Mechanisms of nitric oxide-induced cytotoxicity in normal human hepatocytes.
Chronic exposure of hepatocytes to reactive nitrogen species (RNS) following liver injury and inflammation leads not only to functional and morphological alterations in the liver but also to degenerative liver diseases and hepatocellular carcinoma. Previously, we showed that S-nitroso-N-acetylpenicillamine-amine (SNAP), which generates nitric oxide, and 3-morpholinosydnonimine (Sin-1), which generates equal molar concentrations of superoxide and nitric oxide resulting in peroxynitrite production, exhibited different levels of cytotoxicity to normal human hepatocytes in culture. The aim of the present study was to elucidate some of the molecular and cellular pathways leading to hepatocyte cell death induced by RNS. Following treatment of the 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 alkaline labile, but not sensitive to both formamidopyrimidine-DNA glycosylase (fpg) and endonuclease III, which recognize 8-oxoguanine, thymine glycol, and other oxidized pyrimidines. DNA damage was two- to fivefold greater in mtDNA than in the hprt gene fragment. There was a persistent and marked increase in DNA damage posttreatment that appeared to arise from the disruption of electron transport in the mitochondria, generating reactive species that 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 data support the hypothesis that the functional and morphological changes observed in liver following chronic exposure to RNS are, in part, the result of persistent mitochondrial and nuclear DNA damage.
D'Ambrosio
S M
SM
Department of Radiology, The College of Medicine and Public Health, The Ohio State University, Columbus 43210, USA. dambrosio.1@osu.edu
Gibson-D'Ambrosio
R E
RE
Brady
T
T
Oberyszyn
A S
AS
Robertson
F M
FM
D000328:Adult; D017209:Apoptosis; D015139:Blotting, Southern; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004272:DNA, Mitochondrial / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008981:Molsidomine / Q000031:analogs & derivatives / Q000633:toxicity*; D009152:Mutagenicity Tests; D009569:Nitric Oxide / Q000633:toxicity*; D020030:Nitric Oxide Donors / Q000633:toxicity*; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000633:toxicity*; D017382:Reactive Oxygen Species / Q000378:metabolism
D004272:DNA, Mitochondrial; D020030:Nitric Oxide Donors; D017382:Reactive Oxygen Species; C110881:S-nitro-N-acetylpenicillamine; D009569:Nitric Oxide; C002385:linsidomine; D008981:Molsidomine; D007041:Hypoxanthine Phosphoribosyltransferase; D010396:Penicillamine