CHARACTERIZATION OF HUMAN LIVER CYTOCHROMES P450

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The main goals of this research project are to identify human liver cytochromes P-450 involved in the activation of acetaminophen to a reactive intermediate. Studies in this proposal will focus on modulators of cytochrome P-450 catalyzed acetaminophen activation. We will examine the mechanisms involved in ethanol induction, 7,8 benzoflavone (ANF) stimulation and cimetidine inhibition. Ethanol potentiation of acetaminophen hepatotoxicity has been documented in both human and animal studies, suggesting that modulation by induction of the ethanol enzyme is the mechanism by which potentiation of toxicity occurs. In addition, modulators may be used as a means of identifying isozymes of cytochrome P-450. The use of ANF as a stimulator of acetaminophen activation has allowed us to identify the presence of at least two cytochromes P-450 involved in this reaction in human liver. In addition, we propose to purify the human liver cytochromes and prepare monoclonal antibodies which may be employed in defining which population of individuals may be more susceptible to hepatotoxicity from large doses of acetaminophen. The high degree of specificity exhibited by these antibodies will allow them to be used as specific inhibitors in the acetaminophen assay to verify the role of the cytochromes in the metabolism of acetaminophen. Using needle biopsy samples from individuals exposed to various quantities of ethanol for different lengths of time and subjecting the samples to immunoblots developed with the antibody to the human ethanol P-450, should provide information about induction and de-induction of the enzyme. Finally, we will examine whether human kidney microsomes contribute to acetaminophen activation and identify, by use of the monoclonal antibodies prepared to the liver cytochromes, the cytochromes P-450 which may be involved. Thus, these studies provide a two-fold purpose. First, in identifying human liver cytochromes P-450 that may produce activation of acetaminophen and mechanisms of modulating their metabolism to prevent hepatotoxicity and second, to determine the quantity of ethanol and duration of exposure required for induction of human liver ethanol P-450.