Application of a hepatocyte-erythrocyte coincubation system to studies of cyanide antidotal mechanisms

A coincubation system composed of hepatocytes in primary monolayer culture and erythrocytes suspended in the culture medium was developed and used as a model for investigations of mechanisms of cyanide antidote action at the cellular level. Hepatocyte ATP was used as the cytotoxicity indicator. Treatment of rat hepatocytes in the coincubation system with KCN (1.0 mM) for 10 min at 37 degrees C selectively reduced hepatocyte ATP levels to 33 +/- 15% of control (no KCN added) levels. 4-dimethylaminophenol (DMAP), cobalt(II) chloride, sodium nitrite, sodium thiosulfate, or a combination of the last two antidotes added to the KCN-containing medium significantly reversed ATP depression and the response was concentration dependent. The relative effectiveness, on a molar basis, was estimated to be DMAP greater than CoCl2 much greater than NaNO2 congruent to Na2S2O3. NaNO2 and DMAP induced methemoglobin formation in the absence of cyanide and cyanmethemoglobin formation in its presence; erythrocytes were required in the medium for effectiveness. CoCl2 produced neither cyanmethemoglobin nor thiocyanate in appreciable quantities nor required erythrocytes for antagonism. Na2S2O3 converted cyanide to thiocyanate and reversed ATP depression without erythrocytes in the medium. The addition of erythrocytes increased these rates significantly and to a greater extent than albumin. The overall results are consistent with previously proposed modes of action for these antidotes. However, the enhancement in cyanide metabolism and ATP recovery with Na2S2O3 and erythrocytes in the system was unexpected and raises the possibility that erythrocytes may contribute to cyanide disposition and antagonism in vivo when this antidote is administered.     

血卟啉衍生物对红细胞膜通透性的影响

光敏剂HPD可增加红细胞膜对NaNO_2的通透性。随HPD剂量增加或光照时间延长,通透性呈指数升高。受HPD损伤的红细胞,其通透性随NaNO_2浓度的增加呈指数增长,对温度的敏感性加大。     

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.

     

Comparison of hydroxylamine, 4-dimethylaminophenol and nitrite protection against cyanide poisoning in mice

Intraperitoneal doses of 4-dimethylaminophenol hydrochloride (DMAP), hydroxylamine hydrochloride (H₂NOH) and sodium nitrite (NaNO₂) were found where each converted a maximum of about 37% of the total circulating hemoglobin in mice to methemoglobin. Those doses in mmol/kg were: 0.29 for DMAP, 1.1 for H₂NOH, and 1.1 for NaNO₂. For DMAP and H₂NOH the peak was sharp and at about 7 min after injection whereas for NaNO₂ the peak was much broader and at about 40 min. The i.p. LD₅₀'s in mmol/kg were: 0.48 for DMAP, 1.8 for H₂NOH and 2.3 for NaNO₂. When mice pretreated with each of the methemoglobin-generating agents were challenged with sodium cyanide, the ratios of the LD₅₀'s in protected mice to those in control mice (protection index, PI) were 1.5 for H₂NOH, 2.0 for DMAP and 3.1 for NaNO₂. When sodium thiosulfate was also given in combination with each of the three methemoglobin-generating agents, the protective effect was at least additive. The PI against sodium sulfide was also significantly greater in mice pretreated with NaNO₂ than in mice given H₂NOH. Methemoglobins generated from human and mouse hemoglobins by either NaNO₂ or by H₂NOH had identical binding affinities (dissociation constants) for cyanide. When human red cells containing methemoglobin generated by exposure to either NaNO₂ or H₂NOH were injected into the peritoneal cavity of mice and then followed by cyanide challenges, there was no difference in the PI for the two kinds of methemoglobin. Not only was the PI the same in each case with human cells, but it was also identical with that in mice given NaNO₂ systemically to generate the same total amount of methemoglobin. The difference in PI between NaNO₂ and H₂NOH (or DMAP) in mice appears to be related to the high rate of methemoglobin reductase activity in mouse RBC. It appears likely that cyanmethemoglobin is a substrate for mouse methemoglobin reductase activity, and that NaNO₂ is an inhibitor of mouse methemoglobin reductase. No differences in cyanide antagonism between NaNO₂ and H₂NOH would be anticipated in humans because of the slow rates of methemoglobin reduction in human red cells.     

对二甲氨基酚治疗急性失血合并氰中毒对血液动力学及血液内环境的影响

本实验用犬制备了轻(10％)、中(15％)、重度(20％)急性失血合并ⅳ NaCN 2.5 mg/kg中毒的动物模型,观察了ⅳ DMAP 2.5mg/kg治疗时血液动力学及血液内环境的变化。结果发现,DMAP治疗轻度急性失血合并氰中毒能使心血管功能迅速恢复正常并维持稳定,随失血程度加重DMAP对心血管功能兴奋作用减弱;血气分析及HbFe~(3+)测定结果表明,DMAP治疗急性失血合并氰中毒可造成机体严重缺氧及代谢性酸中毒,并随失血程度的加重而加剧。     

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.     

Operant leverpressing and wheelrunning were differentially reduced by PAPP (p-aminopropiophenone)-induced methemoglobinemia

Cyanide is a potent toxin that binds to cytochrome oxidase blocking electron transfer and the synthesis of adenosine triphosphate (ATP). Many antidotes to cyanide poisoning oxidize hemoglobin to methemoglobin (metHb), which serves as a scavenger of the cyanide anion. However, sufficiently high levels of metHb can be toxic because metHb cannot bind O(2) until it is reduced. The purpose of the proposed study was twofold: (1) Characterize the time course of metHb formation for different doses of p-aminopropiophenone (PAPP), a drug that oxidizes hemoglobin and can be used as an antidote to cyanide intoxication; and (2) Determine whether the effort of an operant response affects the behavioral toxicity of metHb, since more effortful responses presumably are more energetically demanding. In Experiment I, the oral metHb kinetics of p-aminopropiophenone (PAPP) were studied; four doses of PAPP (1, 5, 10, and 20 mg/kg) or the vehicle, polyethylene glycol 200 (PEG200), were delivered via a gavage tube to separate groups of rats. In Experiment II, rats were trained to press a lever or run in an activity wheel at any time during a 12-hour light/dark cycle for their entire daily food intake; five presses or turns were required for the delivery of each food pellet. The same doses of PAPP were delivered p.o. shortly before the onset of darkness, 2100 h. Results from Exp I showed that PAPP induced a dose-dependent rapid increase and relatively slower exponential-like decline in metHb concentration. In Exp. II, the same doses of PAPP induced a dose-dependent reduction in hourly outputs of leverpresses and wheelturns however; wheelturns were reduced significantly more than leverpresses. When the best-fitting metHb curves from Experiment I were superimposed on the time scale for outputs of wheelturns and leverpresses, reduction of output was inversely related to the kinetics of metHb formation. These findings are consistent with the conclusion that PAPP-induced metHb formation reduced the output of wheelrunning more than leverpressing because the more energetically demanding response of wheelrunning was more affected by metHb induced hypoxemia. Furthermore, these data suggest that although certain longacting metHb formers might be useful prophylactics for warfighters, it will be critical to determine the energetic loads of required battlefield activities because even low (10%) therapeutic metHb levels might impair the performance of those activities.     

Operant leverpressing and wheelrunning were differentially reduced by PAPP (p-aminopropiophenone)-induced methemoglobinemia

Cyanide is a potent toxin that binds to cytochrome oxidase blocking electron transfer and the synthesis of adenosine triphosphate (ATP). Many antidotes to cyanide poisoning oxidize hemoglobin to methemoglobin (metHb), which serves as a scavenger of the cyanide anion. However, sufficiently high levels of metHb can be toxic because metHb cannot bind O₂ until it is reduced. The purpose of the proposed study was twofold: (1) Characterize the time course of metHb formation for different doses of p-aminopropiophenone (PAPP), a drug that oxidizes hemoglobin and can be used as an antidote to cyanide intoxication; and (2) Determine whether the effort of an operant response affects the behavioral toxicity of metHb, since more effortful responses presumably are more energetically demanding. In Experiment I, the oral metHb kinetics of p-aminopropiophenone (PAPP) were studied; four doses of PAPP (1, 5, 10, and 20 mg/kg) or the vehicle, polyethylene glycol 200 (PEG200), were delivered via a gavage tube to separate groups of rats. In Experiment II, rats were trained to press a lever or run in an activity wheel at any time during a 12-hour light/dark cycle for their entire daily food intake; five presses or turns were required for the delivery of each food pellet. The same doses of PAPP were delivered p.o. shortly before the onset of darkness, 2100 h. Results from Exp I showed that PAPP induced a dose-dependent rapid increase and relatively slower exponential-like decline in metHb concentration. In Exp. II, the same doses of PAPP induced a dose-dependent reduction in hourly outputs of leverpresses and wheelturns however; wheelturns were reduced significantly more than leverpresses. When the best-fitting metHb curves from Experiment I were superimposed on the time scale for outputs of wheelturns and leverpresses, reduction of output was inversely related to the kinetics of metHb formation. These findings are consistent with the conclusion that PAPP-induced metHb formation reduced the output of wheelrunning more than leverpressing because the more energetically demanding response of wheelrunning was more affected by metHb induced hypoxemia. Furthermore, these data suggest that although certain longacting metHb formers might be useful prophylactics for warfighters, it will be critical to determine the energetic loads of required battlefield activities because even low (10%) therapeutic metHb levels might impair the performance of those activities.     

Effects and biotransformation of 4-dimethylaminophenol in man and dog

The cyanide antidote 4-dimethylaminophenol . HCl (DMAP) was administered orally, i.v., or i.m. to man and dog. Ferrihemoglobin formation and changes of several parameters in human blood were investigated to obtain information on damage to liver, kidney, muscle, and red blood cells; in addition, the metabolism of DMAP was studied. In dogs, the initial rate of ferrihemoglobin production (DMAP, 3.25 mg/kg i.v. or i.m., 15 mg/kg orally) amounted to 28%, 3.5%, and 2% of the total hemoglobin per min; the corresponding values for man were 9%, 2%, and 2% per min. The dogs behaved normally while CPK increased after i.m. injection. In man, only i.m. injection of DMAP (3.25 mg/kg) was followed by increases in LDH, GOT, and CPK of 110, 260, and 490%, resp.; while total bilirubin, conjugated bilirubin, and iron concentration rose by 270, 120, and 50%, respectively. Bilirubin and iron concentration increased also after DMAP i.v. (3.25 mg/kg) or when it was taken orally (600 or 900 mg). The lactate concentration was not influenced while the pyruvate concentration increased by 50%. DMAP produced hemolysis in vitro. Generally, the values determined in vivo approached the starting level within 1 week. Intramuscular injection of DMAP induced reversible subjective and objective symptoms, e.g., local pain, swollen buttock, fever reaction. The urine showed no pathological changes. About 54% of DMAP taken orally was excreted as metabolites in the urine, 41% as glucuronide, 7% as sulfate, and 6% as thioethers. After i.v. administration the total of metabolites was somewhat higher, and the thioether proportion was 15%. The results indicate that DMAP is readily absorbed after oral administration but undergoes significant first pass effect in the liver. Therefore, the 4-fold i.v. dose must be administered orally to achieve the same ferrihemoglobin formation.     

The formation of methaemoglobin by 4-aminopropiophenone (PAPP) and 4-(N-hydroxy) aminopropiophenone.

Oral dosing of rats with the cyanide antidote 4-aminopropiophenone (PAPP), brought about peak methaemoglobin levels at 15-40 min, but peak levels were attained at at 15-25 min after intravenous dosing. After both oral and intravenous administration at equimolar doses, 4-(N-hydroxy)aminopropiophenone (PHAPP), the putative methaemoglobin-producing metabolite of PAPP, produced higher peak levels of methaemoglobin than PAPP. Plasma from rats injected with PAPP was capable of forming methaemoglobin when added to naive rat erythrocytes. The identity of the metabolite responsible is discussed.