Primary hepatocyte apoptosis is unlikely to relate to caspase-3 activity under sustained endogenous oxidative stress.
We previously showed that inhibition of catalase and glutathione peroxidase activities in rat primary hepatocytes by 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS) results in endogenous oxidative stress and apoptosis. For the present study, we determined whether this apoptosis involved activation of caspase-3, which is known to execute apoptosis in many cell types. ATZ and MS increased levels of reactive oxygen species (ROS) from 3-9 h, just before the onset of chromatin condensation (apoptosis) and decreases in protein thiols. Pretreatment with either SKF, a cytochrome P450 inhibitor, or L-ascorbic acid, an antioxidant, completely suppressed the increase in ROS levels and apoptosis, suggesting that the sustained ROS increases may cause the apoptosis. SKF also abolished the decrease in protein thiol content, further supporting the contribution of the P450 system to increased ROS levels. DEVD-CHO, a caspase-3 inhibitor, even at 1 mM had no effect on apoptosis. Caspase-3 activity remained unchanged and pro-caspase-3 processing was not detected during 18 h incubation with ATZ and MS. Moreover, the amount of unoxidized pro-caspase-3 decreased even below the level of untreated hepatocytes. These findings suggest that the sustained oxidative stress is a major cause for the hepatocyte apoptosis, which occurs independently of the caspase-3 related pathway.
Ishihara
Yasuhiro
Y
Department of Biology, Graduate School of Science, Osaka University, Osaka 532-8686, Japan.
Shiba
Dai
D
Shimamoto
Norio
N
D000640:Amitrole / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D001205:Ascorbic Acid / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects* / Q000502:physiology; D008297:Male; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011335:Proadifen / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013870:Thiomalates / Q000037:antagonists & inhibitors / Q000494:pharmacology; D013997:Time Factors
D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D013870:Thiomalates; C046062:2-thiomalic acid; D011335:Proadifen; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D001205:Ascorbic Acid; D000640:Amitrole