ACETAMINOPHEN-INDUCED HEPATOTOXICITY - ANALYSIS OF TOTAL COVALENT BINDING VS SPECIFIC BINDING TO CYSTEINE

Acetaminophen-induced hepatotoxicity is believed to be mediated by cov alent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to essential proteins in liver, it has been shown that the primary re action of this metabolite with hepatic proteins is the formation of 3- (cysteine-S-yl)-acetaminophen adducts. The importance of covalent bind ing to other amino acids that may be formed by reaction of N-acetyl-p- benzoquinone imine with protein is unclear. previously we developed im munochemical assays for the acetaminophen cysteine adducts by immunizi ng animals with the conjugate 3-(N-acetylcystein-S-yl)acetaminophen-ke yhole limpet hemocyanin, wherein the carboxyl group of the N-acetyl-cy steine moiety was coupled to amino groups on the protein. A very sensi tive and specific immunochemical assay was developed for acetaminophen specifically bound to cysteine groups on protein [3-(cystein-S-yl)ace taminophen protein adducts]. Analysis of protein adducts indicated tha t after toxic doses, acetaminophen covalently bound at high levels to cysteine residues on a relatively small number of hepatic proteins. In the present work, a new antiacetaminophen antiserum was prepared by i mmunizing mice with 4-acetamidobenzoic acid coupled to keyhole limpet hemocyanin. Competitive ELISA data indicate that the resulting antiser um has excellent recognition of acetaminophen and related arylacetamid e derivatives. Using this new antiserum. Western blot analyses of live r proteins from acetaminophen-intoxicated mouse livers were performed and compared with similar assays using the anti-3-(cystein-S-yl)acetam inophen antiserum. Visual and densitometric analyses of the Western bl ots indicate that the two antisera detect the same primary acetaminoph en protein adducts; however, minor differences in the intensity of cer tain bands were observed. These differences may represent either diffe rences in antibody accessibility to 3-(cystein-S-yl)acetaminophen addu cts or differences in the proportion of acetaminophen bound to cystein e vs. binding to other amino acids.