ToxSci Advance Access originally published online on May 2, 2008
Toxicological Sciences 2008 104(2):341-351; doi:10.1093/toxsci/kfn088
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DE-71-Induced Apoptosis Involving Intracellular Calcium and the Bax-Mitochondria-Caspase Protease Pathway in Human Neuroblastoma Cells In Vitro
* State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
Department of Biology and Chemistry, City University of Hong Kong, Kowloon, HK SAR, China
1 To whom correspondence should be addressed. Fax: (86) 27-68780123. E-mail: bszhou{at}ihb.ac.cn.
Received February 7, 2008; accepted April 24, 2008
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Abstract |
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Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+]i) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3µM) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of [Ca2+]i in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.
Key Words: pentabrominated diphenyl ether (DE-71); apoptosis; Bax; caspase; calcium; cytochrome c; SK-N-SH.