Promotion of mouse lung tumors by the antioxidant butylated hydroxytolune (BHT; 2,6,-di-tert-butyl-4-methyl phenol) has become a powerful system for understanding how a non-carcinogen enhances tumor growth in a internal organ. This model can test whether putative mechanisms of skin tumor promotion apply to a lung neoplasm which is analogous to a human cancer of dramatically increasing incidence. The BHT/lung tumors system is one of the few where metabolism of the promoter is requisite for function. We propose that stimulation of initiated lung cell proliferation is mediated by generation of electrophilic and/or free radical metabolites of BHT which directly or indirectly lead to altered signal transduction and/or selective cytotoxicity. The following three specific aims test this hypothesis. (1) We will further investigate the current assumption that the principal pathway for metabolic activation of BHT to a promoter involves hydroxylation of a tert-butyl group. We will study the formation of this metabolite, BHT-BuOH in vivo and in isolated Clara cells from the lungs of sensitive (U+B+) and resistant (U+B-) strains of mice, and in established lines of normal and neoplastic lung cells. Mechanisms of resistance to BHB by U+B- mice will be studied by testing the promoting activity of BHT-BuOH in these strains. (2) We will study the bioactivity of products generated from BHT-BuOH in lung microsomes and Clara cells isolated from U+B+ and U+B- strains, and by peroxidases. We will synthesize analogs of BHT-BuOH to elucidate the role of the side-chain hydroxyl group in stimulating the activity of its electrophilic metabolites, and evaluate deuterium isotope effects on the activity of BHT-BuOH. (3) The biochemical actions of BHT metabolites putatively related to tumor promotion we will investigate. This includes a detailed evaluation of the effects of reactive BHT metabolites on the Ca++messenger pathway, focusing on protein kinase C (PKC, Ca++dependent protease (calpain), and an 80K substrate for PKC (p80). We will examine perturbations in the concentration of isozymes of thee proteins and the degree of expression of their genes, using immunoblotting and Northern bloating techniques, following perturbation with BHT and its reactive metabolites of bronchiolar Clara cells isolated from BHT- responsive or insensitive strains of mice and in normal and neoplastic lines of cultured alveolar type 2 cells. The hypothesis that elective cytotoxicity mediate BHT-induced clonal expansion of urethane-initiated lung cells will be examined by measuring the effects of BHT and metabolites on cellular defense mechanisms and radical generation in isolated Clara cells. The possibility of volent binding to critical proteins such as PKC, calpain, or p80 by reactive metabolites of BHT will be examined.
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