The effect of sodium tetrathionate on cyanide conversion to thiocyanate by enzymatic and non-enzymatic mechanisms

Sodium tetrathionate has been proposed as a cyanide antidote despite its reported toxicology and inhibitory effect on rhodanese. We investigated the effect of tetrathionate and an analog, dithionite, on rhodanese activity because of their structural similarity to thiosulfate, a known sulfane sulfur donor for this enzyme. Rhodanese activity of guinea pig liver homogenate was assayed by measuring the formation of ferric thiocyanate complex at 460 nm. With thiosulfate as a substrate, the Km for rhodanese was 6.7 mM and the Vmax was 0.67 mumol thiocyanate min-1 mg-1 protein. The conversion of cyanide to thiocyanate by rhodanese was inhibited in the presence of tetrathionate at the millimolar concentration (e.g. 1 mM) range. Dithionite had a negligible effect on rhodanese activity. Neither thiosulfate (1-100 mM) nor dithionite produced significant amounts of thiocyanate from cyanide spontaneously (i.e. non-enzymatically). However, in the absence of rhodanese, tetrathionate (1.0, 10.0 and 100.0 mM) produced a dose-dependent increase in thiocyanate. These data suggest that tetrathionate detoxifies cyanide non-enzymatically, which may, in part, account for its antidotal effects.     

Cyanide intoxication--I. An oral chronic animal model

The effects of oral chronic cyanide administration to mice were studied. Cyanide intoxication was confirmed by the increased levels of this poison and the concomitant inhibition of cytochrome oxidase activity in liver, brain, heart and blood. The detoxifying enzyme rhodanese was measured. The state of the sulfane sulfur pool was investigated by determination of the cyanide labile-sulfur levels. A clear correlation between rhodanese activity and sulfur content was obtained as a consequence of cyanide action. These results support the belief that rhodanese plays a fundamental role in the detoxification process of cyanide, in preventing cyanide reaching the target tissues.     

The cyanide-metabolizing enzyme rhodanese in human nasal respiratory mucosa

The cyanide-metabolizing enzyme rhodanese is present in rat nasal epithelium at high activity levels. Cyanide is a common environmental pollutant. It is both toxic and an odorant. The high rhodanese activity in rat nasal epithelium may provide a mechanism for detoxicating inhaled hydrogen cyanide and may also play a role in olfaction by limiting the concentrations of cyanide in the nasal epithelium. The objective of this study was to determine whether high levels of rhodanese activity are also present in human nasal epithelium. On a per milligram mitochondrial protein basis, the rhodanese in human nasal tissue exhibited both a lower affinity (higher Km) for cyanide and a lower maximum velocity (Vmax) for cyanide metabolism than did rhodanese from rat nasal tissue. As in human liver, the human nasal enzyme appeared to exhibit substrate activation by cyanide. Rhodanese activity in the maxilloturbinates of nonsmokers was statistically higher than in smokers although only three samples per group were available. The Vmax/Km ratios for rhodanese from the nasal tissue of nonsmokers were consistently greater, thus suggesting the possibility of higher rates of cyanide metabolism in nonsmokers than in smokers.     

Effects of oxygen on the antagonism of cyanide intoxication: cytochrome oxidase, in vitro

Since oxygen was reported to be an effective cyanide antagonist in vivo, particularly in the presence of the classic antidotal combination of sodium nitrite and sodium thiosulfate, in vitro studies were initiated in an attempt to investigate the mechanism of oxygen-mediated cyanide antagonism. The effect of oxygen on cyanide-inhibited cytochrome oxidase with and without cyanide antagonist(s) was investigated in a purified membraneous enzyme system prepared from rat liver mitochondria. Cyanide produced a concentration dependent inhibition of cytochrome oxidase, and 100% oxygen did not alter the inhibition produced by KCN either in the presence or absence of sodium thiosulfate. However, the addition of sodium thiosulfate and rhodanese to the assay reactivated the cyanide-inhibited cytochrome oxidase. Kinetic analysis indicated rhodanese competes with cytochrome oxidase for cyanide, and oxygen had no effect on this coupled reaction. In conclusion, the in vivo antidotal properties of oxygen cannot be attributed to oxygen-mediated reactivation of cyanide-inhibited cytochrome oxidase or an oxygen-mediated acceleration of rhodanese detoxification.     

In vivo studies on rhodanese encapsulation in mouse carrier erythrocytes

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.     

Enzyme therapy in cyanide poisoning: Effect of rhodanese and sulfur compounds

Crystalline bovine liver rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) was evaluated as an antidote in combination with different sulfur compounds against cyanide poisoning in mice. The prophylactic antidote effect, when the antidote was injected i.v. 1 min prior to i.p. injection of cyanide, was dependent on both the dose of the enzyme and the dose of the sulfur compound. An optimal dose of the enzyme of about 2,000 U/kg (3 mg/kg of pure enzyme) was found. This enzyme dose combined with 2 mmol/kg of sodium thiosulfate raised the LD₅₀ for potassium cyanide 7.6 times. When thiosulfate was replaced with equimolar doses of ethanethiosulfonate and propanethiosulfonate, the corresponding values were 10.3 and 9.3 times, respectively. Maximum antidote effect was obtained when the doses of ethanethiosulfonate and propanethiosulfonate were raised to 4 mmol/kg, increasing the LD₅₀ for cyanide 20.8 and 15.4 times, respectively. On the other hand, when given without rhodanese, ethanethiosulfonate and propanethiosulfonate were no better antidotes than thiosulfate.Rhodanese and a sulfur compound given therapeutically to mice when symptoms of cyanide poisoning had occurred, also had a very good antidote effect. The prophylactic antidote effect of rhodanese plus thiosulfate rapidly decreased with increasing time interval between injection of the antidote and cyanide. Thus, when rhodanese and thiosulfate were given 20 min prior to cyanide, the antidote effect was of the same order as that of thiosulfate alone. The antidote effect of the latter did not decrease significantly within the same time interval.Enzyme activity in plasma decreased rapidly after i.v. injection of rhodanese, and enzyme activity in urine was detected following injection. No appreciable inactivation occurred when the enzyme was incubated with whole blood in vitro, but a strong and rapid inhibition, about 85%, of the enzyme occurred in fresh mouse urine in vitro.     

The effect of picrylsulphonic acid on In vitro conversion of cyanide to thiocyanate by 3-mercaptopyruvate sulphurtransferase and rhodanese

This study indicates that 3-mercaptopyruvate sulphurtransferase (MPST; EC 2.8.1.2) activity may serve as a useful in vitro indicator for the analysis of cyanide detoxification to thiocyanate. The time course and capacity of MPST to detoxify cyanide was equal to or exceeded that of rhodanese. Picrylsulphonic acid strongly inhibited purified rhodanese, but in the presence of mercaptopyruvate, it could increase the formation of thiocyanate catalysed by MPST. Formation of thiocyanate by MPST followed a linear time course and had a linear relation to enzyme level. However, substrate dependence did not produce linear Lineweaver-Burk plots when either mercaptopyruvate or cyanide concentration was varied. The differential effect of picrylsulphonic acid on the activities of these two enzymes was confirmed by using a crude kidney extract as the source of both enzymes. Picrylsulphonic acid may provide a useful scientific tool to examine which sulphurtransferase is most responsible for the detoxification of cyanide.     

Histochemical localization of rhodanese activity in rat liver and skeletal muscle

A previously described histochemical technique was applied to the localization of rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) activity in rat skeletal muscle and liver. The physiological function of rhodanese is controversial, but it and other sulfurtransferases can catalyze the conversion of cyanide to the much less toxic thiocyanate. The volume of distribution of cyanide in human and dog is said to correspond roughly to the blood volume. Because of this and other observations, it was hypothesized that sulfurtransferase activity associated with the vascular endothelium on smooth muscle layers of blood vessels might play a role in cyanide detoxification. However, little enzyme activity as identified histochemically was associated with those sites in comparison with others examined. As expected, high activity was found in the liver and moderately high levels were present in skeletal muscle. In muscles sectioned longitudinally, points of rhodanese staining occurred in linear arrays along the lengths of the muscle fiber corresponding to the location of mitochondria within the fiber. The original technique called for incubation of tissue sections with both thiosulfate and cyanide. When thiosulfate was omitted, staining for rhodanese activity was still clearly identifiable in both liver and muscle sections with cyanide alone. In muscle sections the inclusion of both thiosulfate and cyanide resulted in a preferential staining of type I fibers presumably because of their higher content of mitochondria. Thus, this technique is a potential alternative to the NADH dehydrogenase stain for distinguishing between type I and type II muscle fibers. Incubation of tissue sections with only thiosulfate produced results that did not appear to differ from those obtained when both substrates were omitted. From these observations it may be inferred that the endogenous pool of sulfane-sulfur available to sulfurtransferases is larger than any alleged endogenous pool of cyanide. Although sulfurtransferase activity in muscle appeared to be lower than that in liver, the total body muscle mass is greater than the liver mass. Thus, these results support other evidence that skeletal muscle may make a significant contribution to total cyanide biotransformation in the absence of exogenously added thiosulfate.     

In vitro effect of cyanide, thiosulphate and S-adenosyl-L-methionine on the activity of rhodanese and other enzymes.

1. Some in vitro studies were performed to elucidate the action of S-adenosyl-L-methionine (SAM) and thiosulphate on liver rhodanese, delta-amino-levulinic acid dehydratase (Al A-D) and cytochrome oxidase affected by cyanide in the experimental conditions. 2. SAM was unable to interact with the sulfur substituted rhodanese complex suggesting that SAM would blockade the thiosulphate binding sites on rhodanese. 3. Cyanide and thiosulphate inhibited ALA-D activity when both compounds were present in the incubation or the preincubation mixture. Cyanide binding on the enzyme was irreversible. 4. Cyanide inhibited cytochrome oxidase activity and the reversible nature of the binding was demonstrated by gel filtration. 5. SAM had no effect on either ALA-D or cytochrome oxidase activities.     

Nano-intercalated rhodanese in cyanide antagonism

Abstract

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.     

Effect of chlorpromazine on cyanide intoxication

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 effect of cyanide intoxication on hepatic rhodanese kinetics

1. Results of studies on the kinetics of hepatic rhodanese and the effects of S-adenosyl-L-methionine (SAM) on these kinetic parameters in cyanide-treated and non-treated mice are reported here. 2. The enzyme exhibited typical Michaelis-Menten behaviour with cyanide inhibition at concentrations higher than 50 mM. Km values of 4.74 and 0.85 mM were obtained for thiosulphate and cyanide, respectively, in control mice. 3. These results stress the biological importance of the rhodanese reaction for cyanide detoxification. 4. Km values were not significantly modified when the animals were intoxicated with a lethal (20 mg/kg) or a non-lethal (4 mg/kg) dose of cyanide. 5. SAM treatment either in control or in cyanide-poisoned animals doubled the Km's for cyanide.     

The cyanide-metabolizing enzyme rhodanese in rat nasal respiratory and olfactory mucosa

Hydrogen cyanide is a commonly occurring and highly toxic air pollutant. Inhalation of hydrogen cyanide would expose the nasal tissues to its toxic affects unless a detoxicating mechanism were available. Experiments with rat nasal tissues showed that the cyanide-metabolizing enzyme, rhodanese, is present in high concentrations, particularly in the olfactory region. The olfactory tissues had nearly 7-fold more rhodanese on a per mg mitochondrial protein basis than did the liver. These experiments show that nasal metabolism of cyanide may have an important influence on the toxicity of inhaled cyanide and cyanogenic materials.     

Liver and kidney lesions and associated enzyme changes induced in rabbits by chronic cyanide exposure.

The effect of prolonged chronic cyanide exposure on liver and kidney integrity, as well as some associated enzyme and metabolite changes, were investigated in New Zealand white rabbits (initial mean weight 1.52 kg) using a combination of colorimetric, spectrophotometric, enzymatic, gravimetric and histological procedures. Two groups of rabbits were fed for 40 weeks on either pure growers' mash or growers' mash containing 702 ppm inorganic cyanide. Results obtained indicate that the cyanide-fed rabbits had significantly decreased liver activities of alkaline phosphatase, glutamate pyruvate transaminase and sorbitol dehydrogenase relative to controls (P<0.05). On the other hand, there were significant increases (P<0.05) in the serum activities of these enzymes in the cyanide-treated group. Kidney alkaline phosphatase activity was significantly decreased (P<0.05), while serum urea and creatinine were significantly higher (P<0.05) in the cyanide group relative to controls. The cyanide treatment led to significant increases in both tissue and serum activities of lactate dehydrogenase. In addition, liver and kidney rhodanese activities were significantly raised in the cyanide-fed group. There were marked degenerative changes in the liver and kidney sections from the cyanide-treated rabbits. These results suggest that chronic cyanide exposure may be deleterious to liver and kidney functions.     

Encapsulation of thiosulfate: cyanide sulfurtransferase by mouse erythrocytes

Murine carrier erythrocytes, prepared by hypotonic dialysis, were employed in the encapsulation of several compounds including [14C]sucrose, [3H]inulin, and bovine thiosulfate:cyanide sulfurtransferase (rhodanese), a mitochondrial enzyme which converts cyanide to thiocyanate. Approximately 30% of the added [14C]sucrose, [3H]inulin, and rhodanese was encapsulated by predialyzed erythrocytes, and a decrease in the mean corpuscular volume and mean corpuscular hemoglobin was observed. In the encapsulation of rhodanese a recovery of 95% of the erythrocytes was achieved and an 85% equilibrium was established. The addition of potassium cyanide (50 mM) to intact, rhodanese-loaded erythrocytes containing sodium thiosulfate resulted in its metabolism to thiocyanate. These results establish the potential use of erythrocytes as biodegradable drug carrier in drug antagonism.     

Effects of thiosulfate on cyanide pharmacokinetics in dogs

One method to treat cyanide poisoning involves the administration of a combination of sodium thiosulfate and sodium nitrite. Sodium thiosulfate is believed to exert its antidotal effect by serving as a sulfur donor, thereby increasing the rate of rhodanese catalyzed biotransformation of cyanide to thiocyanate. To gain insight into the mechanism of action of thiosulfate on cyanide toxicity, a pharmacokinetic analysis of cyanide distribution and metabolism with and without sodium thiosulfate was conducted in mongrel dogs. A compartmental model for thiocyanate, the major metabolite of cyanide, was developed from plasma concentrations determined at various times after iv administration of thiocyanate; sodium thiosulfate did not alter thiocyanate-model parameters. The model for thiocyanate was coupled to a model for cyanide, and model based equations were fitted to the blood levels of both cyanide and thiocyanate that were measured after iv administration of cyanide. This kinetic analysis showed that thiosulfate increased the rate of conversion of cyanide to thiocyanate over 30-fold. The mechanism of thiosulfate protection appeared to be due to extremely rapid formation of thiocyanate in the central compartment, which thereby limited the amount of cyanide distribution to sites of toxicity.     

Antagonism of cyanide poisoning by chlorpromazine and sodium thiosulfate

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.     

Liver damage does not increase the sensitivity of mice to cyanide given acutely

The major detoxification pathway for cyanide (CN) in many species is a biotransformation to the less toxic thiocyanate (SCN). Hepatic thiosulfate: cyanide sulfurtransferase (rhodanese) is the principal enzyme demonstrating in vitro catalytic activity. Despite the assumed importance of the hepatic enzyme for CN detoxification in vivo, the effects of liver damage (surgical or chemical) on cyanide lethality in animals have not been examined previously. Male CD-1 mice were pretreated with carbon tetrachloride (CCl4, 1 ml/kg, i.p.) 24 h prior to the administration of sodium cyanide (NaCN). In other experiments CCl4 was given in the same doses at both 48 h and 24 h prior to NaCN. Hepatotoxicity was documented by elevated serum glutamicpyruvic transaminase (SGPT) activity, by histologic evaluation of the extent of cellular necrosis, by electron microscopy of the mitochondrial fraction, and by the increased duration of zoxazolamine-induced paralysis. Lethality was not changed by CCl4 pretreatments when NaCN was given alone in doses of 4 or 6 mg/kg or at a dose of 10.7 mg/kg following sodium thiosulfate (Na2S203, 1 g/kg, i.p.). A small but statistically significant protective effect was exhibited by CCl4 when NaCN was given at a dose of 16 mg/kg following the administration of Na2S203. Rhodanese activity as measured in mitochondrial preparations fractionated from the livers of mice pretreated with CCl4 was not different from that in animals given the corn oil vehicle even though electron micrographs showed extensive mitochondrial damage. No difference in CN lethality was evident between sham-operated mice and partially (2/3) hepatectomized mice at 24 h post-surgery. An intact healthy liver does not appear to be essential for cyanide detoxification in mice whether or not thiosulfate is also given. Because rhodanese activity was slightly but significantly higher in mitochondria lysed by Triton X-100 than in intact mitochondria, the mitochondrial membrane may constitute a barrier to Na2S203.     

Cyanide Antagonism with Carrier Erythrocytes and Organic Thiosulfonates

Previous studies reported that resealed erythrocytes containingrhodanese (CRBC) and Na₂S₂O₃ rapidly metabolize cyanide to theless toxic thiocyanate both in vitro and in vivo. This provideda new conceptual approach to prevent and treat cyanide intoxication.Although the rhodanese-containing carrier cells with thiosulfateas the sulfur donor were efficacious, this approach has potentialdisadvantages, as thiosulfate has limited penetration of cellmembrane and product inhibition of rhodanese can occur due toinorganic sulfite accumulation. In order to circumvent substratelimitation and product inhibition by sodium thiosulfate, organicthiosulfonates were explored. These thiosulfonates have higherlipid solubility than thiosulfate and therefore can replenishthe depleted sulfur donor, as they can readily penetrate cellmembranes. Also, product inhibition of rhodanese is less aptto occur. This change in sulfur donors should greatly enhancecyanide detoxication, replenish the sulfur donor, and minimizeproduct inhibition of rhodanese. Present studies demonstratethe enhanced efficacy of exogenous organic thiosulfonates oversodium thiosulfate in the CRBC antidotal system to detoxifythe lethal effects of cyanide either alone or in combinationswith exogenously administered NaNO₂. Murine carrier erythrocytescontaining purified bovine liver rhodanese were administeredintravenously into male Balb/C mice. Subsequently, butanethiosulfonate(BTS) or Na₂S₂O₃ (ip), and NaNO₂ (sc) were co-administered priorto KCN (sc). Potency ratios, derived from the LD₅₀ values, werecompared in groups of mice treated with CRBC-Na₂S₂O₃ or CRBC-BTSeither alone or in combination with NaNO₂. The CRBC-BTS antidotalsystem shows strikingly enhanced protective effect over thatof the CRBC-thiosulfate system either alone or in combinationwith sodium nitrate.