The first long term goal of this proposal is to provide a firm foundation for the use of furafylline in vivo and in vitro as a highly specific probe for studying the role of human cytochrome P450 1A2 in chemical carcinogenesis and toxicity and in drug metabolism. Human cytochrome P450 1A2 is a major constitutive liver enzyme whose levels are induced by exposure to environmental agents such as drugs and constituents of cigarette smoke. Cytochrome P450 1A2 plays an important role in the bioactivation of chemicals and environmental contaminants to mutagenic and carcinogenic compounds. Preliminary data indicates that furafylline is a mechanism based inhibitor (suicide substrate) for 1A2 and an ineffective inhibitor of most other major forms of human hepatic P450. The mechanism and specificity of furafylline will be confirmed and extended in this proposal. Further studies using promutagenic agents activated by human liver preparations to mutagens will be conducted using furafylline and the Ames mutagenicity assay to specifically identify the contribution of 1A2 activation to these processes. The second major goal of this proposal is to make use of furafylline's inhibitory properties to investigate the role of 1A2 in the bioactivation of the potent hepatic carcinogen aflatoxin. Aflatoxin contamination of the human diet is believed to be an important causative agent in human liver cancer, especially in Asia and Africa, and estimates of 250,000 deaths per year have been attributed to it's toxic effects. The mutagenic and carcinogenic properties of aflatoxin appear to be due to activation by hepatic cytochrome P450 isozymes to the 8,9 epoxide metabolite which binds to DNA- Considerable confusion exists over which forms of P450 are involved in the initial premutagenic epoxidation event. Preliminary in vitro data within this application using furafylline as an inhibitor of 1A2 confirms that multiple P450 isoforms are involved in epoxidation. Furthermore, the relative importance of each P450 isoform on aflatoxin epoxidation appears to be dependent on the concentration of aflatoxin. The striking and highly significant observations were made that (1) furafylline almost completely inhibits microsomal epoxidation at low concentrations of aflatoxin and (2) the formation of other major non- mutagenic aflatoxin metabolites is not inhibited by furafylline. Proposed studies designed to confirm these findings may also provide a rationale for the use of furafylline as a safe and potent chemoprotective agent against toxicities caused by metabolic processing of environmental contaminants such as aflatoxin by cytochrome P450 1A2.
