Cyanide-induced lipid peroxidation in different organs: subcellular distribution and hydroperoxide generation in neuronal cells.

To evaluate hydroperoxide generation as a potential mechanism of cyanide neurotoxicity, mice were treated with KCN (7 mg/kg, subcutaneously (s.c.)) and the level of lipid peroxidation (expressed as conjugated dienes) was measured later in various organs. Brain showed elevated conjugated diene levels after cyanide but the liver, which is not considered a target for cyanide toxicity, showed no increase. The heart also showed no increase, whereas kidney conjugated dienes slowly increased to a peak 1 h after cyanide. In vitro studies show elevation of peroxidized lipids in mouse brain cortical slices following incubation with KCN (0.1 mM). Omission of calcium from the medium or pretreatment of brain slices with diltiazem (a calcium channel blocker) prevented formation of conjugated dienes by KCN. Calcium thus appears to play a critical role in cyanide-induced generation of peroxidized lipids in neuronal cells. Subcellular fractionation of brains from mice treated with cyanide showed that lipid peroxidation increased in the microsomal fraction but not in the mitochondrial fraction. Fluorescent studies using 2,7-dichlorofluorescein (a hydroperoxide sensitive fluorescent dye) show that hydroperoxides are generated rapidly after cyanide treatment of PC12 cells, a neuron-like cell, and hydroperoxide levels remain elevated for many minutes in the presence of cyanide. These results suggest that hydroperoxide generation with subsequent peroxidation of lipids may lead to changes in structure and function of certain membranes and contribute to the neurotoxic damage produced by cyanide.

A protein kinase C inhibitor attenuates cyanide toxicity in vivo.

We have examined the effect of pretreatment with a potent protein kinase C (PKC) inhibitor, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), against metabolic alterations induced by sodium cyanide (NaCN), 4.2 mg/kg, in brain of anesthetized male micropigs (6-10 kg). Brain high energy phosphates were analyzed using a 31P nuclear magnetic resonance (NMR) spectroscopic surface coil in a 4.7 Telsa horizontal bore magnet. H-7, 1 mg/kg, was given intravenously (i.v.) 30 min before NaCN challenge (H-7 + CN-). Prior to NaCN, H-7, or H-7 + CN- administration, baseline 31P resonance spectra of 1-min duration were acquired for 5-10 min, and continued for an additional 60 min following i.v. NaCN injection, each animal serving as its own control. Peaks were identified as phosphomonoester (PME), inorganic phosphate (Pi), phosphodiester (PDE), phosphocreatine (PCr) and adenosine triphosphate (ATP), based on their respective chemical shifts. Without H-7 pretreatment, NaCN effects were marked by a rising Pi and a declining PCr peak 2 min after injection, with only 2/5 of the animals surviving the 60 min experiment. Through a pretreatment period of 30 min, H-7 did not affect baseline cell energy profile as reflected by the 31P-NMR spectra, but in its presence, those changes (i.e. diminishing PCr and rising Pi peaks) elicited by NaCN were markedly blunted; 4/5 of the animals in this group survived the NaCN challenge. It is proposed that H-7, a pharmacologic inhibitor of PKC, may be useful in CN- antagonism, underscoring the role of PKC in cyanide intoxication.

Protein kinase C modulation of rhodanese-catalyzed conversion of cyanide to thiocyanate.

Detoxification of cyanide is catalyzed by a sulfurtransferase, rhodanese, a phosphoprotein regulated by unknown protein kinases. In this study, we determined if a Ca2+/phospholipid-modulated phosphotransferase, protein kinase C (PKC) could modify rhodanese activity. Thiocyanate (SCN-) production as an estimate of rhodanese activity in vitro was measured in the presence or absence of exogenously added purified PKC, or 12-O-tetradecanoylphorbol acetate (TPA), a pharmacologic activator of the endogenous PKC. HI-6 (1-(2-(hydroximino)methyl))pyridinium-2-(4-(aminocarbonyl) pyridinium dimethylether) is an oxime that may dephosphorylate phosphoproteins due to the proposed phosphatase-like activity of the oximes. We examined HI-6's effect on rhodanese-catalyzed SCN- production. Bovine kidney rhodanese (0.40 mg/ml protein) was reacted with 4 mM KCN and SCN- production determined spectrophotometrically following the method of Westley (1981). Preincubating rhodanese with 20 or 100 ng of purified PKC (alpha, beta, gamma isozymes) for 5 min before initiating the reaction with 4 mM KCN as the substrate increased SCN- production by 17 or 40%, respectively, over the control (P < 0.05). Rhodanese formation of SCN- decreased when the preincubation was conducted with 1 nM or 100 nM of TPA. With HI-6 at 1 or 10 microM used in place of PKC, or TPA, rhodanese activity was increased by 6 or 14% (P < 0.05), respectively, compared to control. Under the conditions examined, exogenous PKC acting as a possible phosphate acceptor, and HI-6, a potential dephosphorylating compound, increased rhodanese activity. These data are consistent with the observation that rhodanese can exist as a phosphorylated enzyme which is not active and a dephosphorylated form which is active. It is suggested that addition of purified, exogenous PKC may accept phosphate from phosphorylated rhodanese or HI-6 may dephosphorylate rhodanese, both of which stimulate the conversion of cyanide anion to the less toxic SCN-. These observations support the possibility that rhodanese may be regulated by protein phosphorylation and treatments that alter the phosphorylation state of rhodanese may affect cyanide detoxification via SCN- formation.

Cyanide-induced alteration of cytosolic pH: involvement of cellular hydrogen ion handling processes.

Neuronal cells exposed to cyanide rapidly lose the capacity to regulate internal Ca2+ homeostasis, thereby accumulating an excess cytosolic Ca2+ load. The present study was undertaken to examine the effects of KCN on another important ion: hydrogen ion. KCN (1-10 mM) rapidly decreased intracellular pH (pHi) of cultured pheochromocytoma (PC12) cells as indicated by the pH-sensitive fluorescent dye 2',7-bis(carboxyethyl)-5(6)-carboxyfluorescein. Removal of Ca2+ from the media or pretreating the cells with diltiazem (10(-5) M), a calcium channel blocker, delayed the onset and reduced the magnitude of the drop in pHi. Lowering the pH of the incubation medium (pHo) to 6.9 exaggerated the drop in pHi, while raising it to 7.9 attenuated the change in pHi. Removal of Na+ from the media enhanced the cyanide effect. Reintroduction of Na+ or substitution with Li+ reversed the cytosolic acidification, suggesting involvement of the Na+/H+ exchanger in the cyanide action. Pretreatment of cells with amiloride, 0.2 mM, blunted the cytosolic acidification induced by KCN, possibly by decreasing intracellular Na+ accumulation and disrupting H+ efflux. Cyanide thus produces a rapid dysfunction of hydrogen ion handling mechanisms and this may play a role in cyanide neurotoxicity.

Cyanide-induced alteration of the adenylate energy pool in a rat neurosecretory cell line.

Cultures of a rat PC12 pheochromocytoma neurosecretory cell line were used to determine the responsiveness of oxidative energy status of isolated neuronal cells to cyanide exposure. Intracellular levels of ATP and its immediate metabolites, ADP and AMP, were measured in monolayer cultures of PC12 cells incubated for 0-30 min with KCN (10 mM). Over the period 2.5-30 min. cyanide treatment decreased ATP levels by 32-51% but ADP and AMP levels were not altered significantly. Additionally, ATP/ADP and ATP/AMP ratios were significantly reduced in KCN-intoxicated cells. These alterations in energy status may explain the prompt ablation of ion homeostasis reported previously in this model upon exposure to KCN. The energy-depleting actions of cyanide were not modified by pretreatment of cells with diltiazem, a calcium channel antagonist demonstrated to possess cytoprotective activity against histotoxic hypoxia induced by cyanide. Since PC12 cells rapidly respond to cyanide, with predictable depletions of the cell adenylate energy pool, this cell line can serve as a suitable in vitro model for studies of neurotoxicity involving ischemic/hypoxic conditions.

Cyanide-induced neurotoxicity: calcium mediation of morphological changes in neuronal cells.

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.

Cyanide-induced neurotoxicity: mechanisms of attenuation by chlorpromazine.

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.

Calcium mediation of cyanide-induced catecholamine release: implications for neurotoxicity.

Exposure of rat pheochromocytoma (PC12) cells to KCN (1.0-10 mM) over a 30-min period stimulated secretion of dopamine (DA) and decreased intracellular DA content. Addition of KCN (10 mM) to rat frontal cortex slices preloaded with 1-[7-3H]norepinephrine ([3H]NE) increased secretion of NE over a 10- to 30-min incubation period. In PC12 cells release of DA by KCN was nearly abolished in calcium-free media or by prior addition of diltiazem, a calcium channel antagonist. Release of [3H]NE from rat cortical slices by cyanide was only partly inhibited by diltiazem suggesting that intracellular calcium may be involved in this response. In PC12 cells KCN also produced a dose-related release of the DA precursor dihydroxyphenylalanine, without altering intracellular stores. Levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were enhanced at lower concentrations of KCN. These observations indicate cyanide elicits exocytotic release of neurotransmitters in a calcium-dependent manner and also show that cyanide alters catecholamine metabolism. These actions of cyanide may be important in CNS symptoms of intoxication.