Mechanisms of cyanide neurotoxicity: A role for programmed cell death

Edward Michael Mills, Purdue University

Abstract

The central nervous system is the primary organ affected by exposure to cyanide. In a manner not yet fully understood, acute poisoning induces motor neuropathology and Parkinsonism. The overall goal of this project was to identify possible mechanisms of cyanide-induced selective neuronal loss. Based upon cytotoxicity determinations, undifferentiated pheochromocytoma (PC12) cells are relatively insensitive to cyanide exposure. Differentiated cells, however, are quite sensitive to the lethal action of cyanide, as approximately 45 percent of cells died following exposure to 0.10 mM KCN. Programmed cell death (PCD), or apoptosis may be a mechanism of cell death common to many neurodegenerative conditions. Studies were conducted to assess the contribution of apoptosis to KCN-mediated cell loss. Apoptosis often requires transcription. Using the transcriptional inhibitor actinomycin D, it was shown that KCN-induced PC12 cell death requires transcription. Apoptotic DNA fragmentation was observed following KCN exposure to PC12 cells by gel electrophoresis and a modified TdT dUTP nick end labeling (TUNEL) method. Moreover, apoptotic morphology was evident in KCN-treated cells by transmission electron microscopy (TEM). Reactive oxygen species (ROS) have been implicated as mediators of cyanide neurotoxicity and apoptosis. Based upon a fluorometric assay, KCN increased ROS in PC12 cells. ROS appear to play a functional role in KCN neurotoxicity. In a series of experiments, antioxidants reduced cyanide-induced ROS formation, DNA fragmentation, and cytotoxicity. The results of cell culture experiments were compared to those obtained in an established mouse model of cyanide neurotoxicity. Using TUNEL staining as a marker of apoptosis and GFAP immunostaining as a marker of necrosis, it was demonstrated that KCN induces region-specific apoptotic and necrotic lesions in mouse brain. Apoptosis was widespread in cortex following three days KCN exposure and decreased with treatment duration. In contrast, necrosis was evident in the substantia nigra by day three of KCN exposure and increased in intensity up to twelve days exposure. In brain, KCN-induced apoptosis, but not necrosis, appears to be mediated by ROS, as an antioxidant prevented KCN-induced DNA fragmentation in cortex. These results suggest that KCN may be a useful compound in studying the roles of apoptosis and ROS in neurodegeneration.

Degree

Ph.D.

Advisors

Isom, Purdue University.

Subject Area

Toxicology|Neurology|Pharmacology

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