TY - JOUR
T1 - Rotenone-induced apoptosis and role of calcium
T2 - A study on Neuro-2a cells
AU - Swarnkar, Supriya
AU - Goswami, Poonam
AU - Kamat, Pradeep Kumar
AU - Gupta, Sonam
AU - Patro, Ishan K.
AU - Singh, Sarika
AU - Nath, Chandishwar
N1 - Funding Information:
Acknowledgments Authors are thankful to Dr. A. K. Balapure, Head, Tissue and Cell Culture Unit, CSIR-CDRI and his team for providing cells for experimentation. Author SS gratefully acknowledges the Council of Scientific and Industrial Research (CSIR), India, for research fellowship.
PY - 2012/9
Y1 - 2012/9
N2 - 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) onmitochondrial 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 Ca2+/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 antioxidantto 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, alteredmorphological 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 rotenoneinduced apoptosis in neuronal cells.
AB - 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) onmitochondrial 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 Ca2+/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 antioxidantto 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, alteredmorphological 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 rotenoneinduced apoptosis in neuronal cells.
KW - Apoptosis
KW - DNA damage
KW - Melatonin
KW - Neuronal cells
KW - Reactive oxygen species
KW - Rotenone
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U2 - 10.1007/s00204-012-0853-z
DO - 10.1007/s00204-012-0853-z
M3 - Article
C2 - 22526376
AN - SCOPUS:84866095516
SN - 0340-5761
VL - 86
SP - 1387
EP - 1397
JO - Archives of Toxicology
JF - Archives of Toxicology
IS - 9
ER -