The pre-administration of PGE(1) reduced inducible nitric oxide synthase (NOS-2) expression and cell death induced by d-galactosamine (d-GalN) in cultured rat hepatocytes. The present study evaluated the role of nitric oxide (NO) during PGE(1) treatment in fully established d-GalN-induced cytotoxicity in cultured human hepatocytes. Human hepatocytes were isolated from liver resections by classic collagenase perfusion. PGE(1) (1 microM) was administered at 2 h before d-GalN (40 mM), or 2 or 10 h after d-GalN in cultured hepatocytes. The production of NO was inhibited by N-omega-nitroso-l-arginine methyl ester (l-NAME) (0.5 mM). Various parameters related to oxidative and nitrosative stress, mitochondrial dysfunction, NF-kappaB activation, NOS-2 expression and cell death were evaluated in hepatocytes. NO mediated mitochondrial disturbances, nitrosative stress and cell death in d-GalN-treated hepatocytes. The administration of PGE(1) 10 h after d-GalN enhanced NF-kappaB activation, NOS-2 expression and nitrosative stress. Although PGE(1) administered at 2 h before or 2h after d-GalN reduced apoptosis and necrosis, its administration 10 h after d-GalN had no beneficial effect on cell death. In conclusion, the administration of PGE(1) during advanced d-GalN cytotoxicity induced nitrosative stress and lost its cytoprotective properties in cultured human hepatocytes.