The hepatic mixed-function oxidase system with cytochrome P-450 (P-450) as the terminal oxidase plays a major role in detoxifying and toxifying a wide variety of drugs and environmental pollutants, and in hydroxylating endogenous substrates such as steroids and fatty acids. It plays a key role in chemical carcinogenesis, pharmacology, and toxicology. The long term objective of these studies is to understand the function and control of P-450, to utilize its beneficial capabilities and prevent its hazardous activity.
The specific aims are to determine the basis for functional differences between P-450 isozymes, particularly the role of the microsomal milieu and isozyme conformation, and the multiplicity of P-450s in animals and humans. The overall approach is to use a novel, well developed analytical method, where the metabolism of the R and S enantiomers of the anticoagulant drug warfarin to multiple metabolites, together with the inhibition of this metabolism by antibodies against specific P-450 isozymes, will be used as a probe of P-450 isozyme function and composition. Functional studies will be performed on five highly purified rat liver P-450 isozymes. Specific aspects of the role of the microsomal environment which will be studied are the role of lipid vesicles, cytochrome b5, NADPH-cytochrome P-450 reductase, and mixtures of P-450 isozymes. Conformational modifications will be mediated by detergents, urea, thermally and by chemical modification of amino acid residues, and will be monitored by circular dichroism and differential scanning microcalorimetry. Effects of conformational changes on function will be monitored by warfarin metabolism. Multiplicity will be studied in monkey, guinea pig, pig and human liver and compared with previous results with rats, rabbits, and mice. Multiplicity of P-450 will be assessed by comparing P-450 isozyme compositions in these animals and in man based on the regio- and stereoselectivity of warfarin metabolism, its specific inhibition by antibodies to P-450 isozymes, and by immunoelectrophoretic methods. Interindividual differences between human P-450 isozymes will be assessed. Two major benefits related to human health will be the development of a unique noninvasive method for determining human liver P-450 isozyme compositions, and the development of the basis for evaluating extrapolations of animal toxicology data from animals to humans.
