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Hydrogen sulfide is a highly toxic gas-second only to carbon monoxide as a cause of inhalational deaths. Its mechanism of toxicity is only partially known, and no specific therapy exists for sulfide poisoning. We show in several cell types, including human inducible pluripotent stem cell (hiPSC)-derived neurons, that sulfide inhibited complex IV of the mitochondrial respiratory chain and induced apoptosis. Sulfide increased hydroxyl radical production in isolated mouse heart mitochondria and F2-isoprostanes in brains and hearts of mice. The vitamin B12 analog cobinamide reversed the cellular toxicity of sulfide, and rescued Drosophila melanogaster and mice from lethal exposures of hydrogen sulfide gas. Cobinamide worked through two distinct mechanisms: direct reversal of complex IV inhibition and neutralization of sulfide-generated reactive oxygen species. We conclude that sulfide produces a high degree of oxidative stress in cells and tissues, and that cobinamide has promise as a first specific treatment for sulfide poisoning.

Present studies have focused on nano-intercalated rhodanese in combination with sulfur donors to prevent cyanide lethality in a prophylactic mice model for future development of an effective cyanide antidotal system. Our approach is based on the idea of converting cyanide to the less toxic thiocyanate before it reaches the target organs by utilizing sulfurtransferases (e.g., rhodanese) and sulfur donors in a close proximity by injecting them directly into the blood stream. The inorganic thiosulfate (TS) and the garlic component diallydisulfide (DADS) were compared as sulfur donors with the nano-intercalated rhodanese in vitro and in vivo. The in vivo and in vitro experiments showed that DADS is not a more efficient sulfur donor than TS. However, the utilization of external rhodanese significantly enhanced the in vivo efficacy of both sulfur donor-nitrite combinations, indicating the potential usefulness of enzyme nano-delivery systems in developing antidotal therapeutic agents.

The effect of melatonin on potassium cyanide-induced neurotoxicity was investigated in vivo. The ED50 value of potassium cyanide, as measured by induction of tonic and clonic seizures, was significantly increased by 1.5- or 1.8-fold by s.c. preinjection of melatonin (20, 100 or 345 mg/kg) in mice. The preventive effect of melatonin against potassium cyanide-induced seizures was dose dependent. The LD50 value of potassium cyanide, based on 24-h mortality, was also significantly increased by 1.3-fold by preinjection of melatonin. Potassium cyanide (8 mg/kg, s.c.) increased lipid peroxidation in whole brain of mice, and the increased lipid peroxidation was completely abolished when cyanide-induced seizures were stopped by preadministration of melatonin. These results suggest that melatonin, a pineal hormone, may protect against cyanide-induced neurotoxicity with its free radical scavenging effects in mice.

Potassium cyanide (CN) intoxication in mice was found to be effectively antagonized by dihydroxyacetone (DHA), particularly if administered in combination with another CN antidote, sodium thiosulfate. Cyanide-induced convulsions were also prevented by DHA treatment, either alone or in combination with thiosulfate. Injection (i.p.) of DHA (2 g/kg) 2 min after or 10 min before CN (s.c.) increased LD50 values of CN(8.7 mg/kg) by factors of 2.1 and 3.0, respectively. Treatment with a combination of DHA and thiosulfate after CN increased the LD50 by a factor of 2.4. Pretreatment with a combination of DHA and thiosulfate (1 g/kg) increased the LD50 of CN to 83 mg/kg. Administration of alpha-ketoglutarate (2.0 g/kg), but not pyruvate, 2 min after CN increased the LD50 of CN by a factor of 1.6. Brain, heart and liver cytochrome oxidase activities were also measured following in vivo CN treatment with and without DHA. Pretreatment with DHA prevented the inhibition of cytochrome oxidase activity by CN and treatment with DHA after CN accelerated the recovery of cytochrome oxidase activity, especially in brain and heart homogenates. DHA is a physiological agent and, therefore, could prove to be a safe and effective antidote for CN, particularly in cases of fire smoke inhalation in which a combination of CN and carbon monoxide is present. In these cases the normally used antidote, sodium nitrite, to induce methemoglobin so as to trap the CN, is contraindicated because some of the oxygen-carrying capacity of the blood will have already been diminished by carbon monoxide.

In a search for improved cyanide antidotes, the efficacy of isosorbide dinitrate (ISDN), was compared with that of the known cyanide antidote, NaNO2. ISDN was as effective as an optimal dose of NaNO2 in protecting mice against cyanide lethality. To study the mechanism involved, the extent of formation of the cyanide scavenger, methemoglobin, in the action of ISDN was determined. ISDN (300 mg/kg, p.o.) increased methemoglobin from 5 to 10% of total hemoglobin, while, in contrast, NaNO2 (100 mg/kg, i.p.) increased methemoglobin levels to 50% of total hemoglobin. Lowering the dose of NaNO2 to 30 mg/kg reduced methemoglobin levels to approximately 10% of total hemoglobin and in turn nearly abolished its antidotal effect. Decreasing methemoglobin to less than control levels using methylene blue failed to abolish cyanide antagonism by ISDN. Thus, methemoglobin formation by ISDN does not account for its antidotal action. Further studies comparing the respiratory depressant effects of cyanide in the presence of ISDN or NaNO2 also indicated that these two antidotes have different mechanisms of action. Efforts to produce tolerance to the antidotal effect of ISDN against cyanide toxicity were unsuccessful. It is suggested that the well-known ability of ISDN to generate nitric oxide may account for the noted cyanide antagonism.

Murine carrier erythrocytes containing bovine rhodanese and sodium thiosulfate are being explored as a new approach to antagonize the lethal effects of potassium cyanide in mice. Prior studies indicated that these carrier erythrocytes persist in the vascular system for the same length of time as normal erythrocytes and can enhance metabolism of cyanide to thiocyanate. The present studies demonstrate the ability of these carrier red blood cells containing rhodanese and thiosulfate to antagonize the lethal effects of cyanide either alone or in various combinations with sodium nitrite and/or sodium thiosulfate. Potency ratios are compared in groups of mice treated with sodium nitrite, sodium thiosulfate, and carrier erythrocytes containing rhodanese and sodium thiosulfate either alone or in various combinations prior to the administration of potassium cyanide. These results indicate that the administration of carrier erythrocytes containing rhodanese and thiosulfate alone can provide significant protection against the lethal effects of cyanide. These carrier erythrocytes potentiate the antidotal effect of sodium thiosulfate alone or the combination of sodium nitrite and sodium thiosulfate. The mechanisms of cyanide antagonism by these carrier erythrocytes and their broader conceptual significance to the antagonism of other chemical toxicants are discussed.

Previous reports from our laboratory indicated that prophylactic protection against cyanide intoxication in mice can be enhanced by administration of chlorpromazine when it is given with sodium thiosulfate. The mechanism of potentiation of sodium thiosulfate by chlorpromazine was studied alone and in combination with sodium nitrite. Although chlorpromazine was found to induce a hypothermic response, the mechanism of enhancement of the antagonism of cyanide by chlorpromazine does not correlate with the hypothermia produced. Various other possible mechanisms were investigated, such as rate of methemoglobin formation, enzymatic activity of rhodanese and cytochrome oxidase, and alpha-adrenergic blockade. The alpha-adrenergic blocking properties of chlorpromazine may provide a basis for its antidotal effect, since this protective effect can be reversed with an alpha-agonist, methoxamine.

The effects of the administration of methemoglobin (MetHb) prepared in vitro were evaluated in Sprague-Dawley rats given increasing doses of potassium cyanide (KCN). Median lethal dose (LD50) studies were conducted by giving intraperitoneal injections of KCN (in 0.3- to 0.5-ml volumes), then 2 min later administering intravenous (iv) doses of 1000, 1500, or 2500 mg/kg of MetHb through the tail vein. Control rats received an equivalent volume of saline. The resulting LD50 values for KCN were 7.4 +/- 1.1, 11.7 +/- 1.1, 13.9 +/- 1.0, and 14.2 +/- 1.0 mg/kg (mean +/- SD) for the control (no MetHb) and 1000-, 1500-, and 2500-mg/kg dose groups, respectively. Additional groups of rats were given 1000, 1500, or 2500 mg/kg MetHb and submitted for necropsy. The gross finding of darkened kidneys was present in both dose groups, but became consistent and more prominent in the 2500-mg/kg dose group. Evidence of pathologic changes was not present in other organs. Single-dose pharmacokinetic studies were conducted using iv doses of 1600 and 2500 mg/kg MetHb. The elimination half-life was similar in both doses (62.6 min), but the volume of distribution (95.3 +/- 7.2 and 126.3 +/- 5.2 ml/kg, mean +/- SE) and clearance (1.1 +/- 0.1 and 1.5 +/- 0.1 ml/min/kg) were significantly different (P less than 0.05) for the 1600- and 2500-mg/kg dose groups, respectively. From these data we conclude that although MetHb is cleared from the vascular system rapidly, it may be an effective and nontoxic antidote for doses of cyanide up to twice that of the control LD50.