Accelerated Cytotoxic Mechanism Screening of Hydralazine Using an in Vitro Hepatocyte Inflammatory Cell Peroxidase Model

Abstract

Long-term treatment of hypertensive disorders with hydralazine has resulted in some patients developing hepatitis and lupus erythematosus, an autoimmune syndrome. The concentration of hydralazine required to cause 50% cytotoxicity in 2 h (LC₅₀) toward isolated rat hepatocytes was found to be 8 mM. Cytotoxicity was delayed by the P450 inhibitor, 1-aminobenzotriazole, suggesting that P450 catalyzed the formation of toxic metabolites from hydralazine. No hydralazine-induced oxidative stress was apparent as there was little effect on hepatocyte lipid peroxidation, protein carbonyl formation, intracellular H₂O₂, or hepatocyte GSH levels and no effect of butylated hydroxyanisole (BHA) on cytotoxicity. Drug-induced hepatotoxicity in vivo has often been attributed to infiltrating inflammatory cells, for example, neutrophils or resident Kupffer cells whose NADPH oxidase generates H₂O₂, when activated. The effect of a nontoxic continuous infusion of H₂O₂ on hydralazine cytotoxicity was investigated. It was found that H₂O₂ increased hepatocyte susceptibility to hydralazine 4-fold (LC₅₀, 2 mM). Cytotoxicity was still prevented by the P450 inhibitor but now involved some oxidative stress as shown by increased protein carbonyls, endogenous H₂O₂, and GSH oxidation. Lipid peroxidation was not increased, and cytotoxicity was not inhibited by BHA. Cytotoxicity, however, was inhibited by 4-hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (TEMPOL), a ROS scavenger. Because neutrophils or Kupffer cells release myeloperoxidase on activation, the effect of adding peroxidase to the hepatocytes exposed to H₂O₂ on hydralazine cytotoxicity was investigated. It was found that peroxidase/H₂O₂ increased hepatocyte susceptibility to hydralazine 80-fold (LC₅₀, 0.1 mM). Furthermore, cytotoxicity occurred following extensive oxidative stress that included lipid peroxidation, and cytotoxicity that was now prevented by the antioxidant BHA. These results indicate that three cytotoxic pathways exist for hydralazine: a P450-catalyzed pathway not involving oxidative stress, a P450/H₂O₂-catalyzed oxidative stress-mediated cytotoxic pathway not involving lipid peroxidation, and a peroxidase/H₂O₂-catalyzed lipid peroxidation-mediated cytotoxic pathway.