Mechanisms of cyanide neurotoxicity

Edward Uzorezie Maduh, Purdue University

Abstract

Calcium-mediated cellular events underlying cyanide neurotoxicity were investigated in PC12 cells, a cultured neuronal model. Cell suspensions incubated 5-30 min with KCN, 10$\sp{-2}$M, displayed increases in cytosolic Ca$\sp{2+}$ measured by Quin2. An inverse relationship was shown between the rise in free Ca$\sp{2+}$ and cell energy since ATP levels, ATP:ADP and ATP:AMP ratios declined in intoxicated cells from 2.5 min after KCN addition through the 30 min treatment period. This suggests that lack of ATP impairs Ca$\sp{2+}$ homeostasis. The increase in free (Ca) $\sb{\rm i}$ caused by KCN is associated with peroxidation of mouse brain lipids. KCN also caused lipid peroxidation (LP) in the present model system. A cyanide antidote chlorpromazine blocked the Ca$\sp{2+}$ elevation and induction of LP, a finding consistent with the concept that increased Ca$\sp{2+}$ and LP are mediators of cyanide toxicity. By assessing changes in cytosolic pH (pH$\sb{\rm i}$) in cells incubated with KCN in the presence or absence of external Ca$\sp{2+}$ the role of Ca$\sp{2+}$-H$\sp+$ interaction in cyanide toxicity was studied. When bathed in calcium Krebs Ringer (Ca-KR) media pH (pH$\sb{\rm o})$ 7.4, 1-10 mM KCN rapidly decreased pH$\sb{\rm i}$ of cells. This effect was blunted by Ca$\sp{2+}$ removal, pretreating the cells with a Ca$\sp{2+}$ channel antagonist, diltiazem, 10$\sp{-5}$M (DILT) or by raising pH$\sb{\rm o}$ to 7.9. At pH$\sb{\rm o}$ 6.9, the pH$\sb{\rm i}$ acidifying effect of KCN was potentiated. In Ca-KR media free of external Na$\sp+$ the reduction of pH$\sb{\rm i}$ by KCN was amplified and this was reversed by subsequent addition of Na$\sp+$ or Li$\sp+$ ions which suggests that the Na$\sp+$/H$\sp+$ ion exchange mechanism is impaired after cyanide exposure. Cells incubated with 1-10 mM KCN in Ca-KR medium and examined by electron microscopy revealed depletion of secretory granules, loss of cellular processes, bleb formation and mitochondrial swelling, effects which were ameliorated by DILT. Incubation with KCN released NE and DA in a Ca$\sp{2+}$-dependent manner over a 20-30 min period. Membrane integrity deteriorated and cells lost viability as high levels of the cytosolic enzyme, LDH appeared in the media following 60-120 min incubation with 10 mM KCN. Cells pretreated with DILT (10$\sp{-5}$M) 15 min before KCN exposure were protected from these lethal effects. Thus in the PC12 neural system depletion of cell energy is accompanied by disturbance of ion homeostasis marked by pH$\sb{\rm i}$ acidification, cytosolic Ca$\sp{2+}$ rise, loss membrane integrity and cell death. These sequential toxic reactions may explain neuronal damage and dysfunction during cyanide intoxication.

Degree

Ph.D.

Advisors

Isom, Purdue University.

Subject Area

Pharmacology|Neurology

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