Induction of aryl hydrocarbon hydroxylase and demonstration of a specific nuclear receptor for 2,3,7,8-tetrachlorodibenzo- p-dioxin in two human hepatoma cell lines

Abstract

Two established human hepatoma cell lines, Hep3B and HepG2, were examined for aryl hydrocarbon (benzo[ a]pyrene) hydroxylase (AHH) induction and for the presence of the murine-equivalent aromatic hydrocarbon ( Ah) receptor. Both cell lines demonstrated polycyclic aromatic hydrocarbon (PAH)-induced AHH activity; however, assay conditions for induction were different than those established for the control mouse hepatoma cell line, Hepa c1–9. When cytosols from either cell line were exposed to tritiated 2,3,7,8-tetrachlorodibenzo- p-dioxin ([ 3H]TCDD) and analyzed on sucrose gradients with or without prior charcoal treatment, two peaks were observed at positions corresponding to 4–5 S and 8–9 S. The 8–9 S peak was identified as the probable human Ah receptor equivalent since, like the mouse Ah receptor, this peak: (a) was eliminated only by cytochrome P 1-450 inducers; (b) was sensitive to protease digestion; and (c) was thermolabile. Levels of TCDD specifically bound in the 8–9 S peak for HepG2 and Hep3B were 27 and 34 fmol/mg cytosolic protein respectively. The level of TCDD specifically bound was not affected by charcoal treatment or by the addition of sodium molybdate, which is known to stabilize ligand binding to steroid receptors. Incubation of Hep3B or HepG2 cells with [H]TCDD at 37° for 1 hr effected a redistribution of binding from the cytosol 8–9 S peak to a nuclear 6 S peak. The nuclear peaks from both human cell lines demonstrated similar sedimentation properties, temperature-dependence and inducer-specificity, as for the mouse nuclear Ah receptor. Appearance of nuclear 6 S binding is consistent with a temperature-dependent translocation process, supporting the observation that these human hepatoma cell lines contain a binding component which is similar to the mouse Ah receptor in structure and function during AHH induction.