Prenatal toxicity of cyanide in goats--a model for teratological studies in ruminants

Although exposure to cyanogenic plants or cyanide during pregnancy has adverse effects, no teratological study with cyanide has been conducted in goats or any other ruminant. The objective of the present study was to evaluate the effects of the maternal exposure to potassium cyanide (KCN) during pregnancy on both dams and offspring and furthermore, to develop a model for prenatal toxicological studies in ruminants. Twenty-six pregnant goats were allocated into four groups and given 0, 1.0, 2.0, or 3.0mg KCN/kg body weight per day orally (administered via twice-daily gavage) from Day 24 of pregnancy to term. However, one control dam and another from the 3.0mg KCN/kg per day group were sacrificed on Day 120. At birth, the kids were examined carefully for gross abnormalities. Three months after birth, the male kids and one dam from each group were sacrificed for histopathological study. Although clinical signs of poisoning were observed in dams, cyanide treatment did not alter the length of gestation or the number of live kids. Two prognata kids were born in the 3.0mg KCN/kg group, and one dam from the same group aborted two fetuses. There were histological lesions only in the KCN-treated dam (and its fetuses) sacrificed on Day 120; these consisted of an increased number of resorption vacuoles of thyroid follicular colloid, and status spongiosis of nervous white matter. This study proposes a new animal model for teratogenic trials that could be important to evaluate the effects of chemicals throughout pregnancy in goats and potentially other ruminants.     

Organ-distribution of the metabolite 2-aminothiazoline-4-carboxylic acid in a rat model following cyanide exposure

The reaction of cyanide (CN^?) with cystine to produce 2-aminothiazoline-4-carboxylic acid (ATCA) is one of the independent detoxification pathways of cyanide in biological systems. In this report, in vivo production of ATCA and its distributions in plasma and organs were studied after a subcutaneous sublethal dose of 4?mg/kg body weight potassium cyanide (KCN) administration to rats. At this sublethal dose of KCN, ATCA concentration was not significantly increased in the plasma samples, however, it was found significantly increased in liver samples. These results suggested that ATCA might not be a good diagnostic biomarker in plasma for sublethal cyanide exposure; however, liver could serve as the right organ for the detection of ATCA in post-mortem examinations involving cyanide exposure in military, firefighting, industrial and forensic settings.     

Organ-distribution of the metabolite 2-aminothiazoline-4-carboxylic acid in a rat model following cyanide exposure

The reaction of cyanide (CN(-)) with cystine to produce 2-aminothiazoline-4-carboxylic acid (ATCA) is one of the independent detoxification pathways of cyanide in biological systems. In this report, in vivo production of ATCA and its distributions in plasma and organs were studied after a subcutaneous sublethal dose of 4?mg/kg body weight potassium cyanide (KCN) administration to rats. At this sublethal dose of KCN, ATCA concentration was not significantly increased in the plasma samples, however, it was found significantly increased in liver samples. These results suggested that ATCA might not be a good diagnostic biomarker in plasma for sublethal cyanide exposure; however, liver could serve as the right organ for the detection of ATCA in post-mortem examinations involving cyanide exposure in military, firefighting, industrial and forensic settings.     

Biodegradation of cyanide compounds by a Pseudomonas species (S1).

A Pseudomonas sp. (S1), isolated from soil by an enrichment technique was tested for its potential to degrade different cyanide compounds. Further, biodegradation/biotransformation of binary mixtures of the cyanide compounds by the culture was also studied. The results indicated that the culture could grow on the following nitriles by using them as carbon and nitrogen sources: acetonitrile, butyronitrile, acrylonitrile, adiponitrile, benzonitrile, glutaronitrile, phenylacetonitrile, and succinonitrile. Studies on the biodegradation of these cyanide compounds in binary mixtures showed that the presence of acrylonitrile or KCN delayed the degradation of acetonitrile in a mixture, while none of the other cyanide compounds affected the degradation of one another. The transformation products of the nitriles were their corresponding acids, and similarly, KCN was also directly transformed to formic acid. Studies on the transformation of these cyanide compounds showed that the rate of transformation of nitriles to their corresponding carboxylic acids was acrylonitrile > acetonitrile > adiponitrile > benzonitrile > KCN. This culture has the unique characteristic of transforming representatives of saturated aliphatic, aliphatic olefinic, aromatic, and aralkyl nitriles, as well as alkali cyanide, to their corresponding carboxylic acids.     

Carbamylation of hemoglobin in vivo with chronic sublethal dietary cyanide: implications for hemoglobin S

Carbamylation of the hemoglobin in sickle cell anemia has been demonstrated to improve the status of this hemoglobinopathy. Using an animal model of West African human patterns of chronic sublethal dietary cyanide ingestion, 12 miniature swine consuming either 0, 0.4, 0.7, or 1.2 mg of cyanide/kg body weight/day were studied for 24 weeks to determine if this dietary regime could produce irreversible carbamylated hemoglobin. Throughout the study, the hematological status of all animals remained similar; however, the levels of carbamylated hemoglobin as measured by nanomoles of valine hydantoin varied proportionally to dietary sublethal cyanide intakes, indicating that these natural dietary levels could effect an important and presumably permanent modification of the hemoglobin's beta chain. Serum thiocyanate levels were also positively correlated with cyanide ingestion loads (r = 0.83, P less than 0.01). The implications of these findings in swine are important for the millions of humans with hemoglobin S who regularly consume similar levels of dietary cyanide and for our assessments of the biochemical and medical status of hemoglobin S under natural conditions.     

Biodegradation of cyanide by Rhodococcus UKMP-5M

A new bacterial strain, Rhodococcus UKMP-5M isolated from petroleum-contaminated soils demonstrated promising potential to biodegrade cyanide to non-toxic end-products. Ammonia and formate were found as final products during growth of the isolate with KCN as the sole nitrogen source. Formamide was not detected as one of the end-products suggesting that the biodegradation of cyanide by Rhodococcus UKMP-5M may have proceeded via a hydrolytic pathway involving the bacterial enzyme cyanidase. No growth of the bacterium was observed when KCN was supplied as the sole source of carbon and nitrogen even though marginal reduction in the concentration of cyanide was recorded, indicating the toxic effect of cyanide even in cyanide-degrading microorganisms. The cyanide biodegradation ability of Rhodococcus UKMP-5M was greatly affected by the presence of organic nutrients in the medium. Medium containing glucose and yeast extract promoted the highest growth rate of the bacterium which simultaneously assisted complete biodegradation of 0.1 mM KCN within 24 hours of incubation. It was found that growth and cyanide biodegradation occurred optimally at 30°C and pH 6.3 with glucose as the preferred carbon source. Acetonitrile was used as an inducer to enhance cyanide biodegradation since the enzymes nitrile hydratase and/or nitrilase have similarity at both the amino acid and structural levels to that of cyanidase. The findings from this study should be of great interest from an environmental and health point of views since the optimum conditions discovered in the present study bear a close resemblance to the actual scenario of cyanide wastewater treatment facilities.     

The respiratory responses to cyanide of a cyanide-resistant Klebsiella oxytoca bacterial strain

The respiratory system of a cyanide-resistant Klebsiella oxytoca was analyzed by monitoring the changes in the cytochrome contents in response to various inhibitors in the presence of various concentrations of cyanide. The cells grown in the medium without cyanide (KCN) have two terminal oxidases, cytochrome d (Ki = 10(-5) M KCN) and o (Ki = 10(-3) M KCN). When cells were grown on medium with 1 mM KCN, the expression of both b-type cytochrome and cytochrome d in the plasma membranes of the cell decreased by more than 50%, while cytochrome o increased by 70%, as compared with the cells grown in the absence of KCN. Two terminal oxidases with Ki values of about 10(-3) M and 1.7 x 10(-2) M KCN were observed in the plasma membrane fractions of the cells growing on KCN enriched medium. 2-n-Heptyl-4-hydroxyquinoline-N-oxide markedly inhibited the oxidation of NADH by the plasma membranes from the cells grown in the medium without KCN, but not in those plasma membranes from KCN-grown cells. The NADH oxidases in plasma membranes of K. oxytoca grown with and without KCN were equally sensitive to UV irradiation. Adding freshly isolated quinone to the UV-damaged plasma membranes restored the NADH oxidase activity from both types of plasma membranes. From these results, we propose the presence of a non-heme type of terminal oxidase to account for the KCN resistance in K. oxytoca.     

Reversibility of the cyanide inhibition of electron transport in spinach chloroplast thylakoids

The prior treatment of thylakoids with cyanide (30 mM) was shown to inhibit plastocyanin-dependent electron transport reactions. We find that cyanide inhibition of electron flow from either water or diaminodurene to methyl viologen, but not from water to ferricyanide, is partially reversed when the thylakoids are collected by centrifugation and resuspended in a cyanide-free medium. However, methyl viologen reduction in thylakoids pretreated with cyanide is sensitive to cyanide (～1 mM) added to the reaction mixtures, whereas that in control thylakoids is unaffected. The cyanide must be added in the dark. Electron transport to methyl viologen in chloroplasts pretreated with cyanide is also sensitive to inhibition by EDTA and bathocuproine sulfonate. Thus, KCN inhibition of electron transport in thylakoids is partially reversible. Moreover, the accessibility of plastocyanin to various reagents is probably altered by the KCN treatment.     

Effect of alpha-ketoglutarate on neurobehavioral, neurochemical and oxidative changes caused by sub-chronic cyanide poisoning in rats

Recent studies revealed that alpha-ketoglutarate (A-KG) alone or with sodium thiosulfate (STS) provide significant protection against acute and sub-acute cyanide poisoning in rodents. This study addresses the protective effect of A-KG and/or STS in sub-chronic (90 days) cyanide poisoning. Wistar rats were divided into seven groups (n = 10): Control animals, potassium cyanide (KCN) A-KG, STS, KCN + A-KG, KCN + STS and KCN + A-KG + STS. Spontaneous motor activity and motor coordination were recorded every 15th day. Lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) in blood, brain, liver and kidney, and glutamate, aspartate and dopamine in discrete regions of brain were measured following 90 days exposure. Cyanide significantly decreased motor coordination, accompanied by increase in LPO (blood, brain and liver) and dopamine (corpus striatum and cerebral cortex) levels, and depletion in GSH (blood, brain and liver), GPx (brain and liver), SOD (brain and liver), and CAT (blood and brain) levels. Although treatment of A-KG and STS alone significantly blunted the toxicity of KCN, concomitant use of both afforded the maximum protection. This study shows a promising role of A-KG and STS as treatment regime for long term cyanide exposure.     

Reevaluation of the cyanide resistance of seed germination

Although high levels of KCN (53 micromoles per gram fresh weight of seed, corresponding to 3.2 millimolar) failed to block germination of lettuce seeds incubated in covered Petri dishes, the same levels totally blocked germination in sealed dishes. Inhibition was reversed by removing the seal. Placement of KCN remote from seeds also blocked germination in closed systems. Cyanide effectiveness was enhanced by acidifying the KCN solution but negated by the presence of a trap containing strong alkali. Low levels of aqueous HCN (2.6 micromoles HCN per gram, corresponding to 0.16 millimolar) injected into sealed dishes gave maximal inhibition of germination, suggesting that the effectiveness of KCN was due to formation of HCN in KCN solutions. Studies with nine additional crop species generally supported the interpretation that cyanide inhibition of germination has been underestimated in the past due to escape of volatile HCN from open systems.     

The cytochemical demonstration of cyanide resistant peroxidase does not identify eosinophils selectively

Potassium cyanide (KCN) resistant peroxidase is generally accepted by hematologists as a selective stain for the eosinophilic cell line. However, it has been demonstrated biochemically that not only the peroxidases of neutrophils but also those of eosinophils can be inhibited by KCN. Therefore, bone marrow smears of hematologically normal patients were subjected to the peroxidase reaction in the presence of varying concentrations of KCN. It was found that with increasing concentrations of KCN not only neutrophils but also eosinophils were inhibited. Moreover, there were always neutrophilic promyelocytes that were still positive when a considerable number of the eosinophils was already inhibited. Therefore, it can be concluded from our results as well as from biochemical data that there is no concentration of KCN which demonstrates the total of the eosinophilic cell line selectively. The implications of these findings are discussed.     

Molecular interaction of acrylonitrile and potassium cyanide with rat blood

The interaction of acrylonitrile (VCN) with rat blood has been investigated at the molecular level in an attempt to understand the possible mechanism of its toxicity. The results obtained were compared to those with potassium cyanide (KCN), a compound known to liberate cyanide (CN^?) in biologic conditions. The radioactivity derived from K¹⁴CN was eliminated faster than that from [1-¹⁴C]VCN. Up to a maximum of 94% of ¹⁴C from VCN in erythrocytes was detected covalently bound to cytoplasmic and membrane proteins, whereas 90% of the radioactivity from KCN in erythrocytes was found in the heme fraction of hemoglobin. Determination of specific activity showed that binding occurred more in vivo than in vitro which indicated that the VCN molecule was bioactivated inside erythrocytes. These results indicate that KCN interacts mainly through CN^? liberation and binding to heme, whereas VCN, which binds to cytoplasmic and membrane proteins, may cause damage to red cells by mechanisms other than release of CN^?.     

Semi‐quantitative tests of cyanide in foods and excreta of Three Hapalemur species in Madagascar

Three sympatric Hapalemur species (H. g. griseus, H. aureus, and H. (Prolemur) simus) in Ranomafana National Park, Madagascar are known to eat bamboo food parts that contain cyanide. How these lemurs avoid cyanide poisoning remains unknown. In this study, we tested for the presence/absence of cyanide in bamboo lemur foods and excreta to (1) document patterns of cyanide consumption among species with respect to diet, (2) identify routes of elimination of cyanide from the gastrointestinal tract, and (3) determine whether cyanide is absorbed from the diet. We tested 102 food, urine, and fecal samples for hydrogen cyanide (HCN) during two “pre‐dry” seasons (April 2006, May 2007) using commercially available Cyantesmo test strips. The test strips changed color in the presence of HCN, and we recorded color change on a scale of 0 (no change) to 5 (cobalt) at preset intervals with a final score taken at 24 hr. We detected cyanide in bamboo food parts and urine of all three Hapalemur species. Time to color change of the test strips ranged from almost instantaneous to >12 hr incubation. Of the foods tested, only bamboo contained cyanide, but results differed among bamboo species and plant parts of the same species. Specifically, branch shoot and culm pith of the giant bamboo produced strong, immediate reactions to the test paper, whereas parts of liana bamboos produced either weak or no color change. Cyanide was present in almost all urine samples but rarely in fecal samples. This suggests that dietary cyanide is absorbed in the gastrointestinal tract of the Hapalemur species and excreted, at least in part, by the kidneys. Samples from H. griseus exhibited lower, though still detectable, cyanide levels compared with H. simus and H. aureus. Differences among lemur species appear to be related to the specific bamboo parts consumed. Am. J. Primatol. 72:56–61, 2010. © 2009 Wiley‐Liss, Inc.     

Reaction of cyanide with glutathione peroxidase

An oxidized form of ovine erythrocyte GSH peroxidase (Form C) that contains bound glutathione in equimolar ratio to the enzyme selenium is inactivated by cyanide. When Form C was treated with 1 or 10 mM KCN at pH 7.5, there was a rapid increase in ultraviolet absorption at 250 nm, S-cyanoglutathione was released, and the enzyme was reduced, as shown by inactivation with iodoacetate (1 mM, pH 7.5) and uptake of label from [¹⁴C]iodoacetate in equimolar ratio to enzyme selenium. These observations suggest that glutathione is bound to enzyme selenium by a selenenyl-sulfide linkage (E-Se-SG) which is cleaved by cyanide to release a selenol and S-cyanoglutathione; spontaneous oxidation of the selenol to a labile oxidized form of GSH peroxidase leads to irreversible inactivation.     

Nickel-specific, slow-binding inhibition of carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide

The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 microM KCN. Dissociation constants of 8.46 microM (KCN) and 4.70 microM (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.     

A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings response to environmental stress

The effects of potassium cyanide (KCN) pretreatment on the response of cucumber (Cucumis sativus L.) plants to salt, polyethylene glycol (PEG) and cold stress were investigated in the present study. Here, we found that KCN pretreatment improved cucumber seedlings tolerance to stress conditions with maximum efficiency at a concentration of 20 µM. The results showed that pretreatment with 20 µM KCN alleviated stress‐induced oxidative damage in plant cells and clearly induced the activity of alternative oxidase (AOX) and the ethylene production. Furthermore, the structures of thylakoids and mitochondria in the KCN‐pretreated seedlings were less damaged by the stress conditions, which maintained higher total chlorophyll content, photosynthetic rate and photosystem II (PSII) proteins levels than the control. Importantly, the addition of the AOX inhibitor salicylhydroxamic acid (1 mm ; SHAM) decreased plant resistance to environmental stress and even compromised the cyanide (CN)‐enhanced stress tolerance. Therefore, our findings provide a novel role of CN in plant against environmental stress and indicate that the CN‐enhanced AOX might contribute to the reactive oxygen species (ROS) scavenging and the protection of photosystem by maintaining energy charge homoeostasis from chloroplast to mitochondria.     

Pattern of enzyme changes in rabbits administered linamarin or potassium cyanide

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

Isolation and growth of a Pseudomonas species that utilizes cyanide as a source of nitrogen

A simple method of isolating bacteria that utilize cyanide as a source of nitrogen for growth has been developed. This involved supplying hydrogen cyanide as a vapour to glucose-containing minimal-salts agar plates. The bacteria isolated were Gram-negative, oxidase-positive rods producing a fluorescent green pigment and were tentatively identified as strains of Pseudomonas fluorescens. Three organisms were studied further and shown to be P. fluorescens biotype II. One of these (NCIB 11764) was grown in a glucose-containing fed-batch culture with either NH4Cl or KCN as the limiting nutrient. Cyanide-grown bacteria produced stoichiometric amounts of ammonia from cyanide when pulsed with cyanide under aerobic conditions. Stimulation of oxygen uptake was seen on addition of cyanide to suspensions of cyanide-grown but not ammonia-grown bacteria.     

Utilization of cyanide as nitrogenous substrate by Pseudomonas fluorescens NCIMB 11764: evidence for multiple pathways of metabolic conversion.

The growth of Pseudomonas fluorescens NCIMB 11764 on cyanide as the sole nitrogen source was accomplished by use of a modified fed-batch cultivation procedure. Previous studies showing that cyanide metabolism in this organism is both an oxygen-dependent and an inducible process, with CO2 and ammonia representing conversion products, were confirmed. However, washed cells (40 mg ml-1 [dry weight]) metabolized cyanide at concentrations far exceeding those previously described; 85% of 50 mM KCN was degraded in 6 h. In addition, two other C1 metabolites were detected in incubation mixtures; their identities were confirmed as formamide and formate by 13C nuclear magnetic resonance spectrocopy, high-pressure liquid chromatography, radioisotopic trapping experiments, and other analytical means. The relative yields of all four metabolites (CO2, formamide, formate, and ammonia) were shown to be dependent on the KCN concentration and availability of oxygen; at 0.5 to 10 mM substrate, CO2 was the major C1 product, whereas at 20 and 50 mM substrate, formamide and formate were principally formed. The latter two metabolites also accumulated during prolonged anaerobic incubation, suggesting that P. fluorescens NCIMB 11764 can elaborate several pathways of cyanide conversion. One is formally similar to that proposed previously (R. E. Harris and C. J. Knowles, FEMS Microbiol. Lett. 20:337-341, 1983), involving the oxygen-dependent conversion of cyanide to CO2 and ammonia. The other two, occurring in the presence or absence of oxygen, involve separate reactions to yield, respectively, formate plus ammonia or formamide.(ABSTRACT TRUNCATED AT 250 WORDS)