Protective effects of intracellular reactive oxygen species generated by 6-formylpterin on tumor necrosis factor-alpha-induced apoptotic cell injury in cultured rat hepatocytes.
The effects of 6-formylpterin on tumor necrosis factor (TNF)-alpha-induced apoptotic cell injury were studied in cultured rat hepatocytes. The incubation of the hepatocytes with TNF-alpha and actinomycin D (ActD) induced the apoptotic cell injury. The level of aspartate transaminase (AST) in the culture supernatant increased, and the cell viability, estimated by mitochondrial respiration (MTT assay), decreased. The DNA fragmentation and the caspase 3-like activity, which are characterized to apoptosis, increased. When the hepatocytes were incubated with 100-500 microM 6-formylpterin, the intracellular formation of reactive oxygen species (ROS) was observed, and the ratio of reduced and oxidized glutathione (GSH/GSSG) of whole cell lysate decreased. The co-incubation of the TNF-alpha/ActD-treated hepatocytes with 100-500 microM 6-formylpterin attenuated the TNF-alpha/ActD-induced apoptotic cell injury. The level of AST decreased and the cell viability increased. Both the DNA fragmentation and the caspase 3-like activity decreased. The caspases, executors of apoptosis, are known to require a reduced cystein in their active site to function, and the intact intracellular GSH/GSSG is essential for the caspase activation. Therefore, our findings suggest that intracellular ROS generated by 6-formylpterin decline the intracellular redox state to an oxidant state, which suppresses the caspase activity and prevents the apoptotic cell injury of hepatocytes.
Ishii
Hisanari
H
Department of Anesthesia, Kyoto University Hospital, Kyoto 606-8507, Japan.
Arai
Toshiyuki
T
Mori
Hiroko
H
Yamada
Hiroko
H
Endo
Nobuyuki
N
Makino
Keisuke
K
Fukuda
Kazuhiko
K
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001219:Aspartate Aminotransferases / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003609:Dactinomycin / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects; D010084:Oxidation-Reduction; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011622:Pterins / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D011500:Protein Synthesis Inhibitors; D011622:Pterins; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; C005926:2-amino-4-hydroxy-6-formylpteridine; D001219:Aspartate Aminotransferases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione