2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the archetype of a large class of polyhalogenated aromatic compounds that on exposure to human populations evoke a variety of common toxic responses. A sensitive marker of human exposure to TCDD is chloracne, a condition characterized by a thickening of the epidermis, hyperkeratosis, and squamous metaplasia of the epithelial lining of sebacious glands. The long term goal of the proposed research is to determine the biochemical basis underlying the toxicity to human skin evoked by exposure to TCDD. Because the result of this exposure is an alteration of the regulated patterns of epidermal cell proliferation and differentiation programs, emphasis is placed on the actions of TCDD on known biochemical regulators of these processes. These studies will employ a continuous cell line of human keratinocytes as a human model system for the action of TCDD and related dioxins.
Specific aims that are proposed in this project include (1) The determination of the mechanism of increased glucocorticoid binding to specific intracellular receptors in keratinocytes exposed to TCDD. An examination of whether protein synthesis is required to account for the putative two fold in dexamethasone binding will be made. It will be determined whether de novo synthesis of glucocorticoid receptors occurs as a result of TCDD exposure, or activation of pre-existing receptors takes place after cells are challenged with dioxin. These experiments will be performed using specific labelling techniques to examine for protein synthesis and post translational modification. (2) Determination of the mechanism by which TCDD treatment results in """"""""down regulation"""""""" of beta-adrenergic receptors and adenylate cyclase in human keratinocytes. Exposure of human keratinocyte cultures results in a decrease in stimulated adenylate cyclase activity in the presence of isoproterenol, but not unstimulated activity. This action is mimicked by Gpp(NH)p, but does not appear to involve the activie site directly. (3) The human keratinocyte cell line will be validated as a cogent model system by examining the actions of TCDD on selected actions in normal keratinocyte cultures. Emphasis will be on experimerts that will examine whether dioxin and glucocorticoid hormone receptors act in a synergystic fashion to induce cytochrome P450 activity, and to determine whether these receptors interact as reflected in altered binding patterns of steroid hormone to its receptor. The results of these studies will yeild new information and insight into the biochemical basis of the mechanism of toxic action of TCDD and dioxins in humans.
