Ah-receptor-dependent modulation of gene expression by agedand diluted sidestream cigarette smoke

Abstract

Cigarette smoke is known to induce cytochrome P4501A1 expressionand activity in a variety of species. Although the elevation of this isozyme is assumed to be associated with the activation of the CYPlAl gene through a ligand-mediated mechanism involving the Ah-receptor (AhR), this bas not been determined. In this study we have examined the mechanism by which an ambient level of aged and diluted sidestream cigarette smoke (ADSS) induces cytochrome P4501AL Effects of ADSS on C57BL16N and DBA/2N mice were examined. Induction of P4501A1-associated ethoxyresorufin-O-dealkylase (EROD) activity was observed in the lungs of C57BL/6N mice, while there was no induction in DBA/2N mice. ADSS also induced EROD in wild-type mouse hepatoma (Hepa1c1c7) cells (hepal), but not in variant hepal cells defective in the AhR nuclear translocator (ARNT) protein. ADSS exposure of recombinant hepal cells, stably transfected with a reporter plasmid containing the luciferase gene under control of several dioxin responsive enhancers (DREs), resulted in a time- and exposure-dependent induction of luciferase activity. ADSS-mediated induction of luciferase activity was inhibited by a-naphthoflavone (αNF), an Ah-receptor antagonist. Gel retardation analysis demonstrated that exposure to ADSS induced transformation and DNA binding of the AhR complex. In summary, our results not only indicate a role for the AhR in mediating the induction of P4501A1 by ADSS, but also demonstrate that environmentally relevant levels of ADSS must contain AhR ligands at sufficient concentrations to activate gene expression in an AhR-dependent manner.