text: Butylated hydroxytoluene (BHT), a widely used food additive, enhances the development in mice of lung tumors structurally similar to bronchiolo-alveolar tumors in man. We will investigate the mechanisms of this stimulation of tumor multiplicity using research tools developed during the previous grant period. First, evidence indicates a central role of BHT metabolism in tumor promotion. Metabolism in mouse lung homogenates and in lung epithelial cells will be compared 1) in strains of mice that differ in their sensitivity to BHT promotion, 2) in mice treated with agents that alter the pulmonary effects of BHT, and 3) with structurally analogous phenols that do or do not promote lung tumors. These results should enable us to implicate a specific metabolic pathway or pathways in promotion. Second, the biochemical mechanism by which BHT causes enhanced tumor growth will be studied by focusing on BHT-induced changes in Ca++/phospholipid-dependent protein kinase C (Pk-C) and Ca++- dependent protease (calpain) activities, and on the Pk-C catalyzed phosphorylation of an endogenous lung protein substrate (p36). The hypothesis that subcellular translocation and subsequent activation of both Pk-C and calpain follows BHT biotransformation will be, tested using immunoblotting, immunocytochemistry, and partial purification procedures. Correlative tests to evaluate the role of Pk-C catalyzed phosphorylation in promotion will be based on 1) the use of strains of mice susceptible to lung tumor; induction by urethan but resistant to tumor promotion by BHT (U+B- phenotype), 2) an analog of BHT which causes lung damage but does not promote tumors, and 3) an agent which perturbs BHT metabolism without affecting tumor promotion. Third, the lung cells which actually give rise to tumors--the alveolar type 2 cells and bronchiolar Clara cells--will be directly examined to further elucidate the biochemistry of tumor promotion. Clara cells will be isolated from U+B+ and U+B- strains of mice following, treatment with BHT-or BHT analogs, and aspects of the p36 phosphorylation system will be examined to genetically distinguish among biochemical mechanisms in the cells of specific interest. Isolated Clara cells and established lines of both normal and neoplastic type 2 cells will be exposed to BHT metabolites putatively involved in promotion; effects on the p36 phosphorylating system, oxy-radical generation, and cellular protective mechanisms will be examined.