Chronic alcohol consumption has been associated with the toxicity of a number of xenobiotics, especially those that require metabolic activation. The catalyst for the majority of the biotransformations has been suggested to be the ethanol-inducible isozyme of cytochrome P-450, which has been successfuly isolated and characterized from ethanol-treated rabbits and has been termed isozyme 3a. The long term goal of this proposal is to determine the role of isozyme 3a in alcohol-related toxicities developing a specific suicide substrate for the enzyme that can be used in vivo.
The specific aims of this proposal are: 1) to characterize the active site of isozyme 3a using chemical modification with group specific affinity labels; 2) to characterize the suicide inactivation of isozyme 3a with a series of possible substrates that will form reactive products when metabolized by the enzyme; 3) to compare the inactivation of rabbit liver isozyme 3a with the inactivation of an analogous isozyme in the rat, hamster, and mouse; and 4) to determine the specificity of the inactivation using microsomal suspensions and primary hepatocytes as an in vivo model system. The interaction of affinity labels with the active site will be determined spectrally and by competition with substrates for the enzyme. The residue(s) modified will be determined by amino acid analysis and sequence determination of peptides isolated by HPLC. Similar analysis will be done with effective suicide substrates. A series of potential suicide substrates will be examined that will yield Beta, Gamma-unsaturated ketones, Alpha-haloketones, diketones, or ketoaldehydes when oxidized by isozyme 3a. The metabolism of the substrates will be monitored by gas chromatography or HPLC of phenylhydrazone derivatives so that the effectiveness of the inhibition can be monitored by the partition ratio. The affinity of the substrates will be changed by increasing the hydrophobicity and including groups such as pyrazole and imidazole, which are good ligands for the heme. Since the majority of the in vivo effects of ethanol have been examined in other species, the modification of an analogous isozyme in rats, mice, and hamsters will be studied. The modified enzyme will be isolated by immunoaffinity chromatography with antibody to rabbit isozyme 3a and compared to the modified rabbit isozyme. The in vivo specificity of suicide substrates that were effective in vitro will be tested with primary hepatocytes since problems of distribution and metabolism by other tissues can be avoided.
