ACETAMINOPHEN-ARYLATED PROTEINS ARE DETECTED IN HEPATIC SUBCELLULAR-FRACTIONS AND NUMEROUS EXTRAHEPATIC TISSUES IN CD-1 AND C57B1 6J MICE/

To identify acetaminophen (APAP)-bound proteins in addition to the maj or 44 and 58 kDa APAP-binding proteins (Bartolone et al., 1992, Toxico l. Appl. Pharmacol. 113. 19-9; Pumford et al., 1992, Biochem. Biophys. Res. Commun. 182, 1348-1355; Bulera et al., 1995, Toxicol. Appl. Phar macol. 134, 313-320), we investigated subcellular localization of live r proteins and tissue distribution of proteins arylated by a hepatotox ic dose of APAP in CD-1 and C57B1/6J mice. Western blot analysis with affinity-purified, anti-APAP antibodies allowed the detection of coval ently bound proteins in liver mitochondria, nuclei, plasma membrane, c ytosol, and microsomes. Enzyme marker assays revealed that subcellular fractions were 90-98% pure. The lack of contamination from other isol ated subcellular fractions indicates that covalently bound proteins we re specific to the particular subcellular fraction. APAP-arylated prot eins with molecular weights similar to those detected in the liver wer e found in cytosolic fractions from kidney, lung, pancreas, heart, ske letal muscle, and stomach. The presence of arylated proteins in extra- hepatic organs suggests that other organs may be susceptible to APAP t oxicity and may contain critical protein targets that are important in APAP toxicity. In contrast, covalently bound proteins were not detect ed in cytosols isolated from spleen, small intestine, brain, and testi s. The characterization of the APAP-arylated proteins identified in th is study will aid in elucidating the mechanism of APAP-induced toxicit y.