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Age-related changes in toxicity and biotransformation of KCN, an ubiquitous environmental toxicant, have not been previously examined. Male C57BL/6N mice aged 2-3 (young), 10-12 (middle-aged), and 25-30 (old) months were administered KCN at 1, 2, 4, and 6 mg/kg po, and toxic manifestations were monitored for up to 2 hr. The toxic response to KCN (prostration and labored breathing) was significantly greater in 10-12 and 25-30 month vs that in 2-3 month mice at 4 and 6 mg/kg KCN. The basis for this age-related difference in in vivo toxicity was examined by studying biotransformation of KCN to thiocyanate by liver and brain rhodanese (RHO), as well as activity of liver and brain cytochrome oxidase (C-OX), inhibition of C-OX by KCN, and activity of beta-mercaptopyruvate transsulfurase (MT). Tissue and blood levels of CN- following a toxic dose of 6 mg/kg KCN were also measured. No age-related differences were observed in the specific activity of liver and brain RHO, MT, or C-OX. In addition, no differences were observed in the percentage inhibition of C-OX by KCN, or in the Ki for inhibition of brain and liver C-OX. However, activity of brain RHO on a per gram tissue basis was significantly lower in 10-12 and 25-30 months vs that in 2-3 month mice. Liver and blood concentrations of CN- were not significantly different in 2-3 vs 10-12 month mice following treatment with 6 mg/kg KCN; however, significantly greater concentrations of CN- were observed at 4 and 25 min in brains of 10-12 month mice compared to that in 2-3 month mice. These results indicate that increased sensitivity to KCN in older mice may be due in part to a decrease in the amount of brain RHO and altered tissue kinetics of CN- following a toxic dose in older mice.

alpha-Ketoglutaric acid (alpha-KG) has been shown to be an effective antagonist for cyanide-induced lethality. The mechanism of this antagonism is hypothesized to result from alpha-KG binding with cyanide. Several investigative approaches were taken to determine the existence of this binding. First, mixtures of various molar ratios of alpha-KG:cyanide were injected into a high pressure liquid chromatograph. The addition of cyanide reduced the peak area of alpha-KG at a molar ratio of greater than 1:5. Second, blood from naive male ICR mice was spiked with alpha-KG and cyanide. Headspace above these blood samples was injected into a gas chromatograph and analyzed for released hydrogen cyanide. alpha-KG reduced the peak area of hydrogen cyanide released into the headspace at molar ratios of greater than 1:2.5. Third, the effect of cyanide on the ultraviolet spectrum of alpha-KG was determined as an indication of binding. In the presence of cyanide the absorption peak at 316 nm for alpha-KG was eliminated. Inhibition of cytochrome oxidase is an accepted target enzyme for cyanide-induced lethality. Fourth, further evidence of alpha-KG's mechanism was determined by the effect of alpha-KG on brain cytochrome oxidase (BRCYTOX) and its ability to antagonize cyanide-induced inhibition of BRCYTOX. BRCYTOX activity was determined in the presence of alpha-KG and was found to be unaffected between 0.01 and 0.06 M of alpha-KG. Greater concentrations of alpha-KG inhibited BRCYTOX activity. The complete inhibition of BRCYTOX activity by 10(-5) M cyanide was prevented with 0.05 and 0.06 M alpha-KG. Fifth, BRCYTOX activity of animals pretreated with saline and then an LD80 dose (8.5 mg/kg) of cyanide was 80% inhibited, while BRCYTOX activity of animals pretreated with 2 g alpha-KG/kg, i.p., and then an LD80 dose (7.75 mg/kg) of cyanide was not different from control values. Thus, these data suggest that alpha-KG does bind with cyanide, and this binding can account for the antagonism of cyanide-induced lethality.

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.

We proposed a simple method for screening assessment of acute oral and dermal toxicity using only three rats and mice of each sex at each dose level. Animals were first treated with chemicals at a dose of 2000 mg/kg and were carefully observed for compound-related morbidity and mortality. If none of the animals died, the following toxicity tests were suspended. If some of the animals died, toxicity tests at doses of 200 and 20 mg/kg were performed. The approximate LD50 values calculated by this method showed little difference between two separate laboratories and were in good agreement with LD50 values reported in the literature. Our toxicological data also showed that LD50 values were about 2-2.5 times the MNLD (maximum non lethal dose) in acute oral and dermal toxicity. This meant that a chemical could be regarded as having an LD50 of about 4000 mg/kg or higher when there was no mortality at the dose of 2000 mg/kg. A chemical with such low toxicity would not require further testing for lethal effects. Therefore, this simple method combining the fixed-dose procedure with the limit test is suitable for determination of approximate LD50 values of chemicals and for screening for necessity for classical full LD50 test using many animals.

1. Several sub-lethal doses of cyanide were assayed with the aim of obtaining significant differences in the parameters studied. A dose of 4 mg/kg s.c. was selected. 2. Present studies were carried out to determine the time dependence of the effects produced by s.c. administration of a single dose of potassium cyanide as well as the recovery time of some of the toxicity indicative parameters. 3. It was found that cyanide effects continued for at least 3 days; after the parameters had recovered normal values.

1. Present studies were carried out to establish the action of cyanide maintained at a high level during a period as long as desired. 2. One of the earliest effects of cyanide seems to be the inhibition of hepatic rhodanese. These changes do not seem to occur in the blood. 3. Available cyanide labile-sulfur was always altered, unless cyanide tissue levels could not be detected. 4. The inhibitory action of cyanide on enzymatic reaction involving Schiff base intermediates was also corroborated through its effect on delta-aminolevulinate acid dehydratase activity. 5. S-Adenosyl-L-methionine was not able to modify the toxic cyanide action.

Female albino mice were fed sublethal doses of KCN (approx. 10 micrograms/mouse/day) for 7 days, injected intraperitoneally with phenobarbitone (50 mg/kg body wt/day) in the subsequent 3 days, and sacrificed 24 hr after the last injection. Phenobarbitone sleeping time was increasingly shortened (16-27%) daily in cyanide-fed mice in comparison with cyanide-free controls. Both compounds administered singly or simultaneously increased the liver weight/body weight ratios by not more than 10%. Aniline hydroxylase, glucose-6-phosphatase, NADPH- and NADH-cytochrome c reductase activities were similarly increased. Aniline hydroxylase activity was most markedly increased (by a factor of 4). The toxicological implications of these results are discussed.

Protective effects of OP-2507 [15-cis-(4-propylcyclohexyl)-16,17,18,19,20-pentanor-9-deoxy -9 alpha, 6-nitrilo-PGF1 methyl ester] against cerebral anoxia and edema were investigated in a variety of experimental models in mice and rats. OP-2507 given s.c. or p.o. led to a consistent and dose-dependent prolongation of survival time against cerebral anoxia in hypobaric and normobaric hypoxia, KCN-induced anoxia and decapitation-induced gasping. Furthermore, treatment with 0.03-0.1 mg/kg s.c. or 0.3 mg/kg p.o. of OP-2507 was found to be effective against the changes of cerebral energy metabolites and cyclic nucleotides in hypoxic brain. In brain ischemia induced by bilateral ligation of common carotid arteries of rats, a reduction in specific gravity of cortex and an increase in water content of brain were observed, in accordance with changes of cerebral energy metabolites. These edematous and biochemical changes were prevented by the treatment with 0.01-0.03 mg/kg s.c. of OP-2507. These results indicate a potential usefulness of OP-2507 in protecting the brain from oxygen insufficiency resulting from cerebral ischemia.

In order to study the events that follow cyanide-induced inhibition of oxidative metabolism and produce cellular injury, isolated, haemoglobin-free perfused rat livers from fasted rats were exposed to KCN (100 mg/l). KCN reduced the oxygen consumption of the livers by about 80%. Hepatotoxicity was evident by a marked release of enzymes (LDH, SDH) and of glutathione (mainly GSSG) into the perfusate, by a depletion of hepatic glutathione and by an accumulation of calcium in the liver. Cyanide-induced hepatotoxicity could be prevented completely by feeding the rats before preparing the liver as well as by addition of fructose to the perfusate of fasted livers. Both treatments resulted in an increased energy supply from anaerobic glycolysis as evidenced by a large release of lactate + pyruvate into the perfusate. The toxic actions of cyanide were markedly attenuated by deferrioxamine as well as by allopurinol. These antitoxic actions occurred without changes in anaerobic glycolysis. Omission of calcium from the perfusate, however, did not influence cyanide toxicity. Thus, energy supply from anaerobic glycolysis seems to be sufficient for the basic functions of the liver to occur, when oxidative metabolism is inhibited by cyanide. The effects of deferrioxamine and allopurinol indicate the involvement of radical intermediates and/or Fe2+ in cyanide-induced cellular toxicity. An influx of calcium from the extracellular to the intracellular space is not involved in cyanide-induced hepatocellular injury.

Significance of carbamoylation for mutagenic effects of N-nitroso-N-methyl-urea (NMU) on the CHO-AT3-2 cell line of Chinese hamster was studied. True point mutations occurred, due to alkylation. Carbamoylation combined with alkylation, or carbamoylation after alkylation induced the increase in other types of gene mutations as well as micro- and macroaberrations. These effects may be explained by the synergistic effect of alkylation and carbamoylation. Possible mechanisms and levels of interaction between alkylation and carbamoylation are discussed.

Exposure of cultured hepatoma 1c1c7 cells to KCN and iodoacetate, to produce chemical anoxia, caused a rapid and sustained increase in cytosolic-free Ca2+ concentration, which was associated with depletion of intracellular ATP and glutathione. These changes occurred before the loss of cell viability and were accompanied by the appearance of plasma membrane blebs. Pretreatment of the cells with the Ca2+ chelators Quin 2 or BAPTA markedly delayed both the onset of blebbing and loss of cell viability, but did not affect KCN- and iodoacetate-induced loss of ATP and glutathione. Together, these results strongly suggest that a sustained increase in cytosolic Ca2+ concentration plays an important role in killing of hepatoma cells by chemical anoxia.

The heat produced by Ehrlich ascites carcinoma cells was measured microcalorimetrically to investigate the characteristics of the energetics of these cells. 2,4-Dinitrophenol and other inhibitors were used to obtain the standard thermograms. The heat level of starved Ehrlich ascites carcinoma cells (endogenous) was almost constant and linear and depended on the cell number used in the experiments (about 1 cal/1 X 10(8) cells/hr). The endogenous heat generation was suppressed strongly by potassium cyanide but not by iodoacetic acid or 2,4-dinitrophenol. On the other hand, heat evolution was directly related to the amount of glucose added to the medium but not to the number of cells (19 cal/mmol of glucose). The addition of iodoacetic acid markedly suppressed heat generation, but potassium cyanide did not. In contrast, glucose with dinitrophenol increased heat production markedly to about 10% higher than that of the control, possibly due to the uncoupled energy from oxidative phosphorylation. Malonic acid (1 mM) suppressed slightly the endogenous heat but caused a 15% reduction in exogenous heat. The amount of heat produced by the addition of glucose (19 cal/mmol) was equivalent to 40% of the theoretical value of energy released through glycolysis (47 cal/mmol). Thus, the heat produced by exogenous glucose appears to be mainly dependent on glycolysis.

Anti-cyanide action by sodium thiosulfate (ST) was enhanced by prior administration of chlorpromazine (CPZ). However, CPZ (alone) provided no protection against cyanide lethality. To investigate the possibility that CPZ enhances thiocyanate formation in ST-pretreated mice, the effects of CPZ on rhodanese activity and the time course of plasma thiocyanate concentrations were investigated. CPZ did not alter hepatic rhodanese kinetics nor did it enhance plasma thiocyanate concentrations in ST-pretreated mice. The effect of CPZ and ST on the time course of cytochrome oxidase inhibition and recovery, in vivo, was also investigated. At 4 mg KCN/kg, maximal inhibition of brain (40%) and heart (60%) cytochrome oxidase occurred 10 to 20 min post-challenge in control and CPZ-pretreated mice, while no inhibition occurred in ST- and CPZ/ST-pretreated mice. Twenty milligrams KCN/kg caused 100% lethality in control and CPZ-pretreated mice and 6/25 and 4/20 deaths were observed in ST- and CPZ/ST-pretreated mice, respectively. No significant inhibition of brain, heart, and liver cytochrome oxidase activities was observed in surviving ST- and CPZ/ST-pretreated mice challenged with 20 mg KCN/kg. Control and CPZ-pretreated mice died within 5 min of KCN challenge and had almost the same degree of inhibition of brain (35 and 29%, respectively) and heart (60 and 55%, respectively) cytochrome oxidase as did similarly pretreated mice 5 min after challenge with a nonlethal cyanide dose (4 mg/kg). Our results suggest that CPZ does not enhance the formation of thiocyanate in ST-pretreated mice. In addition, the similar degree of cytochrome oxidase inhibition noted after both lethal and nonlethal KCN treatments raises questions as to the ultimate target in cyanide-induced lethality.

Diseases like tropical ataxic neuropathy and endemic goitre have been reported to have definite correlation with a chronic ingestion of cassava (Manihot esculenta Crantz). The toxicity of cassava has been attributed to its two cyanogenic glycosides, linamarin and lotaustralin. In this study, an attempt has been made to understand the pattern of changes in certain clinically significant enzymes brought about by the chronic administration of sublethal doses of linamarin to rabbits. The profound elevation in rhodanese activity observed in the linamarin and cyanide treated rabbits indicated the attempt of the tissues to detoxify cyanide. That intact linamarin could be hydrolysed in vivo was a significant finding from the study. The mode of toxicity of linamarin was similar to that of cyanide by producing a gradual shift from aerobic to anaerobic metabolism.

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.

On the basis of personal results in man and laboratory animal, we examined some aspects of acute and chronic cyanide intoxication: physiopathological aspects, hydroxocobalamin's therapeutic action in treating acute intoxications and neurotoxic sufferings due to cyanide, and hydroxocobalamin's biological action in decreasing the level of free cyanide appearing in blood and urine, a few days after sciatic nerve clamping in rabbits.

Aliphatic nitriles have been postulated to manifest their toxicity through cyanide liberation. We have investigated the signs of toxicity and effect of equitoxic LD50 doses of saturated and unsaturated aliphatic mono- and dinitriles on tissue and blood cyanide levels, tissue glutathione levels and cytochrome c oxidase activities. Signs of toxicity were classified into cholinomimetic effects observed with unsaturated nitriles and central nervous system effects observed with saturated nitriles and potassium cyanide (KCN). Hepatic and blood cyanide levels 1 h after treatment were highest following malononitrile (MCN) and decreased in the order of propionitrile (PCN) greater than KCN greater than butyronitrile greater than acrylonitrile (VCN) greater than allylcyanide greater than greater than fumaronitrile greater than acetonitrile. The order differed in brain where KCN preceded MCN and PCN. Hepatic and brain cytochrome c oxidase were significantly inhibited and corresponded to their cyanide levels. No significant inhibition of cytochrome c oxidase was observed in vitro. VCN was the only nitrile which significantly reduced tissue GSH levels. In conclusion, toxic expression of aliphatic nitriles depends not only upon cyanide release but also on their degree of unsaturation. With unsaturated aliphatic nitriles cyanide release plays a minimal role in their toxicity.

Chronic implantation of surface coils on the skull has been developed to record 31P NMR spectra of the brain in unanesthetized rats. Intraperitoneal sublethal potassium cyanide doses induce strong and reversible changes in high-energy phosphate compounds in the brain, similar in part to those induced by ischemia. These effects are dose-dependent as far as phosphocreatine, inorganic orthophosphates and pH are concerned; ATP does not seem to be altered by KCN doses ranging from 3 to 5 mg/kg but starts decreasing at a dose of 6 mg/kg. The fraction of Mg2+ complexed ATP which could be estimated as about 90% was not affected by KCN intoxication. For high doses (6 mg/kg) a new peak, appearing on the upfield side of the inorganic phosphate peak, may correspond to an acidic compartment, the significance of which is discussed.

Circadian rhythms in mortality and/or survival time following a single intraperitoneal injection of a LD50 of potassium cyanide were studied. In two investigations, different but comparable subgroups of inbred male BALB/cCr mice were treated at 4-h intervals (under conditions standardized for chronobiologic study) during 24-h spans. Mice were observed for exact time-to-death during the first hour after treatment as well as overall mortality during the entire 24-h post-injection span following each KCN treatment timepoint. In both studies, mortality from KCN exhibited a 24-h rhythm. Highest mortality occurred in mice injected at 1600 (80% mortality) in Experiment 1 and 2000 (100% mortality) in Experiment II. Lowest mortality occurred at 0400 (40% mortality) in Experiment I and 0800 (30% mortality) in Experiment II. The need to consider the circadian organization of physiologic function when bioassaying toxicity is discussed.

Carbon monoxide (CO) and hydrogen cyanide are recognized pyrolytic products of plastic materials in fires. Lethal blood concentrations of CO and cyanide are found in fire victims and considered to be the cause of death. However, there are reports of fire victims with sublethal concentrations of both CO and cyanide and no other attributed reason for their deaths. Thus, the purpose of this study was to investigate lethality induced by low concentrations of CO and cyanide. Male ICR mice were used in these studies. Doses of potassium cyanide (4-9 mg/kg, i.p.) were administered to animals pretreated for 3 min with either air or CO (0.63-0.66%). From these data the LD50 value of potassium cyanide (KCN) was determined in these animals pretreated with either air or CO. A significantly lower LD50 value for KCN was found in CO-pretreated animals as compared to air-pretreated animals. In another series of experiments, animals were pretreated with either saline or KCN (1.00-6.35 mg/kg, i.p.) and then placed in the chamber containing a CO atmosphere (0.325-0.375%). Ten to 20% of saline-pretreated animals were dead at the end of the monitoring period. Sublethal doses of KCN (3.5-6.35 mg/kg, i.p.) produced a synergistic lethality as compared to the saline-pretreated animals. Blood was analyzed for CO and cyanide content to determine if there were any changes that could explain this augmented lethality. There was no difference in CO or cyanide blood concentrations between these treatment groups. From these data there was demonstrated a synergistic lethality induced by the combination of CO and cyanide that could not be explained by altered CO or cyanide blood concentrations.

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.

Radiofrequency surface coils were chronically implanted in rats, which were subsequently subjected to 31P nuclear magnetic resonance (NMR) investigations at 4.7 T. The implanted coil allowed study of the animals without need for anesthesia, which is a prerequisite for studies of normal brain metabolism. The animals may be kept in the NMR probe for several hours. During subsequent experiments, they may be placed in the same position, therefore allowing follow-up studies for periods as long as 2 months. This method has been used in the study of sublethal KCN intoxication. KCN, a cytochrome c oxidase inhibitor, induces a blockade of cell respiratory processes, which is reflected, in a dose-dependent manner, by a decrease in phosphocreatine content and pH and an increase in inorganic phosphate content, whereas ATP levels remain constant until high doses of KCN (6 mg/kg i.p.) are reached. 31P NMR allows the time course of these metabolic changes to be followed. For high KCN doses, a new peak, termed X, is observed, which is interpreted as being due to a pool of inorganic phosphate at very low pH (5.65), corresponding to a subset of cells that did not survive KCN injury. Hydroxocobalamine, a specific antidote of KCN, suppresses the metabolic changes due to 6 mg/kg of KCN.

The correlation among hyperammonemia, brain neutral amino acid, and encephalopathy induced by cyanide was investigated in mice. Subcutaneous injection of 10 mg/kg of potassium cyanide increased the levels of blood ammonia by 2.5-fold as compared to those of the corresponding controls and caused loss of consciousness in 100% of the treated mice. alpha-Ketoglutarate (500 mg/kg, ip) completely blocked the development of loss of consciousness and hyperammonemia induced by cyanide. Furthermore, the same doses of potassium cyanide increased by 50-150% the levels of brain neutral and aromatic amino acids such as leucine, isoleucine, tyrosine, and phenylalanine, while the levels of acidic amino acid such as taurine, glutamate, and aspartate did not change. alpha-Ketoglutarate also significantly inhibited the increase of the neutral and aromatic amino acid levels in brain. These findings suggest that the hyperammonemia and the increase of neutral and aromatic amino acids may play an important role in development of loss of consciousness induced by cyanide.

An estimated 35% of all fire victims in the United States have toxicologically significant blood levels of CO and CN. However, the treatment of concurrent CO/CN intoxication has been paid scant attention. The suggestion has been made that these victims should be treated for CN poisoning. The current therapeutic management of CN poisoning in this country includes the utilization of two methemoglobin formers: amyl nitrite and sodium nitrite. This study was undertaken to determine if the administration of methemoglobin formers is advisable, as the victim is already suffering from O2 deprivation due to the presence of carboxyhemoglobin. Groups of 28 male ICR mice (22-24 g) were injected i.p. with 5.0 mg/kg of KCN and then were exposed immediately to 0.35% CO for 8.5 min in a dynamic inhalation chamber. Half of the animals were marked randomly for antidotal intervention, the other 14 animals acted as controls. Treatment of survivors with amyl nitrite (12 mg/l of chamber air) for 1 min increased mortality 43%, whereas treatment for 2 min resulted in a 59% increase in mortality. A 25% increase in mortality was noted among those animals treated with sodium nitrite (80 mg/kg i.p.), as compared to the nontreated control survivors. Treatment with dimethylaminophenol (49 mg/kg i.p.) did not statistically affect mortality.

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.

An experimental model of in situ isolated carotid arteries has been used to evaluate the static mechanical properties of the arterial wall in 12-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). The effects of endothelium removal on carotid compliance were compared with the effects of total abolition of the vascular smooth muscle tone by potassium cyanide. Carotid compliance measured at pressures ranging from 50 to 175 mmHg had maximal values (0.22 +/- 0.07 microliter/mmHg and 0.13 +/- 0.03 microliter/mmHg, respectively, for WKY and SHR; P less than 0.001) for pressure values close to the operating pressure in both groups. Maximal values of carotid compliance were increased by 35 and 45% in WKY and SHR, respectively, after potassium cyanide poisoning (P less than 0.01). Endothelium removal induced a significant increase in carotid compliance compared with control values (WKY +37%, P less than 0.01; SHR +25%, P less than 0.01). The present results suggest that the mechanical properties of the wall of the carotid artery are endothelium-dependent. The differences between the effect of endothelium on the vascular smooth muscle observed in the present experiment and previous results may be due to differences in experimental conditions (in vitro versus in situ) and/or to the specificity of the rat carotid artery.

Mitochondria were isolated from hearts obtained from adult male Sprague-Dawley rats by two-part differential centrifugation of heart homogenates. Time-dependent (0-120 sec) and concentration-dependent (0-10 microM CdCl2) effects of cadmium on pyruvate-malate-supported state 3 and state 4 respiration were measured in a constant temperature reaction chamber at 37 degrees C, according to established procedures. The ID50 for cadmium chloride on state 3 respiration was determined to be 4.2 microM. The inhibition produced by cadmium chloride in heart mitochondria was compared, using identical procedures, to the effects induced by two compounds, sodium atractyloside and potassium cyanide, which are known to alter mitochondrial respiration at specific sites. The calculated ID50 values for these agents in heart mitochondria were 1.8 and 16 microM, respectively. The concentration-dependent inhibition of mitochondrial respiration induced by either cadmium chloride or potassium cyanide was maintained in the presence of 50 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), a known uncoupling agent. In contrast, sodium atractyloside did not block the uncoupling effect of 50 microM CCCP. In addition cadmium chloride was also shown to inhibit CCCP-uncoupled mitochondrial respiration. The cadmium-induced inhibition of mitochondrial respiration was reversed partially by cysteine and completely by 2,3-dimercaptopropanol. The results of the present study indicate that, at all concentrations, cadmium chloride acted solely as an inhibitor of rat heart pyruvate-malate-supported mitochondrial respiration. These findings suggest a possible mechanism for the reported disturbances in myocardial metabolism and function that occur in conjunction with acute and chronic cadmium exposure in humans and experimental animals.

The influence of NKK-105 on the acute toxicity of strychnine nitrate, potassium cyanide, chloral hydrate, atropine, tetrodotoxin, mercuric chloride, cadmium chloride, lead acetate and arsenic trioxide were examined in rats. NKK-105 prolonged the time of death induced by strychnine, potassium cyanide and tetrodotoxin in acute toxicity. Survival time was prolonged in rats treated with the toxic metals by pretreatment with NKK-105. NKK-105 counteracted the lethal effect of strychnine, potassium cyanide, chloral hydrate, mercuric chloride and cadmium chloride. NKK-105 significantly decreased the acute toxicity of these drugs but the inhibitory effect was not observed in the acute toxicity of atropine, tetrodotoxin, lead acetate and arsenic trioxide.

The toxicity of single and multiple fire gases is studied to determine whether the toxic effects of the combustion products from materials can be explained by the toxicological interactions (as indicated by lethality) of the primary fire gases or if minor, more obscure gases need to be considered. LC50 values for Fischer-344 rats have been calculated for the individual gases, carbon monoxide (CO), hydrogen cyanide (HCN), or decreased oxygen (O2), for 30-min exposures plus relevant postexposure periods using the NBS Toxicity Test Method. Combination experiments with CO and HCN indicate that they act in an additive manner. Synergistic effects have been found when the animals are exposed to certain combinations of CO and carbon dioxide (CO2). Five percent CO2 raised the threshold for deaths due to hypoxia and decreased the LC50 of HCN. Decreasing the O2 concentration in the presence of various mixtures of the other major fire gases increased the toxicity even further. A comparison of the concentrations of the major combustion products generated from a number of polymeric materials at their LC50 (30-min exposure plus 14-day postexposure) values with the combined pure gas results indicates that, in most cases, the observed toxicity may be explained by the toxicological interactions of the examined primary toxic fire gases. These results provide necessary information for the computer model currently being developed at the Center for Fire Research to predict the toxic hazard that people will experience under various fire scenarios.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, readily forms complexes with a wide variety of potentially toxic substances, including cyanide (KCN), spermine (SPM), gentamicin (GM), and dopamine (DOP). The role of PLP as an antidote for these toxicants in vivo was studied. Rats were given intraperitoneal injections of the toxicant, followed by injections of either saline or PLP. For KCN, PLP increased the LD50 from 0.088 to 0.188 mmol/kg and extended the survival time at the highest dose employed from a median of 3 min to a median of 60 min. For SPM, PLP increased the LD50 from 0.162 to 0.262 mmol/kg and prevented death from renal failure at all doses employed except the highest. PLP protected against GM-induced neuromuscular paralysis and death. In the case of DOP, results were equivocal, but no deaths were seen in the PLP-treated rats. The combination of individually nontoxic doses of GM, DOP, and SPM caused death with renal tubular necrosis, pulmonary edema and hemorrhages, congestion of the viscera, and a diffuse coagulopathy. Because many seriously ill patients have elevated SPM levels and are given GM and/or DOP, we suggest that further investigation of the potential protective role of PLP in serious illness be undertaken.

An attempt was made to establish hypoxic cultured neurons from chick embryo hemispheres as an in vitro model for testing cerebroprotective drug effects. Hypoxia was induced by adding 1 mmol/l KCN to the incubation medium of the cells. After various time periods (15-120 min), the cyanide-containing medium was replaced by fresh medium so that the cells could recover from hypoxia. High-energy phosphate levels of the cells and the protein content of the cultures were determined as indicators of the cell viability and response. The cells showed a rapid and pronounced decrease in the levels of high-energy phosphates during cytotoxic hypoxia, and after removal of cyanide the energy state was restored again within few minutes. The protein content of the cultures remained unchanged. Pentobarbital inhibited the breakdown of the high-energy phosphates during hypoxia and accelerated their restitution during the recovery period. The results suggest that hypoxic cultured neurons could be a useful model for testing cerebroprotective drug effects.

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.

It was established in experimental normobaric and hypobaric hypoxia and hemic hypoxia induced by carbon monoxide poisoning that zinc compounds administered in a dose of 0.15 mA/kg have a marked prophylactic protective effect. The mechanism of action of zinc compounds consists in changes of oxygen transport blood function. It was shown that interaction of the hemoglobin molecule with zinc ion brings about an increase in Hb affinity for O2 (the left drive of the oxyhemoglobin dissociation curve), a reduction in cooperative interaction of hemoglobin subunits, and a relative decrease in hemoglobin affinity for carbon monoxide. The leading defence mechanism against hypoxic hypoxia is the left drive, the mechanism of defence against carbon monoxide protection consists in the lowering of the "hem-hem" cooperation and of the relative hemoglobin affinity for carbon monoxide.

Flunarizine is a calcium entry blocking drug possessing antihypoxic activity in animal models of cerebral and peripheral ischemia-anoxia and has clinical usefulness in circulatory disorders of both central and peripheral origin. This report compares the activity of flunarizine and verapamil, another calcium entry blocking drug, on the central nervous system (CNS) and peripheral consequences of cytotoxic hypoxia induced by high and low doses of KCN. The lethal effect of KCN (6 mg/kg, i.p.) in rats was prevented by orally administered flunarizine (ED50 = 12 mg/kg with four-hr pretreatment) but not by verapamil (at oral doses up to 80 mg/kg with one-hr pretreatment). Since the lethal effect of KCN involves failure of respiration at the CNS level, these results suggest that flunarizine protects against the hypoxic effect of the cyanide ion by an action in brain tissue. We found also that the stimulant effect of low intravenous doses (0.5 mg/kg/min) of KCN upon respiration rate was not altered in pentobarbital- and chloralose-anesthetized rats treated with oral doses of flunarizine up to 80 mg/kg (with four hr pretreatment). In contrast, KCN-stimulated respiration rate in pentobarbital anesthetized rats was significantly attenuated by verapamil (20 and 40 mg/kg, p.o. with one hr pretreatment). Since low doses of the cyanide ion render respiration quicker and deeper by an action on chemoreceptive cells in peripheral arteries, the effect of verapamil against the hypoxic effect of KCN is mediated by an action in the periphery. In summary, we have shown that the physiological consequences of cytotoxic hypoxia can be affected by calcium entry blocking drugs having site-specific activities. Based on our results, flunarizine is more effective than verapamil against cellular anoxia involving the CNS.