Sensitivity of human hepatocytes in culture to reactive nitrogen intermediates

The cytotoxic effects of 3-morpholinosydnonimine (Sin-1) and S-nitroso-N-acetylpenicillamine-amine (SNAP) on replicatively active human hepatocyte cells in culture was determined as a function of oxidant type. Both Sin-1 which yields nitric oxide and peroxynitrite following the generation of superoxide anion plus nitric oxide, and SNAP which generates nitric oxide, induced dose dependent decreases in the colony forming capabilities of the human hepatocytes. Sin-1 was much more cytotoxic (LD50 = 400 microM) than SNAP (LD50 = 1250 microM). Comparatively, both compounds were much less cytotoxic than H2O2 (LD50 = 96 microM). Sin-1 induced 4-fold higher levels of cellular nitrite than that generated by the chemical in cell free medium. Nitrotyrosine, a marker of peroxynitrite formation in cells, was immunohistochemically detected in hepatocytes treated with both Sin-1 and SNAP. The formation of 3-nitrotyrosine by hepatocytes incubated with SNAP, suggests that hepatocytes generate intracellular superoxide which reacts with the exogenous nitric oxide derived from SNAP to produce intracellular peroxynitrite, resulting in the SNAP cytotoxicity. The enhanced levels of Sin-1 cytotoxicity on the hepatocytes is suggested to be due both to the chemical generation of peroxynitrite and superoxide anion by Sin-1. These data indicate that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication, and that peroxynitirite may play a significant role in the pathogenesis of liver disease.