Hydrogen Sulfide--Mechanisms of Toxicity and Development of an Antidote.

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.

The effect of lipoic acid on cyanate toxicity in different structures of the rat brain.

Cyanate is formed mostly during nonenzymatic urea biodegradation. Its active form isocyanate reacts with protein -NH2 and -SH groups, which changes their structure and function. The present studies aimed to investigate the effect of cyanate on activity of the enzymes, which possess -SH groups in the active centers and are implicated in anaerobic cysteine transformation and cyanide detoxification, as well as on glutathione level and peroxidative processes in different brain structures of the rat: cortex, striatum, hippocampus, and substantia nigra. In addition, we examined whether a concomitant treatment with lipoate, a dithiol that may act as a target of S-carbamoylation, can prevent these changes. Cyanate-inhibited sulfurtransferase activities and lowered sulfide level, which was accompanied by a decrease in glutathione concentration and elevation of reactive oxygen species level in almost all rat brain structures. Lipoate administered in combination with cyanate was able to prevent the above-mentioned negative cyanate-induced changes in a majority of the examined brain structures. These observations can be promising for chronic renal failure patients since lipoate can play a double role in these patients contributing to efficient antioxidant defense and protection against cyanate and cyanide toxicity.

Modulating effect of aqueous extract of Telfairia occidentalis on induced cyanide toxicity in rats.

The effect of lyophilised aqueous extract of Telfairia occidentalis (TO) on induced cyanide toxicity in rats was investigated. Twenty 3-week old male wistar albino rats were randomly distributed into one control and three treatment groups of five rats each: control group (group1), group treated with 3mg/kg body wt of cyanide only (group2), group treated with 3mg/kg body wt. each of cyanide and extract (group3), and a group treated with 3mg/kg Body wt of extract only (group4) were used for the investigation. Cyanide toxicity reduced both food and water intake (p<0.05), while the food intake was improved in group3, this effect of the extract on food was not observed on water intake. Cyanide reduced average body weight of rats significantly (p<0.05). The reduction effect of cyanide on body weight was countered by Telfairia occidentalis extract. The extract did not have an observable effect on rats' body weight. Ocular lesion was observed in 67% of rats in group2 . This ocular effect of cyanide was mitigated significantly by Telfairia occidentalis as only 17% of the rats in group3 had ocular lesion. Cyanide toxicity produced nasal discharge in 39% of the rat population in group2 while there was a partial but considerable reduction (21%) in the severity of nasal discharge in group 3. There was no significant difference (p>0.05) in the organ/body wt.ratio between the treatments and the control groups for all the organs examined in the study. Biochemical analysis of liver enzymes showed that cyanide (group2) damaged the liver as there was significantly elevated presence (p<0.05) of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALP) above those of the control group. The damaging effect of cyanide on the liver was ameliorated by Telfairia occidentalis considerably.Histopathological effect of cyanide toxicity on the organs examined included multifocal degeneration and necrosis of the liver, mild kidney congestion and congestion of the brain. These effects were moderated mildly by Telfairia occidentalis. Group 4, treated with the vegetable alone had none of the observed histopathology in the organs examined. We concluded that lyophilised aqueous extracts of Telfairia occidentalis showed good potential as a safe antidote for cyanide poisoning when administered concomitantly or very shortly after ingestion of sub-lethal dose of cyanide. However, further bioassay guided fractionation and analytical studies are needed to identify the actual chemical compound or molecule in the vegetable responsible for or associated with the observed effects.

Nano-intercalated rhodanese in cyanide antagonism.

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.

Ginkgolides protect primary cortical neurons from potassium cyanide-induced hypoxic injury.

In this study, we investigated the effects of ginkgolides (Gins A, B, C and J), the main constituent of the non-flavone fraction of EGb 761, on hypoxic injury induced by potassium cyanide (KCN) in primary cortical neurons. The neurons were pretreated with or without ginkgolides for 24 h before incubation with KCN for 4 h. Cell viability was then determined by a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyletrazolium bromide] assay and lactate dehydrogenase (LDH) release from neurons into the medium was measured. The morphological changes of neurons were observed under inverse microscopy and electron microscopy. The results demonstrated that KCN (0.05 mmol/l) significantly decreased cell viability and increased LDH release (P < 0.05 versus the control). The characteristic changes of neuronal morphology induced by KCN were observed. However, pretreatment of neurons with 37.5 microg/ml of ginkgolides (ginkgolides + KCN group) led to a significant increase in cell viability, a decrease in LDH release (P < 0.05 versus the KCN group) and a remarkable improvement in cellular morphology in hypoxic neurons compared with the KCN group. The data suggested that ginkgolides have a significant role to protect the primary cortical neurons from hypoxic injury induced by KCN.

Chemical anoxia delays germ cell apoptosis in the human testis.

An understanding of testicular physiology and pathology requires knowledge of the regulation of cell death. Previous observation of suppression of apoptosis by hypoxia suggested a role for ATP in germ cell death. However, the exact effects of ATP production on germ cell death and of apoptosis on the levels of ATP and other adenine nucleotides (ANs) have remained unclear. We investigated the levels of ANs during human testicular apoptosis (analyzed by HPLC) and the role of chemical anoxia in germ cell death (detected by Southern blot analysis of DNA fragmentation, in situ end labeling of DNA, and electron microscopy). Incubation of seminiferous tubule segments under serum-free conditions induced apoptosis and concomitantly decreased the levels of ANs. Chemical anoxia, induced with potassium cyanide (KCN), an inhibitor of mitochondrial respiration, dropped ATP levels further and suppressed apoptosis at 4 h. After 24 h, many of the testicular cells underwent delayed apoptosis despite ATP depletion. Some cells showed signs of necrosis or toxicity. The addition of 2-deoxyglucose, an antimetabolite of glycolysis, did not alter the results obtained with KCN alone, whereas a toxic concentration of hydrogen peroxide switched apoptosis to necrosis. In most of the testicular cells, mitochondrial respiration appears to play a crucial role in controlling primary cell death cascades. In the human testis, there seem to be secondary apoptotic pathways that do not require functional respiration (or ATP).

Effects of melatonin on KCN-induced neurodegeneration in mice.

Melatonin is known to have a neuroprotective effect by preventing epileptic seizures, which are normally induced by cyanide. To demonstrate the neuroprotective function of melatonin, we examined cell death and changes in plasma melatonin level in KCN-treated mice. Neuronal cell death is shown in substantial nigra of KCN-treated groups. In melatonin-treated groups, this cell death decreased in substantia nigra. Plasma melatonin level at 12:00 was significantly decreased to 52.6% after KCN injection as compared to the normal group. In contrast, melatonin level was significantly decreased (74.5%) in KCN + melatonin group. Melatonin level at 24:00 was significantly decreased to 57.0% after KCN injection and also significantly decreased to 81.0% in KCN-melatonin group as compared to the normal group. Results from the present study suggest that melatonin prevents neuronal cell death in KCN-induced brain.

Melatonin attenuates brain mitochondria DNA damage induced by potassium cyanide in vivo and in vitro.

The effect of potassium cyanide on mitochondria DNA (mtDNA) in mouse brain was investigated in vivo and in vitro. When potassium cyanide (0, 0.1, 1.0 or 2.0 mM) was incubated with a crude mitochondria fraction prepared from mouse brain at 37 degrees C for 60 min, the damage of mtDNA was observed in a concentration-dependent manner. However, the mtDNA damage was prevented by a co-treatment with melatonin (1.5 mM), a scavenger of hydroxyl radicals (*OH). Furthermore, a subcutaneous injection of potassium cyanide (7mg/kg) caused both brain mtDNA damage and severe seizures in mouse. The damage of mtDNA and seizures induced by potassium cyanide were abolished by the pre-injection of melatonin (20 mg/kg). Hydrogen peroxide (1.5 mM) inflicted damage to brain mtDNA in the presence of Fe(2+) (3.0 microM). The damage was abolished by the co-treatment with melatonin. Furthermore, when cyanide (0, 0.1 or 1.0 mM) was incubated with the crude mitochondria fraction prepared from mouse brain, the lipid peroxidation was significantly increased in a concentration-dependent manner. The increased lipid peroxidation was completely inhibited by the co-treatment with melatonin (1.0 mM). These results suggest that reactive oxygen species including the *OH may play a cardinal role for mtDNA damage induced by potassium cyanide. Hence, the present study concluded that melatonin protects against DNA damage induced by the *OH produced by cyanide or hydrogen peroxide.

In vitro and in vivo attenuation of experimental cyanide poisoning by alpha-ketoglutarate.

Treatment of cyanide poisoning generally includes methemoglobin forming agents, like amyl nitrite and/or sodium nitrite (SN), in combination with sodium thiosulphate (STS). However, in many instances of cyanide poisoning, use of nitrites are contraindicated due to their strong vasoactive properties. alpha-Ketoglutarate (alpha-KG) antagonises cyanide by cyanohydrin formation and is considered a promising antidote for cyanide poisoning. In the present study, pre-treatment (30 min) and simultaneous treatment (0 min) of alpha-KG (5 mM) was found to confer significant protection against 5 mM potassium cyanide (KCN) induced cytotoxicity in rat thymocytes as measured by eosin Y exclusion and leakage of intracellular lactate dehydrogenase (LDH), but could not prevent the mitochondrial dysfunction (MTT assay), depletion of cellular GSH (reduced glutathione) and DNA damage. The post-treatment (5 or 30 min) of alpha-KG did not offer any protection on any of the above parameters. Results of in vitro studies were also supported by in vivo data. Pre-treatment of peroral (p.o.) alpha-KG (0.125-2.0 g/kg) exhibited dose and time dependent effects and was found to be effective even when given upto 60 min prior to KCN (p.o.). Addition of STS significantly enhanced the protective efficacy of alpha-KG at all the doses and time intervals. A 10 min pre-treatment of alpha-KG increased the LD(50) of KCN 7.6-fold, which was further increased to 25.6-fold by the addition of both SN and STS. Simultaneous treatment of alpha-KG (2.0 g/kg) increased the LD50 of KCN 5.4-fold which was increased to 18.1-fold by the addition of STS. However, addition of SN did not confer any additional protection. In the presence of SN+STS, a decrease in the dose of alpha-KG exhibited a dose-dependent decrease in protection, but still a >10-fold protection could be observed at 1.0 g/kg dose of alpha-KG. Considering the efficacy and safety of peroral alpha-KG, a promising treatment regimen consisting of alpha-KG+STS or alpha-KG+SN+STS is proposed, depending upon the individual situation.

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside.

A new synthetic drug, benzamide riboside (BR) exhibited strong oncolytic activity against leukemic cells in the 5-10 microM range. Higher BR-concentrations (20 microM) predominantly induced necrosis which correlated with DNA strand breaks and subsequent depletion of ATP- and dATP levels. Replenishment of the ATP pool by addition of adenosine prevented necrosis and favoured apoptosis. This effect was not a pecularity of BR-treatment, but was reproduced with high concentrations of all trans-retinoic acid (120 microM) and cyanide (20 mM). Glucose was also capable to suppress necrosis and to favour apoptosis of HL-60 cells, which had been treated with necrotic doses of BR and cyanide. Apoptosis eliminates unwanted cells without affecting the microenvironment, whereas necrosis causes severe inflammation of surrounding tissues due to spillage of cell fluids into the peri-cellular space. Thus, the monitoring and maintenance of cellular energy pools during therapeutic drug treatment may help to minimize nonspecific side effects and to improve attempted drug effects.

Antagonistic effect of melatonin against cyanide-induced seizures and acute lethality in mice.

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.

Antidotal effect of dihydroxyacetone against cyanide toxicity in vivo.

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.

Bcl-2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability.

The protooncogene bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that the mechanism of action of Bcl-2 involves antioxidant activity. The involvement of free radicals in ischemia/reperfusion injury to neural cells has led us to investigate the effect of Bcl-2 in a model of delayed neural cell death. We have examined the survival of control and bcl-2 transfectants of a hypothalamic tumor cell line, GT1-7, exposed to potassium cyanide in the absence of glucose (chemical hypoxia/aglycemia). After 30 min of treatment, no loss of viability was evident in control or bcl-2 transfectants; however, Bcl-2-expressing cells were protected from delayed cell death measured following 24-72 h of reoxygenation. Under these conditions, the rate and extent of ATP depletion in response to treatment with cyanide in the absence of glucose and the rate of recovery of ATP during reenergization were similar in control and Bcl-2-expressing cells. Bcl-2-expressing cells were protected from oxidative damage resulting from this treatment, as indicated by significantly lower levels of oxidized lipids. Mitochondrial respiration in control but not Bcl-2-expressing cells was compromised immediately following hypoxic treatment. These results indicate that Bcl-2 can protect neural cells from delayed death resulting from chemical hypoxia and reenergization, and may do so by an antioxidant mechanism. The results thereby provide evidence that Bcl-2 or a Bcl-2 mimetic has potential therapeutic application in the treatment of neuropathologies involving oxidative stress, including focal and global cerebral ischemia.

Antagonism of cyanide toxicity by isosorbide dinitrate: possible role of nitric oxide.

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.

Oxygen dependence of hepatocyte susceptibility to mitochondrial respiratory inhibitors.

Most zone 3 specific hepatotoxins or their metabolites are mitochondrial toxins, and yet the susceptibility of hepatocytes to respiratory inhibitors at the low O2 concentrations found in zone 3 is not known. Potassium cyanide (CN) and antimycin A (AA) were found to be 5- and 2-fold more cytotoxic at 1% than at 95% O2, respectively. CN also inhibited the respiration of hepatocytes 36% more at 1% O2 than at 95% O2; however, AA inhibited the respiration to the same level at 1% and 95% O2. CN but not AA depleted ATP levels of hepatocytes more extensively at 1% than at 95% O2. The CN-trapping agents dihydroxyacetone, glyceraldehyde, alpha-ketoglutarate and pyruvate prevented CN-induced cytotoxicity more effectively at 95% O2 than at 1% O2. In contrast, thiosulfate was less effective in preventing CN toxicity at 95% than at 1% O2. Hepatocyte thiocyanate formation from CN and thiosulfate was much faster at 1% than at 95% O2, suggesting that rhodanese, the mitochondrial enzyme that forms thiocyanate from CN and thiosulfate, is more effective at 1% O2 than at 95% O2.

Effect of atropine on cyanide-induced acute lethality in mice.

The effects of atropine on acute lethality induced by cyanide were investigated in mice. The LD50 value of cyanide (s.c. injection) was 8.4 (7.6-9.3) mg/kg. However, the LD50 value of cyanide (s.c.) was significantly increased by 1.5-fold when atropine (32 mg/kg) was injected s.c. in mice. Furthermore, the combined administration of atropine (32 mg/kg), Ca2+ (500 mg/kg) and sodium thiosulfate (1 g/kg) tremendously increased the LD50 value by 5.6-fold in mice although sodium thiosulfate or Ca2+ alone increased the LD50 2.5- or 1.5-fold. On the other hand, although the LD50 value of cyanide (intracerebroventricular injection (i.v.t.)) was 52.0 (47.4-57.0) micrograms/brain, the LD50 value of cyanide (i.v.t.) was significantly increased by 1.3- or 1.61-fold in mice 10 min after s.c. injection of atropine (32 mg/kg) or Ca2+ (500 mg/kg). Furthermore, the combined administration of atropine and Ca2+ increased the LD50 value of cyanide by 2.1-fold. These results suggest that atropine inhibits cyanide-induced acute lethality and promotes the antagonistic effect of thiosulfate and Ca2+ in mice.

Patient treated with antidote kit and hyperbaric oxygen survives cyanide poisoning.

A 54-year-old man deliberately drank a potassium-gold cyanide solution that contained approximately 1,650 mg of potassium cyanide. He survived after treatment with the Lilly antidote kit and hyperbaric oxygen.

Antagonism of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanese and sodium thiosulfate.

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.

Phenazoviridin, a novel free radical scavenger from Streptomyces sp. Taxonomy, fermentation, isolation, structure elucidation and biological properties.

Phenazoviridin is a newly discovered free radical scavenger from microorganisms. It was isolated from the culture of Streptomyces sp. HR04. The structure of phenazoviridin was determined as 6-(3-methyl-2-butenyl)phenazine-1-carboxylic acid 6-deoxy-alpha-L-talopyranose ester on the basis of its spectroscopic and physico-chemical properties. The novel substance showed strong inhibitory activity against lipid peroxidation in rat brain homogenate and exhibited antihypoxic activity in mice.

Attenuation of potassium cyanide-mediated neuronal cell death by adenosine.

Glutamate has been shown to play an important role in delayed neuronal cell death occurring due to ischemia. Attenuation of synaptically released glutamate can be accomplished by modulators such as adenosine and baclofen. This study focused on the ability of adenosine to attenuate the excitotoxicity secondary to glutamate receptor activation in vitro after exposure to potassium cyanide (KCN) in hippocampal neuronal cell cultures. For this study, hippocampal cell cultures were obtained from 1-day-old rats and trypan blue staining was used for assessment of cell viability. It was found that the N-methyl-D-aspartate-specific antagonist MK801 (10 microM) attenuated neuronal cell death resulting from exposure to 1 mM KCN for 60 minutes. Adenosine (10 to 1000 microM) decreased neuronal cell death secondary to the same concentration of KCN in a dose-dependent manner. This same neuroprotective effect is mimicked by the adenosine A1-specific receptor agonist N6-cyclopentyladenosine (10 microM). The A1-specific receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (10 to 1000 nM) blocked the neuroprotective effect of adenosine in a dose-dependent manner. Therefore, neuronal cell death produced by KCN in the experimental model described was mediated at least in part by glutamate. This neuronal cell death was attenuated by adenosine via the A1-specific mechanism.