The skin is one of the major interfaces between the body and its environment. Prior research has focused predominantly on the passive """"""""barrier"""""""" function of cutaneous tissue, but for the past decade this laboratory has been studying the skin's barrier function from another viewpoint to test the hypothesis that skin is an active """"""""barrier"""""""" with cytochrome P-450 dependent enzyme activity that can alter the chemical structure and biological activity of endogenous and exogenous substrates. Recently, numerous techniques have been developed for the isolation of P- 450 which are applicable to skin. In this proposal, it is planned to further characterize the P-450 system in mammalian skin. Microsomal fractions will be prepared, solubilized, purified and reconstituted for in vitro assays to define substrates and inducers of the skin P-450 system. Elutriation techniques will be used to isolate subpopulations of epidermal cells for similar studies. P- 450 epidermal isozymes will be resolved by an HPLC methods andy by monoclonal antibodies (MAbs) directed against highly purified hepatic P-450 isozymes. The MAbs will be used to immunopurify the epidermal isozymes which will be further characterized by peptide mapping and NH2-terminal sequence determination. A DNA probe for the hepatic isozyme P-450c which we have shown to be present in epidermal microsomes will be employed to study gene expression of epidermal P-450. P-450 metabolism of the endogenous substrates leukotriene B4 (LTB4) and testosterone will be assessed since P-450-dependent enzymes covert them to inactive metabolites in other tissues. It is then planned to determine whether there is deficient LTB4 metabolism in psoriasis. It is known that specific P-450 isozymes transform inert polyaromatic hydrocarbons (PAHs) such as benzo-(a)pyrene (BP) into reactive metabolites that bind to DNA to initiate cancer. It is planned to study epidermal P-450 isozymes that can activate procarcinogenic PAHs; to use HPLC techniques to define the pattern of P-450 metabolism of PAHs and to assess DNA- adduct formation and repair in a series of coordinated comparative studies in cultured keratinocytes (Balb/C mouse and human), organ cultured epidermis (Balb/C mouse and human) and in an experimental model system whereby human skin can be transplanted to athymic nude Balb/C mice. In aggregate, these studies are designed to broaden the base of existing knowledge concerning the capacity of the skin to metabolize xenobiotics and to define the consequences of such catalytic activity.
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