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.