The long-term goal of this research is to elucidate the underlying mechanisms by which phenolic compounds present in the human diet influence tumorigenesis. Ongoing studies demonstrates that the oxidative metabolism of alkylphenols by cytochromes P450 lead, in many cases, to the formation of quinoid products capable of covalent binding and/or free radical generation in cells. We now propose to extend this work to investigate the involvement of such reactive metabolites in tumor promotion utilizing the most thoroughly characterized model for probing mechanistic aspects of promotion in the lung. This system involves the enhancement of lung tumor development by chronic administration of the food additive butylated hydroxytoluene (BHT) to carcinogen-initiated mice. It has been demonstrated that metabolism of BHT in the target organ is necessary for promotion and it is known that BHT is converted to reactive quinoid metabolites in lung. These findings lead to the hypothesis that promotion depends upon two successive P450 catalyzed oxidations to the ultimate promoting species, a strongly electrophilic quione methide which alkylates one or more critical proteins leading to a disruption of growth control mechanisms. The following specific aims are proposed: (1) Determine the role of metabolism in the differential responsiveness of promotion- sensitive (B+) and promotion-resistant (B-) mice to the lung tumor promoter BHT. Conversion of BHT to a quinone methide and other reactive metabolites, as well as the detoxification of reactive metabolites, will be examined in lung tissues and cells from B+ and B- mice. (2) Investigate alkylation targets of a BHT-derived quinone methide in lung cells that directly or indirectly impair cell-cell signaling. Protein alkylation by a highly reactive quinone methide metabolite of BHT will be examined in cells isolated from the lungs of B= and B- mice, and in tumorigenic and non-tumorigenic cell lines derived from murine lung. Alkylation patterns will be compared by radiochemical and immunochemical methods and selected adducts identified by mass spectrometry and microsequencing. (3) Examine biochemical consequences and oxidative damage in lung cells exposed to reactive metabolites of BHT. Isolated cells from B+ and B- mice and cell lines will be treated with reactive quinoid metabolites of BHT to investigate cytotoxicity, oxidative damage, inhibition of mitochondrial function, and inhibition of enzymes involved in detoxification.
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