The chronic consumption of alcohol is known to be associated with the toxicity of numerous foreign compounds, including drugs, that undergo metabolic activation by cytochrome P-450. This laboratory purified and characterized the ethanol-inducible form of the cytochrome (termed P-450 isozyme 3a on the basis of eleccrophoretic behavior or P-450ALC to indicate high activity in alcohol oxygenation) and has since shown the central role of this catalyst in alcohol-related toxicities.
The specific aims of this proposal are: (a) to complete the characterization of various forms of P-450's involved in alcohol oxidation, including hepatic isozyme 3a, a non-inducible but immunochemically similar cytochrome in nasal membranes, and a variant nasal cytochrome having different electrophoretic behavior and much higher catalytic activities; (b) to determine the mechanism(s) by which diverse agents and treatments cause induction of P-450ALC; and (c) to determine whether the variable responses of different animals to the inducing effects of ethanol can be predicted. The purified enzymes will be sequenced and examined for post-translational modifications, and an attempt will be made to correlate substrate specificity with any structural differences found. Other organelles such as mitochondria and nuclei, and other tissues such as esophagus, stomach, aorta, endocrine tissues, and brain will be examined for interesting new forms of P-450ALC. Variants will also be identified by screening of an appropriate cDNA library. Determination of the level(s) of which P-450ALC biosynthesis is regulated will involve measurement of rates of transcription in isolated nuclei and quantitation of intranuclear precursors and cytoplasmic mRNA transcripts using cloned cDNA as a hybridization probe, as well as quantitation of cellular enzyme levels by radioimmunoassay. The P-450ALC genes will be identified and characterized by restriction mapping, Southern blotting, and sequence determination, and regulatory elements will be identified in the f'-flanking region or elsewhere. With an improved understanding of P-450ALC variants and of the control of P-450ALC gene expression in various tissues, attempts will be made to show by the techniques of molecular genetics whether an animal has been chronically exposed to ethanol and to predict whether a particular animal will be highly induced by such exposure.
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