diethyl-maleate and 3--5--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.

Topics: Acetaminophen; Animals; Carmustine; Dithiothreitol; Glutathione; Glutathione Reductase; In Vitro Techniques; Kinetics; Lipid Peroxides; Liver; Male; Maleates; Rats; Rats, Inbred Strains

Recently, we have reported that 3,5-dialkyl substitution of paracetamol, in contrast to 3-monoalkyl substitution, prevented the paracetamol-induced toxicity in freshly isolated rat hepatocytes without having any effect on its cytochrome P-450 mediated bioactivation to reactive N-acetyl-p-benzoquinone imines (NAPQI). In the present study the mechanism of this prevention of toxicity, with special emphasis on oxidative stress, was studied in more detail in freshly isolated rat hepatocytes, using paracetamol, 3-methyl-, 3,5-dimethyl-paracetamol, synthetic NAPQI and 3,5-dimethyl-NAPQI. 3-Methyl-paracetamol was found to induce glutathione (GSH) depletion, lipid-peroxidation and cytotoxicity in hepatocytes to the same extent as paracetamol. 3,5-Dimethyl-paracetamol, however, even when added in a ten-fold higher concentration when compared to paracetamol, did not induce any of these effects. Similar differences of toxicity were observed between NAPQI and 3,5-dimethyl-NAPQI; 3,5-dimethyl-NAPQI, in contrast to NAPQI, did not reduce protein thiol levels, did not induce GSH depletion, lipid-peroxidation nor cytotoxicity. Only after artificial depletion of GSH levels in the hepatocytes by DEM or BCNU, 3,5-dimethyl-NAPQI was cytotoxic. This effect was accompanied by depletion of protein thiol levels, but not by lipid-peroxidation. Addition of the disulfide reducing agent, dithiothreitol, prevented the artificially created cytotoxicity of 3,5-dimethyl-NAPQI. It is concluded that prevention of paracetamol-induced toxicity by 3,5-dialkyl substitution is primarily due to prevention of irreversible GSH-depletion, presumably caused by the inability of 3,5-dialkyl-NAPQI to conjugate with thiols. As a result, the GSH-dependent cellular defense mechanism against potential oxidative cellular injury by 3,5-dialkyl-NAPQI is left unimpaired. Our observations indicate that a compound, not capable of covalent binding to thiol groups of proteins, can induce toxicity solely as a result of protein thiol oxidation without inducing lipid-peroxidation.

Topics: Acetaminophen; Animals; Benzoquinones; Carmustine; Chemical and Drug Induced Liver Injury; Glutathione; Imines; Lipid Peroxides; Liver; Liver Diseases; Male; Maleates; Oxidation-Reduction; Quinones; Rats; Rats, Inbred Strains; Structure-Activity Relationship