Steroid hormones, drugs, and certain environmental contaminants alter gene expression in target cells. This project is focused on identification of the molecular mechanism by which glucocorticoid hormones and polycyclic aromatic compounds induce distinct species of cytochrome P-450 in mammalian liver. Glucocorticoids and polycyclic aromatic compounds bind to distinct intracellular protein "receptors;" the ligand-receptor complexes interact with DNA in the cell nucleus and appear to affect directly the transcription of cytochrome P450 genes. The major advantage of studying glucocorticoids and polycyclic aromatic compounds is that mutants with defective receptors can be isolated for both systems. During the past year, we have concentrated on the isolation and characterization of mouse hepatoma cell culture mutants that are defective in induction of cytochrome P1-450 by polycyclic aromatic compounds. With the use of mutants that have markedly decreased Ah receptor levels, we have developed and validated a new, rapid assay for the Ah receptor involving anion-exchange HPLC. We have defined the experimental conditions under which [3H]2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) Ah receptor complexes bind to DNA: these studies should provide the basis for purification of the Ah receptor and analysis of its interaction with the P1-450 gene. Human lymphoid cells resistant to glucocorticoid hormones have been analyzed with the use of antibodies to human glucocorticoid receptors. The defective receptor moieties in certain cases can be identified by immunochemical methods. The region affected by mutation can be "mapped" by affinity labeling and limited proteopysis. We have isolated affinity-labeled glucocorticoid receptor with the use of a monoclonal antibody. Although the N-terminus of the receptor is blocked, fragments of the receptor prepared by limited proteolysis may be suitable for determining amino-acid sequence. These data are providing detailed information about the structure of the glucocorticoid receptor.