The in vivo effects of cyanide and its antidotes on rat brain cytochrome oxidase activity.

The in vivo effects of sodium cyanide and its antidotes, sodium nitrite, sodium thiosulfate and 4-dimethylaminophenol (DMAP), as well as the alpha-adrenergic blocking agent phentolamine, on rat brain cytochrome oxidase were studied. The course of inhibition was time-dependent and a peak of 40% was attained between 15 and 20 min after the s.c. injection of 1.3 LD50 (12 mg/kg) of cyanide. Pronounced dose-dependence was observed in the inhibition of the enzyme, at this relatively low, but lethal dose. Further observation was impossible because of rapidly lethal effects of cyanide. In animals artificially ventilated with room air, observation was possible up to 60 min. However, maximum inhibition was also 40%. When antidotes were applied 30 min after 20 mg/kg of cyanide, marked reactivation of cytochrome oxidase activity was observed with all antidotes (particularly with thiosulfate) except for phentolamine which had no effect. Prevention of methemoglobin forming with toluidine blue did not affect the reactivating ability of nitrite or DMAP, thus suggesting more complex protective mechanisms then simple methemoglobin formation. The high efficacy of thiosulfate may be attributed to its rhodanese catalyzed, direct binding to free blood cyanide, leading thus to its dissociation from cytochrome oxidase. The theory that cytochrome oxidase inhibition is a basic mechanism of cyanide toxicity could not be disproved.     

Cyanide intoxication: protection with chlorpromazine.

Protection against cyanide intoxication in mice can be enhanced by the administration of chlorpromazine, providing it is given with sodium thiosulfate, or the sodium thiosulfate-sodium nitrite antidotal combination. Protency ratios which were derived from the LD50 values were compared in groups of mice premedicated with chlorpromazine (10 mg/kg) and/or sodium thiosulfate (1 g/kg) and/or sodium nitrite (100 mg/kg). These results indicate that the administration of chlorpromazine alone provides no protection against the lethal effects of cyanide. Chlorpromazine also does not enhance the protective effect of sodium nitrite; however, it strikingly potentiates the effectiveness of sodium thiosulfate either alone or in combination with sodium nitrite.     

Stereospecific effect of naloxone hydrochloride on cyanide intoxication

Cyanide intoxication in mice can be antagonized by the opiate antagonist, (-)naloxone HCl, alone or in combination with sodium thiosulfate and/or sodium nitrite. Potency ratios, derived from LD50 values, were compared in groups of mice pretreated with sodium nitrite (sc, 100 mg/kg), sodium thiosulfate (ip, 1 g/kg), and (-)naloxone HCl (sc, 10 mg/kg) either alone or in various combinations. These results indicate that naloxone HCl provides a significant protection against the lethal effects of potassium cyanide. The protective effect of sodium thiosulfate, but not sodium nitrite, was enhanced with (-)naloxone HCl. The combined administration of sodium nitrite and sodium thiosulfate was further enhanced with (-)naloxone HCl. This protective effect of naloxone HCl against the lethal effect of cyanide appears to be restricted to the (-)stereoisomer, as the (+)stereoisomer, the inactive opiate antagonist, is also inactive in protecting against the lethal effects of cyanide. The mechanism of antagonism is discussed.     

Antagonism of cyanide poisoning by dihydroxyacetone

Dihydroxyacetone (DHA) effectively antagonized the lethal effect of cyanide in mice and rabbits, particularly if administered in combination with thiosulfate. Oral DHA (2 and 4 g/kg) given to mice 10 min before injection (i.p.) of cyanide increased the LD50 values of cyanide from 5.7 mg/kg to 12 and 17.6 mg/kg, respectively. DHA prevented cyanide-induced lethality most effectively, if given orally 10-15 min before injection of cyanide. A combination of pretreatment with oral DHA (4 g/kg) and post-treatment with sodium thiosulfate (1 g/kg) increased the LD50 of cyanide by a factor of 9.9. Furthermore, DHA given intravenously to rabbits 5 min after subcutaneous injection of cyanide increased the LD50 of cyanide from 6 mg/kg to more than 11 mg/kg, while thiosulfate (1 g/kg) given intravenously 5 min after cyanide injection increased the LD50 of cyanide only to 8.5 mg/kg. DHA also prevented the convulsions that occurred after cyanide intoxication.     

Cyanide intoxication: protection with cobaltous chloride

Protection against the lethal effects of cyanide can be elicited by administration of cobaltous chloride, either alone or in combination with sodium nitrite and/or sodium thiosulfate. Potency ratios derived from the LD50 values were compared in groups of mice premedicated with cobaltous chloride and/or sodium thiosulfate and/or sodium nitrite. Under the conditions of our experiment cobaltous chloride alone is slightly more effective than sodium nitrite; when it is combined with sodium nitrite, an additive effect is obtained. When cobaltous chloride is administered in combination with sodium thiosulfate, a dramatic antagonism of the lethal effects of potassium cyanide is observed. The synergistic antidotal effect of cobaltous chloride may be related to the physiological disposition of the cobaltous ion and its ability to chelate both cyanide and thiocyanate ions.     

Cyanide intoxication in sheep; therapeutics

Various cyanide antidotes were evaluated by comparing the effects of delay in time of therapy following oral administration of sodium cyanide in sheep. Successful therapy of lethal doses of sodium cyanide could be accomplished with the more potent antidotes for up to 30 minutes following administration of sodium cyanide. Either 660 mg/kg sodium thiosulfate or 1 mg/kg p-aminopropriophenone were effective antidotes for moderate lethal doses (7.6 mg/kg) of sodium cyanide. The conventional low dosage nitrite/thiosulfate (6.7 mg/kg and 67 mg/kg) was much less effective. Larger doses (15.2 mg/kg) of sodium cyanide were effectively antagonized by either 660 mg/kg sodium thiosulfate alone or in combination with 1.5 mg/kg p-aminopropriophenone or 22 mg/kg sodium nitrite. At high cyanide dosage, p-aminopropriophenone alone was less effective than sodium thiosulfate alone. Sodium thiosulfate at high dosage appears to be the antidote of choice. This more closely satisfies the requirements of high efficacy and low toxicity for an antidote. Sodium nitrate or other antidotes may be used in conjunction with sodium thiosulfate, but their use is not necessary for high efficacy.     

Antagonism of cyanide intoxication with sodium pyruvate

Cyanide intoxication in mice can be effectively antagonized by sodium pyruvate, particularly if it is administered in combination with the antidotes, sodium nitrite and sodium thiosulfate. Potency ratios derived from the LD50 data were compared in groups of mice treated with sodium nitrite, sodium thiosulfate, and sodium pyruvate either alone or in various combinations. These results indicate that the administration of sodium pyruvate alone does provide minimal, but statistically significant, protection against the lethal effects of cyanide. Sodium pyruvate does not enhance the effect of sodium nitrite; however, it does potentiate the antidotal effect of sodium thiosulfate. The sodium thiosulfate and sodium pyruvate combination is not as effective as the sodium nitrite and sodium thiosulfate combination, but the addition of sodium pyruvate to the sodium nitrite-sodium thiosulfate combination further enhances the antidotal effect. No further enhancement is observed when sodium nitrite, sodium thiosulfate, and sodium pyruvate are combined with oxygen.     

Zur Beurteilung der Blausäure-Antidote

The effect of various antidotes on the exhalation of hydrocyanic acid has been measured in guinea pigs and cats poisoned with cyanide. This procedure permits evaluation of both the speed of action and the capacity of the agents tested to detoxify hydrocyanic acid, and therefore allows an exact judgement as to therapeutic value of various antidotes to cyanide poisoning. The results were as follows:

1. Cobaltous histidine at a dose of 20 mg/kg was distinguished among the compounds tested by its rapid action in both species. Its detoxifying capacity was not adequate however. Treatment of severe cyanide poisoning in man with Co (his)₂ would appear to be reasonable, but only when combined with sodium thiosulfate.
2. The same rapid action as with cobaltous histidine was achieved in cats by intravenous injection of 2.25 mg/kg p-dimethylaminophenole (DMAP) leading to a methemoglobin formation of 30%. A dose of 0.75 mg/kg DMAP forming 10% methemoglobin reduced HCN-exhalation by an equivalent amount only after a 2.4 min delay. The capacity of DMAP to detoxify hydrocyanic acid was considerably greater than that of cobaltous histidine but still was far inferior to that of sodium thiosulfate.
3. The high capacity of sodium thiosulfate to detoxify hydrocyanic acid was likewise demonstrated by the new method employed here in both animal species. However, the onset of its effect was always very delayed. In clinical practice, this agent should never be omitted, but in treatment of severe poisonings it will only be successful when combined with a more rapid-acting antidote such as cobaltous histidine or DMAP.
4. Sodium nitrite, even when applied in relatively high doses, did not act rapidly enough nor did it demonstrate a satisfactory capacity to detoxify hydrocyanic acid. Therefore, it no longer fulfills the requirements that presently should be demanded of an antidote to hydrocyanic acid.

     

In vitro and in vivo evaluation of various carbonyl compounds against cyanide toxicity with particular reference to alpha-ketoglutaric acid

Cyanide is a rapidly acting neurotoxin that necessitates immediate, vigorous therapy. The commonly used treatment regimen for cyanide includes the intravenous administration of sodium nitrite (SN) and sodium thiosulphate (STS). Due to many limitations of these antidotes, a search for more effective, safer molecules continues. Cyanide is known to react with carbonyl compounds to form the cyanohydrin complex. The present study addresses the efficacy of several carbonyl compounds and their metabolites or nutrients with alpha-ketoglutaric acid (A-KG), citric acid, succinic acid, maleic acid, malic acid, fumaric and oxaloacetic acid, glucose, sucrose, fructose, mannitol, sorbitol, dihydroxyacetone, and glyoxal (5 or 10 mM; -10 min) against toxicity of potassium cyanide (KCN; 10 mM) in rat thymocytes in vitro. Six hours after KCN, cell viability measured by MTT assay and crystal violet dye exclusion revealed maximum cytoprotection by A-KG, followed by oxaloacetic acid. A-KG also resolved the leakage of intracellular lactate dehydrogenase, loss in nuclear integrity (propidium iodide staining), and altered mitochondrial membrane potential (rhodamine 123 assay) as a result of cyanide toxicity. Protection Index (ratio of LD(50) of KCN in protected and unprotected animals; PI) of all the compounds (oral; 1.0 g/kg; -10 min) determined in male mice, revealed that maximum protection was afforded by A-KG (7.6 PI), followed by oxaloacetic acid (6.4 PI). Comparative evaluation of various salts of A-KG alone or with STS (intraperitoneal; 1.0 g/kg; -15 min) showed that maximum protection was conferred by disodium anhydrous salt of A-KG, which also significantly prevented the inhibition of brain cytochrome oxidase caused by 0.75 LD(50) KCN. This study indicates the potential of A-KG as alternative cyanide antidote.     

Effect of atropine on cyanide-induced acute lethality in mice

The effects of atropine on acute lethality induced by cyanide were investigated in mice. The LD₅₀ value of cyanide (s.c. injection) was 8.4 (7.6–9.3) mg/kg. However, the LD₅₀ value of cyanide (s.c.) was significantly increased by 1.5-fold when atropine (32 mg/kg) was injected s.c. in mice. Furthermore, the combined administration of atropine (32 mg/kg). Ca²⁺ (500 mg/kg) and sodium thiosulfate (1 g/kg) tremendously increased the LD₅₀ value by 5.6-fold in mice although sodium thiosulfate or Ca²⁺ alone increased the LD₅₀ 2.5- or 1.5-fold. On the other hand, although the LD₅₀ value of cyanide (intracerebroventricular injection (i.v.t.)) was 52.0 (47.4–57.0) μ/brain, the LD₅₀ value of cyanide (i.v.t.) was significantly increased by 1.3- or 1.61-fold in mice 10 min after s.c. injection of atropine (32 mg/kg) or Ca²⁺ (500 mg/kg). Furthermore, the combined administration of atropine and Ca²⁺ increased the LD₅₀ value of cyanide by 2.1-fold. These results suggest that atropine inhibits cyanide-induced acute lethality and promotes the antagonistic effect of thiosulfate and Ca²⁺ in mice.     

Comparison of nitrite treatment and stroma-free methemoglobin solution as antidotes for cyanide poisoning in a rat model

The standard nitrite/thiosulfate regimen for cyanide poisoning was tested in our rat model. By modifying the treatment regimen and the nitrite solution an effective antidote against an LD90 of cyanide could be produced. However, this treatment was effective against two times the LD90 only when administered ten minutes prior to cyanide injection. These results are in marked contrast to our results with stroma-free methemoglobin solutions (SFMS) which showed SFMS to be a highly effective antidote against four times the LD90 when administered 30 seconds after an intravenous injection of cyanide. SFMS proved to be an effective antidote for two times the LD90 when administered up to sixty seconds after the cessation of respiration.     

Nitroprusside intoxication: protection of alpha-ketoglutarate and thiosulphate.

Protection against the lethal effects of sodium nitroprusside (SNP) was observed in mice after treatment with alpha-ketoglutarate (AKG), either alone or in combination with sodium thiosulphate (STS). The LD50 of SNP was 12.0 (11.0-13.0) mg/kg in mice. Ip injection of AFG (500 mg/kg twice in 20 min) increased the LD50 1.7-fold in mice. STS (1 g/kg, ip) alone increased the LD50 5.5-fold. Furthermore, combined administration of AKG and STS increased the LD50 6.9-fold. SNP elicited increased cyanide levels in blood of mice in a dose-dependent manner. SNP (10 mg/kg, sc) administration gave rise to blood cyanide levels of 73.2 +/- 3.0 microM, 30 min after treatment. Ip injection of AKG significantly decreased blood cyanide levels by 30% in mice 30 min after treatment with 10 mg SNP/kg. A single injection of STS (1 g/kg) or a combination of AKG and STS reduced in blood cyanide levels by 88 or 98%, respectively, in mice after treatment with 10 mg SNP/kg. In addition, the increase in blood cyanide levels induced by injection of 50 mg SNP/kg was markedly inhibited by a combination of AKG and STS or (to a lesser extent) by STS alone. These results suggest that the combined administration of AKG and STS, by preventing the increase in blood cyanide levels induced by SNP, may afford protection against the toxic effects of SNP.     

Effect of cyanide antidotes on the metabolic conversion of cyanide to thiocyanate

The excretion of thiocyanate following the administration of equitoxic doses of cyanide to unprotected mice and to animals pretreated with various cyanide antidotes has been studied.The results demonstrate that cyanide given alone or to animals pretreated with thiosulfate is extensively converted to thiocyanate. Animals pretreated with sodium nitrite or a combination of nitrite and sodium thiosulfate excreted even higher amounts of thiocyanate. This demonstrates that cyanide originally detoxified by combination with methemoglobin is ultimately converted to thiocyanate in the animal body.Pretreatment of animals with cobalt compounds (cobaltous chloride or dicobalt-EDTA) or a combination of cobalt compounds and thiosulfate resulted, on the other hand, in a less efficient conversion of cyanide to thiocyanate. The cyanide detoxified by trapping as highly undissociated cobalt-cyanide complexes is instead excreted in the urine, as demonstrated by detection of high amounts of cobalt ions and strongly complex-bound cyanide in the urine from animals treated with cobalt compounds and cyanide. A method for the determination of cyanide present as cobalt-cyanide complexes is described and its forensic application is proposed.     

Protection by sodium thiosulfate and thiourea against lethal toxicity of cis-diamminedichloroplatinum (II) in bacteria and mice

The protective effect of sodium thiosulfate and thiourea on the lethal toxicity of the antitumor drug, cis-diamminedichloroplatinum (II) (cis-DDP), was investigated in bacteria and mice. Initially, the agents capable of antagonizing bactericidal activity of cis-DDP were screened using WP2 uvra, a strain of E. coli sensitive to this drug. Of the ten sulfur-containing compounds tested, thiourea and sodium thiosulfate exhibited potent protecting effects against cis-DDP cytotoxicity in bacteria. Propylthiouracil and methimazole showed intermediate levels of such protection, but the other 6 compounds had little or no protective effects. Thiourea and sodium thiosulfate were then subjected to the acute lethal toxicity test in mice to assess their protective activity in vivo. We found that cis-DDP i.v. lethality against mice can be blocked almost completely by excess amounts of thiourea or sodium thiosulfate. Thiourea protected against cis-DDP toxicity with a narrow range among the effective doses, while sodium thiosulfate was protective with a remarkably wide range of effective doses. The effectiveness of sodium thiosulfate was also indicated in experiments in which the LD50 dose of cis-DDP (16 mg/kg) i.p. increased over the level of greater than 200 mg/kg with concomitant administration of sodium thiosulfate i.p.     

Effect of chlorpromazine on cyanide intoxication

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.     

The role of cyanide liberation in the acute toxicity of aliphatic nitriles

The acute ip LD50 values for a series of six aliphatic nitriles were determined in mice and compared with acetone cyanohydrin and sodium cyanide. When sodium thiosulfate was given in multiple injections, it protected mice against death by acetonitrile, propionitrile, n-butyronitrile, malononitrile, or succinonitrile. In contrast, multiple injections of sodium nitrite protected mice against death by acrylonitrile, n-butyronitrile, and malononitrile, but not against acetonitrile, propionitrile, or succinonitrile. Single prophylactic doses of either thiosulfate or nitrite protected mice against death by either acetone cyanohydrin or sodium cyanide. Only sodium cyanide and acetone cyanohydrin predictably produced death within 5 min. All other nitriles produced death at widely varying intervals from a few minutes to many hours. Only acetone cyanohydrin and cyanide inhibited the activity of purified preparations of cytochrome c oxidase in vitro and in equimolar concentrations. Pretreatment of mice with carbon tetrachloride protected them against death from all nitriles except acetone cyanohydrin. Elevated concentrations of cyanide were found in the livers and brains of mice given lethal doses of all of the nitriles, acetone cyanohydrin, or sodium cyanide. The tissue concentrations of cyanide were substantially reduced in all cases when thiosulfate was also given or when nitriles were given to carbon tetrachloride-pretreated mice. Cyanide was liberated when n-butyronitrile or succinonitrile were incubated with mouse liver slices or NADPH-fortified mouse hepatic microsomal preparations. This reaction was inhibited when the livers were taken from mice pretreated with carbon tetrachloride or when SKF-525A was added in vitro to normal liver slices. Acetone cyanohydrin in all systems tested behaved qualitatively and quantitatively like its molar equivalent in cyanide. The results suggested that the other nitriles examined here possess little, if any, acute toxicity in the absence of normal hepatic function and that these nitriles were activated by hepatic mechanisms to release cyanide which can account for their major acute toxic effects.     

Acute, sublethal cyanide poisoning in mice is ameliorated by nitrite alone: complications arising from concomitant administration of nitrite and thiosulfate as an antidotal combination

Sodium nitrite alone is shown to ameliorate sublethal cyanide toxicity in mice when given from ～1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. An optimum dose (12 mg/kg) was determined to significantly relieve cyanide toxicity (5.0 mg/kg) when administered to mice intraperitoneally. Nitrite so administered was shown to rapidly produce NO in the bloodsteam as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. It is argued that antagonism of cyanide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity rather than the methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood resulted in the detection of sulfidomethemoblobin by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase, the effects of the two inhibitors being essentially additive under standard assay conditions rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sublethal levels of cyanide intoxication.     

Promising role of alpha-ketoglutarate in protecting against the lethal effects of cyanide

Treatment of cyanide poisoning generally includes amyl nitrite and/or sodium nitrite (SN) in combination with sodium thiosulphate (STS). However, in many instances of cyanide poisoning, use of nitrites is contraindicated due to their strong vasoactive properties. alpha-Ketoglutarate (alpha-KG) antagonizes cyanide poisoning by cyanohydrin formation. Protective efficacy of graded doses of alpha-KG (p.o.) as pretreatment, simultaneous treatment or post-treatment was evaluated against acute potassium cyanide (KCN) poisoning (p.o.) in female rats. Pretreatment with alpha-KG (0.125-2.0 g/kg) exhibited dose- and time-dependent effects and was found to be effective even when given up to 60 min prior to KCN. Addition of STS significantly enhanced the protective efficacy of alpha-KG at all the doses and time intervals. A 10-min pretreatment with alpha-KG increased the LD50 of KCN by 7-fold, which was further increased 28-fold by the addition of both SN and STS. Simultaneous treatment with alpha-KG (2.0 g/kg) increased the LD50 of KCN by 7-fold, which was doubled by the addition of STS. However, addition of SN did not confer any additional protection. Post-treatment with alpha-KG + STS minimized the mortality by 50% but did not significantly extend the survival time in KCN (2 LD50)-administered rats. KCN (0.75 LD50) inhibited rat brain cytochrome oxidase, which was significantly protected by pretreatment or simultaneous treatment with alpha-KG and STS. Considering the efficacy and safety of peroral alpha-KG, a promising treatment regimen consisting of alpha-KG + STS or alpha-KG + SN + STS is proposed depending upon the situation.     

Protection against cyanide-induced convulsions with alpha-ketoglutarate

Protection against convulsions induced by cyanide was observed after treatment with alpha-ketoglutarate, either alone or in combination with sodium thiosulfate, a classical antagonist for cyanide intoxication. However, sodium thiosulfate alone did not protect against cyanide (30 mg/kg)-induced convulsions. gamma-Aminobutyric acid (GABA) levels in brain were decreased by 31% in KCN-treated mice exhibiting convulsions. The combined administration of alpha-ketoglutarate and sodium thiosulfate completely abolished the decrease of GABA levels induced by cyanide. Furthermore, sodium thiosulfate alone also completely abolished the decrease of GABA levels. These results suggest that the depletion of brain GABA levels may not directly contribute to the development of convulsions induced by cyanide. On the other hand, cyanide increased calcium levels by 32% in brain crude mitochondrial fractions in mice with convulsions. The increased calcium levels were completely abolished by the combined administration of alpha-ketoglutarate and sodium thiosulfate, but not affected by sodium thiosulfate alone. These findings support the hypothesis proposed by Johnson et al. (Toxicol. Appl. Pharmacol., 84 (1986) 464) and Robinson et al. (Toxicology, 35 (1985) 59) that calcium may play an important role in mediating cyanide neurotoxicity.