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Cyanide is a potent neurotoxicant that can produce dopaminergic neuronal death in the substantia nigra and is associated with a Parkinson-like syndrome. In this study involvement of Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a BH3-only Bcl-2 protein, in cyanide-induced death of dopaminergic cells was determined in mice and Mes 23.5 cells. Treatment of mice with cyanide up-regulated BNIP3 and Bax expression in tyrosine hydroxylase (TH)-positive cells of the substantia nigra, and progressive loss of TH-positive neurons was observed over a 9-day period. In Mes 23.5 dopaminergic cells, cyanide stimulated translocalization of BNIP3 to both endoplasmic reticulum (ER) and mitochondria. In ER, BNIP3 stimulated release of Ca(2+) into the cytosol, followed by accumulation of mitochondrial Ca(2+), resulting in reduction of mitochondrial membrane potential (Deltapsi(m)) and eventually cell death. Cyanide also activated Bax to colocalize with BNIP3 in ER and mitochondria. Forced overexpression of BNIP3 activated Bax, whereas gene silencing reduced Bax activity. Knockdown of Bax expression by small interfering RNA blocked the BNIP3-mediated changes in ER and mitochondrial Ca(2+) to block cyanide-induced mitochondrial dysfunction and cell death. These findings show that BNIP3-mediates cyanide-induced dopaminergic cell death through a Bax downstream signal that mobilizes ER Ca(2+) stores, followed by mitochondrial Ca(2+) overload.

To study oxidative mechanisms in cyanide toxicity, cyanide-induced generation of intracellular oxidant species was determined by microfluorescence in cerebellar granule cells loaded with the oxidant-sensitive fluorescence dye 2,7-dichlorofluorescin. KCN produced a concentration-dependent (25-200 microM) generation of intracellular oxidant species that was blocked by N-methyl-D-aspartate receptor antagonists (MK-801 or AP5) or by removal of extracellular Ca++ from the incubation medium. To determine the relative contribution of NO and reactive oxygen species (ROS) to the increase of cellular fluorescence after KCN, a selective inhibitor of nitric oxide synthase, a NO scavenger and enzymes that metabolize ROS were added to the incubation medium. Interference with the nitric oxide system (reduced hemoglobin as a NO scavenger or [N(G)-nitro-L-arginine methyl ester [L-NAME] reduced fluorescence by 50%). Addition of enzymes that metabolize peroxide (catalase or superoxide dismutase [SOD]) also reduced fluorescence by nearly 50%. Combination of SOD with hemoglobin or L-NAME provided additional attenuation of the fluorescence and it was concluded that both NO and ROS are generated concurrently after KCN. Furthermore a correlation was observed between NO and ROS formation and levels of malonaldehyde (MDA), a marker of lipid peroxidation. Pretreatment with MK-801 blocked KCN-induced MDA formation, whereas L-NAME partially diminished MDA production. Treatment with a combination of SOD/catalase and L-NAME blocked the KCN-induced lipid peroxidation. In cytotoxicity studies cyanide-induced cell death was blocked by MK-801, whereas partial attenuation was produced by L-NAME; SOD/catalase treatments did not protect the cells. However, significant protection from cyanide-induced cytotoxicity was observed when L-NAME was combined with SOD/catalase. It is concluded that cyanide activates N-methyl-D-aspartate receptors to simultaneously generate both NO and ROS, which may lead to formation of the cytotoxic peroxynitrite anion.

Suspensions of proximal tubular and distal tubular (DT) cells from rat kidney were treated with iodoacetate and potassium cyanide (IAA+KCN) as a model to assess injury from ATP depletion. Cells were also incubated under N2/CO2 to assess if they respond similarly to ATP depletion due to hypoxia. Based on cytotoxic effects of IAA (lactate dehydrogenase [LDH] release, protein sulfhydryl depletion) and inhibition of lactate formation, 20 microM IAA was chosen with 1 mM KCN to inhibit cellular ATP generation. DT cells exhibited significantly greater LDH release due to both IAA + KCN and hypoxia than PT cells. Mechanisms of cellular injury and the ability of various strategies to protect against (IAA+KCN)-induced injury were then studied in isolated renal DT cells to investigate factors responsible for the enhanced susceptibility of this renal cell population, about which little metabolic and toxicological information is known. IAA+KCN produced marked depletion of ATP, only minimal changes in cellular content of glutathione, but significantly decreased cellular content of glutathione disulfide, suggesting generation of a proreductant environment. Extracellular acidosis (pH 6.2 vs. 7.4) completely prevented the increase in LDH release during 2-hr incubations with IAA+KCN and partially prevented ATP depletion. Similarly, preincubation with glutathione, glycine, ATP, or adenosine significantly protected DT cells from injury. Complete restoration of cellular ATP content was not required for protection, although viability correlated better with cellular content of total adenine nucleotides. These studies are the first to explore cellular energetics and cytotoxicity in renal DT cells and demonstrate that these cells are highly sensitive to injury from ATP depletion due to either IAA+KCN or hypoxia.

"Graded" metabolic stress was induced chemically in an ex vivo preparation of retina to examine the early events following metabolic inhibition that lead to acute toxicity. Toxicity was assessed histologically and by quantitation of endogenous gamma-aminobutyric acid (GABA) release. Blockade of glycolysis with iodoacetate or electron transport with potassium cyanide for 30 min ("mild" metabolic stress) produced histopathology and GABA release similar to that seen with glutamate agonist treatment. These effects were completely prevented by the N-methyl-D-aspartate (NMDA) antagonist [(+)-5-methyl-10,11-dihydro-5H-dibenzo (a,d)cyclohepten,5,10-imine maleate (MK-801) and occurred in the absence of any net increase in extracellular glutamate or aspartate. More "severe" compromise of metabolism (iodoacetate plus potassium cyanide for 30 min) caused greater swelling and GABA release, which was only partially attenuated by competitive or noncompetitive NMDA antagonists and was accompanied by elevations in extracellular excitatory amino acids. Temporal studies of "severe" metabolic inhibition and the rise in excitatory amino acids demonstrated that, like "mild" metabolic inhibition, the early acute pathology was mediated exclusively by the NMDA receptor and occurred before elevation in excitatory amino acids. Tetrodotoxin and 6-nitro,7-cyanoquinoxaline,2,3-dion (CNQX) had little effect per se on reducing GABA release under conditions of "severe" metabolic inhibition; however, CNQX or tetrodotoxin in combination with MK-801 afforded greater protection than did MK-801 alone. Thus, activity at kainate/AMPA receptors and voltage-sensitive Na+ channels may be additional factors contributing to acute toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

An estimated 35% of all fire victims in the United States have toxicologically significant blood levels of CO and CN. However, the treatment of concurrent CO/CN intoxication has been paid scant attention. The suggestion has been made that these victims should be treated for CN poisoning. The current therapeutic management of CN poisoning in this country includes the utilization of two methemoglobin formers: amyl nitrite and sodium nitrite. This study was undertaken to determine if the administration of methemoglobin formers is advisable, as the victim is already suffering from O2 deprivation due to the presence of carboxyhemoglobin. Groups of 28 male ICR mice (22-24 g) were injected i.p. with 5.0 mg/kg of KCN and then were exposed immediately to 0.35% CO for 8.5 min in a dynamic inhalation chamber. Half of the animals were marked randomly for antidotal intervention, the other 14 animals acted as controls. Treatment of survivors with amyl nitrite (12 mg/l of chamber air) for 1 min increased mortality 43%, whereas treatment for 2 min resulted in a 59% increase in mortality. A 25% increase in mortality was noted among those animals treated with sodium nitrite (80 mg/kg i.p.), as compared to the nontreated control survivors. Treatment with dimethylaminophenol (49 mg/kg i.p.) did not statistically affect mortality.