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Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H2O2 generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H2O2 generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H2O2 accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.

1Alpha,25-dihydroxyvitamin D(3) (VD(3)) is a neuroprotectant that can reduce cytotoxicity produced by a variety of toxicants. The mechanism of the neuroprotection was studied in rat primary cortical cells in which Wy14,643, an agonist of peroxisome proliferator activated receptor-alpha (PPARalpha), enhances cyanide (KCN) neurotoxicity. In this cell model, Wy14,643 pretreatment enhanced cyanide-induced cell death, and the increased cell death was linked to up-regulation of uncoupling protein-2 (UCP-2). VD(3) reversed cyanide-induced mitochondrial dysfunction in cells pretreated with Wy14,643, as reflected by restoration of cellular ATP and mitochondrial membrane potential (DeltaPsi(m)). Analysis of cellular state 4 oxygen consumption showed increased mitochondrial uncoupling accompanied by up-regulation of UPC-2. The uncoupling was attenuated by prior treatment with VD(3). The interaction of VD(3) with UCP-2 was attributed to increased expression of IkappaB, an inhibitor of NF-kappaB (transcription factor that regulates UCP-2 expression). The increased IkappaB levels lead to reduced nuclear translocation and DNA binding of nuclear factor-kappaB. The role of oxidative stress in the response was then evaluated. Cotreatment with Wy14,643 and cyanide markedly increased reactive oxygen species generation and decreased reduced glutathione levels. The oxidative stress was blocked by VD(3) pretreatment. It was concluded that VD(3) blocks Wy14,643 enhancement of cyanide neurotoxicity by suppressing the redox-mediated transcriptional up-regulation of UCP-2, resulting in reduced mitochondrial proton leak and stabilization of mitochondrial function., (2008 Wiley-Liss, Inc.)

Acute cyanide toxicity is attributed to inhibition of cytochrome c oxidase (CcOX), the oxygen-reducing component of mitochondrial electron transport; however, the mitochondrial action of cyanide is complex and not completely understood. State-3 oxygen consumption and CcOX activity were studied in rat N27 mesencephalic cells to examine the functional interaction of cyanide and nitric oxide (NO). KCN produced a concentration-dependent inhibition of cellular respiration. Cyanide's median inhibitory concentration (IC50) of oxygen consumption (13.2 +/- 1.8microM) was higher than the CcOX IC50 (7.2 +/- 0.1microM). Based on respiratory threshold analysis, 60% inhibition of CcOX was necessary before oxygen consumption was decreased. Addition of high levels of exogenous NO (100microM S-nitroso-N-acetyl-DL-penicillamine) attenuated cyanide inhibition of both respiration and CcOX. On the other hand, when endogenous NO generation was blocked by an NOS inhibitor (N(omega)-monomethyl-L-arginine ester), the cyanide IC50 for both respiration and CcOX increased to 59.6 +/- 0.9microM and 102 +/- 10microM, respectively, thus showing constitutive, low-level NO production enhanced cyanide inhibition. Laser scanning cytometry showed that cyanide elevated mitochondrial NO, which then was available to interact with CcOX to enhance the inhibition. It is concluded that the rapid, potent action of cyanide is due in part to mitochondrial generation of NO, which enhances inhibition of CcOX. At low mitochondrial oxygen tensions, the cyanide-NO interaction would be increased. Also, the antidotal action of sodium nitrite is partly explained by generation of high mitochondrial levels of NO, which antagonizes the CcOX inhibition.

Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a Bcl-2 homology domain 3 (BH3) domain only protein, has been identified as a mitochondrial mediator of hypoxia-induced cell death. Since cyanide produces histotoxic anoxia (chemical hypoxia), the present study was undertaken in primary rat cortical cells to determine involvement of the BNIP3 signaling pathway in cyanide-induced death. Over a 20 h exposure KCN increased BNIP3 expression, followed by a concentration-related apoptotic death. To determine if BNIP3 plays a role in the cell death, expression was either increased with BNIP3 cDNA (BNIP3+) or knocked down with small interfering RNA (RNAi). In BNIP3+ cells, cyanide-induced apoptotic death was markedly enhanced and preceded by reduction of mitochondrial membrane potential (delta psim), release of cytochrome c from mitochondria and elevated caspase 3 and 7 activity. Pretreatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Ala-Asp-fluoromethyl ketone (zVAD-fmk) suppressed BNIP3+-mediated cell death, thus confirming a caspase-dependent apoptosis. On the other hand, BNIP3 knockdown by RNAi or antagonism of BNIP3 by a transmembrane-deleted dominant-negative mutant (BNIP3 delta TM) markedly reduced cell death. Immunohistochemical imaging showed that cyanide stimulated translocation of BNIP3 from cytosol to mitochondria and displacement studies with BNIP3 delta TM showed that integration of BNIP3 into the mitochondrial outer membrane was necessary for the cell death. In BNIP3+ cells, cyclosporin-A, an inhibitor of mitochondrial pore transition, blocked the cyanide-induced reduction of delta psim and decreased the apoptotic death. These results demonstrate in cortical cells that cyanide induces a rapid upregulation of BNIP3 expression, followed by translocation to the mitochondrial outer membrane to reduce delta psim. This was followed by mitochondrial release of cytochrome c to execute a caspase-dependent cell death.

Cyanide produces degeneration of the nervous system in which different modes of cell death are activated in the vulnerable brain areas. In brain, the mechanism underlying the cell death is not clear. In this study, an immortalized dopaminergic cell line was used to characterize the cell death signaling cascade activated by cyanide. Cyanide-treated cells exhibited a time- and concentration-dependent apoptosis that was caspase independent. Cyanide induced a rapid surge of intracellular reactive oxygen species (ROS) generation, followed by p38 mitogen-activated protein kinase (MAPK) activation and nuclear accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). Activation of p38 MAPK and HIF-1alpha accumulation were attenuated by N-acetyl-L-cysteine (antioxidant), catalase (hydrogen peroxide scavenger), or a selective p38 MAPK inhibitor (SB203580). Cyanide activated the hypoxia response element (HRE) promoter, which was also blocked by the antioxidants and SB203580. HRE activation was followed by increased BNIP3 gene transcription, as reflected by elevated BNIP3 mRNA and protein levels. BNIP3 upregulation was reduced by selective RNAi knockdown of HIF-1alpha. Overexpression of BNIP3 produced mitochondrial dysfunction (reduced membrane potential), caspase-independent apoptosis, and sensitization of the cells to cyanide-induced toxicity. Expression of a dominant-negative mutant or RNAi knockdown of BNIP3 protected the cells from cyanide. It was concluded that cyanide activated the HIF-1alpha-mediated pathway of BNIP3 induction through a redox-sensitive process. Increased BNIP3 expression then served as an initiator of mitochondrial-mediated death.

We have previously shown in rat primary cultured mesencephalic cells that cyanide induces a high level of oxidative stress and necrotic death. To evaluate the mechanism of the cytotoxicity, the effects of cyanide on intracellular glutathione (GSH) pools and inducible nitric oxide synthase (iNOS)-mediated reactive nitrogen species (RNS) generation were studied. Cyanide rapidly depleted intracellular GSH. Restoration of GSH blocked cell death, whereas depletion of GSH by synthesis inhibition increased the necrosis. Selective depletion of mitochondrial GSH (mtGSH) increased oxidative stress and enhanced cell death, whereas the cytoplasmic pool was not critical to cell survival. These actions were accompanied by increased iNOS expression as determined by Western blot analysis, RT-PCR and immunohistochemistry. Up-regulation of iNOS led to increased generation of NO as reflected by elevated nitrite levels (an end product of NO metabolism). It was determined by use of a selective inhibitor that up-regulation of iNOS expression was transcriptionally regulated by activation of nuclear factor-kappaB, a redox-sensitive transcription factor. It was concluded that, in cyanide-mediated neurotoxicity, mtGSH is a vital component of the cellular antioxidant defense, and its depletion can lead to oxidative stress-mediated iNOS up-regulation, thus enhancing RNS generation and necrosis., (Copyright 2006 Wiley-Liss, Inc.)

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear factor PPAR family that regulates a variety of cellular functions, including lipid metabolism, cellular oxidative stress defense, and inflammatory responses. Based on the report that Wy14,643, a PPARalpha agonist, can upregulate uncoupling protein-2 (UCP-2), this study was conducted in primary cortical cells to determine if PPARalpha activation enhances cyanide-induced neurotoxicity through changes in the level of UCP-2. PCR and Western blot analysis showed that Wy14,643 upregulated UCP-2 transcriptionally over a 12-h period. This response was mediated by PPARalpha since it was blocked by MK886, a selective PPARalpha antagonist. The effect of UCP-2 upregulation on the cytotoxic response to cyanide was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (apoptosis) and propidium iodide staining (necrosis). Wy14,643 switched the mode of cyanide-induced cell death from apoptosis to necrosis. Cell death was preceded by marked mitochondrial dysfunction, as reflected by depletion of ATP and reduction of the mitochondrial membrane potential (DeltaPsim). Knock down of UCP-2 expression by RNA interference blocked the Wy14,643-mediated enhancement of cyanide-induced mitochondrial dysfunction and the switch of the cell death mode, thus confirming that the response was mediated by upregulation of UCP-2. This study shows that PPARalpha activation can upregulate UCP-2 expression, which in turn enhances cyanide-induced necrotic cell death through an increase of mitochondrial dysfunction.

Uncoupling protein 2 (UCP-2) regulates mitochondrial function by increasing proton leak across the inner membrane to dissociate respiration from ATP synthesis and reduce reactive oxygen species generation. A number of studies have shown that UCP-2 expression protects cells from oxidative stress mediated injuries. In the current study, we show UCP-2-mediated reduction in mitochondrial function contributes to the mitochondrial dysfunction and the necrotic death of primary cultured mesencephalic cells (MCs) after exposure to cyanide, a complex IV inhibitor. The necrotic cell death was directly related to the level of mitochondrial dysfunction, as shown by reduction in ATP levels and decreased mitochondrial membrane potential. Treatment with cyanide for 6 h or longer upregulated UCP-2 expression. Blockade of up-regulation with a transcription or a translational inhibitor reduced the response to cyanide. Knockdown with RNAi or transfection with a UCP-2 dominant-negative interfering mutant reduced the cyanide-induced mitochondrial dysfunction and cell death, showing that constitutive expression of UCP-2 plays a role in the response to cyanide. Overexpression of UCP-2 by transfection with human full-length cDNA potentiated the cyanide toxicity. These findings indicate that UCP-2 can serve as a regulator of mitochondria-mediated necrotic cell death, in which enhanced expression can increase the vulnerability of primary MCs to injury due to complex IV-mediated inhibition by cyanide.

Uncoupling protein 2 (UCP-2) is expressed in the inner mitochondrial membrane and modulates mitochondrial function by partially uncoupling oxidative phosphorylation, and it has been reported to modulate cell death. Cyanide is a potent neurotoxin that inhibits complex IV to alter mitochondrial function to induce neuronal death. In primary rat cortical cells KCN produced an apoptotic death at 200-400 microM. Higher concentrations of potassium cyanide (KCN) (500-600 microM) switched the mode of death from apoptosis to necrosis. In necrotic cells, ATP levels were severely depleted as compared to cortical cells undergoing apoptosis. To determine if UCP-2 expression could alter KCN-induced cell death, cells were transiently transfected with full-length human UCP-2 cDNA (UCP-2+). Overexpression switched the mode of death produced by KCN (400 microM) from apoptosis to necrosis. The change in cell death was mediated by impaired mitochondrial function as reflected by a marked decrease of ATP levels and reduction in mitochondrial membrane potential. RNA interference or transfection with a dominant interfering mutant blocked the necrotic response observed in UCP-2+ cells. Additionally, treatment of UCP-2+ cells with cyclosporin A blocked necrosis, indicating the involvement of mitochondrial permeability pore transition in the necrotic death. These results show that increased expression of UCP-2 alters the response to a potent mitochondrial toxin by switching the mode of cell death from apoptosis to necrosis. It is concluded that UCP-2 levels influence cellular responses to cyanide-induced mitochondrial dysfunction.

Cyanide is generated in neurons and this report examines the two different receptors which mediate cyanide formation in neuronal tissue. An opiate receptor blocked by naloxone increases cyanide production both in rat brain and in rat pheochromocytoma (PC12) cells. A muscarinic receptor in PC12 cells releases cyanide and the effect is blocked by atropine. In rat brain, in vivo, a muscarinic agonist inhibits cyanide generation, possibly by acting on receptor subtypes different from those in PC12 cells. Cyanide generation by a muscarinic agonist in PC12 cells is blocked by pertussis toxin but that caused by an opiate is not. Thus, two different receptors and two different second messenger systems can mediate cyanide generation in PC12 cells. In parallel with the in vivo data, cultured primary rat cortical cells also show decreased cyanide release following muscarinic stimulation. Both blockade of cyanide generation by muscarinic receptor activation and cyanide release by opiate agonists from cortical cells are pertussis toxin insensitive. Similarly, little cyanide generation was seen following cholera toxin treatment. These data indicate that opiate receptors increase and muscarinic receptors decrease cyanide production in rat brain tissue by G-protein independent mechanisms. This work supports the suggestion that the powerful actions of cyanide may be important for neuromodulation in the CNS.

In cyanide-induced apoptosis, an increase in cytosolic free Ca2+ and generation of reactive oxygen species are initiation stimuli for apoptotic cell death. Previous studies have shown that cyanide-stimulated translocation of Bax (Bcl-associated X protein) to mitochondria is linked with release of cytochrome c and subsequent activation of a caspase cascade [Shou, Li, Prabhakaran, Borowitz and Isom (2003) Toxicol. Sci. 75, 99-107]. In the present study, the relationship of the cyanide-induced increase in cytosolic free Ca2+ to activation of Bad ( Bcl-2/Bcl-X(L)- antagonist, causing cell death) was determined in cortical cells. Bad is a Ca2+-sensitive pro-apoptotic Bcl-2 protein, which on activation translocates from cytosol to mitochondria to initiate cytochrome c release. In cultured primary cortical cells, cyanide produced a concentration- and time-dependent translocation of Bad from cytosol to mitochondria. Translocation occurred early in the apoptotic response, since mitochondrial Bad was detected within 1 h of cyanide treatment. Mitochondrial levels of the protein continued to increase up to 12 h post-cyanide exposure. Concurrent with Bad translocation, a Ca2+-sensitive increase in cellular calcineurin activity was observed. Increased cytosolic Ca2+ and calcineurin activation stimulated Bad translocation since BAPTA [bis-(o-aminophenoxy)ethane-N, N, N', N'-tetra-acetic acid], an intracellular Ca2+ chelator, and cyclosporin A, a calcineurin inhibitor, significantly reduced translocation. BAPTA also blocked release of cytochrome c from mitochondria as well as apoptosis. Furthermore, treatment of cells with the calcineurin inhibitors cyclosporin A or FK506 blocked the apoptotic response, linking calcineurin activation and the subsequent translocation of Bad to cell death. These observations show that by inducing a rapid increase in cytosolic free Ca2+, cyanide can partially initiate the apoptotic cascade through a calcineurin-mediated translocation of Bad to mitochondria.

Dopamine (DA)-induced neurotoxicity is potentiated when cellular metabolism is compromised. Since cyanide is a neurotoxin that produces mitochondrial dysfunction and stimulates intracellular generation of reactive oxygen species (ROS), KCN was used to study DA-induced apoptosis in primary cultured mesencephalon cells. Treatment of neurons with DA (300 microM) for 24h produced apoptosis as determined by TUNEL staining, DNA fragmentation and increased caspase activity. Pretreatment with KCN (100 microM) 30min prior to DA increased the number of cells undergoing apoptosis. When added to the cells alone, this concentration of KCN did not induce apoptosis. DA stimulated intracellular generation of ROS, and treatment with KCN enhanced ROS generation. Treatment of cells with glutathione or uric acid (antioxidants/scavengers) attenuated both the increase in ROS generation and the apoptosis, demonstrating that ROS are initiators of the cytotoxicity. Studies on the sequence of events mediating the response showed that DA-induced depolarization of the mitochondrial membrane was dependent on ROS generation and KCN enhanced this action of DA. Following changes in mitochondrial membrane potential, cytochrome c was released from mitochondria, leading to caspase activation and eventually cell death. These results demonstrate that oxidative stress and mitochondrial dysfunction are initiators of DA-induced apoptosis. Subsequent cytochrome c release activates the caspase effector component of apoptosis. Cyanide potentiates the neurotoxicity of DA by enhancing the generation of ROS and impairing mitochondrial function.

A comparative study was conducted in rat primary cortical (CX) and mesencephalic (MC) neurons to investigate intracellular cascades activated during cyanide-induced injury and to determine the point at which the cascades diverge to produce either apoptosis or necrosis. Cyanide treatment (400 microM) for 24 h produced primarily apoptosis in CX cells, whereas the same concentration of cyanide induced predominantly necrosis in MC cells as indicated by increased propidium iodide staining and cellular lactate dehydrogenase efflux. Cyanide increased generation of cellular reactive oxygen species (ROS) in both CX and MC cells, but the rate of formation and nature of the oxidative species varied with cell type. Catalase decreased cyanide-induced ROS generation in CX but not in MC cells. Nitric oxide generation was more prominent after cyanide treatment of MC compared with CX cells. N-Methyl-D-aspartate receptors were more involved in CX apoptosis than in MC necrosis. Mitochondrial membrane potential decreased moderately in CX cells on exposure to cyanide, whereas MC cells responded with a more pronounced reduction in potential. In CX cells cyanide produced a concentration-dependent release of cytochrome c from mitochondria and increased caspase activity, whereas little change was seen in MC neurons. Thus, cyanide-induced necrosis of MC cells involved generation of excessive amounts of nitric oxide and superoxide accompanied by mitochondrial depolarization. In contrast cyanide causes a lower level of oxidative stress in CX cells, involving mainly hydrogen peroxide and superoxide, and a moderate change in mitochondrial membrane potential that lead to cytochrome c release, caspase activation, and apoptosis.

Cyanide induces apoptosis through cytochrome c activated caspase cascade in primary cultured cortical neurons. The underlying mechanism for cytochrome c release from mitochondria after cyanide treatment is still unclear. In this study, the roles of endogenous Bcl-2 proteins in cyanide-induced apoptosis were investigated. After cyanide (100-500 microm) treatment for 24 h, two pro-apoptotic Bcl-2 proteins, Bcl-X(S) and Bax were up-regulated as shown by western blot and RT-PCR analysis. The expression levels of two antiapoptotic Bcl-2 proteins, Bcl-2 and Bcl-X(L), remained unchanged after cyanide treatment, whereas the mRNA levels of Bcl-X(S) and Bax began to increase within 2 h and their protein levels increased 6 h after treatment. NF-kappaB, a redox-sensitive transcription factor activated after cyanide treatment, is responsible for the up-regulation of Bcl-X(S) and Bax. SN50, which is a synthetic peptide that blocks translocation of NF-kappaB from cytosol to nucleus, inhibited the up-regulation of Bcl-X(S) and Bax. Similar results were obtained using a specific kappaB decoy DNA. NMDA receptor activation and reactive oxygen species (ROS) generation are upstream events of NF-kappaB activation, as blockade of these two events by MK801, l-NAME or PBN inhibited cyanide-induced up-regulation of Bcl-X(S) and Bax. Up-regulation of pro-apoptotic Bcl-X(S) and Bax contributed to cyanide-induced cytochrome c release, because SN50 and a specific Bax antisense oligodeoxynucleotide significantly reduced release of cytochrome c from mitochondria as shown by western blot analysis. It was concluded that NF-kappaB-mediated up-regulation of Bcl-X(S) and Bax is involved in regulating cytochrome c release in cyanide-induced apoptosis.

In a study of the mechanism by which cyanide is produced in neural tissue, it was hypothesized that nerve cells generate cyanide in a manner similar to that in leukocytes. As in white blood cells, glycine addition enhanced cyanide production in rat pheochromocytoma cells. Because myeloperoxidase catalyses cyanide production in leukocytes, a selective myeloperoxidase inhibitor (aminobenzoic acid hydrazide) was tested and found to inhibit opiate agonist-induced cyanide production in pheochromocytoma cells and also in rat brain. In addition, hydrogen peroxide enhanced cyanide release in pheochromocytoma cells, further suggesting that the process is oxidative in nature. Sonicated rat pheochromocytoma cells did not generate cyanide in response to an agonist acting on surface receptors even though disrupted cells responded to glycine. The mitochondrial fraction from rat brain produced more cyanide in response to glycine than any other fraction. Thus glycine seems to act at an intracellular site to enhance cyanide production and the process seems to involve a peroxidase mechanism similar to that reported for white blood cells., (Copyright 2000 Wiley-Liss, Inc.)

Activation of NMDA receptors plays an important role in cyanide neurotoxicity. Cyanide indirectly activates the receptor by inducing neuronal release of glutamate and also enhances receptor-mediated responses by a direct interaction with the receptor complex. This study investigated the mechanism in cerebellar granule cells by which cyanide enhances NMDA-induced Ca2+ influx. Cyanide (50 microM) increased the influx of Ca2+ over the NMDA concentration range of 0.5-500 microM. Experiments showed that cyanide does not interact with the receptor's glycine or PKC mediated phosphorylation regulatory sites. N-ethylmaleimide, a thiol alkylating agent which inactivates the redox regulatory sites of the receptor, blocked the enhancing effect of cyanide. Pretreatment of cells with 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), a compound that oxidizes the receptor redox sites, had no effect on the response to cyanide. On the other hand, the nonpermeant reducing agents, dithiothreitol or cysteine, further increased the cyanide effect. These observations can be explained by cyanide interacting with redox sensitive disulfide groups that are not accessible to the non-permeant reducing agents. It is proposed that cyanide interacts with a redox site(s) located either on the intracellular receptor domain or in the transmembrane hydrophobic domain. Furthermore the enhancement by cyanide of the excitotoxic actions of NMDA involves receptor sites that are sensitive to oxidation/reduction and this interaction contributes to the neurotoxic action of cyanide.

The effect of cyanide on the N-methyl-D-aspartate (NMDA)-stimulated increase in cytosolic free calcium ([Ca++]i) was studied by microfluorescence in fura-2-loaded cerebellar granule cells. The response to NMDA was enhanced by NaCN over a concentration range of 20 to 100 microM. These concentrations of NaCN in the absence of NMDA had no effect on basal [Ca++]i. In comparison, NaCN did not affect K+-depolarization-induced [Ca++]i elevation. The NaCN potentiation of NMDA-evoked [Ca++]i elevation was blocked by addition of Mg++ and by the NMDA receptor antagonists 2-amino-5-phosphono-valeric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate. Pretreatment of the cells with pregnenolone sulfate or arachidonate, known modulators of the NMDA receptor, enhanced NaCN action. The voltage-sensitive calcium channel blockers nifedepine and diltiazem did not affect the NaCN-induced potentiation. Additionally, the NaCN action was not altered when tetrodotoxin was used to block Na+ channel-mediated glutamate release. In patch-clamp studies, NaCN increased the amplitude and duration of NMDA-stimulated whole-cell currents. NaCN also enhanced the NMDA receptor response in single-channel patch-clamp experiments. In the outside-out patch recording configuration, NaCN increased the NMDA receptor channel opening frequency without affecting single-channel conductance or mean channel open time. These results indicate that cyanide interacts directly with the NMDA receptor channel complex to enhance receptor-mediated responses.

Cyanide's actions are complex and cannot be attributed solely to inhibition of oxygen utilization. Recent mechanistic studies show that cyanide inhibits multiple enzymes and alters several vital intracellular processes to produce the intoxication syndrome. By understanding the intracellular targets and the mechanisms underlying the toxicity, it is proposed that more effective antidotal regimens can be achieved. A mechanistic based, multi-step in vitro model was developed for screening potential cyanide antidotes. A series of compounds was screened for their ability to reverse the effect of cyanide on six neurochemical markers in the PC12 cell line (neuronal cell model). Each compound was assigned a composite score based on the six assays; several compounds were identified which then exhibited efficacy in animal testing. Additional mechanistic based studies show that antioxidants and nitric oxide generators have promise as anti-cyanide agents. It is concluded that mechanistic based antidote design can be used to identify new compounds for testing in animal models.

Excitatory amino acid (EAA)-like and excitotoxic properties of the secondary metabolite of cyanide, 2-iminothiazolidine-4-carboxylic acid, (2-ICA) were evaluated because of its possible role in cyanide-induced neurotoxicity. Intracerebroventricular (i.c.v.) injections of 2-ICA in mice produced wild-running seizures that were qualitatively and quantitatively similar to seizures observed with glutamate. 2-ICA, kainate and proline seizures were prevented by both the NMDA and non-NMDA antagonists, MK-801 and CNQX, respectively. In contrast, NMDA-induced seizures were prevented by MK-801, but not CNQX. When infused i.c.v. in rats over a seven day period, 2-ICA produced extensive and selective loss of CA-1 pyramidal neurons of the hippocampus. In hippocampal slices preloaded with D-[3H]aspartate, 2-ICA superfusion did not evoke release nor significantly augment potassium stimulated release of the radiolabeled transmitter. These findings indicate 2-ICA has excitotoxic properties and its role in cyanide neurotoxicity deserves further study.

Incubation of rat pheochromocytoma (PC12) cells with cyanide (1-10 mM) for 30 min produced a compound which eluted between dopamine (DA) and 3,4-dihydroxyphenylacetic acid in HPLC-EC analysis. Generation of the compound was rapid, concentration-dependent and blocked by pretreatment with clorgyline, a monoamine oxidase A inhibitor. In cell free incubation, the compound formed rapidly when cyanide was added to a mixture of monoamine oxidase and DA, but was not detected when cyanide was included with DA alone. The compound was radiolabelled when 14C-KCN was added to the incubation mixture. Based on these results, it is proposed that the deaminated metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), reacts non enzymatically with cyanide to form the cyanohydrin adduct 2-hydroxy-3-(3,4-dihydroxyphenyl) propionitrile (HPN). HPN was confirmed by spectral analysis and co-elution with synthetic HPN. Incubation of mouse brain slices with cyanide (1 mM) generated 0.98 ng HPN/100 mg wet wt. over a 10 min period and HPN was detected in brains of mice after injection of cyanide (15.6 micrograms) into the lateral brain ventricle. Repeated doses of KCN (6 mg/kg, s.c., five times) produced 0.14 +/- 0.03 ng/100 mg of tissue in striatum. Incubation of PC12 cells for 60 min with 500 microM HPN killed 23% of the cells and increased DA release from the cells by 39.8% over untreated cells. Uptake of HPN into the cells was partially blocked by the catecholamine uptake inhibitor imipramine. These results indicate cyanide reacts with the DA metabolite DOPAL to generate a biologically active cyanohydrin adduct which may contribute to the neurotoxic response to cyanide.

Since cyanide is a reactive chemical substance and has the potential of forming a variety of adducts in biological systems, the rate of accumulation of labeled cyanide was studied in neural tissue, a major target for the toxic action of cyanide. Accumulation of 14CN in mouse brain slices and in rat pheochromocytoma (PC12) cells was about five times more rapid in the first few minutes than at later times. In both tissues, incubation at low temperature (4 degrees C) decreased the secondary phase without affecting the initial phase. In PC12 cells, determination of the subcellular distribution of cyanide revealed that the cytoplasmic fraction was the least sensitive to temperature and therefore may represent the initial rapid phase of cyanide accumulation. Accumulation of cyanide in mitochondrial and microsomal fractions was temperature sensitive. Cyanide accumulation is proportional to the concentration in the medium (0.1-1 mM) in both mouse brain and PC12 cells suggesting that the cyanide sinks in neural tissue have a large capacity and are nonspecific. Cyanide interaction with neural tissue is not uniform since cyanide accumulated more in hypothalamus than in other brain areas. Despite this, mitochondrial enzyme activity was no greater in hypothalamus than in other brain areas. No increase in thiocyanate, the main metabolite of cyanide, could be detected in brain tissue incubated 30 min with 1 mM cyanide, showing that accumulated 14C is not in the form of thiocyanate. Cyanide appears to equilibrate rapidly across the plasma membrane and then slowly accumulates in mitochondria and membrane elements of the neuronal cell. Competition for cyanide among subcellular elements may be a factor in the toxic action of cyanide.

The effect of cyanide on NMDA-activated ion current and MK801 binding was studied in cultured rat hippocampal neurons. In microfluorometric analysis using fura-2, removal of extracellular Mg2+ resulted in a five-fold increase in NMDA-induced peak of [Ca2+]i. One mM NaCN enhanced the peak NMDA responses in the presence, but not in the absence of extracellular Mg2+. Cyanide enhanced the immediate rise in [Ca2+]i produced by NMDA, followed over a 1-5 min period by a gradual increase of [Ca2+]i. Similar results were obtained in whole-cell patch clamp recordings from hippocampal neurons. One mM KCN enhanced the NMDA-activated current in the presence, but not in the absence of extracellular Mg2+. This effect was independent of cyanide-mediated metabolic inhibition since the recording pipette contained ATP (2 mM). In binding assays NaCN (1 mM) increased the binding affinity of [3H]MK-801 to rat forebrain membranes in the presence of Mg2+, whereas in the absence of Mg2+, NaCN did not influence binding. These results indicate that cyanide enhances NMDA-mediated Ca2+ influx and inward current by interacting with the Mg2+ block of the NMDA receptor. The effect of cyanide can be explained by an initial interaction with the Mg2+ block of the NMDA receptor/ionophore which appears to be energy-independent, followed by a gradual increase in Ca2+ influx resulting from cellular energy reserve depletion.

The effect of acute cyanide intoxication on levels of transcriptional regulatory proteins Fos and c-Jun in rat cortex, hippocampus, cerebellum and brain stem was studied. Western blot analysis showed a differential effect of cyanide on Fos levels in the selected brain areas. The most prominent changes were seen 60 min. following ip. injection of KCN in all brain areas except the brain stem, which showed the maximal change 120 min. following cyanide. Fos levels were doubled in cortex and cerebellum and decreased to below 70% of the control levels in hippocampus. Levels of c-Jun were not altered 60 min. following cyanide treatment. Pretreatment with the NMDA receptor antagonist, MK-801, prevented the cyanide-induced changes of Fos. The differential effect of cyanide on Fos levels in different brain areas and the blockade of these changes by MK-801 suggest involvement of multiple neuronal pathways, including the excitatory amino acid (EAA) neurotransmitter system. It is concluded that cyanide alters levels of the transcriptional regulatory protein Fos through activation of the EAA neurotransmitter system and, thus, may affect gene expression in neuronal or glia cells.

A series of six biochemical markers of cyanide toxicity (dopamine release, hydroperoxide generation, cytosolic-free calcium levels, catalase activity, cytochrome oxidase activity, and superoxide dismutase activity) in cultured rat pheochromocytoma (PC12) cells were used to establish a screen for evaluation of potential anticyanide compounds. Thirty-nine substances, including anticonvulsants, adrenergic blockers, antioxidants, and antipsychotics were tested and ranked according to the results. Based on the composite scoring in all six assays, carbamazepine, mannitol, allopurinol, and phenytoin were ranked as the most effective anticyanide compounds. Additionally, known cyanide antidotes (e.g., pyruvate, mercaptopyruvate, alpha-ketoglutarate, naloxone, and flunarizine) obtained relatively high ranking in the PC12 cell screen. Furthermore, a significant correlation was found between protective effects (based on LD50s) of cyanide antidotes in mice and ranking in the in vitro screen. This study illustrates that by assaying a series of biochemical markers in a neuronal-type cell line, a rapid, cost-effective in vitro toxicological screen is possible. Several compounds have been identified which inhibit the biochemical effects of cyanide and may be used to enhance effectiveness of the standard cyanide antidotes.

Calcium channel blockade decreases the elevation of brain calcium as well as the tremors produced by cyanide in mice. To determine if cyanide-induced morphological changes could also be inhibited by calcium channel blockade, the effect of diltiazem was studied in cultured rat pheochromocytoma (PC12) cells, a neuronal model. Incubation with KCN (1 to 10 mM for 1 to 2 hr) caused depletion of secretory granules, alignment of remaining granules along the plasma membrane, and mitochondrial swelling. All these effects were inhibited by pretreatment with 0.01 mM diltiazem. Scanning electron microscopy revealed that cyanide (1 to 10 mM for 1 to 2 hr) produced loss of microvilli and bleb formation in PC12 cells. These changes were partially inhibited by preincubation with 0.01 mM diltiazem. Incubation of cells with 10 mM cyanide increased release of lactic dehydrogenase (LDH) into the culture media at 60 and 120 min. A decrease in cell viability, as determined by trypan blue dye exclusion, paralleled the release of LDH. At 120 min of cyanide incubation, 24% of the cells excluded dye. Both the release of LDH and decreased cell viability were attenuated by pretreatment with diltiazem. The results indicate that the influx of extracellular calcium is an important factor mediating cyanide-induced morphologic changes in neuronal cells.

The mechanism of action of nitrite-thiosulfate (Chen et al., 1933a ,b; Hug , 1933) in the antagonism of the lethal effects of cyanide is much more complex than proposed 50 years ago. Some of the recent findings concerning the mechanism of nitrite action have conceptual theoretical and practical significance, as the development of newer cyanide antagonists are dependent on the elucidation of the basic mechanism of antidotal action. There are preliminary evidence which suggest a vasogenic action rather than methemoglobin formation is the primary action of nitrite, as a cyanide antagonist. Various vasogenic compounds have been uncovered and they may play an important role in the future development of a new class of cyanide antagonists. Also recent development in thiol detoxication of cyanide suggest that rhodanese may play a more complex role. The detoxification of cyanide may be viewed from a considerably more complex perspective with the elucidation of recent mechanisms. It also may provide a newer conceptual basis for a more rational development of future compounds to antagonize the lethal effects of cyanide.

Attempts were made to evaluate the effects of pretreatment with air and oxygen either alone or in various combinations with sodium nitrite and/or sodium thiosulfate on the physiological disposition of 14C-labeled sodium cyanide in mice. The radioactive respiratory excretion was studied by radiorespirometry, and the effects of various combinations of cyanide antagonists were compared. Oxygen either alone or in combination with sodium thiosulfate significantly enhanced the respiratory excretion when compared with air. Sodium thiosulfate accelerated the initial rate, but not the total amount of radioactivity excreted. The cumulative recovery of radioactive gases was significantly greater with groups receiving oxygen either alone or with sodium thiosulfate. When sodium nitrite was employed as an antidote either alone or with sodium thiosulfate, no difference in the respiratory excretion was noted between air and oxygen. The use of the sodium nitrite-sodium thiosulfate combination either with air or oxygen resulted in a marked decrease in the initial rate as well as the total amount of respiratory radioactivity excreted. No significant differences between various experimental groups were noted in the total amount of urinary radioactivity excreted or the total body retention of radioactivity.

Since oxygen was reported to be an effective cyanide antagonist in vivo, particularly in the presence of the classic antidotal combination of sodium nitrite and sodium thiosulfate, in vitro studies were initiated in an attempt to investigate the mechanism of oxygen-mediated cyanide antagonism. The effect of oxygen on cyanide-inhibited cytochrome oxidase with and without cyanide antagonist(s) was investigated in a purified membraneous enzyme system prepared from rat liver mitochondria. Cyanide produced a concentration dependent inhibition of cytochrome oxidase, and 100% oxygen did not alter the inhibition produced by KCN either in the presence or absence of sodium thiosulfate. However, the addition of sodium thiosulfate and rhodanese to the assay reactivated the cyanide-inhibited cytochrome oxidase. Kinetic analysis indicated rhodanese competes with cytochrome oxidase for cyanide, and oxygen had no effect on this coupled reaction. In conclusion, the in vivo antidotal properties of oxygen cannot be attributed to oxygen-mediated reactivation of cyanide-inhibited cytochrome oxidase or an oxygen-mediated acceleration of rhodanese detoxification.

The role of oxidative metabolism in the disposition of potassium cyanide (KCN), was investigated in mice administered KCN, (4.6 mg/kg, s.c.) containing 4.5 microCi [14C]KCN. The expired pulmonary metabolites, [14C]hydrocyanic acid (HCN) and 14CO2, were collected and analyzed. Approximately 1% and 2% of the KCN dose was expired as [14C]HCN and 14CO2, respectively. Expiration of the pulmonary metabolites was decreased following pretreatment with sodium nitrite, sodium thiosulfate, oxygen, or a combination of cyanide antidotes. Treatment with hydrogen peroxide lowered the amount of [14C]HCN expired and did not alter the expiration of 14CO2. Treatment with 3-amino-1,2,4-triazole (catalase inhibitor), superoxide dismutase, or diethyldithiocarbamic acid (superoxide dismutase inhibitor) did not change the amount of [14C]HCN expired. However, superoxide dismutase significantly increased the amount of 14CO2 expired, whereas diethyldithiocarbamic acid decreased 14CO2 expiration. The results from these studies suggest that in vivo cyanide can be oxidized to CO2 and treatment with agents that alter the availability of endogenous superoxide and/or hydrogen peroxide can alter the rate of cyanide oxidation.

A sensitive and specific radiolabel method for measuring expired hydrogen cyanide (HCN) or carbon dioxide (CO2) derived from cyanide was developed. An ethanol collecting solution containing a 10(-2) M of cobalt chloride trapped 88% of the H14CN passed through the solution following acid volatilization of a known amount of K14CN. The range of linearity, r = 0.998, exceeded the 0.01 to 0.1 mumoles tested for measuring the pulmonary metabolites. The cobalt chloride collecting solution trapped less than 0.02% of the 10 to 100 mumoles of 14CO2 generated by acid hydrolysis of 14C-sodium bicarbonate (NaH14CO3). Introduction of a second collecting solution specific for CO2, composed of ethanol: ethanolamine (2:1, v/v), was used to collect the CO2 derived from cyanide. Following the subcutaneous (sc) administration of 4.6 mg/kg of potassium cyanide (KCN) and 4.5 microcuries (microCi) K14CN, 1 to 2% and 2 to 3% of the dose was expired as H14CN and 14CO2, respectively.

Previous reports from our laboratory indicated that prophylactic protection against cyanide intoxication in mice can be enhanced by administration of chlorpromazine when it is given with sodium thiosulfate. The mechanism of potentiation of sodium thiosulfate by chlorpromazine was studied alone and in combination with sodium nitrite. Although chlorpromazine was found to induce a hypothermic response, the mechanism of enhancement of the antagonism of cyanide by chlorpromazine does not correlate with the hypothermia produced. Various other possible mechanisms were investigated, such as rate of methemoglobin formation, enzymatic activity of rhodanese and cytochrome oxidase, and alpha-adrenergic blockade. The alpha-adrenergic blocking properties of chlorpromazine may provide a basis for its antidotal effect, since this protective effect can be reversed with an alpha-agonist, methoxamine.

Lipid peroxidation of brain lipids as determined by the conjugated diene method was observed in mice following administration of sublethal doses of potassium cyanide (KCN). Conjugated diene production was dose- and time-dependent; 10 mg/kg KCN produced detectable levels of conjugated dienes at 30 min post cyanide, whereas, 15 mg/kg produced marked levels of conjugated dienes over a 10-60-min period after KCN. Pretreatment of mice with either diltiazem (600 micrograms/kg, i.v.) or allopurinol (25 mg/kg, i.v.) blocked the generation of conjugated dienes. These results suggest lipid peroxidation of neuronal membranes play a role in cyanide intoxication and this action is related to altered regulation of neuronal calcium homeostasis and activation of xanthine oxidase.

The status of brain antioxidant enzymes and glutathione levels in mice intoxicated with KCN were correlated with lipid peroxidation in brain membranes. KCN (7 mg/kg, s.c.) rapidly increased conjugated dienes in brain lipids, with peak levels observed 30 min after cyanide treatment. At 60 min post cyanide, conjugated diene levels were only slightly elevated above controls. Temporal changes in activity of most antioxidant enzymes corresponded with the observed time course of cyanide-induced membrane lipid peroxidation. Thirty minutes after KCN, brain catalase (CA), glutathione peroxidase (GPX) and glutathione reductase (GR) activities were significantly reduced (percent inhibition compared to control: CA 44%, GPX 30%, and GR 41%). At 60 min, CA and GPX enzyme activity returned to control levels, whereas GR was elevated 34% above control activity. Superoxide dismutase was not significantly inhibited 30 min after KCN, but declined to 71% of control activity at 60 min. Brain levels of reduced glutathione declined 42% below control 30 min after cyanide and returned to within 9.4% of control at 60 min. At 30 and 60 min after cyanide, oxidize glutathione levels were not significantly changed from control levels. These studies suggest that membrane lipid peroxidation and subsequent membrane dysfunction observed in cyanide intoxication is related in part to a compromised antioxidant defense.

The effect of cyanide on whole-brain calcium levels was determined in mice administered KCN and correlated with the neurotoxic signs manifested during acute cyanide poisoning. KCN (10mg/kg, sc) significantly increased whole-brain total calcium levels from 48.1 +/- 1.8 to 66.5 +/- 3.9 micrograms/g dry wt within 15 min after administration. The levels remained elevated for 3 hr and returned to control readings after 12 hr. Dose-response studies revealed KCN, at doses of 10-15 mg/kg, produced significant elevations of whole-brain calcium 30 min after administration. No measurable effect was obtained from lower doses which suggested a threshold effect. Pretreatment 15 min before KCN with diltiazem, a calcium channel blocker, prevented the cyanide-induced rise in whole-brain total calcium. Cyanide-induced tremors, which are centrally mediated symptoms of intoxication, were quantified and correlated with the observed changes in whole-brain calcium. Tremors were detected at 10 and 12 mg/kg KCN and peak intensity was observed at 15 min postcyanide. Pretreatment with diltiazem markedly attenuated the cyanide-induced tremors. It appears that a correlation exists between cyanide-induced change in whole-brain calcium and tremors. This study suggests that intraneuronal calcium may play an important role in mediating cyanide neurotoxicity and calcium channel blocking agents may be useful in limiting the severity of the centrally mediated symptoms of acute cyanide intoxication.

Chlorpromazine (CPZ) is an effective cyanide antidote, with its greatest efficacy displayed when combined with the antidotes, sodium nitrite and sodium thiosulfate. Since the central nervous system is a primary target organ in cyanide toxicity, the objective of the present study was to determine the mechanisms by which CPZ prevents cyanide-induced damage in neural systems. KCN (10 mM) increased cytosolic free calcium in rat pheochromocytoma (PC12) cells as indicated by the fluorescent dye quin 2. This was blocked by addition of CPZ (0.1 mM) to the cells 15 min prior to addition of KCN. Incubation of cells with KCN (0.1 mM) increased the levels of lipid conjugated dienes and this was blocked by addition of CPZ (1 microM). Peroxidation of brain lipids in mice administered KCN (7-15 mg/kg, sc) was also attenuated by pretreatment with CPZ. Furthermore, production of lipid peroxidation in fresh mouse brain slices, following incubation with 0.1 mM KCN, was blocked by simultaneous addition of CPZ. These observations indicate CPZ prevents cyanide-induced calcium influx and decreases peroxidation of membrane lipids. Thus the antidotal activity of CPZ in cyanide toxicity appears to be related to maintenance of cellular calcium homeostasis and membrane integrity.

The effect of KCN on cytosolic Ca2+ levels was measured in PC12 cells using Quin II/AM, a fluorometric calcium indicator. The resting cytosolic Ca2+ concentration, 115.0 +/- 4.9 nm, increased gradually and steadily over a 30-min time period following addition of 10(-4), 10(-3), or 10(-2)M KCN to the cells. After 15 min, 10(-3) and 10(-2)M KCN produced a three- and sixfold increase in the cytosolic Ca2+ concentration, respectively. In K+-depolarized cells, KCN induced a more rapid rise of intracellular calcium than in cells treated with KCN or KCI alone. KCN and/or K+-induced accumulation of cytosolic Ca2+ was blocked when the cells were pretreated with 10(-5)M diltiazem (a calcium channel blocker). These results demonstrate in a cell model that cyanide induces an accumulation of cytosolic Ca2+ and this additional cytosolic Ca2+ load appears to originate primarily from the extracellular compartment. This study supports previous reports implicating calcium as an intracellular mediator of cyanide toxicity.

The effect of cyanide on release of catecholamines was evaluated in isolated bovine adrenal glands stimulated with 4 different agonists. Cyanide (0.1-1 mM) increased catecholamine release induced by barium or cadmium 2-3-fold. Acetylcholine or potassium induced secretion of adrenal catecholamines was also enhanced by cyanide, but only to the extent of 30-50%. These data suggest that cyanide acts by multiple mechanisms to enhance evoked catecholamine release. The above results may be partly explained by the fact that cyanide inhibited 45Ca efflux from stimulated bovine adrenals. Changes in plasma membrane permeability may be crucial in the alterations of ion flux and evoked catecholamine release caused by cyanide.