ACETAMINOPHEN BINDING/HEPATOCYTE HOMEOSTASIS-TOXICITY

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Acetaminophen (APAP) is a widely used, safe, antipyretic analgesic which, when taken in excessive doses, causes severe hepatic necrosis. Hepatotoxicity is the result of microsomal activation of APAP to an electrophile which first depletes glutathione reserves and then covalently binds to critical cell components. The mechanism whereby APAP or its metabolite initiate and perpetuate the pathological sequence which results in cell death remains unknown. Recent studies with APAP have demonstrated selective early covalent binding to a single microsomal protein band. APAP also resulted in ultrastructural and biochemical changes in endoplasmic reticulum, mitochondria, lysosomes and plasma membrane. The relationship between the selective arylation of proteins and the observed cellular effects of APAP has not yet been established. The proposed studies will focus upon the nature and importance of covalent binding to specific proteins in APAP-induced hepatotoxicity. This will be addressed by studying the relationships among 1) the modification of specific proteins by selective covalent binding and/or oxidation, 2) the alterations in specific organelle functions, and 3) the initiation and progression of the hepatotoxicity. The design will involve coordination of studies of mouse hepatocytes, in culture with in vivo studies in mice. We have developed an affinity purified antibody which is specific for covalently bound APAP. It will be used extensively in the proposed studies to permit identification of organelle proteins to which APA binds and determination of the relationship between APAP's specific binding and the alterations in homeostatic processes as well as the relationship of the alterations to the hepatotoxicity. Liver damage will be verified biochemically, histopathologically and ultrastructurally. The degree of toxicity will be varied through careful selection of APAP dosage and time combinations as well as through experimental enhancement and antagonism. This will permit assessment of the importance of covalent binding and altered protein sulfhydryl levels to the derangement of specific organelle functions in APAP hepatotoxicity. Clarification of the nature of the APAP induced changes and identification of the altered homcostatic functions which are critical to the toxic process will permit a clearer understanding of the biochemical mechanisms underlying the hepatotoxicity of APAP and similarly acting hepatotoxins. This will facilitate the eventual development of rational alternative therapies for its prevention.