cDNA Cloning and Characterization of a High Affinity Aryl Hydrocarbon Receptor in a Cetacean, the Beluga, Delphinapterus leucas

• Published in: Toxicological sciences Vol. 64; no. 1; pp. 41 - 56
• Main Authors: Jensen, Brenda A., Hahn, Mark E.
• Format: Journal Article
• Language: English
• Published: Cary, NC Oxford University Press 01.11.2001
• Subjects: Amino Acid Sequence; Animal, plant and microbial ecology; Animals; Applied ecology; aryl hydrocarbon receptor; Base Sequence; beluga; Biological and medical sciences; Cell Fractionation; cetacean; Cloning, Molecular; COS Cells; dissociation constant; DNA, Complementary; Ecotoxicology, biological effects of pollution; Effects of pollution and side effects of pesticides on vertebrates; Enhancer Elements, Genetic; Fundamental and applied biological sciences. Psychology; Genes, Reporter; Humans; in vitro expression; Liver - chemistry; Liver - metabolism; Mammalia; Marine; Molecular Sequence Data; Polychlorinated Dibenzodioxins - metabolism; Protein Structure, Tertiary; Radioligand Assay; Receptors, Aryl Hydrocarbon - chemistry; Receptors, Aryl Hydrocarbon - genetics; Receptors, Aryl Hydrocarbon - metabolism; Sequence Alignment; species-specific; susceptibility; TCDD; Transcription, Genetic; Whales - genetics; Whales - metabolism; Nucleotide sequence; Pollutant behavior; Toxicity; Species specificity; Polycyclic aromatic compound; Delphinapterus leucas; Planar molecule; Marine environment; Persistence; Ah receptor; Vertebrata; Biological fixation; Mammalia; Halogenated hydrocarbon; Affinity; Cetacea; Molecular cloning
• ISSN: 1096-6080; 1096-0929
• DOI: 10.1093/toxsci/64.1.41

Some cetaceans bioaccumulate substantial concentrations of planar halogenated aromatic hydrocarbons (PHAHs) in their tissues, but little is known about the effects of such burdens on cetacean health. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related PHAHs cause toxicity via activation of the aryl hydrocarbon receptor (AHR), a member of the bHLH-PAS family of transcription factors. Differences in AHR structure and function are known to contribute to species-specific differences in susceptibility to PHAH toxicity. To ascertain the potential for PHAH effects in a cetacean, we characterized an AHR from the beluga whale,Delphinapterus leucas. The 3.2 kb cDNA encodes an 845-amino acid protein with a predicted size of 95.5 kDa. Overall, the beluga AHR shares 85% amino acid sequence identity with the human AHR and 75% identity with the mouse AHR Ahb–1allele. Beluga AHR protein synthesized in a rabbit reticulocyte lysate system demonstrated specific, high-affinity [3H]TCDD binding. Saturation binding analysis was used to compare the [3H]TCDD binding affinity of thein vitro-expressed beluga AHR with affinities ofin vitro-expressed AHRs from a dioxin-sensitive mouse strain (Ahb–1allele) and humans. The beluga AHR bound [3H]TCDD with an affinity (Kd= 0.43 ± 0.16 nM) that was at least as high as that of the mouse AHR (Kd= 0.68 ± 0.23 nM), and significantly greater than that of the human AHR (Kd= 1.63 ± 0.64 nM). In electrophoretic mobility shift assays, the beluga AHR exhibited sequence-specific, Arnt-dependent binding to a dioxin responsive enhancer (DRE). Upon transient transfection into mammalian cells, the beluga AHR activated transcription of a luciferase reporter under control of a DRE-containing fragment of the mouseCyp1a1promoter. These results show that in anin vitrosystem, the beluga AHR possesses characteristics similar to those of AHRs from other mammals that are considered sensitive to toxic effects of PHAHs. Together, these results demonstrate that the use ofin vitro-expressed proteins is a promising approach for addressing molecular and biochemical questions concerning PHAH toxicity in endangered or protected species.