Effect of oxygen on the antagonism of cyanide intoxication--cytochrome oxidase, in vivo

The inhibition and recovery of brain and liver cytochrome oxidase in mice pretreated in an air or oxygen atmosphere were measured after the administration of KCN with and without sodium nitrite and sodium thiosulfate pretreatment. Inhibition of cytochrome oxidase in both brain and liver reached a maximum within 5 min after cyanide administration, and cytochrome oxidase activity was restored more rapidly in liver than in brain. Also, this enzymatic activity returned more rapidly in oxygen than in air. In the animals pretreated with sodium nitrite and sodium thiosulfate, brain but not liver cytochrome oxidase was inhibited by cyanide. The effect of administering varying doses of KCN to mice maintained in air or oxygen resulted in a dose-dependent inhibition of both brain and liver cytochrome oxidase. Oxygen treatment produced a shift to the right in the dose-response curve when compared to the air treatment group. No significant difference was detected in rhodanese activity in air and oxygen in mice receiving varying doses of cyanide.     

Diagnosis of cyanide intoxication by measurement of cytochrome c oxidase activity

Cytochrome c oxidase (CCO), a mitochondrial enzyme, is inactivated by cyanide or carbon monoxide (CO) intoxication. We measured CCO activity, in the major organs of the rat at various times after death caused by cyanide intoxication. Tissue samples were homogenized, and the CCO activity in the mitochondrial fraction was measured using ferrous cytochrome c as the substrate. The CCO activity inhibition was highest in the brain, although the cyanide concentration was lowest level. As a result of this and the clinical symptoms displayed, we consider the brain to be the primary organ of cyanide intoxication. As cyanide is highly toxic to humans, in small amounts and many patients and victims have already had some medical care, it is difficult to detect cyanide in criminal investigations. The CCO activities in various organs remained significantly low for 2 days after the cyanide intoxication, suggesting that the diagnosis may be possible by measuring not only the cyanide concentration but also the CCO activity.     

氰化氢复合中毒对大鼠血中氰离子和细胞色素氧化酶活力的影响

火灾烟雾中有毒气体的成分复杂,但在火灾现场能达到足够浓度引起急性中毒或致死效应的只有一氧化碳(CO)和氰化氢(HCN)气体[1],而其他主要引起感官和肺的刺激效应,或产生一些迟发性的中毒效应。现代装璜和家具中大量高分子聚合物的使用,燃烧烟雾中剧毒HCN气体的产生有了明显增加     

The efficacy of alpha-ketoglutaric acid in the antagonism of cyanide intoxication

It has been reported that compounds containing carbonyl groups can readily react with cyanide. Pyruvic acid, an alpha-ketocarboxylic acid, has been shown to antagonize the lethal effects of cyanide. It is suggested that its mechanism of action rests in its ability to react with or "bind" cyanide. In this study, alpha-ketoglutaric acid, also an alpha-ketocarboxylic acid, was evaluated for its ability to counteract the lethal effects of cyanide. alpha-Ketoglutaric acid increased the LD50 value of cyanide (6.7 mg/kg) by a factor of five, a value statistically equivalent to that ascertained in mice pretreated with sodium thiosulfate and sodium nitrite. The combination of alpha-ketoglutaric acid and sodium thiosulfate increased the LD50 value of cyanide to 101 mg/kg. Addition of sodium nitrite to the alpha-ketoglutaric acid/sodium thiosulfate regimen increased the LD50 value of cyanide to 119 mg/kg. Unlike sodium nitrite, no induction of methemoglobin formation was observed with alpha-ketoglutaric acid pretreatment. It is apparent from these studies that the administration of alpha-ketoglutaric acid in conjunction with sodium thiosulfate resulted in fewer animal deaths than sodium nitrite and sodium thiosulfate without the dangerous formation of methemoglobin.     

Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity.

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.     

The effect of methemoglobin on the inhibition of cytochrome c oxidase by cyanide, sulfide or azide.

Under our experimental conditions sulfide was a more potent inhibitor of a particulate preparation of cytochrome oxidase than was cyanide; azide proved to be a relatively weak inhibitor, all of which is in agreement with the observations of others. The undissociated species (H₂S) appeared to be more inhibitory than the anionic species (HS^?) in accord with the conclusions of others about HCN and HN₃. Addition of methemoglobin to the oxidase inhibited by cyanide or sulfide restored the activity of the enzyme system, but the addition of methemoglobin to the azide-inhibited oxidase under the same conditions had little or no effect. Our results suggest that sulfide produces death in animals by inhibition of cytochrome oxidase, but such a mechanism seems unlikely in the case of azide.     

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.     

Effects of acute lethal cyanide intoxication on central dopaminergic pathways.

In rats treated with sodium cyanide (NaCN), 20 mg/kg intraperitoneally, the striatal dopamine (DA) level was decreased within 60 sec. compared to controls injected with NaCl 0.9%. Treatment with NaCN also increased the naturally occurring L-DOPA in the striatum, but not in the other brain regions studied. Decreased DA levels but increased L-DOPA accumulation were also seen in cyanide-treated animals after inhibition of neuronal L-aromatic amino decarboxylase. In rats given a non-lethal dose of NaCN, 2.5 mg/kg intraperitoneally, 30 min. before sacrifice and L-DOPA, 100 mg/kg intraperitoneally, 25 min. before sacrifice, regional L-DOPA levels were not significantly changed, but the striatal DA levels were diminished compared to controls. Decreased DA levels might indicate that cyanide inhibits the synthesis of brain DA. However, both increased L-DOPA and increased accumulation of L-DOPA after neuronal decarboxylase were observed after cyanide. Furthermore, we have earlier reported that lethal doses of NaCN decreased the DA metabolite HVA in the striatum but did not significantly change the oxidatively deaminated metabolite of DA, DOPAC. Inhibition of L-aromatic amino acid decarboxylase appears to play a minor role in causing decreased striatal DA levels. However, our findings might be compatible with cyanide-produced inhibition of the energy-demanding granular uptake and/or release of DA.     

In vitro activation of dibromoacetonitrile to cyanide: role of xanthine oxidase

Dibromoacetonitrile (DBAN) is a disinfection byproduct of chlorination of drinking water. Epidemiological studies indicate that it might present a potential hazard to human health. The present work provides evidence for DBAN activation to cyanide (CN(-)) by the hypoxanthine (HX)/xanthine oxidase (XO)/iron (Fe) system in vitro. Optimum conditions for the oxidation of DBAN to CN(-)were characterized. Addition of the sulfhydryl compounds glutathione, N-acetyl- L-cysteine or dithiothreitol significantly enhanced the rate of CN(-)release. A high positive correlation existed between hydroxyl free radical ((*)OH) generation and CN(-) formation. Addition of the (*)OH scavengers mannitol or dimethylthiourea to the reaction mixtures resulted in a significant decrease in the rate of DBAN oxidation. Addition of the antioxidant enzymes catalase or superoxide dismutase resulted in a significant decrease in the rate of DBAN oxidation. The iron chelator desferrioxamine significantly decreased CN(-) formation. The maximum velocity (V(max)) and Michaelis-Menten constant (K(m)) of the reaction were assessed. Allopurinol competitively inhibited the reaction, while folic acid uncompetitively inhibited the reaction. In conclusion, (*)OH generated by the HX/XO/Fe system are implicated in DBAN oxidation. The present results represent a novel pathway for DBAN activation and might be important in explaining DBAN-induced toxicity.     

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.     

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 nitric oxide toward the inhibition of cytochrome c oxidase by carbon monoxide and cyanide

The principle mitochondrial target where the respiratory inhibitors CO, CN(-), and NO act in the execution of their acute toxic effects is complex IV of the electron-transport chain, cytochrome c oxidase. However, there is a paucity of studies in the literature regarding the concerted effects of such poisons. Accordingly, the combined inhibitory effects of CO + CN(-), NO + CN(-), and NO + CO on the activity of cytochrome c oxidase preparations are reported. Only in the case of CO + CN(-) do the effects of the two inhibitors seem to be additive as expected. NO appears to be antagonistic toward the effects of the other two inhibitors; that is, the effects of both CO an CN(-) on enzyme activity are ameliorated by NO when present. To further clarify these observations, the ligand substitutions of heme-bound CN(-) by NO in cytochrome c oxidase and hemoglobin have also been briefly investigated. These results suggest that displacement of CN(-) from the ferric hemoproteins by NO is rate-limited by heme reduction-and in the case of the enzyme, the presence of nonligand-binding electron-transfer centers facilitates the reaction. The findings are discussed in relation to the idea that NO does not behave as a classic reversible (by dissociation) inhibitor.     

The effect of cyanide intoxication on hepatic rhodanese kinetics

1. Results of studies on the kinetics of hepatic rhodanese and the effects of S-adenosyl-L-methionine (SAM) on these kinetic parameters in cyanide-treated and non-treated mice are reported here. 2. The enzyme exhibited typical Michaelis-Menten behaviour with cyanide inhibition at concentrations higher than 50 mM. Km values of 4.74 and 0.85 mM were obtained for thiosulphate and cyanide, respectively, in control mice. 3. These results stress the biological importance of the rhodanese reaction for cyanide detoxification. 4. Km values were not significantly modified when the animals were intoxicated with a lethal (20 mg/kg) or a non-lethal (4 mg/kg) dose of cyanide. 5. SAM treatment either in control or in cyanide-poisoned animals doubled the Km's for cyanide.     

Zinc,copper and manganese in the organs of rats after sublethal cyanide intoxication

Single doses of sodium cyanide (60 mol/kg body weight s.c.) were administered to male Sprague-Dawley rats. The effect of this poison on the content of the trace elements zinc, copper and manganese was investigated in various organs after 30 min, 2 h, 24 h, 48 h and 1 week. The zinc content in the liver was elevated 24 h after this sublethal cyanide dose (by approximately 20%). In contrast, the copper content in the kidneys was lowered (by approximately 15%) at the same time. Almost similar changes were observed in the same organs after daily administration of the poison for 5 days. For comparison, another group of rats was allowed to respire for 30 min the air that contained only 10% oxygen. The above changes in the trace element concentrations were not observed under these conditions.After sublethal cyanide poisoning there seemed be slight but specific alterations in the trace element concentrations in the liver and kidneys of rats. On the other hand, there were no alterations in serum, heart, lung, brain, muscle, bone or testes. Up to now there is no clearcut explanation for the development and the possible biochemical importance of these results.     

In vitro and in vivo comparison of sulfur donors as antidotes to acute cyanide intoxication.

Antidotes for cyanide (CN) intoxication include the use of sulfane sulfur donors (SSDs), such as thiosulfate, which increase the conversion of CN to thiocyanate by the enzyme rhodanese. To develop pretreatments that might be useful against CN, SSDs with greater lipophilicity than thiosulfate were synthesized and assessed. The ability of SSDs to protect mice against 2LD50 of sodium cyanide (NaCN) administered either 15 or 60 min following administration of an SSD was assessed. To study the mechanism of action of the SSD, the candidate compounds were examined in vitro for their effect on rhodanese and 3-mercaptopyruvate sulfurtransferase (MST) activity under increasing SSD concentrations. Tests were conducted on nine candidate SSDs: ICD1021 (3-hydroxypyridin-2-yl N-[(N-methyl-3-aminopropyl)]-2-aminoethyl disulfide dihydrochloride), ICD1022, (3-hydroxypyridin-2-yl N-[(N-methyl-3-aminopropyl)]-2-aminoethyl disulfide trihydrochloride), ICD1584 (diethyl tetrasulfide), ICD1585 (diallyl tetrasulfide), ICD1587 (diisopropyl tetrasulfide); ICD1738 (N-(3-aminopropyl)-2-aminoethyl 2-oxopropyl disulfide dihydrochloride), ICD1816 (3,3'-tetrathiobis-N-acctyl-L-alanine), ICD2214 (2-aminoethyl 4-methoxyphenyl disulfide hydrochloride) and ICD2467 (bis(4-methoxyphenyl) disulfide). These tests demonstrated that altering the chemical substituent of the longer chain sulfide modified the ability of the candidate SSD to protect against CN toxicity. At least two of the SSDs at selected doses provided 100% protection against 2LD50 of NaCN, normally an LD99. All compounds were evaluated using locomotor activity as a measure of potential adverse behavioral effects. Positive hypoactivity relationships were found with several disulfides but none was found with ICD1584, a tetrasulfide. Separate studies suggest that the chemical reaction of potassium cyanide (KCN) and cystine forms the toxic metabolite 2-iminothiazolidine-4-carboxylic acid. An alternative detoxification pathway, one not primarily involving the sulfur transferases. may be important in pretreatment for CN intoxication. Although studies to elucidate the precise mechanisms are needed. it is clear that these newly synthesized compounds provide a new rationale for anti-CN drugs, with fewer side-effects than the methemoglobin formers.