The Specific Aims of this proposal are to elucidate the mechanism by which phenolic antioxidants and certain natural products; namely, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), 5,6-benzoflavone, and certain coumarin derivatives effectively inhibit monooxygenase activity in vitro and in vivo. These agents are known to be effective cancer chemoprevention agents and past work has indicated that they may exert their effect at the level of the cytochrome P-450-dependent monooxygenases required for metabolic activation of a number of carcinogens. Some evidence exists to indicate that certain metabolites of these cancer chemopreventive agents serve as redox-active agents which can prevent electron transfer to cytochrome P-450, but upon reduction by NADPH-cytochrome P-450 reductase can reduce molecular oxygen to hydrogen peroxide (i.e., stimulate NADPH oxidase activity). Standard chromatographic and analytical chemistry methods will be used to elucidate whether redox-active agents account for the action of these compounds or whether they directly interact with the monooxygenases. Integration of this information with the known biochemical mechanisms of the enzyme (heme reduction, oxygen activation, alteration of oxidase/oxygenase activity) will provide the conceptual framework for understanding their mechanism of action. For those compounds which inhibit monooxygenase activity, but do not stimulate the NADPH-oxidase activity of microsomes, we will establish how they affect the biochemical mechanism of cytochrome P-450. Subsequently, assays for monooxygenase function with carcinogens (metabolism, DNA or protein adduct formation, DNA damage) in the presence of these agents will be performed in more biologically integrated systems, such as isolated liver or lung cells to test the hypothesis of their mechanism of action. Such information may allow the design of therapeutic regimens to protect against the effects of many chemical carcinogens.
