Source of early reactive oxygen species in the apoptosis induced by transforming growth factor-beta in fetal rat hepatocytes.
Transforming growth factor-beta (TGF-beta) induces an oxidative stress process in hepatocytes that mediates its apoptotic activity. To determine the cellular source of the early reactive oxygen species (ROS) generated by fetal rat hepatocytes in response to TGF-beta, we used inhibitors that block different ROS-producing systems. Diphenyleneiodonium, which inhibits NADPH oxidase and other flavoproteins, completely blocked the increase in ROS induced by TGF-beta, coincidently with an impairment of caspase-3 activation and cell death. Rotenone, an inhibitor of the NADH dehydrogenase in mitochondrial complex I, attenuated, but did not completely inhibit, ROS-production, caspase activation, and cell death mediated by TGF-beta. No significant protection was observed with inhibitors of other ROS-producing systems, such as cytochrome P450 (metyrapone), cyclooxygenase (indomethacin), and xanthine oxidase (allopurinol). Additional experiments have indicated that two different mechanisms could be involved in the early ROS production by TGF-beta. First, an inducible (cycloheximide-inhibited) NADPH oxidase-like system could account for the extramitochondrial production of ROS. Second, TGF-beta could increase ROS by a rapid downregulation of antioxidant genes. In particular, intramitochondrial ROS would increase by depletion of MnSOD. Finally, glutathione depletion is a late event and it would be more the consequence than the cause of the increase in ROS induced by TGF-beta.
Herrera
Blanca
B
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Murillo
Miguel M
MM
Alvarez-Barrientos
Alberto
A
Beltrán
Jesús
J
Fernández
Margarita
M
Fabregat
Isabel
I
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002374:Catalase / Q000235:genetics; D002478:Cells, Cultured; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000196:embryology; D018613:Microscopy, Confocal; D019255:NADPH Oxidases / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D024502:alpha-Tocopherol / Q000494:pharmacology
D009861:Onium Compounds; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016212:Transforming Growth Factor beta; D012402:Rotenone; C007517:diphenyleneiodonium; D002374:Catalase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D005978:Glutathione; D024502:alpha-Tocopherol