An important goal for toxicology is the prediction of the toxic effects of environmental contaminants. The most commonly used criteria for assessment of this risk is carcinogenicity. Many Compounds are activated to carcinogens by oxidation to a reactive intermediates which then bind to DNA, interfering with replication of critical sequences and leading to eventual death (genotoxicity). Many different indirect tests are utilized to detect this genotoxic effect. About one third of compounds found to be noncarcinogenic are genotoxic in vitro. This discrepancy is attributed to oxidative metabolism that occurs in the rodent test animals. Understanding this metabolic oxidation could play an important role in eliminating concerns about genotoxic compounds as potential carcinogens. Conversely knowledge of oxidative metabolism could also play an important role in predicting which xenobiotics will be activated to carcinogens. This application addresses the need for a better understanding of how chemicals interact with enzymes of oxidation (cytochrome P450s). Knowledge of cytochrome P450 interaction. when coupled with structural information of known mutagens and carcinogens (structural alerts), will serve as essential information for predicting oxidative interactions. We propose to produce recombinant human cytochrome P450s that have been modified to display easily measured characteristics (i.e. serve as reporters) when incubated with compounds of unknown toxicological properties. To serve as reporters the cytochrome P450 proteins will be engineered to be expressed as fusions with either a reductase subunit or an affinity binding domain. Using these recombinant fusion enzymes, spectral assay systems will be developed to measure their interaction with unknown compounds. The interactions to be examined will be: 1.) Binding, 2.) Ability to serve as a substrate and 3.) Inhibition of substrate oxidation. Initially assays will be developed and tested to be performed in a spectrophotometer at visible wavelengths. Further refinements will be made to create a system that can be performed with the use of a microtiter plate reader. The ultimate application of this proposal would be the production of a series of microtiter plate assays containing a series of cytochrome P450s that will rapidly produce a profile of any series of compounds. The profile of a given compound could include: what form(s) of cytochrome P450 it bound to and with what affinity; what form(s) were inhibited by the compound; and what forms the compound was metabolized by. This information will be summarized as a metabolite alert profile (MAP) that can be integrated into existing databases of genotoxicity and carcinogenicity. We will utilize bacterial expression technology to produce a panel of several important human cytochrome P450s in large quantities: 1. CYP1A1; 2. CYP1A2; 3. CYP2G8 and CYP2G9; 4. CYP2D6; 5. CYP2E1; 6 CYP3A4; 7. CYP3A5. We anticipate the future development of additional human cytochrome P450s for these assays (In particular the GYP2A6, CYP2B6, CYP2C18, CYP2C19 cDNAs and their variants as well as GYP2D6 variants). As progress develops in the field of human cytochrome P45O genetic research and new genes and allelic variants are identified the panel of recombinant cytochrome P450s will be increased.
