ACETAMINOPHEN--COVALENT BINDING AND HEPATOXICITY

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Acute overdosage with the antipyretic analgesic, acetaminophen (APAP) can cause severe, fatal hepatic necrosis. Hepatotoxicity is the result of APAP activation to an electrophile which covalently binds to liver proteins. Although, the relationship between APAP's protein covalent binding and toxicity is not clear, selective arylation of a 58 kDa APAP binding protein (58-ABP) is better associated with toxicity than is total covalent binding. The 58-ABP is the most prominent target detected with anti-APAP antibody in mouse and human liver during APAP hepatotoxicity. In mice, 58-ABP arylation was most closely associated with target tissue damage (liver, lung and kidney) in varied experimental paradigms. The arylated protein is also detected in mouse plasma after toxic APAP exposures, and this may mirror the detection of APAP-protein adducts in human plasma during poisoning. Our working hypothesis is that arylation of the 58-ABP is important for APAP-induced target organ toxicity. Whether is plays an initiating or protective role cannot presently be resolved. Recent results have raised new questions which are the basis of the proposed research. 1) Is there a threshold for 58-ABP binding which, if exceeded, is associated with toxicity? 2) Is the 58-ABP also targeted by other agents which bind covalently to proteins? 3) What is the toxicological significance of the appearance of arylated 58-ABP in plasma? The proposed research will extensively use anti-APAP and anti-58, a new antibody prepared against the 58-ABP. This will permit quantitative assessment of the existence of a covalent binding threshold. Antibodies will be developed against bound bromobenzene to permit comparison of its binding with APAP's and with bromobenzene toxicity. Immunoprecipitation with anti-58 will facilitate detection of 58-ABP binding by other xenobiotics. In vitro studies will determine if other agents bind at the same site on the 58-ABP and APAP, and culture and in vivo studies will reveal if such binding can cause toxic interactions during simultaneous exposures. We will also determine if the appearance of 58-ABP in plasma after APAP or other xenobiotics reflects cellular secretion of arylated protein or incidental loss as a result of cell death. Studies with agents which bind but do not cause toxicity will also be included to determine if such agents target the same proteins as the toxic compounds. Collectively such studies will provide new insight into the importance of selective 58-ABP arylation by APAP and other toxic chemicals for target organ toxicity. They will also determine if the appearance of 58-ABP in plasma can become a useful diagnostic marker for xenobiotic-induced target organ damage.