Detection and characterization of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin binding to Ah receptor in a rainbow trout hepatoma cell line.
Ah receptor was identified and characterized in cytosol and nuclear extracts from the rainbow trout hepatoma cell line RTH-149. The cytosolic receptor was detectable with both halogenated ([3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) and non-halogenated ([3H]3-methylcholanthrene and [3H]benzo[a]pyrene) aromatic hydrocarbons and sedimented at approximately 9 S after velocity sedimentation on sucrose gradients. The apparent binding affinity (kd) of cytosolic Ah receptor was always less than 1 nM as derived from Scatchard or Woolf plot analyses. The same analyses indicated a concentration of Ah receptor in the RTH-149 cells of approximately 20 fmol/mg cytosolic protein or approximately 4400 receptor sites per cell. Thus, this trout hepatoma cell line has a low concentration of high-affinity binding sites in comparison to Ah receptor concentrations in cytosol obtained from rodent tissues. Incubation of whole cells with the radioligand [3H]TCDD resulted in transformation of the cytosolic Ah receptor to a nuclear binding form which could be detected as a specifically labeled peak sedimenting at approximately 6 S on sucrose gradients. Aryl hydrocarbon hydroxylase was induced after exposure of RTH-149 cells to TCDD or benz[a]anthracene for 24 hr in culture. These data demonstrate the existence of the Ah receptor in a cell line derived from a nonmammalian species and provide an additional step toward understanding the mechanisms by which fish respond to specific aquatic contaminants.
Lorenzen
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Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada.
Okey
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D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000096:biosynthesis; D003600:Cytosol / Q000737:chemistry; D004790:Enzyme Induction / Q000187:drug effects; D008114:Liver Neoplasms, Experimental / Q000737:chemistry*; D008982:Molybdenum / Q000494:pharmacology; D000072317:Polychlorinated Dibenzodioxins / Q000378:metabolism* / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug / Q000032:analysis* / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000032:analysis; D014337:Trout / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011955:Receptors, Drug; D011965:Receptors, Glucocorticoid; D008982:Molybdenum; C024687:sodium molybdate(VI); D001189:Aryl Hydrocarbon Hydroxylases