Selective protein arylation by acetaminophen and 2,6-dimethylacetaminophen in cultured hepatocytes from phenobarbital-induced and uninduced mice. Relationship to cytotoxicity.

To evaluate the mechanistic importance of covalent binding in acetaminophen (APAP)-induced hepatotoxicity, we compared the effects of 2,6-dimethylacetaminophen (2,6-DMA) to those of APAP in primary cultures of mouse hepatocytes. Immunochemical analysis of electrophoretically separated proteins has shown that the majority of covalent binding after a cytotoxic dose of APAP occurs on two major bands of 44 and 58 kD (Bartolone et al., Biochem Pharmacol 36: 1193-1196, 1987). At equimolar concentrations, 2,6-DMA bound proteins only 15% as extensively as did APAP and was not cytotoxic in hepatocytes from uninduced mice. However, when the hepatocytes were obtained from phenobarbital-induced mice, APAP administration resulted in increased protein arylation and a more rapid onset of cytotoxicity. Furthermore, in the cells from phenobarbital-induced mice, 2,6-DMA not only resulted in increased binding but also in overt cytotoxicity. Since our affinity-purified anti-APAP antibody did not cross-react with 2,6-DMA, a new antibody specific for 2,6-DMA was prepared and, after affinity purification, was used to detect 2,6-DMA protein adducts by Western blotting. Results indicated that, in hepatocytes from both phenobarbital-induced and non-induced mice, the binding of 2,6-DMA was also highly selective with the most prominent target being the 58 kD cytosolic protein. However, by contrast to APAP, only minimal binding to the 44 kD protein was detected after 2,6-DMA treatment. Although several additional protein adducts were increased in treated cells from phenobarbital-induced mice, the 58 kD protein was clearly the most prominently arylated target associated with both APAP and 2,6-DMA cytotoxicity. These data suggest that both the specificity of covalent binding as well as the extent of binding to the major targets may play an important role in the ensuing toxicity.