title: Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis.
abstract: Tumor necrosis factor alpha (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H(2)O(2), either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H(2)O(2) also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis -- H(2)O(2), antimycin, diamide, BCNU -- all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IkappaB-alpha phosphorylation and NF-kappaB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-kappaB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range -- in which redox changes can inhibit NF-kappaB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.
authors.affiliation: University of Southern California Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90089-9121, USA. derickh@usc.edu
authors.forename: Derick
authors.initials: D
authors.lastname: Han
authors.forename: Naoko
authors.initials: N
authors.lastname: Hanawa
authors.forename: Behnam
authors.initials: B
authors.lastname: Saberi
authors.forename: Neil
authors.initials: N
authors.lastname: Kaplowitz
mesh_terms: D000818:Animals; D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002330:Carmustine / Q000494:pharmacology; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D010084:Oxidation-Reduction; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C032456:antimycin; D000968:Antimycin A; D006861:Hydrogen Peroxide; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D002330:Carmustine