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Rotenone-induced apoptosis and role of calcium: a study on Neuro-2a cells

  • Molecular Toxicology
  • Published:
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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 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 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.

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Abbreviations

DNA:

Deoxyribonucleic acid

CaMKII:

Ca2+/calmodulin-dependent protein kinase II

PI:

Propidium iodide

BSA:

Bovine serum albumin

EDTA:

Ethylenediamine tetraacetic acid

PBS:

Phosphate buffer saline

RNS:

Reactive nitrogen species

ROS:

Reactive oxygen species

SCGE:

Single-cell gel electrophoresis

LMP:

Low melting point

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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.

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Authors and Affiliations

  1. Division of Toxicology, CSIR-Central Drug Research Institute, P.O.Box 173, Lucknow, 226001, U.P., India

    Supriya Swarnkar, Poonam Goswami, Sonam Gupta, Sarika Singh & Chandishwar Nath

  2. Division of Pharmacology, CSIR-Central Drug Research Institute, Lucknow, 226001, U.P., India

    Pradeep Kumar Kamat

  3. School of Studies in Neuroscience, Jiwaji University, Gwalior, 474011, M.P., India

    Ishan K. Patro

Authors
  1. Supriya Swarnkar
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  2. Poonam Goswami
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  3. Pradeep Kumar Kamat
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  4. Sonam Gupta
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  5. Ishan K. Patro
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  6. Sarika Singh
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  7. Chandishwar Nath
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Correspondence to Chandishwar Nath.

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Swarnkar, S., Goswami, P., Kamat, P.K. et al. Rotenone-induced apoptosis and role of calcium: a study on Neuro-2a cells. Arch Toxicol 86, 1387–1397 (2012). https://doi.org/10.1007/s00204-012-0853-z

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  • DOI: https://doi.org/10.1007/s00204-012-0853-z

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