title: Rotenone-induced apoptosis and role of calcium: a study on Neuro-2a cells.
abstract: Rotenone causes cytotoxicity in astrocytic cell culture by glial activation, which is linked to free radical generation. The present study is an investigation to explore whether rotenone could also cause cellular toxicity in mouse neuroblastoma cells (Neuro-2a) under treatment similar to astroglial cells. The effect of rotenone (0.1, 1, and 10 μM) on mitochondrial dehydrogenase enzyme activity by MTT reduction assay, PI uptake, total reactive oxygen species (ROS)/superoxide levels, nitrite levels, extent of DNA damage (by comet assay), and nuclear morphological alteration by Hoechst staining was studied. Caspase-3 and Ca⁺²/calmodulin-dependent protein kinase II (CaMKIIα) gene expression was determined to evaluate the apoptotic cell death and calcium kinase, respectively. Calcium level was estimated fluorometrically using fura-2A stain. Rotenone decreased mitochondrial dehydrogenase enzyme activity and generated ROS, superoxide, and nitrite. Rotenone treatment impaired cell intactness and nuclear morphology as depicted by PI uptake and chromosomal condensation of Neuro-2a cells, respectively. In addition, rotenone resulted in increased intracellular Ca⁺² level, caspase-3, and CaMKIIα expression. Furthermore, co-exposure of melatonin (300 μM), an antioxidant to cell culture, significantly suppressed the rotenone-induced decreased mitochondrial dehydrogenase enzyme activity, elevated ROS and RNS. However, melatonin was found ineffective to counteract rotenone-induced increased PI uptake, altered morphological changes, DNA damage, elevated Ca⁺², and increased expression of caspase-3 and CaMKIIα. The study indicates that intracellular calcium rather than oxidative stress is a major factor for rotenone-induced apoptosis in neuronal cells.
authors.affiliation: Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow 226001, UP, India.
authors.forename: Supriya
authors.initials: S
authors.lastname: Swarnkar
authors.forename: Poonam
authors.initials: P
authors.lastname: Goswami
authors.forename: Pradeep Kumar
authors.initials: PK
authors.lastname: Kamat
authors.forename: Sonam
authors.initials: S
authors.lastname: Gupta
authors.forename: Ishan K
authors.initials: IK
authors.lastname: Patro
authors.forename: Sarika
authors.initials: S
authors.lastname: Singh
authors.forename: Chandishwar
authors.initials: C
authors.lastname: Nath
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001253:Astrocytes / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020013:Calcium Signaling / Q000187:drug effects*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D053144:Cell Nucleus Shape / Q000187:drug effects; D004249:DNA Damage; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D008550:Melatonin / Q000494:pharmacology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D010575:Pesticides / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000037:antagonists & inhibitors / Q000494:pharmacology*
chemical_list: D000975:Antioxidants; D009419:Nerve Tissue Proteins; D010575:Pesticides; D012333:RNA, Messenger; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D012402:Rotenone; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C517671:Camk2a protein, mouse; C505409:Casp3 protein, mouse; D053148:Caspase 3; D008550:Melatonin