Acetaminophen-induced hepatotoxicity. Analysis of total covalent binding vs. specific binding to cysteine

Acetaminophen-induced hepatotoxicity is believed to be mediated by covalent binding of the reactive metabolite N-acetyl-p-benzoquinone imine to essential proteins in liver. It has been shown that the primary reaction of this metabolite with hepatic proteins is the formation of 3-(cysteine-S-yl)-acetaminophen adducts. The importance of covalent binding to other amino acids that may be formed by reaction of N-acetyl-p-benzoquinone imine with protein is unclear. Previously, we developed immunochemical assays for the acetaminophen cysteine adducts by immunizing animals with the conjugate 3-(N-acetylcystein-S-yl)acetaminophen-keyhole limpet hemocyanin, wherein the carboxyl group of the N-acetyl-cysteine moiety was coupled to amino groups on the protein. A very sensitive and specific immunochemical assay was developed for acetaminophen specifically bound to cysteine groups on protein [3-(cystein-S-yl)acetaminophen protein adducts]. Analysis of protein adducts indicated that 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 immunizing mice with 4-acetamidobenzoic acid coupled to keyhole limpet hemocyanin. Competitive ELISA data indicate that the resulting antiserum has excellent recognition of acetaminophen and related arylacetamide derivatives. Using this new antiserum, Western blot analyses of liver proteins from acetaminophen-intoxicated mouse livers were performed and compared with similar assays using the anti-3-(cystein-S-yl)acetaminophen antiserum. Visual and densitometric analyses of the Western blots indicate that the two antisera detect the same primary acetaminophen protein adducts; however, minor differences in the intensity of certain bands were observed. These differences may represent either differences in antibody accessibility to 3-(cystein-S-yl)acetaminophen adducts or differences in the proportion of acetaminophen bound to cysteine vs. binding to other amino acids.