Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543
Received March 9, 2001; accepted August 2, 2001
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ABSTRACT |
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Key Words: aryl hydrocarbon receptor; TCDD; in vitro expression; beluga; cetacean; species-specific; susceptibility; dissociation constant.
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INTRODUCTION |
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Among the several classes of POPs, the planar halogenated aromatic hydrocarbons (PHAH) are known to be especially toxic to vertebrate animals. PHAHs include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), other polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and certain non- and mono-ortho-substituted PCBs. These compounds cause a number of toxic and biochemical effects in laboratory rodents, including immunosuppression, endocrine dysfunction, enzyme induction, cancer, and reproductive/developmental toxicity (Pohjanvirta and Tuomisto, 1994; Poland and Knutson, 1982
; Schmidt and Bradfield, 1996
). Although the mechanisms by which TCDD and other PHAHs cause toxicity are not completely understood, it is known that these compounds exert their toxic and gene-regulatory effects via the aryl hydrocarbon receptor (AHR) (Hahn, 1998a
; Poland et al., 1976
; Schmidt and Bradfield, 1996
), a soluble, ligand-activated transcription factor and a member of the basic helix-loop-helix family of transcription factors (Gu et al., 2000
). Studies in AHR-null mice have shown that this protein is required for TCDD toxicity (Fernandez-Salguero et al., 1996
; Hundeiker et al., 1999
; Mimura et al., 1997
; Peters et al., 1999
; Thurmond et al., 2000
). In addition, strain- and species-specific differences in sensitivity to the effects of TCDD have been shown to depend, at least in part, on the properties of the respective AHRs, including their ligand-binding affinities (Ema et al., 1994b
; Poland et al., 1994
; Sanderson and Bellward, 1995
).
Several laboratories have provided evidence for a functional AHR signal transduction pathway in cetaceans. Cytochrome P450 (CYP) forms in the 1A and 1B subfamilies, which in other mammals are regulated by the AHR, have been identified by catalytic, immunochemical, and molecular assays in cetaceans (Godard et al., 2000; Goksøyr et al., 1986
, 1988
; Teramitsu et al., 2000
; White et al., 1994
). A strong correlation between hepatic levels of CYP1A1 and blubber concentrations of non-ortho-substituted PCBs provided indirect evidence for CYP1A1 induction in beluga from the Arctic (White et al., 1994
). The presence of a cetacean AHR that is capable of specific binding to dioxins was confirmed using dolphin kidney cell lysates (Carvan et al., 1994
) and beluga liver cytosol (Hahn et al., 1994
).
To assess the potential susceptibility of cetaceans to PHAHs, we are characterizing the AHR signaling pathway of the beluga (Hahn et al., 1994; Jensen, 2000
; White et al., 1994
, 2000
). This species was chosen for several reasons: (1) the existence of distinct stocks that vary in their exposure to PHAHs (Muir et al., 1990
); (2) the postulated role of PHAH in the decline of the St. Lawrence beluga stock (De Guise et al., 1995
; Martineau et al., 1994
; Sargeant and Hoek, 1988
); (3) the recent proposal of beluga as a "model odontocete cetacean species" by a working group of marine mammal toxicologists convened by the Marine Mammal Commission (Marine Mammal Commission, 1999
); and (4) the potential for follow-up research on captive populations. In the present report, we describe the cloning, in vitro expression, and initial functional analysis of a beluga AHR. We compare this beluga AHR to a relatively high affinity AHR, the product of the mouse Ahb1 allele (Burbach et al., 1992
; Ema et al., 1992
; Poland et al., 1994
), as well as to the human AHR (Dolwick et al., 1993a
). This is the first molecular characterization of an AHR in a marine mammal. The results of these analyses suggest that beluga may be sensitive to the effects of PHAHs. More generally, our results show that cDNA cloning with in vitro characterization of proteins involved in mechanisms of toxicity is a promising approach for gathering species-specific data that may contribute to an improved understanding of the contaminant sensitivity of protected species, for which in vivo dosing experiments are ethically, legally, and logistically impossible.
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MATERIALS AND METHODS |
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Tissue collection.
Beluga liver was collected from Mackenzie River Delta, NWT, Canada, as described earlier (White et al., 1994). Liver tissue was also collected from a subsistence hunt of Chukchi Sea beluga in Alaska during the summer of 1997. Liver tissue was snap frozen in liquid nitrogen approximately 34 hours after death. Tissues were maintained at liquid nitrogen temperatures throughout transport, storage, and powdering with mortar and pestle. The C57BL/6J mouse was killed by cervical dislocation and the liver was extracted from the animal and snap frozen minutes after death.
RNA isolation.
Total RNA was isolated from liver tissue using the guanidinium isothiocyanate method (Clemens, 1984). PolyA+ RNA was isolated using oligo dT columns (Collaborative Research, Bedford MA) as described by Maniatis et al. (1982). The quality of the total and polyA+ RNA was confirmed by visualization on ethidium bromide-stained agarose minigels and quantity was determined by UV absorbance.
Oligonucleotide primers.
Primers were synthesized by National Biosciences, Life Technologies, or Integrated DNA Technologies. The degenerate primers AHR-A1, AHR-A2, AHR-B1, and AHR-B2 have been described previously (Hahn and Karchner, 1995; Karchner and Hahn, 1996
). The gene-specific primers used are presented in Table 1
.
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PolyA+ mRNA isolated from the Alaskan beluga was used with a GeneAmp RT/PCR kit (Applied Biosystems), with the following conditions in the Perkin Elmer 2400 thermocycler: 94°/5:00 (95°/0:15, 60°/0:30, 72°/1:00) for 35 cycles. Degenerate primers AHR-A1 and AHR-B1 containing inosines amplified a 648-bp fragment that was sequenced with a Licor4000 automated sequencing system using an Excel II cycle sequencing kit (Epicentre Technology).
Gene-specific primers were then designed for 5'- and 3'-RACE. The 5' gene-specific primers Dlb538 and Dlb467 and the 3' gene-specific primers Dlf152, Dlf258, Dlf467, and Dlf538 were used with the Marathon gene amplification kit (Clontech). To maximize specificity, "touchdown" PCR (Don et al., 1991) was used for all RACE reactions: 94°/0:30 min (94°/0:05, 72°/2:00) (5 cycles); (94°/0:05,70°/2:00) (5 cycles); and (94°/0:05, 68°/2:00) (25 cycles). Nested 5'-RACE was carried out on the Dlb538 product using Dlb467. The 3'-RACE products were too large to sequence outright, so an additional PCR was performed to obtain the remaining sequence of the 3'-RACE clone using DLExon9F and DlUTR1rev with conditions 95°/5:00 (95°/0:30,56°/0:30,72°/0:30) (35 cycles); 72°/7:00. All sequencing was carried out on the Licor4000 automated sequencing system using an Excel II cycle sequencing kit. RT-PCR and RACE products were assembled to determine the consensus beluga AHR sequence. This sequence was based on 44 individual fragments; 38 were obtained by cycle sequencing of 21 separate clones and 6 sequences were from direct sequencing of PCR products in forward and reverse directions. The minimum number of sequences that were used to form a consensus at each base was 4 within the coding region and 2 in the untranslated regions (UTRs).
Multiple alignment of beluga, mouse, and human AHR was done with the ClustalW program contained in the MacVector software, version 6.5.3 (Oxford Molecular).
Expression vector construction.
Once sequencing revealed the start and stop codons, primers were designed to amplify the full-length cDNA. Two 5' primers were designed, one with the native sequence (5nat-hind3) and the other with a Kozak sequence (5koz-hind3) (AG switch at +4) (Kozak, 1987
). Both contained HindIII restriction sites outside of the coding region. The full-length beluga AHR was amplified using Klentaq DNA polymerase (Clontech). Primer pairs 5nat-hind3/xba1-utr and 5koz-hind3-/xba1-utr were used in touchdown PCR: 94°/1:00 (94°/0:05, 70°/2:00) (5 cycles); (94°/0:05, 68°/2:00) (5 cycles); (94°/0:05, 66°/2:00) (25 cycles); and 68°/7:00.
The full-length AHR products derived from primer pairs 5nat-hind3/xba1-utr and 5koz-hind3-/xba1-utr were cloned into the HindIII and XbaI sites of pcDNA3.0 (Invitrogen) and pSP64 Poly(A) (Promega) vectors. This generated constructs containing native and Kozak 5' ends, each under control of either the T7 or SP6 promoter. The 4 constructs were tested for expression efficiency. Proteins were synthesized using the TNT Quick Coupled Transcription and Translation system (Promega) for T7 or SP6 promoters in the presence of [35S]-methionine. The pSP64belAHRnat construct under control of the SP6 promoter expressed 2- to 3-fold higher levels of protein compared to the pcDNA constructs. The pcDNAbelAHRkoz construct expressed at higher levels compared with the pcDNAbelAHRnat sequence. Thus, the pSP64belAHRnat was used for in vitro translation studies, and the pcDNAbelAHRkoz was used for transfection experiments. To ensure that the pSP64belAHRnat expression plasmid (hereafter referred to as pSP64belAHR) and the pcDNAbelAHRkoz (hereafter referred to as pcDNAbelAHR) were free of PCR errors, each was completely sequenced and confirmed to match the belAHR consensus sequence that had been determined by RT-PCR and RACE.
Cytosol preparation.
Liver cytosol was prepared as described in Hahn et al. (1994). Cryo-preserved liver was first powdered while under liquid nitrogen, then homogenized in MEEDGM (25 mM MOPS, 1 mM EDTA, 5 mM EGTA, 0.02% NaN3, 20 mM Na2MoO4, 10% (v:v) glycerol, 1 mM DTT, pH 7.5) containing protease inhibitors (100 u/ml aprotinin, 7 µg/ml pepstatin A, 5 µg/ml leupeptin, 20 µM tosyl-L-phenylalanine chloromethyl ketone, and 0.1 mM phenylmethylsulfonyl fluoride). After serial centrifugations of 750g, 12,000g, and 100,000g, the supernatants were frozen in liquid nitrogen until analysis.
In vitro protein synthesis.
Beluga, mouse and human AHR proteins were synthesized using the TNT Quick coupled Reticulocyte Lysate Systems (Promega) in the presence or absence of [35S]-labeled methionine. The sizes of the proteins were confirmed by SDS-PAGE with 2 µl of a 25-µl TNT reaction containing [35S]-labeled AHR (approximately 25 µg total protein), followed by fluorography and autoradiography.
Western blotting.
Two µl of in vitro (TNT) synthesized mouse, human, and beluga AHRs and 100 µg beluga liver cytosolic protein were loaded onto a Tris-acetate gel (Novex). The gel was blotted onto 0.22 µm PVDF membrane, and probed with SA-210 rabbit antimouse AHR polyclonal antibody (Biomol) and secondary goat antirabbit antibody (Schleicher & Schuell). Bands were visualized with CSPD chemiluminescent substrate (Tropix).
Velocity sedimentation.
In vitro-expressed beluga, mouse, and human AHR were analyzed by velocity sedimentation on sucrose gradients in a vertical tube rotor (Tsui and Okey, 1981). For each AHR or unprogrammed lysate control, two identical TNT reactions (100 µl total) were combined and diluted 1:2 with MEEDMG buffer (described above). Each sample was split into 2 aliquots and incubated with [3H]TCDD (2 nM) ± TCDF (400 nM) for 8 h at 4°C in glass tubes. Following incubation, the expressed proteins were treated with 1.5 mg/ml dextran-coated charcoal (DCC) (charcoal:dextran 10:1 w:w) in a polypropylene tube. Cytosols were diluted to 5 mg protein/ml in MEEDMG buffer and incubated under the same conditions as the expressed proteins except that they were not washed with DCC. The [3H]TCDD concentration was verified by sampling each tube for total counts. Samples were analyzed on 1030% sucrose gradients as described earlier (Karchner et al., 1999
). Specific binding was defined as the difference between total binding (incubations containing [3H]TCDD) and nonspecific binding (incubations containing [3H]TCDD plus a 200-fold excess of TCDF).
[3H]TCDD saturation binding analysis.
The specific binding of [3H]TCDD to in vitro-expressed AHRs was measured using a modification of the DCC-based binding assay of Poland et al. (1976). The beluga, mouse, and human AHR proteins were synthesized by in vitro transcription and translation as for the velocity sedimentation assay. The reactions were then diluted 1:8 in MEEDGM with protease inhibitors. Diluted TNT-expressed proteins were incubated in 16 x 100-mm glass tubes for 78 h with each of 9 concentrations (0 to 8 nM nominal) of [3H]TCDD in DMSO. Duplicate aliquots were taken at the beginning of the incubation to measure the actual concentrations of [3H]TCDD in each tube. After the incubation, 30 µl were transferred in triplicate aliquots to standard, 1.5-ml polypropylene tubes containing 30 µl of 2.3 mg DCC/ml MEEDGM. Tubes were vortexed x 3 for 5 s, with a 5-min incubation on ice between each vortexing. After a short spin to sediment the DCC, 40 µl of the supernatant were counted to measure bound [3H]TCDD. Total counts and total binding were measured on a Beckman 5000 scintillation counter.
A difference in the assay used here as compared to the original method (Poland et al., 1976) is our use of "unprogrammed" TNT lysate (UPL; TNT lysate plus an empty expression vector) to determine nonspecific binding. In tissue-based AHR binding assays, specific binding is determined from the difference between total [3H]TCDD binding and the [3H]TCDD binding measured when excess cold ligand (usually TCDF) is used to block specific binding sites. The use of in vitro-expressed proteins provides the opportunity to measure nonspecific binding directly, by using a blank reaction containing UPL. Sucrose gradient analysis showed that UPL lacks specific binding when assayed in the presence and absence of excess TCDF (see Results and Karchner et al., 1999
). Therefore, analysis of unprogrammed TNT lysate provides a convenient means for measuring nonspecific binding without the need for duplicate tubes for each species tested. This method also avoids potential artifacts associated with the use of high concentrations of TCDF, which is poorly soluble in aqueous solutions.
All curves were plotted as "free" [3H]TCDD (nM) vs. bound [3H]TCDD. The amount of bound [3H]TCDD was determined directly from measured radioactivity after DCC treatment. Free [3H]TCDD was determined by subtracting the bound [3H]TCDD concentration from the total [3H]TCDD concentration for each tube. The binding of [3H]TCDD to UPL (nonspecific binding) was also plotted as a function of the free [3H]TCDD concentration and fit to a linear model; this relationship was used to calculate the predicted nonspecific binding at each concentration of free [3H]TCDD in incubations containing in vitro-synthesized AHRs. This calculated value was then subtracted from the total binding in each tube to obtain the specific binding. The specific binding points were fit by nonlinear regression to the equation for the Langmuir binding isotherm:
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Electrophoretic mobility shift assays.
Electrophoretic mobility shift assays were performed using wild type and mutant DRE-containing oligonucleotides (Yao and Denison, 1992). Oligonucleotides were: wild-type (5'-GAT CTG GCT CTT CTC ACG CAA CTC CG-3' and 5'-CGG AGT TGC GTG AGA AGA GCC AGA TC- 3' ) and mutant (5'- GAT CTG GCT CTT CTC ACA CAA CTC CGG ATC- 3' and 5'- GAT CCG GAG TTG TGT GAG AAG AGC CAG ATC-3') (core sequence in bold; mutations underlined). To generate labeled dioxin-responsive enhancer (DRE)-containing probes, one strand of the wild-type DRE oligonucleotide was radiolabeled with [
32P]-ATP using T4 kinase according to the manufacturer's recommendations (Promega). After the labeling reaction, the complementary strand was added and annealed to the labeled strand by heating the combined oligonucleotides to 90°C and allowing them to slowly cool to room temperature. Unincorporated radionucleotides were removed by spinning the product through a Centri-Spin 20 column (Princeton Separations). For competition experiments, a double-stranded, unlabeled wild-type DRE oligonucleotide and a mutant DRE oligonucleotide that contains a single base pair substitution within the DRE core consensus sequence were used.
Prior to initiation of the in vitro expression reaction, the TNT Master Mix (Promega) was incubated with 2.5 µg DCC per µl Master Mix for 10 min on ice (Karchner et al., 1999; Powell et al., 1999
). The DCC was then pelleted, and the Master Mix was removed and used in the in vitro expression reactions with pSP64belAHR, pSPORTmoAHR, and pSPORTArnt. Three µl of either the beluga or mouse AHR reactions were mixed with 3 µl human Arnt reaction and MEEDG (10 mM MOPS buffer, pH 7.5 with 1 mM dithiothreitol, 1 mM EDTA, 5 mM EGTA, 0.02% NaN3, and 10% glycerol). Where AHR or Arnt was left out of the reaction, an equal volume of unprogrammed lysate was substituted. Acetone or 40 nM TCDD was then added, and the mixture incubated for 2 h. For the competition assays, 3 µl unlabeled DRE, 3 µl mutant DRE, 0.6 µg anti-moAHR antibody (Biomol), or 0.6 µg rabbit IgG was added for an additional 15-min incubation. Finally, a master mix containing NaCl (165 mM final), pdIdC (1.25 µg/µl), glycerol (10%), and labeled DRE probe (50,000 cpm/rxn) was added to all tubes, and the incubation was continued for an additional 30 min. All incubations were at room temperature. The samples were resolved on a 4.5% TBE gel, and the gel was dried and exposed to X-ray film overnight.
Transfection assays.
COS-7 monkey kidney cells, which express no AHR and very little Arnt (Ema et al., 1994a,b
) were transfected with human Arnt and beluga or mouse AHR, together with pGudLuc6.1 and a renilla luciferase plasmid (pRL-TK, Promega), to serve as the transfection control. pGudLuc6.1 contains the firefly luciferase gene under control of an MMTV promoter regulated by 4 DREs within a 480-bp fragment derived from the murine Cyp1A1 promoter (Long et al., 1998
). Cells were transfected with 0, 10, or 50 ng of pcDNAbelAHR or pSPORTmoAHR, 0 or 50 ng pSPORTArnt, 20 ng GudLuc6.1, and 3 ng pRL-TK. The empty vector pcDNA3.1 (100290 ng) was used to bring the total amount of DNA transfected to approximately 300 ng.
The transfection protocol was as follows. First, COS-7 cells were plated at 40,000 cells/well of a 48-well plate. Twenty-four hours later, the medium was replaced with serum-free medium and the cells were transfected using the Lipofectamine 2000 reagent (Gibco) as described by the manufacturer. Five hours later, cells were dosed with DMSO or 10 nM TCDD. Eighteen hours after dosing, the cells were harvested and luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) on a Turner TD-20/20 luminometer (Turner Designs).
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RESULTS |
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Characterization of beluga AHR synthesized in vitro.
We analyzed the beluga AHR protein expressed in a rabbit reticulocyte lysate system under control of the SP6 and T7 promoters, with and without modification to create Kozak sequences near the start codon. An autoradiogram of a polyacrylamide gel containing the proteins made with [35S]methionine revealed that the construct containing the native AHR sequence, under control of the SP6 promoter (pSP64belAHR), was the most efficiently expressed (not shown). The apparent molecular weight of the in vitro-expressed protein was 110 kDa, consistent with its apparent size as observed by photo-affinity labeling of beluga hepatic cytosol (Hahn et al., 1994
). All functional assays of in vitro (TNT)-expressed protein were henceforth conducted using the pSP64belAHR plasmid.
The relative sizes of the expressed beluga, human, and mouse proteins synthesized in the presence of [35S]methionine are consistent with their relative predicted molecular weights of 95.5, 90.6, and 96.0 kDa for pSP64belAHR, pSPORTmoAHR, and pSPORThuAHR products, respectively (Fig. 4A). Analysis by Western blot (Fig. 4B
) confirmed the identity of the expressed proteins as AHRs, and showed that in each case the size of the expressed protein is the same as that derived from tissue cytosol. The relative intensities of the bands do not necessarily reflect quantitative differences between the beluga and mouse AHR expression in liver, because (1) the antibody we used was polyclonal and raised against the N-terminus of the mouse AHR, so equal cross-reactivity cannot be assumed, and (2) the beluga liver was sampled under suboptimal conditions.
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Relative binding affinities of beluga, mouse, and human AHRs expressed in vitro.
Having established the ability of the beluga AHR to specifically bind [3H]TCDD, we next sought to compare the TCDD-binding affinity (Kd) of the in vitro-expressed beluga AHR with the affinities of in vitro-expressed mouse and human AHRs. We used a saturation-binding assay modified from the DCC-based cytosolic-binding assay described by Poland et al. (1976). Critical to these experiments is the achievement of an abundance of "free" ligand relative to bound ligand, so that the [3H]TCDD concentrations used to measure the dissociation constant are only minimally reduced by associations with lower-affinity binding sites. A satisfactory balance of detectable specific binding ( 500 dpm) and fraction of "free" [3H]TCDD (
80%) for the beluga AHR was achieved with an 8- to 10-fold dilution of the lysate (Jensen, 2000
). After incubation with [3H]TCDD, bound radioligand was separated from "free" radioligand with the use of DCC, and the amount of bound [3H]TCDD was plotted as a function of the concentration of free [3H]TCDD. Since the UPL does not bind [3H]TCDD specifically (Fig. 5
), it was used to determine the amount of nonspecific binding, as described in Materials and Methods. This eliminated the need for parallel incubations with [3H]TCDD plus excess unlabeled competitor. Binding to UPL increased linearly with increasing amount of [3H]TCDD in the concentration range used (Fig. 6
), consistent with the theoretical properties of nonspecific binding sites (low affinity, high capacity, and not saturable).
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DISCUSSION |
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One approach for assessing the potential toxicological significance of PHAHs in a given species is to examine proteins involved in the mechanism of toxicity. We chose to examine the AHR, a protein known to interact directly with PHAHs and to play an essential role in PHAH toxicity (Fernandez-Salguero et al., 1996; Mimura et al., 1997
; Peters et al., 1999
). However, biochemical examination requires intact proteins from high quality tissue samples, which are difficult to collect from marine mammals because of the paucity of sampling opportunities, delays in excising tissues (leading to autolysis), and the in vitro lability and low abundance of the AHR. To circumvent these and other problems associated with the sampling and analysis of marine mammal tissues, we cloned a beluga AHR, expressed it in vitro, and characterized the TCDD-binding affinity and other functional properties of the expressed protein in comparison with AHRs from mouse and human. The results of our analyses provide suggestive evidence that beluga, and possibly other cetaceans, may be sensitive to AHR agonists, thus implying that PHAHs have the potential to affect cetacean health.
Beluga AHR Shares High Sequence Identity with Other Mammalian AHRs
Cloning and sequence analysis of a full-length cetacean AHR confirms and extends earlier observations of proteins in cytosols from beluga liver and a dolphin cell line that bind dioxin specifically (Carvan et al., 1994; Hahn et al., 1994
). The beluga AHR possesses major functional domains that are characteristic of AHRs, including the bHLH, PAS A, PAS B, and glutamine-rich regions. As with other AHRs, the N-terminus of the beluga AHR is highly conserved, while the C-terminus is much less so, and might be termed "hypervariable," as noted by others (Dolwick et al., 1993a
). Phylogenetic analyses (not shown) demonstrate that the beluga AHR amino acid sequence groups with the "AHR1 clade" rather than the "AHR2 clade" recently identified in some vertebrates (Hahn et al., 1997
; Karchner et al., 1999
). Overall, the beluga AHR sequence is most closely related to the human AHR; these 2 proteins differ by only 3 residues in length and share 85% amino acid identity. The beluga AHR shares 75% identity with the mouse Ahb1 allele (Burbach et al., 1992
), although within the bHLH and PAS domains this identity is much higher. The closer relationship of the beluga AHR to the human AHR, as compared to the mouse AHR, is unexpected in light of the phylogenetic relationships between cetacea, rodentia, and primates determined from other gene sequences (Madsen et al., 2001
; Murphy et al., 2001
). The basis and significance of this observation are not yet clear.
Despite the high degree of sequence identity among mammalian AHRs, subtle changes in the amino-acid sequence can cause remarkable changes in AHR function (Pohjanvirta et al., 1998; Poland et al., 1994
; Sun et al., 1997
) that could be the basis for species-specific differences in sensitivity to AHR ligands. A much greater understanding of the AHR structure-function relationship is required before function may be deduced accurately from AHR sequence data. For this reason, we characterized the in vitro binding affinity and other properties of the beluga AHR.
Beluga Whales Possess a High Affinity AHR
In order to conduct a comparison of the beluga AHR with AHRs from terrestrial mammals that have been reasonably well characterized, we chose a relatively "high affinity" and a relatively "low affinity" AHR to use alongside the beluga AHR in our experimental system to determine comparative TCDD-binding affinities. The high affinity AHR was the mouse Ahb1 allele (Burbach et al., 1992). The representative low affinity AHR that we used was the human AHR that was cloned from the HepG2 hepatoma cell line (Dolwick et al., 1993a
). In vitro, the HepG2 AHR, like the mouse Ahd allele, has a 45-fold lower affinity for TCDD than does the mouse Ahb1 allele (Ema et al., 1994b
; Poland et al., 1994
).
Determination of the Kd for in vitro-expressed beluga, mouse, and human AHRs by saturation binding showed that the beluga AHR is a high affinity AHR. Absolute binding affinities are highly dependent on the protein concentrations used in the incubations, with apparent binding affinities decreasing with increasing protein concentrations (Bradfield et al., 1988). Therefore, Kd values are best compared among those determined under similar experimental conditions and protein concentrations. Table 3
shows several intralaboratory data sets for which Kds for mouse (Ahb1 or Ahd allele) and human AHRs were reported. When mouse and human AHR Kd values are compared within laboratories, the affinity of the mouse AHR (product of the Ahb1 allele) is greater than that of the human AHR in each case. Regardless of whether the samples were tissue cytosols, cell-line cytosols, or in vitro-expressed proteins, the human AHR Kd values were 3- to 8-fold greater than those of the mouse. The robustness of this difference in binding affinities between experimental conditions and laboratories indicates a real functional difference between the mouse and human AHRs. Our findings show a 2.4-fold difference in binding affinity between mouse and human AHRs and a nearly 4-fold difference between beluga and human AHRs (p < 0.05, Table 2
). Thus, the beluga AHR allele described here encodes a high affinity AHR.
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The Beluga AHR Binds DREs and Is Transcriptionally Active
To more completely assess the function of the beluga AHR, we examined its ability to participate in sequence-specific protein-DNA interactions with a murine DRE and to activate transcription of a reporter gene under control of a murine enhancer element containing multiple DRE sequences. These properties are characteristic of rodent and human AHRs (Burbach et al., 1992; Dolwick et al., 1993a
,b
; Fukunaga et al., 1995
; Whitelaw et al., 1994
) but had not yet been examined in a cetacean or other marine mammal. The results of these assays clearly demonstrated that the beluga AHR is capable of high affinity, sequence-specific, and Arnt-dependent interactions with mammalian DREs, and that this receptor is able to activate transcription of target genes, presumably through the glutamine-rich region or other putative transactivation domains in the C-terminal half of this protein. The demonstrated functionality of the beluga AHR in vitro is consistent with evidence for inducible CYP1A1 expression in vivo (White et al., 1994
; 2000).
Significance of a High Affinity AHR in Cetaceans
Establishing a cause and effect relationship between contaminant burdens and disease in marine mammals is difficult. Because controlled dosing experiments in cetaceans are usually not feasible, a "weight of evidence" approach has been proposed for investigating the existence of contaminant-induced toxic effects in marine mammals (Ross, 2000). Currently, 4 lines of research are seen as contributing to this approach. These include (1) epidemiological and descriptive studies of wild populations, (2) mechanistic studies in laboratory rodents, (3) feeding studies in captive animals (possible only in limited cases), and (4) feeding studies involving rodents exposed to marine mammal diets or contaminated tissues.
Our results suggest a fifth type of data that can contribute to the weight-of-evidence approach: species-specific cloning and characterization of proteins involved in mechanisms of toxicity. This approach has been used recently to characterize the AHR and other receptors in nonmammalian species (Abnet et al., 1999; Karchner et al., 1999
, 2000
; Matthews and Zacharewski, 2000
), but has not previously been applied to marine mammals. Such data can provide a link between mechanistic studies in laboratory rodents and epidemiological findings in wildlife. In this context, our study has demonstrated that belugas possess an AHR that shares a high degree of sequence identity with other mammalian AHRs, shares key functional properties with these AHRs, and binds TCDD with an affinity that is at least as high as a high affinity AHR from a dioxin-sensitive strain of mouse.
It may be informative to compare the binding affinity of the in vitro-expressed beluga AHR with the concentration of AHR ligands in beluga tissues, keeping in mind that such comparisons are complicated by the many differences that exist between in vitro and in vivo systems. The concentrations of "TCDD equivalents" (TEQs) in livers of St. Lawrence belugas are in the range of 0.01 to 0.13 nM (Gauthier et al., 1998; Metcalfe et al., 1999
); Muir et al., 1996).3 Using the Kd of 0.43 nM for the in vitro-expressed beluga AHR (Table 2
) and extrapolating directly to whole liver, these TEQ concentrations would be predicted to result in 2% to 23% receptor occupancy. At such levels of AHR occupancy, some effects might be expected, especially if belugas possess "spare" AHR capacity as shown in other systems (Hestermann et al., 2000
).
The presence of a high affinity AHR in a cetacean is consistent with a role for the AHR and PHAHs in the toxic effects observed in environmentally exposed cetaceans. Additional factors that will influence the sensitivity of cetaceans to PHAHs include the PHAH-AHR structure-binding relationships and the tissue- and cell-specific pattern of AHR expression. These questions are currently being addressed using the cloned beluga AHR cDNA and probes derived therefrom (Jensen, 2000). Together, these results demonstrate that the use of in vitro-expressed proteins is a promising approach for addressing molecular and biochemical questions concerning PHAH toxicity in endangered or protected species, in which logistical and ethical concerns preclude testing in live animals.
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ACKNOWLEDGMENTS |
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NOTES |
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1 Present address: Boston University School of Public Health, R-405, 715 Albany St., Boston, MA 02118.
2 To whom correspondence should be addressed at the Biology Department, WHOI, Redfield 340, MS 32, Woods Hole, MA 02543. Fax: (508) 457-2134. Email: mhahn{at}whoi.edu.
The value of 0.01 nM TEQ was calculated directly from the data provided by Gauthier et al. (1998) for a single neonatal beluga from the St. Lawrence estuary. The value of 0.13 nM TEQ was calculated for adult male beluga from the St. Lawrence estuary, using the data of Muir et al. (1996), as follows. These authors reported blubber TEQ values of 1400 ng/kg lipid. Assuming equilibration of PHAHs across tissues on a lipid basis, and assuming that the liver of these animals contained 3% lipid as shown for other St. Lawrence beluga (Metcalfe et al., 1999), the hepatic concentration of TEQs in these beluga is estimated to be 42 ng/kg wet weight, which is equivalent to 0.13 nM TCDD.
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