2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the archetype of a large class of polyhalogenated compounds that evoke a variety of common toxic responses on exposed human populations. The long term goal of the proposed research is to determine the biochemical basis underlying the toxicity evoked by exposure to TCDD. Many of the toxic responses of cells exposed to TCDD involve modification of normal patterns of epithelial cell proliferation and differentiation. Because the result of exposure to TCDD is an alteration of the regulated patterns of epidermal cell proliferation and differentiation programs, emphasis is placed on the actions of TCDD as a modulator of known signal transducing systems that regulate epidermal cell growth. The proposed studies will use primary, and continuous cultures of human keratinocytes as the model system, in which to study TCDD toxicity.
Specific Aims that are proposed for this project are: (1) Determine the mechanism of modulation of growth factor synthesis in keratinocytes exposed to TCDD. Preliminary experiments have shown that levels of transforming growth factor-alpha (TGF-alpha) in the culture medium of normal keratinocytes (HuE) and immortalized, but non-tumorigenic (SCC 12F) cultures increases significantly after the cells are exposed to TCDD. Experiments will be conducted to determine whether synthesis of the peptide growth factor increases, or secretion of already synthesized TGF- alpha is modulated after exposure to TCDD. (2) Determine whether dioxins modulate the rate of synthesis or decrease the rate of degradation of TGF- alpha mRNA. Nuclear runoff transcription analysis will be conducted using a cDNA probe specific for TGF-alpha to analyze the accumulation of TGF- alpha-specific mRNA in HuE and SCC 12F human keratinocyte cells exposed to TCDD. (3) Determine whether TGF-alpha is the only inducible component responsible for TCDD-modulated toxicity, and whether other components in medium from keratinocyte cultures exposed to dioxins modulate binding of TGF-alpha to the EGF receptor. Presence of TGF-beta and other cytokines in the medium from TCDD-exposed cultures will be determined using western immunoblot analysis. Competition binding experiments will be undertaken to determine whether components other than TGF-alpha are present in the medium from cultures exposed to TCDD that will transmodulate EGF receptor in keratinocytes exposed to dioxins.
