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Uncoupling protein 2 (UCP-2) is an inner mitochondrial membrane proton carrier that modulates mitochondrial membrane potential (DeltaPsi(m)) and uncouples oxidative phosphorylation. We have shown that up-regulation of UCP-2 by Wy14,643, a selective peroxisome proliferator-activated receptor-alpha (PPARalpha) agonist, enhances cyanide cytotoxicity. The pathway by which Wy14,643 up-regulates UCP-2 was determined in a dopaminergic cell line (N27 cells). Since dopaminergic mesencephalic cells are a primary brain target of cyanide, the N27 immortalized mesencephalic cell was used in this study. Wy14,643 produced a concentration- and time-dependent up-regulation of UCP-2 that was linked to enhanced cyanide-induced cell death. MK886 (PPARalpha antagonist) or PPARalpha knock-down by RNA interference (RNAi) inhibited PPARalpha activity as shown by the peroxisome proliferator response element-luciferase reporter assay, but only partially decreased up-regulation of UCP-2. The role of oxidative stress as an alternative pathway to UCP-2 up-regulation was determined. Wy14,643 induced a rapid surge of ROS generation and loading cells with glutathione ethyl ester (GSH-EE) or pre-treatment with vitamin E attenuated up-regulation of UCP-2. On the other hand, RNAi knockdown of PPARalpha did not alter ROS generation, suggesting a PPARalpha-independent component to the response. Co-treatment with PPARalpha-RNAi and GSH-EE blocked both the up-regulation of UCP-2 by Wy14,643 and the cyanide-induced cell death. It was concluded that a PPARalpha-mediated pathway and an oxidative stress pathway independent of PPARalpha mediate the up-regulation of UCP-2 and subsequent increased vulnerability to cyanide-induced cytotoxicity.

Execution of apoptosis can involve activation of the caspase family of proteases. Recent studies show that caspase inhibition can switch the morphology of cell death from apoptotic to necrotic without altering the level of death among cell populations. In the present study, the effect of caspase inhibition on cortical (CX) cell death induced by cyanide was investigated. In primary cultured CX cells exposed to cyanide (400 microM), death was primarily apoptotic as indicated by positive TUNEL staining. Reactive oxygen species (ROS) generation and subsequent caspase activation mediated the apoptosis. Inhibition of the caspase cascade with zVAD-fmk switched the apoptotic response to necrotic cell death, as assessed by increased cellular efflux of LDH and propidium iodide uptake by the cells. The change in death mode was accompanied by a marked increase in poly (ADP-ribose) polymerase-1 (PARP-1) activity, reactive oxygen species (ROS) generation, a reduction in the mitochondrial membrane potential (Delta psi(m)), and reduced cellular ATP. Prior treatment of cells with 3-aminobenzamide (3-AB), a PARP-1 inhibitor, prevented the cells from undergoing necrosis and preserved intracellular ATP levels. These findings indicate that apoptosis and necrosis share common initiation pathways and caspase inhibition can switch the apoptotic response to necrosis. Inhibition of PARP-1 preserves cellular ATP levels and in turn blocks execution of the necrotic death pathway.

Cyanide (KCN)-induced generation of reactive oxygen species (ROS) involves cyclooxygenase-2 (COX-2)-mediated reactions in some neurons. The present study examines the extent to which COX isoforms are involved in KCN-induced apoptotic cell death processes of cultured cortical cells. After treatment with KCN (10-300 microM), COX-2 was expressed in a time- and concentration-dependent manner increasing markedly over a 4-h period. However, no significant changes were observed in COX-1 levels at any cyanide concentration. Correlated with COX-2 up-regulation, KCN induced a time-dependent apoptotic death. TUNEL staining showed that the COX-2 inhibitor NS-398 (30 microM) blocked KCN-induced apoptosis, whereas the selective COX-1 inhibitor valeryl salicylate did not affect the level of apoptotic cell death. Exposure of cells to KCN (300 microM) for 24 h resulted in DNA fragmentation, which was also reduced by NS-398. Prostaglandin E(2) (PGE(2)) accumulation in cell culture supernatants was increased by KCN and NS-398 blocked PGE(2) generation. PCR studies further confirmed that COX-2 expression was increased by KCN. Antioxidants phenyl-N-test-butylnitrone, superoxide dismutase, and catalase significantly inhibited KCN-induced COX-2 up-regulation and subsequent apoptosis. N(G)-nitro-L-arginine methylester an inhibitor of nitric oxide synthase, blocked KCN-induced PGE(2) production and apoptosis, but not COX-2 expression. Increased nitric oxide levels caused by cyanide may directly activate the COX-2 enzyme. These data show that cyanide treatment of cortical cells involves increased COX-2 expression, PGE(2) accumulation, and ROS generation, resulting in apoptotic cell death.

The central nervous system is one of the main target organs in cyanide toxicity. Primary cultured cortical neurons were used to study the cellular mechanisms underlying cyanide-induced cytotoxicity. After exposure to KCN (100-300 microM) for 24 h, cortical neurons underwent apoptosis as characterized by positive TUNEL staining. Reactive oxygen species (ROS) play an important role in cyanide-induced neuronal apoptosis; immediately after cyanide (100-300 microM) treatment, ROS generation was observed and continued to be elevated for up to 3 h. NMDA receptor activation and subsequent Ca(2+) influx contribute in part to cyanide-induced ROS formation, since the selective NMDA receptor antagonist MK801 and intracellular Ca(2+) chelator BAPTA blocked ROS generation. Interestingly, caspases, recently reported to be involved in neuronal apoptosis, play a role in the late phase of ROS production after cyanide stimulation. Z-VAD, a nonspecific caspase inhibitor, blocked ROS generated 1 h after cyanide treatment, but it had no effect on ROS generated immediately after cyanide treatment. Nuclear factor kappaB (NF-kappaB), a redox-sensitive transcription factor, was activated dose dependently after cyanide treatment. Blockade of ROS generation by MK801, Z-VAD, and various antioxidants also blocked the activation of NF-kappaB. SN50, a synthetic peptide which inhibits the nuclear translocation of NF-kappaB, blocked cyanide-induced apoptotic cell death. These results indicate that NF-kappaB plays an important role in cyanide-induced apoptosis in cortical neurons, and the caspases may contribute in part to the activation of NF-kappaB after cyanide treatment by inducing the late phase of ROS generation., (Copyright 2000 Academic Press.)

We have demonstrated that cyanide (KCN) induces selective degeneration of dopaminergic neurons in mice and apoptotic cell death in cultured neurons. In the present study the mode of cyanide-induced cell death was determined in the susceptible brain areas. Mice were treated with KCN (6 mg/kg ip) or vehicle (saline) twice daily for 1 to 12 days. After 3 days of KCN treatment, two separate lesions were observed in coronal brain sections. Widespread DNA fragmentation in parietal and suprarhinal regions of the motor cortex was observed by the in situ terminal deoxynucleotide transferase nick-end labeling (TUNEL) technique. Pyknosis and chromatin condensation, morphological hallmarks of apoptotic cells, were observed in TUNEL-positive regions. On the other hand, in the substantia nigra (SN), KCN produced a progressive, bilateral necrotic lesion that was evident by 3 days of treatment. The SN lesion was circumscribed by a prominent ring of glial infiltration, as determined by glial-acidic fibrillary protein (GFAP) immunostaining. The extent of the SN lesion steadily increased with treatment duration, and DNA fragmentation was not observed over the 1- to 12-day period. On the other hand, cortical apoptosis was not associated with necrotic cell loss or astrogliosis. Pretreatment of animals with the antioxidant alpha-phenyl-tert-butyl nitrone (PBN) for 7 days prior to and during 3 days of KCN administration markedly reduced cortical DNA fragmentation whereas the PBN treatment did not influence the SN necrosis or astrocytic gliosis. Except for moderate GFAP immunostaining in corpus callosum, other brain areas were not affected by cyanide. It is concluded that KCN-induced neuronal loss involves selective activation of necrosis or apoptosis in different neuronal populations, and involves divergent mechanisms and sensitivity to antioxidants., (Copyright 1999 Academic Press.)

WY-14,643, a lipid-lowering drug, increases basal rates of oxygen uptake in perfused livers. Because peroxisomes consume oxygen for H2O2 production and are induced by WY-14,643 treatment, it is possible that peroxisomal beta-oxidation can account for some of this increase in cellular respiration. Therefore, cyanide, an inhibitor of mitochondrial cytochrome oxidase, was infused into livers of WY-14,643-fed rats (0.1% WY-14,643 in laboratory rat chow for 1, 21, and 105 days) to assess peroxisomal cyanide-insensitive respiration. As expected, the addition of cyanide abolished oxygen uptake nearly completely; however, after approximately 20 min oxygen consumption unexpectedly returned to basal levels in 105-day WY-14,643-treated animals but not in untreated controls. Urea synthesis, a process dependent upon ATP, was decreased and remained low during cyanide infusion in livers from both groups, indicating that mitochondria were not responsible for this unusual increase in oxygen uptake in the presence of cyanide. Methanol metabolism, which requires oxygen to form H2O2, was decreased from 37 +/- 5 to 6 +/- 1 micromol/g/hr in all groups treated with cyanide; however, it was increased significantly about 20 min later to 25 micromol/g/hr in livers from WY-14,643-treated rats, indicating that oxygen for peroxisomal H2O2 production is involved in cellular respiration in the presence of cyanide. Fasting abolished the recovery of both oxygen uptake and methanol metabolism in WY-14,643-fed rats, suggesting that ATP for acyl CoA synthetase, an enzyme which metabolizes fatty acids to acyl CoA compounds, is provided by glycolysis. Indeed, oleate significantly increased methanol metabolism in fed control rats from 8 +/- 4 to 26 +/- 3 micromol/g/hr in the presence of cyanide, indicating that fatty acid supply is necessary for peroxisomal respiration. Taken together, these experiments demonstrate that when mitochondrial respiration is inhibited, livers from rats fed WY-14,643 chronically have the unique ability of metabolizing fatty acids through the peroxisome using glycolytic ATP.

Methanol is an ocular toxicant which causes visual dysfunction often leading to blindness after acute exposure. The physiological and biochemical changes responsible for this toxicity are poorly understood. Previously, we reported that the folate-reduced (FR) rat is an animal model which mimics the characteristic human methanol toxicities. The present study examines the hypothesis that depletion of ATP after methanol administration is the initiating event in methanol-induced retinal toxicity. ATP is reduced in retinae of methanol-treated FR rats to the same extent as is seen in retinae of FR and folate-sufficient (FS) rats treated with the Müller cell (retinal glial cell) toxin alpha-aminoadipic acid. Changes in the electroretinogram and the response of Müller cells to a potassium stimulus are also similarly eliminated in methanol-treated FR rats and alpha-aminoadipic acid-treated FR and FS rats. These results suggest that the Müller cell may be the initial target in methanol-induced visual system toxicity.

Previous reports have shown that dopamine (DA) is depleted in the brains of animals treated with cyanide. To develop a model for studying the mechanisms of cyanide-induced changes in dopaminergic systems, mice were treated with cyanide (KCN, 6 mg/kg, sc) twice a day for 7 days and 16 hr after the last dose neurochemical, histological, or behavioral parameters were evaluated. DA levels in KCN-treated animals decreased in the striatum (41%), hippocampus (30%), and cerebral cortex (13%) as compared to saline-treated controls. In striatal and hippocampal tissues, but not in cerebral cortex, malondialdehyde levels increased 43 and 57%, respectively, as compared to controls, indicating that peroxidation of lipids occurred in these brain areas. Over 30% of the treated mice exhibited decreased locomotor activity and akinesia, which were suppressed by l-DOPA (100 mg/kg, ip). Tyrosine hydroxylase (TH) immunohistochemical examination of brains from cyanide-treated animals showed a reduced number of TH-positive cells in substantia nigra, indicating a loss of dopaminergic neurons. In contrast, acute cyanide (KCN, 6 mg/kg, sc) did not produce significant neurochemical or behavioral changes. Under these treatment conditions, cyanide produces a central dopaminergic toxicity which is characterized by decreased DA levels in select brain areas, impaired locomotor activity, and neuronal damage.

Resealed erythrocytes containing sodium thiosulfate and rhodanese (CRBC) are being employed as a new approach in the antagonism of cyanide intoxication. In earlier in vitro studies, the behavior of red blood cells containing rhodanese and sodium thiosulfate was investigated with regard to their properties and their capability of metabolizing cyanide to thiocyanate. The present studies are concerned with the properties of these rhodanese-containing carrier erythrocytes in the intact animal. These carrier erythrocytes were administered intravenously and the survival of the encapsulated enzyme was compared with the administration (iv) of free exogenous enzyme. Also, the amount of leakage of the encapsulated rhodanese from the red blood cell was determined. The survival of the carrier red blood cell. prepared by hypotonic dialysis, was found to be characterized by a biphasic curve. There was an initial rapid loss of approximately 40 to 50% of the carrier cells with a t1/2 = 2.5 hr. Subsequently the remaining resealed annealed carrier erythrocytes persisted in the vascular system with a t1/2 = 8.5 days. When free exogenous rhodanese was administered directly into the vascular system, it was rapidly eliminated with a t1/2 = 53 min. Red blood cells containing sodium thiosulfate and rhodanese apparently are effective in vivo in the biotransformation of cyanide. In animals pretreated with encapsulated rhodanese and sodium thiosulfate, blood cyanide concentrations are appreciably decreased with a concomitant increase in thiocyanate ion, a metabolite of cyanide. When erythrocytes, which contained no rhodanese or sodium thiosulfate, were subjected to hypotonic dialysis, cyanide was not metabolized to any appreciable extent. Furthermore, carrier erythrocytes containing rhodanese and sodium thiosulfate were found to increase the protection against the lethal effects of cyanide by approximately twofold. The ability of these carrier erythrocytes alone to metabolize cyanide and to antagonize the lethal effects of cyanide reflects the potential of this new antidotal approach in the antagonism of chemical toxicants.

Subcutaneous injection of sodium dichromate into male Sprague-Dawley rats immediately produced a variety of metabolic changes in a dose-dependent manner. Serum lactate and glucose were significantly increased after dichromate treatment, reaching maximum levels at 15 and 30 min, respectively. Then, the toxicity progressively diminished. In contrast, a steady increase in blood urea nitrogen (BUN) concentration was caused by dichromate, reaching maximum levels at 60 min after the administration; elevated BUN levels were sustained for several hours thereafter. Unlike KCN (5 mg/kg, ip) and As2O3 (5 mg/kg, ip), dichromate rapidly decreased serum insulin within 15 min after intoxication in doses of 20 and 40 mg/kg; hypoinsulinemia lasted 60 min. However, insulin levels returned to the normal range at 120 min after treatment. Dichromate-induced metabolic disturbance was also observed in the 24 hr-fasted rats, the response of which was similar to normal rats except for later hyperglycemia. In both cases, the duration time was short (30 to 60 min). Adrenalectomy and insulin pretreatment had no effect on dichromate-induced hyperglycemia. These results suggest that dichromate-induced metabolic disturbance results from the concomitant effects of a sudden decrease in serum insulin level and its direct inhibitory effect on carbohydrate metabolism. In addition, the characteristic biphasic pattern of metabolic disturbance might be related to metabolic fate of dichromate in vivo.

It has long been known that a number of tissue hypoxicants are generated in the fire scenario. However, until recently few investigators have undertaken to correlate smoke inhalation deaths with the simultaneous exposure to histotoxic hypoxicants. Carbon monoxide and hydrogen cyanide are two histotoxic hypoxicants that are generated in nearly every fire. Prior studies performed in our laboratory have demonstrated that death can result from concomitant exposure to otherwise sublethal concentrations of carbon monoxide and cyanide. Since most smoke inhalation victims exhibit acid/base anomalies, we sought to investigate whether the death associated with simultaneous exposure to these two hypoxicants, at concentrations widely held to be nonlethal, could be explained by acid/base imbalances. Male ICR mice were exposed to 0.35% carbon monoxide immediately after having been injected ip with potassium cyanide solution, or were challenged with either agent alone. Animals challenged with cyanide or carbon monoxide alone demonstrated significant hypoxia. However, animals challenged with both agents demonstrated much greater hypoxia than could be explained by an additive effect alone. Controls demonstrated no alteration in acid/base homeostasis. Blood pH perturbations were found to be due to severe lactic acidosis coupled with inadequate respiratory compensation. Thus, it appears that the synergistic lethal effect of simultaneous administration of carbon monoxide and cyanide are related to a precipitous decrease in blood pH, the tissue hypoxia and its resulting complications.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.