Investigation of mechanisms of acetaminophen toxicity in isolated rat hepatocytes with the acetaminophen analogues 3,5-dimethylacetaminophen and 2,6-dimethylacetaminophen.

The toxicity of acetaminophen (4'-hydroxyacetanilide), 3,5-dimethylacetaminophen (3'-5'-dimethyl-4'-hydroxyacetanilide), and 2,6-dimethylacetaminophen (2',6'-dimethyl-4'-hydroxyacetanilide) was investigated in hepatocytes isolated from phenobarbital-pretreated rats. At a concentration of 5 mM, acetaminophen was found to be the most cytotoxic of the three analogues. Inhibition of cellular glutathione reductase by pretreatment of hepatocytes with BCNU enhanced the toxicity of 3,5-dimethylacetaminophen without affecting the toxicity of either acetaminophen or 2,6-dimethylacetaminophen. In contrast, pretreatment with diethylmaleate preferentially enhanced the toxicity caused by 2,6-dimethylacetaminophen and, to a lesser extent, acetaminophen, without measurably affecting the toxicity of 3,5-dimethylacetaminophen. All three hydroxyacetanilides depleted cellular glutathione concentrations, but only the 3,5-dimethyl analogue caused measurable formation of glutathione disulfide. However, the cytotoxicity of all analogues could be decreased by the administration of the thiol agent, dithiothreitol. Moreover, all three analogues had antioxidant properties, and their ability to decrease cellular malondialdehyde formation correlated with their half-wave (E1/2) oxidation potentials. The administration of the ferric ion chelator, desferrioxamine, which completely inhibited lipid peroxidation as measured by malondialdehyde formation, had no significant effects on cytotoxicity caused by acetaminophen or 3,5-dimethylacetaminophen, but partially protected against cytotoxicity caused by 2,6-dimethylacetaminophen, the poorest antioxidant of the three analogues. Covalent protein binding of all three analogues was measured. Whereas both acetaminophen and 2,6-dimethylacetaminophen bound to hepatocyte proteins under conditions where they were cytotoxic, 3,5-dimethylacetaminophen did not. Dithiothreitol was found to decrease the binding of radiolabel from both acetaminophen and its 2,6-dimethyl analogue, whereas desferrioxamine had no effect. These data indicate that the three analogues cause their cytotoxic effects by different mechanisms, although toxicity in all cases is probably mediated through their oxidation products, the quinone imines, which have as a common feature their ability to deplete cellular thiols.