ToxSci Advance Access originally published online on November 25, 2008
Toxicological Sciences 2009 107(2):440-450; doi:10.1093/toxsci/kfn240
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Recombinant Transthyretin Purification and Competitive Binding with Organohalogen Compounds in Two Gull Species (Larus argentatus and Larus hyperboreus)
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* National Wildlife Research Centre, Carleton University, Ottawa, Ontario, K1A 0H3, Canada
Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada
Department of Biology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway
Norwegian Polar Institute, Tromsø NO-9296, Norway
1 To whom correspondence should be addressed at Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada. Fax: (613) 998-0458. E-mail: robert.letcher{at}ec.gc.ca.
Received July 11, 2008; accepted November 14, 2008
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Abstract |
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Glaucous gulls (Larus hyperboreus) from Svalbard, Norway (marine), and herring gulls (Larus argentatus) from the Laurentian Great Lakes (freshwater) of North America are differentially exposed to persistent and bioaccumulative anthropogenic contaminants, such as polychlorinated biphenyls (PCBs) and polybrominated diphenyl ether (PBDE) flame retardants and metabolic products. Such compounds can potentially perturb hormone transport via binding interactions with proteins such as transthyretin (TTR, prealbumin). In this present study, we isolated, cloned and sequenced TTR cDNA from the brain and liver of two species (herring and glaucous gull), which, to our knowledge, is the first report describing the TTR nucleic acid and amino acid sequences from any gull species. Identical TTR nucleotide and amino acid sequences were obtained from both gull species (liver and brain). Recombinant TTR (rTTR) was expressed and purified, and determined as a monomer of 18 kDa and homodimer of 36 kDa that putatively is comprised of the two protein monomers. Concentration dependent, competitive TTR-binding curves with each of the natural TTR ligands 3,5,3'-triiodothyronine (T3) and thyroxine (T4) were generated as well as by treatment with a range of concentrations (10–3–105nM) of 2,2',3,4',5,5',6-heptaCB (CB187), 2,2',4,4'-tetrabromoDE (BDE47), and hydroxyl- (OH) and methoxyl (MeO)-containing analogs (i.e., 4-OH-CB187, 6-OH-BDE47, 4'-OH-BDE49, 4-MeO-CB187, and 6-MeO-BDE47). Relative to the nonsubstituted BDE47 and CB187 and their MeO-substituted analogs, the OH-substituted analogs all had lower Ki and Kd values, indicating greater affinity and more potent competitive binding to both T3 and T4. The OH-substitution position and/or the diphenyl ether substitution of the four bromine atoms resulted in more potent, greater affinity, and greater relative potency for 4'-OH-BDE49 relative to 6-OH-BDE47. CB187 was more comparable in binding potency and affinity to 4-OH-CB187, then was 6-OH-BDE47 and 4'-OH-BDE49 relative to BDE47 where the binding potency and affinity was several orders of magnitude greater for 6-OH-BDE47 and 4'-OH-BDE49. This indicated that the combination of the more thyroid hormone–like brominated diphenyl ether backbone (relative to the chlorinated biphenyl backbone), and in combination of having an OH-group, results in a more effective competitive ligand on gull TTR relative to both T3 and T4. Known circulating levels of 4-OH-CB187, 6-OH-BDE47, and 4'-OH-BDE49 in the plasma of free-ranging Svalbard glaucous gulls were comparable to the concentration of in vitro competitive potency of T3 and T4 with gull TTR. These results suggest that environmentally relevant and selected OH-containing PCB, and to a lesser extent PBDE congeners have the potential to be physiologically effective in these gull species via perturbation of T4 and T3 transport.
Key Words: thyroid hormones; Larid birds; transthyretin cloning and expression; competitive binding; PCBs; PBDEs and metabolites.
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