Abstract

The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) protein were evaluated in the Hepa 1c1c7 (Hepa-1) cell line by indirect immunofluorescence microscopy and Western blot analysis. Wild-type (WT) Hepa-1 cells stained for AhR show intense cytoplasmic fluorescence with minimal nuclear reactivity. WT cells treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show a time-dependent decrease in cytoplasmic AhR staining and a concomitant increase in nuclear fluorescence. WT cells stained for Arnt show nuclear fluorescence with minimal cytoplasmic reactivity, a pattern unchanged after TCDD treatment. Hepa-1 type II variants express normal levels of AhR but are defective in TCDD-mediated induction of cytochrome P4501A1. Type II variants stained for Arnt show reduced nuclear fluorescence, compared with WT cells, and express minimal levels of Arnt protein, as determined by Western blot analysis. Type II variants stained for the AhR show intense cytoplasmic fluorescence that becomes nuclear after TCDD treatment. Detailed evaluation by immunoelectron microscopy of the AhR and Arnt present in the nuclear compartment of WT cells shows that both proteins are uniformly distributed and do not appear to be associated with nuclear pores, membranes, or nucleoli. Western blot analysis of nuclei isolated from WT Hepa-1 cells fractionated with Nonidet P-40 shows that minimal levels of AhR or Arnt are retained in the nuclear fraction after TCDD treatment. Collectively, these results indicate that the unliganded AhR resides in the cytoplasm, Arnt is localized to the nucleus, and Hepa-1 cells defective in Arnt expression exhibit TCDD-mediated nuclear accumulation of the AhR.