xenobiotic response elements; XREs) and alters the expression of target genes (reviewed in Hankinson, 1995
; Schmidt and Bradfield, 1996
). The AhR complex may also cause changes in gene expression patterns through complex interactions with other signaling pathways (reviewed in Carlson and Perdew, 2002
; Puga et al., 2002
). AhR-mediated changes in gene expression are thought to play a mechanistic role in HAH toxicity. cDNA microarray studies have documented changes in the expression of hundreds of genes in cultured human hepatoma cells exposed to TCDD; these include mRNAs regulated both directly and indirectly by AhR signaling (Frueh et al., 2001
; Puga et al., 2000b
). The best-characterized AhR-regulated gene is cytochrome P4501A1 (CYP1A1), which is strongly induced (reviewed in Hankinson 1995
; Schmidt and Bradfield, 1996
). Numerous studies in animals (including mammals, birds, and fish) and cell lines emphasize that properties of the AhR signaling pathwayspecifically the expression or functional properties of the AhR itselfoften underlie the wide variations in HAH sensitivity observed in different animal groups (reviewed in Hahn, 1998
).
We are using X. laevis as a model system for probing the mechanistic role of AhR function in the HAH insensitivity of developing frogs. X. laevis is of particular interest because of its widespread use as a general model of vertebrate development. It is also used in FETAX (Frog embryo teratogenesis assayXenopus) and similar bioassays of the developmental toxicity of chemicals, mixtures, and environmental samples (ASTM, 1998; Bantle, 1996
). X. laevis is known to have an active AhR signaling pathway, including two CYP1A genes (Fujita et al., 1999
), two ARNT genes (Bollerot et al., 2001
; Rowatt et al., 2003
), and an AhR (Ohi et al., 2003
). We report the identification of a second AhR paralog (AhR1
) and characterize the expression patterns and TCDD-responsiveness of both AhRs. Understanding the molecular mechanisms of TCDD insensitivity in developing frogs is important for determining the human health relevance of frog embryo toxicity assays such as FETAX. Moreover, the unique features of frog AhR signalingincluding gene number and orthology, expression patterns, and function during embryogenesis and metamorphosismay ultimately provide a novel perspective on the relationship between the mechanisms of TCDD toxicity and the endogenous functions of AhRs during vertebrate development.
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MATERIALS AND METHODS |
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Oligonucleotide primers.
Primers were synthesized by Qiagen/Operon and used as described below.
cDNA cloning and plasmid construction.
Initially, partial cDNAs encoding both X. laevis and X. tropicalis AhRs were amplified from stage 46 total RNA using reverse transcriptase-polymerase chain reaction (RT-PCR) with degenerate primers Qf and AhR-B1, as described previously (Hahn et al., 1997). The 5' and 3' end sequences of the X. laevis cDNAs were determined by RACE (rapid amplification of cDNA ends) PCR (Frohman et al., 1988
), using the SMART RACE cDNA amplification kit and Advantage HF-2 PCR (Clontech). For 5' RACE of AhR1
, the gene-specific primer sequence was: 5'-CAGATTGCTGGAAACCCAGGTAG-3'; for 3' RACE: 5'-AGAAAGGGAAAGATGGGTCCACG'-3'. For 5' RACE reactions of AhR1ß, the primer sequences was 5'-AGCTAACACCTGAGTCTAAGCACG-3'; and for 3' RACE, 5'-GCAGAGCAAGACAGATGGTAACGGC-3'. Finally, cDNAs containing the entire open reading frames of the X. laevis AhRs were amplified using Platinum Pfx DNA polymerase (Invitrogen) and cloned into pCMVTNT (Promega). A single clone corresponded to the amino acid sequence encoded by each AhR contiguous sequence, a finding that was verified by sequencing each position in at least three individual clones.
Sequence alignment and phylogenetic analysis.
Multiple alignments of the indicated amino acid sequences were performed with CLUSTAL-X (Thompson et al., 1997). Aligned amino acid sequences comprising the well-conserved PAS domain were used to construct phylogenetic trees by maximum parsimony (PAUP 4.0b10 [Swofford, 1998
]) and the Neighbor-Joining (NJ) algorithm (Saitou and Nei, 1987
). Alignment positions with gaps were excluded. Bootstrap analysis (Felsenstein, 1985
) was performed to assess relative confidence in the topologies.
Semi-quantitative RT-PCR.
Expression of AhR, ARNT, and CYP1A mRNAs was assessed in adult organs, at various developmental stages, and in A6 cells via RT-PCR, essentially as described previously (Powell et al., 2000; Rowatt et al., 2003
). Total RNA was treated with DNase I (DNA-free; Ambion) to eliminate contamination by genomic DNA and reverse transcribed to cDNA using Omniscript reverse transcriptase (Qiagen) primed by random hexamers (2.5 µM). Aliquots of the reverse transcription reactions (cDNA from 225 ng total RNA) were used as templates for PCR with specific primers for (0.15 µM each). The linear range of detection for the various PCR products was determined by varying the cycle number from 25 to 45 in 3-cycle increments and measuring relative band intensities on 2% agarose gels with a ChemImager 4000 low-light imaging system (Alpha Innotech) with automatic background subtraction (data not shown). Cycling conditions were: 94°C, 15 s; 50°, 30 s; 68°, 1 min for 28 cycles. Primer sequences for AhR1
were 5'-CCCTTCAATCCTGGAGATACGAA-3' and 5'-GGCTTTCTCCATTCCTTGTGCTTC-3'; for AhR1ß, 5'-TCTACGGCGAGAAAAAGGAGC-3' and 5'-GAGGCAACCACCAAGACAAATCC-3'; and for ß-actin, 5'-GCACCCCTGAATCCTAAAGC-3' and 5'-CAATGATGAAGAAGAGGCAGC-3'. Primer sequences for amplifying CYP1A6 were 5'-CAGTATGGACTAACAATG-3' and 5'-GGTAGAGAGACAATGATC-3'; for CYP1A7, 5'-CAGTATGGACTAACAATG-3' and 5'-CAATGATGAAGAAGAGGCAGC-3'. In experiments to detect CYP1A transcripts, only 25 cycles were employed.
In vitro protein synthesis.
TNT Quick Coupled Reticulocyte Lysate Systems (Promega) were used according to the manufacturer's directions to synthesize unlabeled or 35S-labeled proteins in 25 µl reactions. Aliquots of the TNT reactions were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), followed by fluorography (using Amplify [Amersham]) and autoradiography. Mouse AhR (high affinity, b-1 allele; Burbach et al., 1992) and human ARNT were synthesized in the same fashion using pSPORTAhR and pSPORTARNT, gifts from Dr. C. A. Bradfield (University of Wisconsin).
Cytosolic extracts.
Cytosolic extracts were prepared from pools of whole embryos or tadpoles (5 to 50 animals) according to the method of Hahn et al. (1993). Briefly, flash-frozen tadpoles were powdered under liquid nitrogen, dissolved in MEDMG buffer (25 mM MOPS, pH 7.5, 1 mM EDTA, 5 mM EGTA, 20 mM Na2MoO4, 0.02% NaN3, 10% glycerol 1 mM DTT) containing protease inhibitors (20 µM tosyl-L phenylalanine chloromethyl ketone), 5 µg/ml leupeptin, 100 U/ml aprotinin, 7 µg/ml pepstatin A, and 0.1 mM phenylmethylsulfonyl fluoride) and homogenized. Homogenates were centrifuged at 750 x g, 12,000 x g, and 100,000 x g, and the final supernatant was frozen in liquid nitrogen.
Western blotting.
25 µg of cytosolic protein or 2 µl of a TNT reaction were subjected to SDS-PAGE and blotted to nitrocellulose. Blots were probed with dilution of a monoclonal antibody SA210 (Biomol; 300 µg/ml), directed against the N-terminal half of mouse AhR (Pollenz et al., 1994).
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assays were performed as described previously (Karchner et al., 1999; Powell et al., 1999
), using proteins synthesized in TNT reactions. Prior to protein synthesis, TNT lysates were extracted with dextran-coated charcoal (DCC; 1.0 mg/ml Norit N decolorizing charcoal [Fisher]); 0.1 mg/ml dextran [Sigma] in MEDG), as described previously to reduce specific, background binding to the xenobiotic response element (XRE) probe (Karchner et al., 1999
; Powell et al., 1999
).
Velocity sedimentation analysis.
Specific TCDD binding was detected by velocity sedimentation on sucrose gradients in a vertical tube rotor using 1,6-3H-TCDD [33.1 Ci/mmol; >99% radiopurity; Chemsyn (Lenexa, KS)] as described previously (Karchner et al., 1999). Mouseb1 and frog AhRs were synthesized in TNT reactions, diluted 1:2 in MEDMG buffer (25 mM MOPS, pH 7.5, 1 mM EDTA, 5 mM EGTA, 20 mM Na2MoO4, 0.02% NaN3, 10% glycerol 1 mM DTT), split into two 100-µl aliquots, and incubated for 18 h at 4°C with 4 nM 3H-TCDD. Nonspecific binding was determined by reactions containing an empty vector (unprogrammed lysate [UPL]).
Saturation binding analysis.
The binding affinity of X. laevis AhRs was measured in DCC-based saturation binding assays modified from Poland et al. (1976) and Jensen and Hahn (2001)
. Mouseb1 and frog AhRs were synthesized in TNT reactions, diluted 1:4 in MEDG buffer (25 mM MOPS, pH 7.5, 1 mM EDTA, 5 mM EGTA, 0.02% NaN3, 10% glycerol 1 mM DTT), and incubated with graded concentrations of 3H-TCDD in DMSO for 2.5 h at 4°C in glass test tubes. Next, 5-µl aliquots were taken from each mixture to measure the actual concentrations of 3H-TCDD in each tube. Duplicate 30-µl aliquots were then mixed with 30 µl of DCC in polypropylene tubes. Tubes were vortexed briefly three times and incubated on ice for 5 min between each vortexing. The DCC was pelleted by centrifugation for 5 min at 12,000 x g. Bound 3H-TCDD was measured in 50 µl of each supernatant. Total and bound radioactivity were measured directly with a Beckman LS6500 scintillation counter. The concentration of free 3H-TCDD was determined by subtracting the bound concentration of 3H-TCDD from the total concentration.
Nonspecific binding was measured in unprogrammed TNT lysates, plotted against the concentration of free 3H-TCDD, and fit to a linear equation. This equation was used to calculate the predicted nonspecific binding in reactions containing AhR proteins at each free 3H-TCDD concentration. Specific binding was determined by subtracting the calculated nonspecific binding from the total binding measured in each reaction. Specific binding data were fit by nonlinear regression to the equation describing the Langmuir binding isotherm (Kenakin, 1999). Curve fits and statistics were accomplished using GraphPad Prism version 4.
Transactivation assays.
The TCDD-dependent transcriptional activity of each AhR was measured in luciferase reporter gene assays using COS-7 monkey kidney cells (ATCC; Manassas, VA) co-transfected with pGudLuc 6.1 (XRE-containing firefly luciferase reporter; Garrison et al., 1996; Long et al., 1998
), pRL-TK (Renilla luciferase transfection control; Promega), and AhR and ARNT expression constructs, essentially as described previously (Karchner et al., 2002
). Transfections were carried out 24 h after plating 30,000 cells in triplicate wells of a 48-well plate. For each well, a total of 300 ng of DNA was complexed with 1 µl of Lipofectamine 2000 (Invitrogen). The amounts of transfected AhR and ARNT DNA were adjusted to optimize the fold-inducibility of pGudLuc6.1 over basal reporter expression. AhR1
and AhR1ß cDNAs were in pCMVTNT, while mouse AhR and human ARNT were in pSPORT (gifts from Dr. C. Bradfield), all driven by the CMV promoter. Transfected DNA amounts were 50 ng of AhR, 50 ng of ARNT, 20 ng of pGudLuc 6.1, and 3 ng of pRL-TK. The total amount of transfected DNA was kept constant by addition of pCMVTNT vector with no insert. Cells were treated 5 h after transfection with either DMSO or TCDD at 0.5% final DMSO concentration. 18 h after dosing, cells were lysed and luminescence measured using the Dual Luciferase Assay kit (Promega) in a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA). Luminescence values were determined as a ratio of the firefly luciferase units to the Renilla luciferase units. The fractional response was then determined for each AhR at each TCDD concentration by subtracting the relative luminescence of vehicle-treated cells and determining the ratio of each value to the maximal responsiveness level in the concentration-response curve (Poland and Glover, 1975
).
X. laevis cell culture.
X. laevis A6 kidney epithelial cells (ATCC; Manassas, VA; Rokaw et al., 1996) were grown under the recommended conditions (26°C; 5% CO2 atmosphere; NCTC 109 medium plus 10%; fetal bovine serum, and 200 mM L-glutamine/penicillin/streptomycin) in 25 cm2 flasks pretreated with purified human fibronectin (BD Biosciences). At 85% confluence, cells were exposed for 24 h with graded concentrations of TCDD dissolved in DMSO. Control cultures were exposed to an equal volume of DMSO (0.5% total). Total RNA was extracted with QIAshredder spin columns and RNeasy kits (Qiagen) prior to use in RT-PCR.
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RESULTS |
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Expression of AhR1 and AhR1ß.
The relative mRNA expression of AhR1 and AhR1ß was assessed by semi-quantitative RT-PCR using primers specific to each cDNA. The two AhRs exhibit similar expression patterns, both in the adult animal, where expression is widespread (Fig. 3a), and during development (Fig. 3b), when mRNAs were detectable at stage 12 and after but not at stage 8. One possible difference in expression may be in the adult brain and eye, where AhR1ß mRNA appears to be more abundant than AhR1
. Notably, immunoreactive bands co-migrating with AhR1
and AhR1ß proteins could be resolved and detected on western blots of cytosolic extracts derived from whole tadpoles, suggesting that both proteins are expressed in vivo (Fig. 3c).
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Xenopus laevis AhRs exhibit low TCDD affinity and transcriptional responsiveness: Ligand Binding and Transactivation Properties of AhR1 and AhR1ß.
The ability of frog AhRs to bind TCDD specifically was directly demonstrated by velocity sedimentation analysis on sucrose density gradients. Both AhR1 and AhR1ß exhibited detectable peaks of 3H-TCDD binding eluting from the gradient at a position similar to the mouse AhRb1 (Fig. 5; Burbach et al., 1992
). However, the degree of binding was much lower with the frog AhRs at the same concentration of TCDD, consistent with the electrophoretic mobility shift assays, in which DNA-binding activity of frog AhRs exceeded background levels only at relatively high concentrations of TCDD (Fig. 4b and data not shown). Taken together with the low toxicity of TCDD in frogs, these data suggest that TCDD may bind AhR1
and AhR1ß with low affinity. We tested this hypothesis by measuring the binding of affinity of each receptor for 3H-TCDD in saturation binding assays. AhR proteins were synthesized in TNT reactions (Fig. 4a), and nonspecific binding was assessed using an equal volume of unprogrammed TNT lysate. X. laevis AhR1
exhibited an apparent Kd of 47.2 nM (Fig. 6a), whereas AhR1ß was not saturable, even with free TCDD concentrations exceeding 25 nM, precluding ultimate quantification of TCDD affinity (Fig. 6b). In contrast, the mouse AhR exhibited an apparent Kd of only 2.4 nM (Fig. 6c).
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Technical issues confounding the accurate estimation of equilibrium dissociation constants for AhR proteins in saturation binding assays have been well documented in previously published studies. To control for the potential effects of high lipophilic ligand concentration, high total protein concentration (Bradfield et al., 1988), and potential artifacts of cell-free protein synthesis (Ramadoss and Perdew, 2004
), we sought to confirm and further quantify results of the saturation binding analyses with transfection-based reporter gene assays. COS-7 cells were co-transfected with expression plasmids for a single AhR and human ARNT1 as well as pGudLuc6.1, a luciferase reporter gene containing a 480-bp portion of the mouse CYP1A1 regulatory region (Garrison et al., 1996
; Long et al., 1998
). The relative responsiveness of AhR1
, AhR1ß, and mouse AhR was assessed with graded concentrations of TCDD (Fig. 7). Differences in transcriptional activity were readily apparent. Although the X. laevis AhRs activated greater overall luciferase activity than the mouse protein, the response mediated by mouse AhR was saturated at much lower levels (Fig. 7a). The relative potencies of TCDD are more readily visualized by plotting the fractional induction against TCDD concentration for each AhR, dissecting dose responsiveness from efficacy (Fig. 7b). Using non-linear regression analysis, we estimated the EC50 for mouse AhR at 0.13 nM, whereas EC50's for X. laevis AhR1
and AhR1ß were estimated at 3.3 nM and 7.4 nM, respectively. Thus, the EC50 values estimated in these experiments reflect the relationship between the Kd's estimated by saturation binding analysis: TCDD exhibits at least 25-fold lower potency with either X. laevis AhR than with the mouse receptor.
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DISCUSSION |
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Variations in AhR affinity for TCDD are known to correlate with TCDD sensitivity differences in other species and strains of animals. For example, Sanderson and Bellward (1995) showed that pigeons, great blue herons, and cormorants exhibit TCDD affinities around 15 nM in hepatic cytosol, with EC50's for CYP1A induction ranging from 3 µg to 20 µg/kg egg. Chickens, which exhibit 10-fold higher affinity for TCDD (Kd = 1.2 nM) showed an EC50 for CYP1A induction 10- to 100-fold lower than the less sensitive birds (0.2 µg/kg egg; Sanderson and Bellward, 1995
). In mice, a single point mutation (A375V) in the AhR is associated with a 45-fold reduction in TCDD affinity in the AhRd allele compared to the AhRb1 allele (Ema et al., 1994
; Poland et al., 1994
; Ramadoss and Perdew, 2004
) and an 8- to 24-fold reduction in TCDD toxicity in AhRd/d animals compared with those homozygous for the b-1 allele (Birnbaum et al., 1990
). Like AhRd, the lower affinity human AhR also contains valine at this position (Ema et al., 1994
; Poland et al., 1994
; Ramadoss and Perdew, 2004
). Notably, although they bind TCDD with low affinity, both X. laevis AhR1s resemble the high-affinity mouse allele with an alanine in the corresponding position. Given the great deal of divergence between the frog and mammalian proteins (
55% overall amino acid identity), the large differences in affinity and transcriptional activity seen between the frog and mouse AhRs likely result from a number of structural and functional variations in the proteins.
Saturation Binding Curves versus AhR Responsiveness in Cells
The saturation binding assays performed in these studies (Fig. 6) likely underestimate the absolute affinity of the receptors for TCDD. In saturation binding assays with mouse and human AhRs, apparent binding affinities are known to vary inversely with overall protein concentration (Bradfield et al., 1988; Ramadoss and Perdew, 2004
). Detection of specific binding activity of the X. laevis AhRs required lower dilutions of the TNT lysates (and hence higher overall protein concentrations) than those used in previous studies (e.g., Jensen and Hahn, 2001
), and the conditions for the mouse AhR binding assays were adjusted to match. Concomitantly, Kd estimates for the mouse AhRb1 protein were 3- to 10-fold higher that those reported in previous studies of TNT proteins (Ema et al., 1994
; Jensen and Hahn 2001
; Poland et al. 1994
), suggesting that the affinity of the frog AhRs for TCDD was also greater than measured. However, because assays were performed in parallel under identical conditions, the relative affinities determined here nonetheless provide compelling evidence for low-affinity TCDD binding by X. laevis AhRsa property that likely underlies low TCDD toxicity.
Importantly, the relative TCDD affinities of frog and mouse AhRs were reflected consistently in the activity of each protein in the luciferase reporter gene assays (Fig. 7). These transfection-based assays are perhaps a more relevant indication of the TCDD responsiveness of all three AhRs than the saturation binding curves, providing an integrated measure of ligand binding affinity and intrinsic efficacy. Recent studies comparing ligand binding by AhR in intact cells and in cell lysates suggest that receptors retain greater activity in intact cells, offering a more accurate reflection of their true ligand-binding properties (Ramadoss and Perdew, 2004). Furthermore, in studies of several cell lines, the EC50 for CYP1A induction was typically well below the apparent Kd of the cytosolic receptor for TCDD (Hestermann et al., 2000
; Pollenz, 1996
; Pollenz and Necela, 1998
), a phenomenon that relates to the existence of "spare receptors" in the system (Hestermann et al., 2000
). We observed a similar relationship between in vitro binding affinity and the dose response of luciferase reporter gene induction by X. laevis AhR1
and AhR1ß, conceivably due to both technical and physiological factors.
Evolution and AhR Gene Multiplicity
The existence of multiple AhR genes in a single species is not without precedent. Many fish harbor two AhR genes, AhR1 and AhR2, that arose from an ancient gene duplication event predating the divergence of cartilaginous fish from the vertebrate lineage (Hahn et al., 1997). Phylogenetic analysis of X. laevis AhRs reveals that they arose from a much more recent gene duplication event, likely associated with a duplication of the entire genome, a common occurrence in members of the Xenopus genus. Consistent with this interpretation, phylogenetic analysis reveals that both AhR1
and AhR1ß are orthologous to the AhR found in Xenopus tropicalis (Fig. 2), a true diploid species that diverged from the common Xenopus lineage prior to the genome duplication event associated with X. laevis. Numerous recently diverged paralogous genes have been documented in X. laevis (Hughes and Hughes, 1993
), including those encoding other nuclear receptors (Grun et al., 2002
; Moore et al., 2002
; Wu et al., 2003
). The existence of AhR2 orthologs in X. laevis remains a possibility. However, BLAST searches of the recently re-annotated X. tropicalis genome revealed only one AhR gene, identical to the sequence we report here. This suggests that the more ancient AhR2 paralog may have been lost in the Xenopus lineage, as it apparently has in mammals (Hahn et al., 1997
; Karchner et al., 1999
).
X. laevis AhR1 and AhR1ß are somewhat reminiscent of AhR2
and AhR2ß, closely related AhR paralogs in rainbow trout (Oncorhyncus mykiss), another pseudotetraploid species (Abnet et al., 1999a
), although the X. laevis AhR1 paralogs share fewer amino acid identities (86% versus 97%). The rainbow trout AhR2 paralogs are both capable of binding TCDD, but they show distinct promoter and ligand preferences in reporter gene assays (Abnet et al., 1999b
), a subtly different expression pattern, and differentially induced expression by TCDD exposure in some animal tissues (Abnet et al., 1999a
), suggesting that they have distinct physiological functions. Our studies reveal more similarities than differences in X. laevis AhR1
and AhR1ß. They are very similar in size and sequence and share comparable expression patterns and responsiveness to TCDD. However, functional differences may well exist. The relative abilities of each protein to bind other AhR ligands, activate transcription in different promoter contexts, interact with X. laevis ARNT1 and ARNT2, and function in conjunction with each other are currently under investigation.
Significance of Multiple, Low-Affinity AhRs in X. laevis
In addition to their historical use as a model of vertebrate development, X. laevis embryos are used in FETAX, a standardized test of developmental toxicity (ASTM, 1998; Bantle, 1996
). Substantial effort has been invested by research groups (e.g., Bantle et al., 1990
, 1994
, 1996
, 1999
; Fort et al., 1989
, 2001a
, 2001b
, 1995
, 1998
) and government panels (FETAX, 2000) to validate this test and to adapt it for use in the screening of chemicals and environmental samples. With the discovery of two X. laevis AhRs with low affinity for TCDD, this study identifies an important mechanistic basis for the differences in HAH toxicity between the frog embryo model and other vertebrates, including humans, which should help to evaluate and refine the use of FETAX in conjunction with HAH-containing samples. Low affinity of X. laevis AhRs for other ligands might also explain the low toxicity of polynuclear aromatic hydrocarbons in FETAX, and it may underlie the historically reported low expression levels of cytochromes P-450 during early frog development (ASTM, 1998
; Bantle, 1996
; Bantle et al., 1991
; Fort et al., 1991
, 2001a
, 2001b
).
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ACKNOWLEDGMENTS |
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