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.

Endoplasmic Reticulum Stress Instigates the Rotenone Induced Oxidative Apoptotic Neuronal Death: a Study in Rat Brain

“…Histopathological changes induced by rotenone administration were also correlated with the biochemical alterations in striata and midbrain regions of rat brain [8]. Our previous studies in neuronal and astrocytes cells have also shown that rotenone treatment caused oxidative stress and apoptosis in both neuronal as well as astroglial cells [10,11]. Involvement of astroglial activation and apoptosis are also reported as the major features in rotenone-induced dopaminergic neuronal death in rat brain [12].…”

“…Numbers of studies have suggested the involvement of oxidative stress-mediated death of neurons after rotenone administration. However, the antioxidant treatment provides only partial but significant protection in the rotenone-induced neuronal death [10]; therefore, further investigations are required.…”

Rotenone exerts developmental neurotoxicity in a human brain spheroid model

“…This could affect the synaptic function, since Ca 2+ is required in all the processes related to neurotransmitter release and absorption. The increase of intracellular Ca 2+ levels after rotenone treatment has been already described (Swarnkar et al, 2012). This increase of Ca 2+ seems to be related to the increased opening of voltage-gated Ca 2+ channels (Wang and Xu, 2005).…”

The Mitochondrial Complex I Inhibitor Rotenone Induces Endoplasmic Reticulum Stress and Activation of GSK-3β in Cultured Rat Retinal Cells

“…44,45 This could drastically affect retinal neurons in the cultures, which have a relatively high density of mitochondria, and thus also provide a further drive to rapid nonapoptotic death. Calpain-l, as a calcium-activated cysteine protease, can deactivate or break down cytoskeletal proteins such as a-spectrin, and is thus predominantly associated with the process of cell death via necrosis.…”