Detection and characterization of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin binding to Ah receptor in a rainbow trout hepatoma cell line

Abstract

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.