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.     

Determination of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine and plasma by gas chromatography-mass spectrometry

The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATCA) is a promising biomarker for cyanide exposure because of its stability and the limitations of direct determination of cyanide and more abundant cyanide metabolites. A simple, sensitive, and specific method based on derivatization and subsequent gas chromatography-mass spectrometry (GC-MS) analysis was developed for the identification and quantification of ATCA in synthetic urine and swine plasma. The urine and plasma samples were spiked with an internal standard (ATCA-d(2)), diluted, and acidified. The resulting solution was subjected to solid phase extraction on a mixed-mode cation exchange column. After elution and evaporation of the solvent, a silylating agent was used to derivatize the ATCA. Quantification of the derivatized ATCA was accomplished on a gas chromatograph with a mass selective detector. The current method produced a coefficient of variation of less than 6% (intra- and interassay) for two sets of quality control (QC) standards and a detection limit of 25 ng/ml. The applicability of the method was evaluated by determination of elevated levels of ATCA in human urine of smokers in relation to non-smokers for both males and females.     

Dose and Time‐Dependent Effects of Cyanide on Thiosulfate Sulfurtransferase, 3‐Mercaptopyruvate Sulfurtransferase,and Cystathionine λ‐Lyase Activities

We assessed the dose‐dependent effect of potassium cyanide (KCN) on thiosulfate sulfurtransferase (TST), 3‐mercaptopyruvate sulfurtransferase (3‐MPST), and cystathionine λ‐lyase (CST) activities in mice. The time‐dependent effect of 0.5 LD50 KCN on cyanide level and cytochrome c oxidase (CCO), TST, 3‐MPST, and CST activities was also examined. Furthermore, TST, 3‐MPST, and CST activities were measured in stored mice cadavers. Hepatic and renal TST activity increased by 0.5 LD50 KCN but diminished by ≥2.0 LD50. After 0.5 LD50 KCN, the elevated hepatic cyanide level was accompanied by increased TST, 3‐MPST, and CST activities, and CCO inhibition. Elevated renal cyanide level was only accompanied by increased 3‐MPST activity. No appreciable change in enzyme activities was observed in mice cadavers. The study concludes that high doses of cyanide exert saturating effects on its detoxification enzymes, indicating their exogenous use during cyanide poisoning. Also, these enzymes are not reliable markers of cyanide poisoning in autopsied samples.     

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.     

High-dose ascorbic acid decreases detoxification of cyanide derived from amygdalin (laetrile): studies in guinea pigs

Cysteine, a sulphur-containing amino acid, is required to metabolize ascorbic acid (as ascorbate sulphate) and detoxify cyanide (to thiocyanate). In guinea pigs, conjoint use of laetrile (a cyanogenic glycoside) and ascorbic acid (in large doses) decreases the detoxification of cyanide derived from laetrile through diminishing the availability of cysteine, but not impairing hepatic rhodanese activity, which is involved in the detoxification of cyanide to thiocyanate. These results agree with the symptoms of a sublethal dose of KCN toxicity manifested by the animals. The studies, therefore, indicate that individuals taking megadoses of ascorbic acid concurrently with laetrile may be subject to self-poisoning.     

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.     

Physiologically based pharmacokinetic modeling of hydrogen cyanide in humans following the oral administration of potassium cyanide and cyanogenic glycosides from food

Abstract

Cyanogenic glycosides (CGs) are commonly found in some edible plants and seeds. After ingestion, CGs can release toxic hydrogen cyanide (HCN) in humans. At present, unfortunately, there is no tool capable of predicting the cyanide concentration in human blood and organs following oral administration of CG-containing food. The aim of this study was to develop a physiologically based pharmacokinetic (PBPK) model of cyanide following the ingestion of CG-containing food in humans. To develop this model, pharmacokinetic data concerning cyanide concentration levels in humans exposed to potassium cyanide (KCN) and CG-containing foods (persipan paste, linseed, cassava, and bitter apricot kernels) were obtained from published data. This study created a model structure consisting of four organ compartments including the lungs, kidneys, liver, and slowly perfused tissues by employing Berkeley Madonna software to extract three unknown parameters including the maximum velocity of rhodanese, the absorption rate constant, and the bioavailability for oral administration of KCN and the four CG-containing foods (equivalent a 6.8?mg dose of cyanide). The model was then validated by comparing the simulated results for the concentration-time courses of cyanide levels in venous blood with data from two clinical studies covering the oral administration of KCN and linseed at three other doses.     

Nerve Growth Factor Protects the Cortical Neurons from Chemical Hypoxia-induced Injury

Previous studies showed that nerve growth factor (NGF) exerts protective effects on cultured neurons against various kinds of damage. However, a recent publication reported that exposure of NGF-treated PC12 cells to physical hypoxia resulted in a higher cell death rate when compared to the untreated controls. In the present study, we therefore investigated the effects of NGF on the hypoxic cortical neurons induced by potassium cyanide (KCN). We demonstrated that NGF at a higher concentration can significantly increase neuronal viability, decrease the release of lactate dehydrogenase and improve cellular morphology in the hypoxic cortical neurons. However, we also found that pretreatment of NGF was not able to completely revise the decreased cell viability and the increased leakage of lactate dehydrogenase (LDH) induced by KCN, although the indexes in the neurons treated with NGF and KCN were significantly higher than those in the neurons treated with KCN only. Analysis of the data showed that the incomplete revision of NGF should be not due to the dosage of NGF we used. It might be induced by the inability of NGF to inhibit all injury pathways induced by potassium cyanide.     

Kinetic analysis of anaerobic metabolism in rats during acute cyanide poisoning

In order to investigate the changes in energy metabolism during acute anoxia, blood levels of various metabolites were analysed in cyanide-poisoned rats. After intraperitoneal injection of a sublethal dose of potassium cyanide (5 mg/kg), blood samples were obtained by cervical dislocation at intervals of 5 min until 30 min. Lacatate and lactate/pyruvate ratio (L/P) in plasma concomitantly changed with cyanide; increased rapidly at 5 min, remained fairly constant until 20 min and then began to decrease at 25 min. In contrast, the products of ATP degradation, oxypurines and inorganic phosphate (Pi), increased gradually until 25 min and then began to decrease at 30 min. Allantoin in plasma scarcely increased throughout the experiments. The results indicate that the rapid activation of anaerobic ATP formation by glycolysis was followed by the increase in ATP degradation in cyanide-poisoned rats. Thus, increase in plasma oxypurines could be regarded as an indicator for severe anoxic states in tissues with massive ATP degradation.     

Photobiomodulation directly benefits primary neurons functionally inactivated by toxins: role of cytochrome c oxidase

Far red and near infrared (NIR) light promotes wound healing, but the mechanism is poorly understood. Our previous studies using 670 nm light-emitting diode (LED) arrays suggest that cytochrome c oxidase, a photoacceptor in the NIR range, plays an important role in therapeutic photobiomodulation. If this is true, then an irreversible inhibitor of cytochrome c oxidase, potassium cyanide (KCN), should compete with LED and reduce its beneficial effects. This hypothesis was tested on primary cultured neurons. LED treatment partially restored enzyme activity blocked by 10-100 microm KCN. It significantly reduced neuronal cell death induced by 300 microm KCN from 83.6 to 43.5%. However, at 1-100 mm KCN, the protective effects of LED decreased, and neuronal deaths increased. LED significantly restored neuronal ATP content only at 10 microm KCN but not at higher concentrations of KCN tested. Pretreatment with LED enhanced efficacy of LED during exposure to 10 or 100 microm KCN but did not restore enzyme activity to control levels. In contrast, LED was able to completely reverse the detrimental effect of tetrodotoxin, which only indirectly down-regulated enzyme levels. Among the wavelengths tested (670, 728, 770, 830, and 880 nm), the most effective ones (830 nm, 670 nm) paralleled the NIR absorption spectrum of oxidized cytochrome c oxidase, whereas the least effective wavelength, 728 nm, did not. The results are consistent with our hypothesis that the mechanism of photobiomodulation involves the up-regulation of cytochrome c oxidase, leading to increased energy metabolism in neurons functionally inactivated by toxins.     

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.     

Biochemical effects of sublethal doses of cypermethrin on the sixth-instar larvae of Tribolium castaneum (Herbst.)

Effects of sublethal doses ie, 2, 4, 10, and 20 ppm of cypermethrin, were studied on the sixth-instar larvae of Tribolium castaneum (Herbst.). Of all the biochemical parameters tested, the free amino acids and cholesterol content and the activity of amylase were found to be the most sensitive components. Glutamate pyruvate transaminase activity was elevated at all doses except 2 ppm. The activities of alkaline phosphatase and glutamate oxaloacetate transaminase and glucose content were raised significantly at doses of 10 and 20 ppm. Acid phosphatase activity and the soluble protein content increased at a dose of 20 ppm. Total lipid and triglycerides, however, decreased significantly at this sublethal dose. Other biochemical parameters, such as cholinesterase and lactate dehydrogenase activities and the total protein, urea, glycogen, DNA, and RNA contents, were not significantly affected by exposure to different doses of cypermethrin.     

Cyanide toxicity and interference with diet selection in quail (Coturnix coturnix)

Cyanide is a ubiquitous substance in the environment. Most of the cyanide absorbed by an animal is detoxified by enzymatic combination with sulfur, thus the detoxification process imposes a nutritional cost. In mammals, interactions among nutrients and toxics may influence the composition of the diet and food intake, as a function of positive or negative post-ingestive feedback. The present work aimed to describe the toxic effects of cyanide, and to determine whether cyanide interferes with diet selection in quail (Coturnix coturnix). A toxicological study was performed with 27 female quails that were assigned to three groups that received by gavage 0, 1.0 or 3.0 mg of KCN/kg/day, for 7 consecutive days. The diet selection trial was conducted with 20 female quails, that had access to two separate rations: a conventional quail ration and the same ration supplemented with 1% NaSO₄. During the toxicological study, clinical signs of poisoning and death occurred in a quail treated with cyanide. Histological changes were found only in animals dosed with cyanide, and these consisted of mild hepatic periportal vacuolation, an increased number of vacuoles in the colloid of the thyroid glands, and spongiosis in the mesencephalon. No clinical signs were found in any quail throughout the diet selection trial. There were no significant differences in food consumption or ration preference. In conclusion, exposure to cyanide promotes damage to the liver and central nervous system in quails. In contrast, the ingestion of sulfur by quail was not affected by exposure to cyanide.     

Hepatic effects of phenobarbitone in female mice fed sublethal levels of dietary cyanide

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.     

Hepatic radiation injury in the rat.

The whole livers of rats were exposed intraoperatively to graded doses (0 to 75 Gy) of 137Cs gamma radiation. At various times (0 to 155 days) after liver irradiation, minimally invasive, nondestructive tests (rose bengal retention and plasma alkaline phosphatase, glutamic-oxaloacetic acid transaminase, glutamic-pyruvic transaminase) were performed on at least half the surviving animals in each dose group to assess developing liver injury. Liver histology was done on animals sacrificed 96 to 100 days after irradiation. Radiation damage to the stomach killed approximately 50% of the animals 30 to 60 days after exposure to doses of 25 Gy or higher. These deaths were significantly reduced when care was taken to shield the stomach during irradiation. Stomach injury did not, however, appreciably affect liver function as measured by rose bengal retention. Whole-liver irradiation to 15 Gy resulted in reduced liver size and minimal histological changes, but did not result in increased rose bengal retention or plasma alkaline phosphatase concentration. The next highest dose group studied (25 Gy) showed significant histological abnormalities and liver injury as measured by increased rose bengal retention and liver enzymes. The latent period for development of hepatic injury, as measured by increased rose bengal retention, was 35 to 42 days and was relatively invariant over the 25- to 75-Gy dose range. Hepatic vein lesions and cellular necrosis were the most prominent histological lesions observed in 25-Gy-irradiated liver.     

Chromosome aberrations reduced in whole-body irradiated mice by pretreatment with cyanide.

Male mice exposed to single, whole-body 60Co irradiation, were injected intraperitoneally with a non-toxic dose of KCN, 2 min or 20 min prior to irradiation. Bone-marrow cells were examined for chromatid breaks and chromosome aberrations (CA) at different times post-irradiation. The 2 min but not the 20 min treated mice had a marked reduction in chromatid breaks and chromosome aberrations. A study was made of mice exposed to 3.0 Gy (1.8 Gy/min), treated with KCN 2 min prior to irradiation and examined 5 min to 30 d post-irradiation. After 5 min there were no significant changes in frequency of CA. Subsequently, the incidence of CA in the KCN-treated group was reduced compared to the irradiated controls. By the 30th day, however, CA frequencies had returned to control levels in all groups. No effect of KCN treatment was observed in the white or red blood cells. The cytogenetic results were posited to be a function of the relative inhibition and recovery times of cyanide affected cytochrome oxidase, DNA synthesis, and ATP.     

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.     

Accumulation, distribution and toxicology of dietary nickel in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush)

An 18-day experiment was conducted to investigate the uptake and sublethal toxicity of dietary Ni in adult lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush) fed diets containing 0, 1000 and 10000 microg Ni/g, prepared with and without brine shrimp. The results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed to lower dietary Ni doses. In the present study feed refusal was observed in LT and LWF fed 10000 microg Ni/g, after three and 4-5 feedings, respectively. LT fed Ni-contaminated diets exhibited different patterns of Ni accumulation than LWF. Increased Ni concentrations in all LWF tissues, except the intestine, were associated with increased doses of Ni. Copper and Zn concentrations in kidney and liver of LWF were altered. Metallothionein concentrations in kidneys of LT fed 1000 microg Ni/g and 10000 microg Ni/g and LWF fed 10000 microg Ni/g and in livers of LWF fed 10000 microg Ni/g (diet without shrimp only) increased significantly. Increased lipid peroxide production in the plasma of LT fed 10000 microg Ni/g was observed. Blood glucose and electrolytes were affected by Ni exposure. Histopathological alterations were observed in kidneys of LWF fed low and high dose diets, livers of whitefish fed high dose diets, and intestines of LWF fed high dose diets and LT fed low and high dose diets. LT fed high dose diets exhibited significant decreases in weight.     

Cyanide-induced changes in the levels of neurotransmitters in discrete brain regions of rats and their response to oral treatment with alpha-ketoglutarate

Cyanide is a potential suicidal, homicidal and chemical warfare agent. It produces histotoxic hypoxia following inhibition of cytochrome c oxidase, a terminal respiratory chain enzyme. The profound metabolic changes lead to neurotoxicity including alterations in the levels of neurotransmitters. The present study addressed the effect of acute exposure of lethal and sub-lethal doses of potassium cyanide (KCN; 0.75 or 2.0 LD50; po) on the levels of neurotransmitters in discrete brain regions of rats and its response to treatment with alpha-ketoglutarate (alpha-KG; 0.5 g/kg; po; -10 min) alone or with sodium thiosulphate (STS; 1.0 g/kg; ip; -15 min). KCN significantly decreased norepinephrine, dopamine and 5-hydroxytryptamine levels in different brain regions which were resolved by alpha-KG and/or STS. Corpus striatum and hippocampus were more sensitive as compared to cerebral cortex and hypothalamus. alpha-KG, a potential cyanide antidote alone or with STS showed neuroprotective effects against cyanide.     

Experimentally induced chromosome abberrations in plants. II. The effect of cyanide and other heavy metal complexing agents on the production of chromosome aberrations by x-rays

The discovery of Lilly and Thoday, that the presence of potassium cyanide (KCN) increases the production of chromosome aberrations by x-rays in anoxia, but has no effect on the production of chromosome aberrations by x-rays in air, was confirmed. In the presence of cyanide, the effect of a given dose of x-rays in nitrogen was found to be even greater than the effect of the same dose of x-rays in air. The cyanide effect on x-ray breakage in nitrogen was obtained at cyanide concentrations as low as 2 x 10(-5)M. The breakage obtained after the combined x-ray-cyanide treatments was of the x-ray type, as evidenced by the distribution of breaks within and between the chromosomes. A number of other heavy metal complexing agents as well as some other compounds were tested for their ability to increase x-ray breakage in nitrogen and air. Of these compounds only cupferron proved to be effective. The results are discussed and it is concluded that the increased x-ray breakage in the presence of cyanide or cupferron cannot be due to an accumulation of peroxides. Instead it is suggested that the cyanide effect may be due to a complex formation between the active agents and heavy metals, presumably iron, within the chromosomes. The consequences of this hypothesis on the concept of the "oxygen effect," are discussed.