Mechanisms of nitric oxide-induced cytotoxicity in normal human hepatocytes

Chronic exposure of hepatocytes to reactive nitrogen species (RNS) Followin g liver injury and inflammation leads nor only to functional and morphologi cal alterations in the liver but also to degenerative liver diseases and he patocellular carcinoma. Previous ly, we showed th at S-nitroso-N-acetylpeni cillamine-amine (SNAP), which generates nitric oxide, and 3-morpholinosydno nimine (Sin-1), which generates equal molar concentrations of superoxide an d nitric oxide resulting in peroxynitrite production, exhibited different l evels of cytotoxicity to normal human hepatacytes in culture. The aim of th e present study was to elucidate some of the molecular and cellular pathway s leading to hepatocyte cell death induced by RNS. Following treatment of t he hepatocytes with SNAP or Sin-1, gene-specific DNA damage was measured in mtDNA and a hprt gene fragment using a quantitative Southern blot analysis . Both agents induced dose- dependent increases in DNA damage that was alka line labile, but not sensitive to both formamidopyrimidine-DNA glycosylase (fpg) and endonuclease III, which recognize 8-oxoguanine, thymine glycol, a nd other oxidized pyrimidines. DNA damage was two- to fivefold greater in m tDNA than in the hprt gene frog ment. There was a persistent and marked inc rease in DNA damage posttreatment that appeared to arise from the disruptio n of electron transport in the mitochondria, generating reactive species th at saturated the repair system. DNA damage induced by Sin-1 and SNAP led to cell-cycle arrest in the S-phase, growth inhibition, and apoptosis. The da ta support the hypothesis that the functional and morphological changes obs erved in river following chronic exposure to RNS are, in part the result of persistent mitochondrial and nuclear DNA damage. Environ. Mel. Mutagen. (C ) 2001 Wiley-Liss, Inc.