A COMPARATIVE-STUDY OF MOUSE-LIVER PROTEINS ARYLATED BY REACTIVE METABOLITES OF ACETAMINOPHEN AND ITS NONHEPATOTOXIC REGIOISOMER, 3'-HYDROXYACETANILIDE

Acetaminophen (4'-hydroxyacetanilide), a widely used analgesic/antipyr etic drug, is hepatotoxic in large doses, whereas the m-hydroxy isomer of acetaminophen, 3'-hydroxyacetanilide, is not hepatotoxic. Both are oxidized by mouse liver cytochromes P-450 to reactive metabolites tha t bind covalently to hepatic proteins. Because previous studies have s hown that peak levels of liver protein adducts formed after the admini stration of each of these compounds to mice are nearly equivalent, and because liver protein adduct formation correlates with hepatotoxicity caused by acetaminophen in mice, we investigated the abundance and pa tterns of protein adducts formed by acetaminophen and its regioisomer for significant differences. Hepatotoxic doses of acetaminophen to mic e significantly altered the abundances of several liver proteins 2 h a fter dosing as revealed by densitometric analysis of two-dimensional e lectrophoretic patterns of these proteins. The same analysis after the administration to mice of 3'-hydroxyacetanilide indicated that this n onhepatotoxic regioisomer of acetaminophen caused several similar chan ges in protein patterns, but also revealed some significant difference s. Binding of radiolabeled acetaminophen and 3'-hydroxyacetanilide to hepatic proteins corroborated and extended these results. Two hours af ter the administration of C-14-labeled analogs of these two compounds to mice, at a time when their extent of total covalent binding to hepa tic proteins is approximately equivalent, there are many similarities but also some differences in selectivity of proteins that are adducted , as revealed by both one-dimensional and two-dimensional gel electrop horesis followed by phosphorimage analysis of radiolabel bound to prot ein bands. Moreover, protein adducts formed from 3'-hydroxyacetanilide were found to be less stable than those formed from acetaminophen und er the conditions of electrophoretic analysis. Furthermore, a comparis on of radiodetection and immunodetection of protein adducts formed fro m acetaminophen with an antibody specific for acetaminophen protein ad ducts indicates that the antibody detects most of the same proteins th at are radiolabeled and that the relative quantitative contribution of various adducts to the overall covalent binding of acetaminophen to p roteins is approximately the same by both methods. Thus, 3'-hydroxyace tanilide should prove to be a useful tool to aid in the discrimination of hepatic acetaminophen protein adducts that may be critical or nonc ritical to survival of hepatocytes.