title: Reactive oxygen species and c-Jun N-terminal kinases contribute to TEMPO-induced apoptosis in L5178Y cells.
abstract: The biological consequences of exposure to piperidine nitroxides is a concern, given their widespread use in manufacturing processes and their potential use in clinical applications. Our previous study reported that TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl), a low molecular weight free radical, possesses pro-oxidative activity in L5178Y cells. In this study, we investigated and characterized the role of reactive oxygen species (ROS) in TEMPO-induced toxicity in L5178Y cells. We found that TEMPO induced time- and concentration-dependent intracellular ROS production and glutathione depletion. TEMPO also induced apoptosis as demonstrated by increased caspase-3/7 activity, an increased proportion of annexin V stained cells, and decreased expression of anti-apoptotic proteins including Bcl-2, Bcl-xL and Mcl-1. N-acetylcysteine, a ROS scavenger, attenuated the ROS production and apoptosis induced by TEMPO. Moreover, Western blot analyses revealed that TEMPO activated γ-H2A.X, a hallmark of DNA damage, and c-Jun N-terminal kinases (JNK), a key member in the mitogen-activated protein kinase (MAPK) signaling pathway. Addition of SP600125, a JNK-specific inhibitor, blocked TEMPO-mediated JNK phosphorylation and also attenuated TEMPO-induced apoptosis. These findings indicate that both ROS production and JNK activation are involved in TEMPO-induced apoptosis, and may contribute to the toxicity of TEMPO in L5178Y cells.
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Guo
authors.affiliation: Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States.
authors.forename: Si
authors.initials: S
authors.lastname: Chen
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States; Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Zhuhong
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States.
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States.
authors.forename: Stacey L
authors.initials: SL
authors.lastname: Dial
authors.affiliation: Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States.
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR 72079, United States. Electronic address: nan.mei@fda.hhs.gov.
authors.forename: Nan
authors.initials: N
authors.lastname: Mei
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D045744:Cell Line, Tumor; D003497:Cyclic N-Oxides / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: C494812:Bcl2l1 protein, mouse; D003497:Cyclic N-Oxides; C579094:Mcl1 protein, mouse; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D017382:Reactive Oxygen Species; D051020:bcl-X Protein; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D053148:Caspase 3; D053179:Caspase 7; D005978:Glutathione; C003959:TEMPO; D000111:Acetylcysteine
keywords: Apoptosis; MAPK pathway; Nitroxide; Reactive oxygen species; TEMPO