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Background: Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism., Methods: Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L -1 , corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy., Results: The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants., Conclusions: The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms., Competing Interests: The authors declare that they have no competing interests., (© 2024 Harshkova et al.)

Ethnopharmacological Relevance: Different parts of Antiaris africana Englea (Moraceae) are used traditionally for the treatment of various diseases, including epilepsy and other nervous system disorders., Aims of This Study: The current study was designed to evaluate the neuroprotective activity of flavonoids isolated from A. africana against potassium cyanide (KCN)-induced oxidative damage in brain homogenate., Materials and Methods: Dried and ground leaves of A. africana were extracted with methanol and fractioned into n-hexane (HFA), dichloromethane (DFA), ethyl acetate (EFA) and methanol (MFA). Each fraction was assessed for neuroprotective potential by anticholinesterase activity test. The fraction with the best anticholinesterase activity was subjected to various chromatographic techniques through bioassay-guided fractionation to isolate the bioactive compounds. The protective ability of the extract, fractions and compounds against Potassium cyanide (KCN)-induced mitochondrial damage in rat brain homogenate was evaluated. Structures of the isolated compounds were determined using 1D and 2D NMR, mass spectrometry and by comparison with literature data., Results and Discussion: The ethyl acetate fraction showed the best anticholinesterase activity with an IC 50 of 23.23 ± 1.12 μg/ml. Quercetin and a biflavonoid glucoside identified as 3'-4'-bisquercetin-3β-D-diglucoside from this fraction displayed a remarkable antioxidant activity in the DPPH assay and showed significant (P < 0.05) increase in the activity of dehydrogenase inhibited by KCN in a concentration dependent manner. However, quercetin was more effective in reducing the MDA level and acetylcholinesterase activity that were elevated by KCN., Conclusion: Quercetin and the bisquercetin-diglucoside isolated from the leaves of A. Africana for the first time, are major contributors to the observed neuroprotective property of the plant which supports its folkloric usage in the management of seizures, epilepsy and other neurological disorders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Cyanide (CN) is a metabolic poison that is capable of intoxicating individuals through accidental or intentional means. With high concentration exposures, death can occur in minutes. In cases of mass casualty exposures, there is a need for a rapid-acting countermeasure capable of being administered in a short period of time in a pre-hospital setting to treat victims., Objective: These studies evaluate the safety and efficacy of a novel aqueous formulation of dimethyl trisulfide (DMTS) as an intramuscular (IM) CN countermeasure using non-anesthetized rodent models., Methods: Non-anesthetized rodents (mice and rats) were exposed to hydrogen cyanide (HCN) or potassium cyanide (KCN) along with immediate IM 10% DMTS treatment or vehicle treatment. Survival and other parameters, such as the time to recovery and assessment of clinical toxic signs (e.g., gasping, loss of righting reflex, convulsions, etc.), were quantified to determine the effectiveness of 10% DMTS treatment (12.5, 25, 75 mg/kg IM) compared to vehicle control treatment. A rat KCN delayed-treatment model with a 15-minute treatment delay was also utilized to simulate a real-life exposure/treatment scenario with 10% DMTS treatment. The stability of the 10% DMTS formulation was also assessed., Results: A 25 mg/kg IM dose of 10% DMTS exhibits potent efficacy against subcutaneous (SC) KCN challenge in both mice and rats and inhalational HCN exposure in mice. 10% DMTS treatment also shortens the time to recovery in rats using a delayed-treatment model., Conclusion: IM treatment with 10% DMTS improves survival and clinical outcomes in non-anesthetized rodent models of acute CN toxicity. Additionally, the use of an SC KCN delayed-treatment model in rats is advised to assess the performance of a candidate CN countermeasure in a more realistic exposure/treatment scenario.

The aim of the present study was to determine whether methylene blue (MB) could directly oppose the neurological toxicity of a lethal cyanide (CN) intoxication. KCN, infused at the rate of 0.375 mg/kg/min intravenously, produced 100% lethality within 15 min in unanaesthetized rats (n = 12). MB at 10 (n = 5) or 20 mg/kg (n = 5), administered 3 min into CN infusion, allowed all animals to survive with no sequelae. No apnea and gasping were observed at 20 mg/kg MB (P < 0.001). The onset of coma was also significantly delayed and recovery from coma was shortened in a dose-dependent manner (median of 359 and 737 seconds, respectively, at 20 and 10 mg/kg). At 4 mg/kg MB (n = 5), all animals presented faster onset of coma and apnea and a longer period of recovery than at the highest doses (median 1344 seconds, P < 0.001). MB reversed NaCN-induced resting membrane potential depolarization and action potential depression in primary cultures of human fetal neurons intoxicated with CN. MB restored calcium homeostasis in the CN-intoxicated human SH-SY5Y neuroblastoma cell line. We conclude that MB mitigates the neuronal toxicity of CN in a dose-dependent manner, preventing the lethal depression of respiratory medullary neurons and fatal outcome., (© 2020 New York Academy of Sciences.)

Cyanide-induced chemical hypoxia is responsible for pronounced oxidative damage in the central nervous system. The disruption of mitochondrial oxidative metabolism has been associated with upregulation of uncoupling proteins (UCPs). The present study addresses the dose- and time-dependent effect of sub-acute cyanide exposure on various non-enzymatic and enzymatic oxidative stress markers and their correlation with inducible-nitric oxide synthase (iNOS) and uncoupling protein-2 (UCP-2) expression. Animals received (oral) triple distilled water (vehicle control), 0.25 LD50 potassium cyanide (KCN) or 0.50 LD50 KCN daily for 21 d. Animals were sacrificed on 7, 14 and 21 d post-exposure to measure serum cyanide and nitrite, and brain malondialdehyde (MDA), reduced glutathione (GSH), glutathione disulfide (GSSG), cytochrome c oxidase (CCO), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CA) levels, together with iNOS and UCP-2 expression, and DNA damage. The study revealed that a dose- and time-dependent increase in cyanide concentration was accompanied by corresponding CCO inhibition and elevated MDA levels. Decrease in GSH levels was not followed by reciprocal change in GSSG levels. Diminution of SOD, GPx, GR and CA activity was congruent with elevated nitrite levels and upregulation of iNOS and UCP-2 expression, without any DNA damage. It was concluded that long-term cyanide exposure caused oxidative stress, accompanied by upregulation of iNOS. The upregulation of UCP-2 further sensitized the cells to cyanide and accentuated the oxidative stress, which was independent of DNA damage.

Background: Cyanide is a deadly compound used as a terrorist agent. Current FDA approved antidotes require intravenous administration, limiting their utility in a mass casualty scenario. Dimethyl trisulfide (DMTS), a sulfur-based molecule, binds cyanide converting it to the less toxic by-product thiocyanate. Studies evaluating efficacy in rodents have been performed, but a large, clinically relevant animal model has not been reported., Objective: This study evaluates the efficacy of intramuscular DMTS on survival and clinical outcomes in a swine model of acute, severe cyanide toxicity., Methods: Anesthetized swine were instrumented for continuous monitoring of hemodynamics. Prior to potassium cyanide infusion animals were acclimated and breathing spontaneously. At 5-minutes post-apnea animals were treated with DMTS or saline. Vital signs, hemodynamics, and laboratory values were evaluated at various time points., Results: Baseline values and time to apnea were similar in both groups. Survival in the DMTS treated group was 83.3% and 0% in saline controls (p = .005). The DMTS group returned to breathing at a mean time of 19.3 ± 10 min after antidote, control animals did not return to breathing (CI difference 8.8, 29.8). At the end of the experiment or time of death, mean lactate was 9.41 mmol/L vs. 4.35 mmol/L (CI difference -10.94,0.82) in the saline and DMTS groups, respectively and pH was 7.20 vs. 7.37 (CI difference -0.04, 0.38). No adverse effects were observed at the injection site., Conclusion: Intramuscular administration of DMTS improves survival and clinical outcomes in our large animal swine model of acute cyanide toxicity.

Cyanide is a potent neurotoxic substance that can initiate series of intracellular reactions leading to oxidativestress. To evaluate effect of sublethal administration of potassium cyanide (KCN) on sensorimotor functions and long termvisuo-spatial learning and memory in adult Swiss mice and possible ameliorative role of vitamins A and E. These vitaminsA and E (dietary) are antioxidants that have scavenging properties against free radicals and reactive oxygen species as aresult of oxidative stress induced by cyanide. Thirty-five mice weighing between 18-22 g were used for the study. Theanimals were randomly divided into five groups (n = 7) and exposed to sublethal concentration of potassium cyanide (10%LD50; 1.5 mg/kg). KCN was administered orally while vitamin A (25 mg/kg) and vitamin E (50 mg/kg) were administeredintra-peritoneal (IP) once daily for 28 days. Potassium cyanide (KCN) was first administered and after 10 minutes intervals,followed by vitamin A and then E after 5 minutes, vitamin E were administered across the different treatment groups. Micewere examined for signs of toxicity. Vitamins pre-treatment ameliorated toxic signs. In the dynamics of wire grid, coathanger and stationary beam test, the latency to fall in weeks 2 and 4 were statistically significant. In acquisition and retention,using elevated plus maze (EPM), KCN treated group recorded high transfer latencies in seconds (50.40±1.72 secs) and(57.60±0.93 secs) as compared to group IV (29.40±0.68 secs; 5.60±0.60 secs). Cyanide is a neurotoxin that affects motorfunctions with progressive decline in motor strength and coordination. KCN affects acquisition and retention memory whilepre-treatment with antioxidant vitamins A and E ameliorated these deficits.

This study was aimed at determining the efficacy of epinephrine, followed by chest compressions, in producing a return of spontaneous circulation (ROSC) during cyanide (CN)- or hydrogen sulfide (H 2 S)-induced toxic cardiac pulseless electrical activity (PEA) in the rat. Thirty-nine anesthetized rats were exposed to either intravenous KCN (n = 27) or H 2 S solutions (n = 12), at a rate that led to a PEA within less than 10 min. In the group intoxicated by CN, 20 rats were mechanically ventilated and received either epinephrine (0.1 mg/kg i.v. n = 10) followed by chest compressions or saline (n = 10, "control CN") when in PEA. PEA was defined as a systolic pressure below 20 mmHg and a pulse pressure of less than 5 mmHg for 1 min. In addition, seven spontaneously breathing rats were also exposed to the same CN protocol, but infusion was stopped when a central apnea occurred; then, as soon as a PEA occurred, epinephrine (0.1 mg/kg IV) was administered while providing manual chest compressions and mechanical ventilation (CPR). Finally, 12 rats were intoxicated with H 2 S, while mechanically ventilated, and received either saline (n = 6, "control H 2 S") or epinephrine (n = 6) with CPR when in PEA. None of the control-intoxicated animals resuscitated (10 rats in the control CN group and 6 in the control H 2 S group). In contrast, all the animals intoxicated with CN or H 2 S that received epinephrine followed by chest compressions, returned to effective circulation. In addition, half of the spontaneously breathing CN-intoxicated animals that achieved ROSC after epinephrine resumed spontaneous breathing. In all the animals achieving ROSC, blood pressure, cardiac output, peripheral blood flow and [Formula: see text]O 2 returned toward baseline, but remained lower than the pre-intoxication levels (p < 0.01) with a persistent lactic acidosis. Epinephrine, along with CPR maneuvers, was highly effective in resuscitating rodents intoxicated with CN or H 2 S. Since epinephrine is readily available in any ambulance, its place as an important countermeasure against mitochondrial poisons should be advocated. It remains critical to determine whether the systematic administration of epinephrine to any victims found hypotensive following CN or H 2 S intoxication could prevent PEA, decrease post-ischemic brain injury and increase the efficacy of current antidotes by improving the circulatory status.

Cyanide is a readily available and potentially lethal substance. Oral exposure can result in larger doses, compared with other routes. Currently, there are no antidotes specific for use in the treatment of oral cyanide poisoning, and studies cannot be done in humans. We report on a new large animal model of oral cyanide toxicity to evaluate potential antidotes. Six female swine ( Sus scrofa ; weight, 45 to 55 kg) were anesthetized, intubated, and instrumented. Animals received a KCN bolus of either 5 or 8 mg/kg delivered via orogastric tube. Time to apnea was recorded; parameters monitored included heart rate, respiratory rate, blood pressure, pulse oximetry, end-tidal CO2, arterial blood gasses, and lactate concentrations. The Welch t test was used to calculate confidence intervals, mean, and standard deviation, and a Kaplan-Meier survival curve was used to compare survival between the 2 groups. At baseline, all animals in both groups were similar. Animals in the 5-mg/kg group had a more rapid time to apnea (5.1 ± 2.1 min), longer time to death (48.5 ± 38.1 min), and a greater rate of survival than the 8-mg/kg group (apnea, 10.6 ± 10.7 min; death, 26.1 ± 5.8 min). All animals displayed signs of toxicity (acidemia, hyperlactatemia, hypotension, apnea). We here report a large animal (swine) model of oral cyanide poisoning with dose-dependent effects in regard to time to death and survival rate. This model likely will be valuable for the development of medical countermeasures for oral cyanide poisoning.

The aim of the present study was to determine the deleterious effects of prolonged oral cyanide insult on various organs and tissues in rabbits. For this purpose, 12 locally bred adult male rabbits were allocated into two groups of 6 viz. control and experimental. Rabbits in control group were offered feed only while the rabbits in experimental group received feed plus potassium cyanide (KCN) at 3 mg/kg body weight orally for a period of 40 days. None of the rabbit in both the groups demonstrated any of the gross changes in any organ on postmortem examination. Liver was normal in size, shape, texture and color. Kidneys were also normal in size and color. Histopathological examination revealed severe hepatocyte vacuolation and degeneration in liver of rabbits in experimental group. There was also excessive congestion in liver and bile duct of rabbits in experimental group. Kidneys of rabbits in experimental group demonstrated severe glomerular and tubular necrosis and congestion. In the tubular epithelial cells, pyknotic nuclei were also present. On the other hand, heart and pancreas of rabbits in both control and experimental group did not show any histopathological change in microscopic structures. In conclusion, prolonged oral cyanide administration could have harmful effects on liver and kidney functions.

To evaluate the removal of potassium cyanide (KCN) and its toxicity in algae, an initial comprehensive analysis was performed with Chlorella vulgaris. The algae showed potential removal capability for KCN, with the maximal removal rate of 61%. Moreover, effects of KCN on growth, cellular morphology and antioxidant defense system of C. vulgaris were evaluated. Cell number and chlorophyll a content decreased in most cases, with the maximal inhibition rates of 48% and 99%, respectively. The 100 mg L - 1 KCN seriously damaged the algal cell membrane. Additionally, activity of superoxide dismutase (SOD) was promoted by KCN exposure among 0.1-50 mg L - 1 and inhibited by 100 mg L - 1 KCN, while the malondialdehyde (MDA) content gradually decreased in C. vulgaris with increasing exposure concentration compared to the control. The present study reveals that C. vulgaris is useful in bio-treatment of cyanide-contaminated aquatic ecosystem, except in high concentrations which would cause overwhelming effects.

Transmembrane proteins of the ABC family contribute to a multiple xenobiotic resistance (MXR) phenotype in cells, driving the extrusion of toxic substances. This phenotype promotes a high degree of protection against xenobiotics. The present study provides a better understanding of the MXR activity in the podal disk cells of Bunodosoma cangicum exposed to copper, and further establishes the relationship between protein activity (measured by accumulation of rhodamine-B) and bioaccumulation of copper in these cells. Sea anemone cells were exposed for 24h to copper (0, 7.8 and 15.6μg/L) in presence and absence of MXR blocker (verapamil 50μM). Results indicate that copper exposure increases intracellular metal content when ABC proteins were blocked, causing an increase in cellular death. The present study also verified the relationship between MXR activity, ATP depletion, and general metabolic activity (by MTT). MXR activity decreased in treatment groups exposed to copper concentrations of 15.6μg/L and 10mM energy depleting potassium cyanide. Metabolic activity increased in cells exposed to 7.8μgCu/L, but 15.6μgCu/L was similar to 0 and 7.8μg/L. The presence of copper decreased the ABC proteins expression. The present study improves the knowledge of MXR in anemone cells and shows that this activity is closely associated with copper extrusion. Also, the copper exposure is able to modify the metabolic state and to lead to cytotoxicity when cells cannot defend themselves., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The UK's Initial Operational Response (IOR) is a revised process for the medical management of mass casualties potentially contaminated with hazardous materials. A critical element of the IOR is the introduction of immediate, on-scene disrobing and decontamination of casualties to limit the adverse health effects of exposure. Ad hoc cleansing of the skin with dry absorbent materials has previously been identified as a potential means of facilitating emergency decontamination. The purpose of this study was to evaluate the in vitro oil and water absorbency of a range of materials commonly found in the domestic and clinical environments and to determine the effectiveness of a small, but representative selection of such materials in skin decontamination, using an established ex vivo model. Five contaminants were used in the study: methyl salicylate, parathion, diethyl malonate, phorate and potassium cyanide. In vitro measurements of water and oil absorbency did not correlate with ex vivo measurements of skin decontamination. When measured ex vivo, dry decontamination was consistently more effective than a standard wet decontamination method ("rinse-wipe-rinse") for removing liquid contaminants. However, dry decontamination was ineffective against particulate contamination. Collectively, these data confirm that absorbent materials such as wound dressings and tissue paper provide an effective, generic capability for emergency removal of liquid contaminants from the skin surface, but that wet decontamination should be used for non-liquid contaminants., Competing Interests: The authors have declared that no competing interests exist.

Selective stimulation of carotid chemoreceptors by intravenous infusion of low doses of potassium cyanide (KCN) produces short-lasting escape responses that have been proposed as a model of panic attack. In turn, preclinical studies suggest that facilitation of the endocannabinoid system attenuate panic-like responses. Here, we compared the effects of cannabinoid-related compounds to those of alprazolam, a clinically effective panicolytic, on the duration of the escape reaction induced by intravenous infusion of KCN (80μg) in rats. Alprazolam (1, 2, 4mg/kg) decreased escape duration at doses that did not alter basal locomotor activity. URB597 (0.1, 0.3, 1mg/kg; inhibitor of anandamide hydrolysis), WIN55,212-2 (0.1, 0.3, 1mg/kg; synthetic cannabinoid), arachidonoyl-serotonin (1, 2.5, 5mg/kg; dual TRPV1 and anandamide hydrolysis inhibitor), and cannabidiol (5, 10, 20, 40mg/kg; a phytocannabinoid) did not decrease escape duration. Alprazolam also prevented the increase in arterial pressure evoked by KCN, while bradycardia was unchanged. This study reinforces the validity of the KCN-evoked escape as a model of panic attack. However, it does not support a role for the endocannabinoid system in this behavioral response. These results might have implications for the screening of novel treatments for panic disorder., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Potassium cyanide (KCN) is an inhibitor of cytochrome C oxidase causing rapid death due to hypoxia. A well-characterized model of oral KCN intoxication is needed to test new therapeutics under the Food and Drug Administration Animal Rule. Clinical signs, plasma pH and lactate concentrations, biomarkers, histopathology, and cyanide and thiocyanate toxicokinetics were used to characterize the pathology of KCN intoxication in adult and juvenile mice. The acute oral LD50s were determined to be 11.8, 11.0, 10.9, and 9.9 mg/kg in water for adult male, adult female, juvenile male, and juvenile female mice, respectively. The time to death was rapid and dose dependent; juvenile mice had a shorter mean time to death. Juvenile mice displayed a more rapid onset and higher incidence of seizures. The time to observance of respiratory signs and prostration was rapid, but mice surviving beyond 2 hours generally recovered fully within 8 hours. At doses up to the LD50, there were no gross necropsy or microscopic findings clearly attributed to administration of KCN in juvenile or adult CD-1 mice from 24 hours to 28 days post-KCN challenge. Toxicokinetic analysis indicated rapid uptake, metabolism, and clearance of plasma cyanide. Potassium cyanide caused a rapid, dose-related decrease in blood pH and increase in serum lactate concentration. An increase in fatty acid-binding protein 3 was observed at 11.5 mg/kg KCN in adult but not in juvenile mice. These studies provide a characterization of KCN intoxication in adult and juvenile mice that can be used to screen or conduct preclinical efficacy studies of potential countermeasures., (© The Author(s) 2016.)

The Food and Drug Administration Animal Rule requires evaluation of cardiovascular and central nervous system (CNS) effects of new therapeutics. To characterize an adult and juvenile mouse model, neurobehavioral and cardiovascular effects and pathology of a single sublethal but toxic, 8 mg/kg, oral dose of potassium cyanide (KCN) for up to 41 days postdosing were investigated. This study describes the short- and long-term sensory, motor, cognitive, and behavioral changes associated with oral dosing of a sublethal but toxic dose of KCN utilizing functional observation battery and Tier II CNS testing in adult and juvenile mice of both sexes. Selected tissues (histopathology) were evaluated for changes associated with KCN exposure with special attention to brain regions. Telemetry (adult mice only) was used to evaluate cardiovascular and temperature changes. Neurobehavioral capacity, sensorimotor responsivity or spontaneous locomotor activity, and rectal temperature were significantly reduced in adult and juvenile mice at 30 minutes post-8 mg/kg KCN dose. Immediate effects of cyanide included bradycardia, adverse electrocardiogram arrhythmic events, hypotension, and hypothermia with recovery by approximately 1 hour for blood pressure and heart rate effects and by 2 hours for body temperature. Lesions consistent with hypoxia, such as mild acute tubular necrosis in the kidneys corticomedullary junction, were the only histopathological findings and occurred at a very low incidence. The mouse KCN intoxication model indicates rapid and completely reversible effects in adult and juvenile mice following a single oral 8 mg/kg dose. Neurobehavioral and cardiovascular measurements can be used in this animal model as a trigger for treatment., (© The Author(s) 2016.)

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.

Objectives: The objective of this study is to measure mitochondrial respiration using intact cells from whole blood exposed to cyanide as a new biomarker for mitochondrial inhibition., Methods: A single nontourniqueted venous blood sample was collected from 10 healthy volunteers after informed consent. Venous lactate was measured immediately following blood collection. Half of the remaining blood sample was then incubated with 100 mM of potassium cyanide (KCN) for 5 min, and half of the sample remained unexposed. Repeat lactate measurements were performed from blood exposed and not exposed to KCN. Measurement of mitochondrial respiration: intact PBMCs were placed in a 2-mL chamber at a final concentration of 2-3 × 10(6) cells/mL. Measurements of oxygen consumption were performed at 37°C in a high-resolution oxygraph (Oxygraph-2k Oroboros Instruments, Innsbruck, Austria). Oxygen flux (in pmol O2/s/10(6) cells), which is directly proportional to oxygen consumption, was recorded continuously using DatLab software 6 (Oroboros Instruments)., Results: There were significance differences in the relevant key parameters of mitochondrial respiration: Of the parameters measuring mitochondrial respiration, four of the six demonstrated a statistically significant mean difference between control and cyanide: for routine respiration (mean difference [control-cyanide]: 8.9 pmol O2/s/10(6) cells; 95% CI: 5.6-12.2, p < 0.0001); Proton Leak (mean difference: 0.73 pmol O2/s/10(6) cells; 95% CI: -0.33-1.79, p = 0.157); Maximal respiration (mean difference: 21.7 pmol O2/s/10(6) cells; 95% CI: 16.0-27.6, p < 0.0001); Residual oxygen consumption (mean difference 0.25 pmol O2/s/10(6) cells; 95% CI: -0.68-1.18, p = 0.557). There was a significant difference in spare respiratory capacity (SRC) and adenosine triphosphate (ATP)-linked respiration with the control samples demonstrating a higher SRC and ATP-linked respiration. Finally, there is a statistically significant difference in lactate (mean difference -0.32, 95% CI: -0.41 to -0.23, p < 0.0001), though clinically similar level, with a higher lactate concentration in the cyanide samples., Conclusions: In this ex vivo model, the measurements of key parameters in mitochondrial respiration may be a more sensitive measure of cellular function when compared to lactate.

A novel total toxicity detection method based on a mediated biosensor system with flow injection (MB-FI) was developed to rapidly and reliably detect respiration inhibitors (i.e., As2O3, KCN, salicylic acid (SA), 2,4-dintirophenol (DNP)) in water. The mediated biosensor toxicity assessment using microorganisms immobilized in calcium alginate filaments can greatly simplify the testing process and save time. In the MB-FI system, ferricyanide together with a respiration inhibitor was injected into the bioreactor, inhibiting the respiration of the immobilized microorganisms. The degree of inhibition was measured by determining the ferrocyanide generated in the effluent, expressed as the 50% inhibition concentration (IC50). The IC50 values for the four respiration inhibitors obtained using this method were comparable to those obtained using the classic method, confirming that this approach is an alternative alert method. More importantly, this constructed biosensor system with flow injection will facilitate the application and commercialization of this toxicity monitoring technology., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Embryos, unlike adults, are typically sessile, which allows for an increase in the available metrics that can be used to assess chemical toxicity. We investigate Daphnia magna development rate and oxygen consumption as toxicity metrics and compare them to arrested embryo development using four different techniques with potassium cyanide (KCN) as a common toxicant. The EC50 (95 % CI) for arrested development was 2,535 (1,747-3,677) μg/L KCN. Using pixel intensity changes, recorded with difference imaging, we semi-quantitatively assessed a decrease in development rate at 200 μg/L KCN, threefold lower than the arrested development lowest observed effect concentration (LOEC). Respirometry and self-referencing (SR) microsensors were two unique techniques used to assess oxygen consumption. Using respirometry, an increase in oxygen consumption was found in the 5 μg/L KCN treatment and a decrease for 148 μg/L, but no change was found for the 78 μg/L KCN treatment. Whereas, with SR microsensors, we were able to detect significant changes in oxygen consumption for all three treatments: 5, 78, and 148 μg/L KCN. While SR offered the highest sensitivity, the respirometry platform developed for this study was much easier to use to measure the same endpoint. Oxygen consumption may be subject to change during the development process, meaning consumption assessment techniques may only be useful only for short-term experiments. Development rate was a more sensitive endpoint though was only reliable four of the six embryonic developmental stages examined. Despite being the least sensitive endpoint, arrested embryo development was the only technique capable of assessing the embryos throughout all developmental stages. In conclusion, each metric has advantages and limitations, but because all are non-invasive, it is possible to use any combination of the three.

Twelve adult rabbits bred locally were divided into two equal groups of 6; experimental and control groups. Rabbits in the experimental group were orally dosed with KCN at 3mg/kg body weight for 40 consecutive days. Members in control group were given placebo (distilled water) for the same period. Animals in both groups were offered feed at 90gm/kg/day while ample drinking water was available ad lib. Feed consumption and body weight of rabbits in both the groups were recorded. Blood samples were also drawn to determine various hematological parameters. Statistical analysis revealed a non-significant difference of total and daily feed intakes in rabbits of experimental and control groups. Whereas the feed efficiency of rabbits in the experimental group were significantly reduced (P<0.05) compared to controls. Likewise a significant decrease in body weight gain of rabbits in experimental group (P<0.05) was observed. A non-significant difference (P>0.05) was observed in leukocyte count, differential leukocyte count and platelets of rabbits in both the groups. Erythrocyte count, hemoglobin concentration, packed cell volume and mean corpuscular hemoglobin were significantly decreased in treated rabbits. It was concluded that chronic cyanide intake had a deleterious effect on feed efficiency, growth rate and blood components of rabbits.

Glia activation and neuroinflamation are major factors implicated in the aetiology of most neurodegenerative diseases (NDDs). Several agents and toxins have been known to be capable of inducing glia activation an inflammatory response; most of which are active substances that can cause oxidative stress by inducing production of reactive oxygen species (ROS). Neurogenesis on the other hand involves metabolic and structural interaction between neurogenic and glia cells of the periventricular zone (PVZ); a region around the third ventricle. This study investigates glia activation (GFAP), cell proliferation (Ki-67) and neuronal metabolism (NSE) during neurogenesis and oxidative stress by comparing protein expression in the PVZ against that of the parietal cortex. Adult Wistar Rats were treated with normal saline and 20 mg/Kg KCN for 7 days. The tissue sections were processed for immunohistochemistry to demonstrate glia cells (anti Rat-GFAP), cell proliferation (anti Rat-Ki-67) and neuronal metabolism (anti Rat-NSE) using the antigen retrieval method. The sections from Rats treated with cyanide showed evidence of neurodegeneration both in the PVZ and cortex. The distribution of glia cells (GFAP), Neuron specific Enolase (NSE) and Ki-67 increased with cyanide treatment, although the increases were more pronounced in the neurogenic cell area (PVZ) when compared to the cortex. This suggests the close link between neuronal metabolism and glia activation both in neurogenesis and oxidative stress.

Background: Hydrogen cyanide possesses a high acid-dissociation constant of 9.14, favoring its vaporization and depletion from the culture media at physiological pH, which may cause the cyanide toxicity unstable in vitro., Objective: We investigated whether adjustment of culture medium pH stabilizes cyanide concentration and decreases the effective concentration of potassium cyanide (KCN)., Methods: Murine fibroblast cells were exposed to different concentrations of KCN in media maintained at pH 7.4 or 9.2, in the presence or absence of hydroxocobalamin. After incubation for 1 h, we evaluated medium pH, cyanide concentration, cytochrome activity, and cell viability., Results: Cyanide concentration decreased to 18.8% in pH 7.4 medium compared to 83.2% in pH 9.2 medium. A significant decrease in cytochrome activity was observed at 40 mM and 1.25 mM KCN in pH 7.4 and pH 9.2 media, respectively. In pH 9.2 medium, dose-dependent cytotoxicity of KCN and antidotal effects of hydroxocobalamin were observed., Conclusion: Adjustment of culture medium pH to 9.2 could stabilize cyanide concentration and decrease the effective concentration of KCN, allowing stable evaluation of KCN toxicity and antidotal efficacy.

While exposure to chemical mixtures is an everyday reality, an understanding of their combined effects, and any potential prediction thereof, is extremely limited. Realistic exposures potentially consist of hundreds to thousands of chemicals per day, but even relatively simple binary mixture interactions can be inherently difficult to predict based upon the lack of temporal and spatial mechanisms for the individual constituents. To this end, we explore the concept of capitalizing on downstream convergence of intracellular signal transduction to experimentally simplify the means of determining xenobiotics that, when combined, could result in increased or unexpected toxicity. In a proof of principle study, we exposed HepG2 cells to deguelin, a natural isoflavonoid, alone and in combination with KCN, and determined the relative post-translational phosphorylation responses to several key proteins related to mitochondrial outer membrane permeabilization. Dose-dependent phosphorylation activity provides a clear identification of threshold response to low-level exposures, and crosstalk amongst selected proteins correctly forecasts mixtures interactions that may lead to increased toxicity. We then used Bliss Independence to determine if the experimentally measured mixture phosphorylation responses could be predicted with individual responses. Independence accurately predicted mixture interactions for deguelin and KCN (87.5%). To more fully exhaust independence as a model for determining potential pharmacodynamic interactions, we exposed HepG2 cells to deguelin and staurosporine, a broad kinase inhibitor; independence accurately predicted these mixture responses (77.5%). In this study, we demonstrate the potential of a new in vitro approach for the prediction of toxic mixtures interactions that is fundamentally driven by the interdependence of signal transduction and apoptosis., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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.

Poisoning by cyanide can be verified by analysis of the cyanide detoxification product, α-ketoglutarate cyanohydrin (α-KgCN), which is produced from the reaction of cyanide and endogenous α-ketoglutarate. Although α-KgCN can potentially be used to verify cyanide exposure, limited toxicokinetic data in cyanide-poisoned animals are available. We, therefore, studied the toxicokinetics of α-KgCN and compared its behavior to other cyanide metabolites, thiocyanate and 2-amino-2-thiazoline-4-carboxylic acid (ATCA), in the plasma of 31 Yorkshire pigs that received KCN (4mg/mL) intravenously (IV) (0.17 mg/kg/min). α-KgCN concentrations rose rapidly during KCN administration until the onset of apnea, and then decreased over time in all groups with a half-life of 15 min. The maximum concentrations of α-KgCN and cyanide were 2.35 and 30.18 μM, respectively, suggesting that only a small fraction of the administered cyanide is converted to α-KgCN. Although this is the case, the α-KgCN concentration increased >100-fold over endogenous concentrations compared to only a three-fold increase for cyanide and ATCA. The plasma profile of α-KgCN was similar to that of cyanide, ATCA, and thiocyanate. The results of this study suggest that the use of α-KgCN as a biomarker for cyanide exposure is best suited immediately following exposure for instances of acute, high-dose cyanide poisoning., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) catalyses the N-methylation of nicotinamide to 1-methylnicotinamide (MeN). We have previously shown that the ectopic expression of NNMT in SH-SY5Y human neuroblastoma cells increased adenosine triphosphate synthesis and complex I activity, effects of which were replicated by the addition of MeN. In this study, we investigated whether NNMT expression in SH-SY5Y conferred protection against mitotoxicity induced by rotenone, potassium cyanide (KCN), 2,4-dinitrophenol, and 6-hydroxydopamine, and whether any effects observed were mediated via increased MeN production. NNMT expression abolished the toxic effects of KCN, 2,4-dinitrophenol, and 6-hydroxydopamine, and reduced that of rotenone. In contrast, although MeN significantly reduced the toxicity of rotenone, it had no effect upon the toxicity of KCN, 2,4-dinitrophenol, and 6-hydroxydopamine. These data show that NNMT is cytoprotective against toxins that inhibit various aspects of mitochondrial function, and that these are not mediated solely via increased MeN production, but in combination with other unidentified mechanisms., (© 2013 Wiley Periodicals, Inc.)

Background: Mitochondrial dysfunction is an important factor contributing to tissue damage in both severe traumatic brain injury and ischemic stroke. This experimental study explores the possibility to diagnose the condition bedside by utilising intracerebral microdialysis and analysis of chemical variables related to energy metabolism., Methods: Mitochondrial dysfunction was induced in piglets and evaluated by monitoring brain tissue oxygen tension (PbtO2 ) and cerebral levels of glucose, lactate, pyruvate, glutamate, and glycerol bilaterally. The biochemical variables were obtained by microdialysis and immediate enzymatic analysis. Mitochondrial function was blocked by unilateral infusion of NaCN/KCN (0.5 mol/L) through the microdialysis catheter (N = 5). As a reference, NaCl (0.5 mol/L) was infused by intracerebral microdialysis in one group of animals (N = 3)., Results: PbtO2 increased during cyanide infusion and returned to baseline afterwards. The lactate/pyruvate (LP) ratio increased significantly following cyanide infusion because of a marked increase in lactate level while pyruvate remained within normal limits. Glutamate and glycerol increased after cyanide infusion indicating insufficient energy metabolism and degradation of cellular membranes, respectively., Conclusion: Mitochondrial dysfunction is characterised by an increased LP ratio signifying a shift in cytoplasmatic redox state at normal or elevated PbtO2 . The condition is biochemically characterised by a marked increase in cerebral lactate with a normal or elevated pyruvate level. The metabolic pattern is different from cerebral ischemia, which is characterised by simultaneous decreases in intracerebral pyruvate and PbtO2 . The study supports the hypothesis that cerebral ischemia and mitochondrial dysfunction may be identified and separated at the bedside by utilising intracerebral microdialysis., (© 2013 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Cytochemical analysis of acid phosphatase was used to evaluate lysosomal membranes stability under oral intake of potassium cyanide by rats over one month in daily doses of 1.30 mg/kg (1/10 LD50) and 0.65 mg/kg (1/20 LD50). The authors demonstrated phase-related dose-dependent changes in the lysosomal state, and the main response feature was associated with functional activation that usually followed the membrane alteration.

This study evaluates the toxic effects of five substances (atropine, fenitrothion, potassium cyanide, mercuric chloride and lead nitrate) on the yeast Saccharomyces cerevisiae. It describes a new biological toxicity test based on inhibition of S. cerevisiae viability and compares it with two standard toxicity tests based on Daphnia magna mobility inhibition (EN ISO 6341) and Vibrio fischeri bioluminiscence inhibition (EN ISO 11348-2). The new biological test -S. cerevisiae lethal test - is cheaper and 24 times faster than the D. magna test. The test speed is comparable with the V. fischeri test but the new test is more sensitive for some substances. The test indicates reliably the presence of all used toxicants in water in concentrations which are significantly lower than the concentration in toxic or lethal doses for man. Therefore, this new toxicity test could be proposed for rapid detection of toxic substances in water., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

Oxygen is the ultimate electron acceptor for mitochondrial respiration, a process catalyzed by cytochrome c oxidase (COX). In mammals, oxygen concentration regulates gene transcription of COX subunit IV isoforms. Here, we demonstrate that chemical hypoxia, i.e. inhibition of mitochondrial respiration by application of the COX inhibitors cobalt, cyanide, and azide, affects COX isoform IV-1 and IV-2 transcription in a gender- and brain region-specific way. After treatment with cyanide and cobalt, female cortical and mesencephalic astrocytes, respectively, revealed an up-regulation of COX IV-2 which was accompanied by increased ROS production and necrotic cell death. In male astrocytes, the ratio of COX IV-1/COX IV-2 was lowest after treatment with cobalt and paralleled by highest levels of ROS production and necrosis. These results support the view of a causal correlation of COX IV-2 transcription with cellular oxidative stress and cell death and highlight a gender specificity of these effects. By comparing three toxins, cobalt represented the most potent inducer of overall cell death and resembled most closely the previously observed effects of oxygen deprivation on decreasing the cox4i1/cox4i2 ratio. Overall, an increased sensitivity of male compared with female cell viability towards the toxins was detected. These regulatory responses might be causative for the known gender specificity of toxic and neurodegenerative processes in the brain.

Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research.

This study was proposed to investigate the potential protective effect of alpha-lipoic acid (α-LA) against potassium cyanide (KCN)-induced seizures and lethality in mice. The intraperitoneal ED(50) value of KCN, as measured by induction of clonic and tonic seizures was increased by pretreatment of mice with α-LA (25, 50 and 100 mg/kg) intraperitoneally in a dose-dependent manner. Similarly, the intraperitoneal LD(50) value of KCN, based on 24h mortality, was increased by pretreatment with α-LA in a dose-dependent manner. Intraperitoneal injection of the estimated ED(50) of KCN (4.8 mg/kg) into mice increased, 1h later, nitric oxide (NO) production and brain glutamate and malondialdehyde (MDA) levels. The estimated ED(50) of KCN also decreased brain intracellular reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity in these animals. Administration of the estimated LD(50) of KCN (6 mg/kg) produced, 24h later, similar marked biochemical alterations in surviving animals. Pretreatment of mice with α-LA inhibited; dose-dependently KCN (ED(50) and LD(50))-induced an increase in NO production and brain MDA level as well as a decrease in brain intracellular GSH level and GSH-Px activity. The elevation induced by KCN in brain glutamate level was not inhibited by α-LA. It can be concluded that the protective effect of α-LA against KCN-induced seizures and lethality may be due to inhibition of NO overproduction and maintenance of intracellular antioxidant defense mechanisms., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

Growing concern over environmental pollution by chemicals that may directly or indirectly impact human and ecosystem health make it increasingly necessary to establish a biological early warning system to minimize these impacts. To achieve this, simple and reliable approaches and criteria for environmental risk assessment must be developed. We propose a new approach for detecting behavioral responses to toxicants in flowing water. We conducted a series of tests to determine toxic responses of Japanese medaka (Oryzias latipes) to lethal concentrations of potassium cyanide (KCN; 10 mg L(-1)) and phenol (25 mg L(-1)). Fish movements were tracked by three-dimensional (3D) biomonitoring under flow-through conditions. The 3D data were used to evaluate behavioral responses in terms of swimming speed, vertical position in the water, and entropies of these parameters. Fish exposed to KCN surfaced more frequently than unexposed fish, whereas fish exposed to phenol stayed near the bottom. Maximum swimming speed significantly increased after exposure to both test chemicals, whereas median swimming speed significantly decreased after exposure. Despite large variations in swimming behavior between individual fish, toxic behavioral responses could be identified by the significant decrease in the entropy of vertical position after 10-30 min of exposure to the test chemicals. We conclude that the use of entropy can contribute to the development of a biological early warning system for detecting toxicants at early stages of pollution. To use the present approach to detect lower concentrations of toxicants, further studies should be conducted using long-term monitoring of fish exposed to sublethal concentrations., (© 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.)

Bioreactors containing sessile bacteria (biofilms) grown on hollow fiber membranes have been used for treatment of many wastestreams. Real time operational control of bioreactor performance requires detailed knowledge of the relationship between bulk liquid water quality and physiological transport at the biofilm-liquid interface. Although large data sets exist describing membrane-aerated bioreactor effluent quality, very little real time data is available characterizing boundary layer transport under physiological conditions. A noninvasive, microsensor technique was used to quantify real time (≈1.5 s) changes in oxygen and proton flux for mature Nitrosomonas europaea and Pseudomonas aeruginosa biofilms in membrane-aerated bioreactors following exposure to environmental toxins. Stress response was characterized during exposure to toxins with known mode of action (chlorocarbonyl cyanide phenyl-hydrazone and potassium cyanide), and four environmental toxins (rotenone, 2,4-dinitrophenol, cadmium chloride, and pentachlorophenol). Exposure to sublethal concentrations of all environmental toxins caused significant increases in O(2) and/or H(+) flux (depending on the mode of action). These real time microscale signatures (i.e., fingerprints) of O(2) and H(+) flux can be coupled with bulk liquid analysis to improve our understanding of physiology in counter-diffusion biofilms found within membrane aerated bioreactors; leading to enhanced monitoring/modeling strategies for bioreactor control.

Bacteriophages are viruses that infect bacteria and contribute significant changes in the overall bacterial community. Prophages are formed when temperate bacteriophages integrate their DNA into the bacterial chromosome during the lysogenic cycle of the phage infection to bacteria. The prophage (phage DNA integrated into bacterial genome) on the bacterial genome remains dormant, but can cause cell lysis under certain environmental conditions. This research examined the effect of various environmental stress factors on the ammonia oxidation and prophage induction in a model ammonia oxidizing bacteria Nitrosospira multiformis ATCC 25196. The factors included in the study were pH, temperature, organic carbon (COD), the presence of heavy metal in the form of chromium (VI) and the toxicity as potassium cyanide (KCN). The selected environmental factors are commonly encountered in wastewater treatment processes, where ammonia oxidizing bacteria play a pivotal role of converting ammonia into nitrite. All the factors could induce prophage from N. multiformis demonstrating that cell lysis due to prophage induction could be an important mechanism contributing to the frequent upset in ammonia oxidation efficiency in full scale treatment plants. Among the stress factors considered, pH in the acidic range was the most detrimental to the nitrification efficiency by N. multiformis. The number of virus like particles (VLPs) increased by 2.3E+10 at pH 5 in 5h under acidic pH conditions. The corresponding increases in VLPs at pH values of 7 and 8 were 9.67E+9 and 1.57E+10 in 5h respectively. Cell lysis due to stress resulting in phage induction seemed the primary reason for deteriorated ammonia oxidation by N. multiformis at lower concentrations of Cr (VI) and potassium cyanide. However, direct killing of N. multiformis due to the binding of Cr (VI) and potassium cyanide with cell protein as demonstrated in the literature at higher concentrations of these toxic compounds was the primary mechanism of cell lysis of N. multiformis. Organics represented by the chemical oxygen demand (COD) did not have any effect on the phage induction in N. multiformis. This AOB remained dormant at low temperature (4 degrees C) without any phage induction. Significant decrease in the number of live N. multiformis cells with a corresponding increase in the number of VLPs was recorded when the temperature was increased to 35 degrees C. Death of N. multiformis at 45 degrees C was attributed to the destruction of cell wall rather than to the phage induction., ((c) 2010 Elsevier Ltd. All rights reserved.)

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.

Cyanide is a mitochondrial poison, which is ubiquitously present in the environment. Cyanide-induced oxidative stress is known to play a key role in mediating the neurotoxicity and cell death in rat pheochromocytoma (PC12) cells. PC12 cells are widely used as a model for neurotoxicity assays in vitro. In the present study, we investigated the protective effects of alpha-ketoglutarate (A-KG), a potential cyanide antidote, and N-acetyl cysteine (NAC), an antioxidant against toxicity of cyanide in PC12 cells. Cells were treated with various concentrations (0.625-1.25 mM) of potassium cyanide (KCN) for 4 hours, in the presence or absence of simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM). Cyanide caused marked decrease in the levels of cellular antioxidants like superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). Lipid peroxidation indicated by elevated levels of malondialdehyde (MDA) was found to be accompanied by decreased levels of reduced glutathione (GSH) and total antioxidant status (TAS) of the cells. Cyanide-treated cells showed notable increase in caspase-3 activity and induction of apoptotic type of cell death after 24 hours. A-KG and NAC alone were very effective in restoring the levels of GSH and TAS, but together they significantly resolved the effects of cyanide on antioxidant enzymes, MDA levels, and caspase-3 activity. The present study reveals that combination of A-KG and NAC has critical role in abbrogating the oxidative stress-mediated toxicity of cyanide in PC12 cells. The results suggest potential role of A-KG and NAC in cyanide antagonism.

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 3.0mg 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% NaSO(4). 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.

Chemoreflex afferent fibers terminate in the nucleus tractus solitarii (NTS), but the specific location of the NTS neurons excited by peripheral chemoreflex activation remains to be characterized. Here, the topographic distribution of chemoreflex sensitive cells at the commissural NTS was evaluated. To reach this goal, Fos-immunoreactive neurons (Fos-ir) were accounted in rostro-caudal levels of the intermediate and caudal commissural NTS, after intermittent chemoreflex activation with intravenous injection of potassium cyanide [KCN (80microg/kg) or saline (0.9%, vehicle), one injection every 3min during 30min]. In response to intermittent intravenous injections of KCN, a significant increase in the number of Fos-ir neurons was observed specifically in the lateral intermediate commissural NTS [(LI)NTS (82+/-9 vs. 174+/-16, cell number mean per section)] and lateral caudal commissural NTS [(LC)NTS (71+/-9 vs. 199+/-18, cell number mean per section)]. To evaluate the influence of baroreceptor-mediated inputs following the increase in blood pressure during intermittent chemoreflex activation, we performed an intermittent activation of the arterial baroreflex by intravenous injection of phenylephrine [1.5microg/kg iv (one injection every 3min during 30min)]. This procedure induced no change in Fos-ir in (LI)NTS (64+/-6 vs. 62+/-12, cell number mean per section) or (LC)NTS (56+/-15 vs. 77+/-12, cell number mean per section). These data support the involvement of the commissural NTS in the processing of peripheral chemoreflex, and provide a detailed characterization of the topographical distribution of activated neurons within this brain region.

Cyanide is a potent neurotoxicant that can produce dopaminergic neuronal death in the substantia nigra and is associated with a Parkinson-like syndrome. In this study involvement of Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a BH3-only Bcl-2 protein, in cyanide-induced death of dopaminergic cells was determined in mice and Mes 23.5 cells. Treatment of mice with cyanide up-regulated BNIP3 and Bax expression in tyrosine hydroxylase (TH)-positive cells of the substantia nigra, and progressive loss of TH-positive neurons was observed over a 9-day period. In Mes 23.5 dopaminergic cells, cyanide stimulated translocalization of BNIP3 to both endoplasmic reticulum (ER) and mitochondria. In ER, BNIP3 stimulated release of Ca(2+) into the cytosol, followed by accumulation of mitochondrial Ca(2+), resulting in reduction of mitochondrial membrane potential (Deltapsi(m)) and eventually cell death. Cyanide also activated Bax to colocalize with BNIP3 in ER and mitochondria. Forced overexpression of BNIP3 activated Bax, whereas gene silencing reduced Bax activity. Knockdown of Bax expression by small interfering RNA blocked the BNIP3-mediated changes in ER and mitochondrial Ca(2+) to block cyanide-induced mitochondrial dysfunction and cell death. These findings show that BNIP3-mediates cyanide-induced dopaminergic cell death through a Bax downstream signal that mobilizes ER Ca(2+) stores, followed by mitochondrial Ca(2+) overload.

The logistics involved in obtaining and maintaining large numbers of corals hampers research on the toxicological effects of environmental contaminants for this ecologically and economically important taxon. A method for creating and culturing single-cell suspensions of viable coral cells was developed. Cell segregation/separation was based on specific cell densities and resulting cell cultures were viable for at least 2 mos. Low-density cells lacking symbiotic zooxanthallae and rich in mitochondria were isolated and cultured for toxicity studies. Cells were exposed to differing degrees or concentrations of heat stress, rotenone, cyanide, sulfide, and cuprous oxide. Cells were assayed for mitochondrial membrane potential using the fluorescent probe, JC-9, and for overall viability using the MTT/formazan spectrophotometric viability assay. Significant differences were observed between controls and treatments and the efficacy of this method was validated; only 2 cm(2) of tissue was required for a seven-point concentration-exposure series.

Cyanide is a highly toxic cellular poison that requires immediate and aggressive treatments. Combination of sodium nitrite (SN) and sodium thiosulfate (STS) is the treatment of choice but oral treatment of alpha-ketoglutarate (A-KG) has also been shown to significantly antagonize cyanide poisoning in laboratory animals. This study reports the efficacy of various treatment regimens as: (i) repeated doses of A-KG after simultaneous treatment of A-KG and STS, (ii) repeated doses of A-KG after pre-treatment of SN, STS and A-KG, (iii) repeated doses of STS after pre-treatment of SN, STS and A-KG, and (iv) repeated doses of A-KG and STS after pretreatment of SN, STS and A-KG on mortality of female rats exposed to massive doses of potassium cyanide. A maximum of 40-folds protection was observed when A-KG at 1.0 g kg(-1) after 2 hr and 0.5 g kg(-1) after 4 hr was repeated following the pre-treatment of SN (0.025 g kg(-1); subcutaneous;-45 min), STS (1.0 g kg(-1); intraperitoneal; -15 min) andA-KG (2.0 g kg(-1); oral; -10 min). Similar protection was also conferred by repeating 0.5 g kg(-1) each of A-KG and STS 2 hr after pre-treatment of SN, STS and A-KG. Also, 38-folds protection after simultaneous administration of 20 g kg(-1) A-KG and 1.0 g kg(-1) STS, followed by 2.0 g kg(-1) A-KG after 2 hr was noteworthy The results indicate that repeated treatment of A-KG alone after simultaneous treatment of A-KG and STS or repeated treatment of A-KG alone or with STS after pre-treatment of A-KG, SN and STS have immense potential in challenging extremely high doses of cyanide as compared to the antidotes given once. The study has implications in the development of A-KG as an alternate treatment for cyanide poisoning.

Currently, no reliable noninvasive methods exist for monitoring the severity of in vivo cyanide (CN) toxicity, treatment, and resulting physiological changes. We developed a broadband diffuse optical spectroscopy (DOS) system to measure bulk tissue absorption and scattering. DOS was used to optically monitor CN toxicity and treatment with sodium nitrite (NaNO2). To perform experiments, the DOS probe was placed on the hind leg of rabbits. A sodium CN solution was infused intravenously. DOS and concurrent physiologic measurements were obtained. After completion of CN infusion, NaNO2 was infused to induce methemoglobinemia (MetHb). During infusion of CN, blood gas measurements showed an increase in venous partial pressure of oxygen (pO2), and following reversal, venous pO2 values decreased. DOS measurements demonstrated corresponding changes in hemoglobin oxygenation states and redox states of cytochrome-c oxidase (CcO) during CN infusion and NaNO2 treatment. Therefore, DOS enables detection and monitoring of CN toxicity and treatment with NaNO2.

Cyanide is a rapidly acting mitochondrial poison that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia followed by cell death. Cyanide is predominantly a neurotoxin but its toxic manifestations in non-neuronal cells are also documented. This study addresses the oxidative stress mediated cytotoxicity of cyanide in Rhesus monkey kidney epithelial cells (LLC-MK2). Cells were treated with various concentrations of potassium cyanide (KCN) for different time intervals and cytotoxicity was evidenced by increased leakage of intracellular lactate dehydrogenase, mitochondrial dysfunction (MTT assay) and depleted energy status of cells (ATP assay). Cytotoxicity was accompanied by lipid peroxidation indicated by elevated levels of malondialdehyde (MDA), reactive oxygen species (ROS) and reactive nitrogen species (RNS) (DCF-DA staining), diminished cellular antioxidant status (reduced glutathione (GSH), glutathione peroxidase, superoxide dismutase and catalase). These cascading events triggered an apoptotic kind of cell death characterized by oligonucleosomal DNA fragmentation and nuclear fragmentation (Hoechst 33342 staining). Apoptosis was further confirmed by increased caspase-3 activity. Cyanide-induced cytotoxicity, oxidative stress, and DNA fragmentation were prevented by alpha-ketoglutarate (A-KG) and N-acetyl cysteine (NAC). A-KG is a potential cyanide antidote that confers protection by interacting with cyanide to form cyanohydrin complex while NAC is a free radical scavenger and enhances the cellular GSH levels. The study reveals cytotoxicity of cyanide in cells of renal origin and the protective efficacy of A-KG and NAC.

Different studies have supported neuroprotective effects of Corticotropin-releasing hormone (CRH) against various excitotoxic and oxidative insults in vitro. However, the physiological mechanisms involved in this protection remain largely unknown. The present study was undertaken to determine the impact of CRH administration (at concentrations ranging from 200 fmol to 2 nmol) before and at delayed time intervals following potassium cyanide (KCN)-induced insult in rat primary cortical neurons. A second objective aimed to determine whether kappa and delta opioid receptor (KOR and DOR) blockade, using nor-binaltorphimine and naltrindole respectively (10 microM), could alter CRH-induced cellular protection. Our findings revealed that CRH treatments before or 3 and 8 h following KCN insult conferred significant protection against cortical injury, an effect blocked in cultures treated with alpha-helical CRH (9-41) prior to KCN administration. In addition, KOR and DOR blockade significantly reduced CRH-induced neuronal protection observed 3 but not 8 h post-KCN insult. Using western blotting, we demonstrated increased dynorphin, enkephalin, DOR and KOR protein expression in CRH-treated primary cortical neurons, and immunocytochemistry revealed the presence of opioid peptides and receptors in cortical neurons. These findings suggest protective effects of CRH against KCN-induced neuronal damage, and the contribution of the opioid system in modulating CRH actions.

Oxidative stress plays a crucial role in mediating cyanide toxicity. The present study addresses the effect of cyanide on activity and gene-expression profile of certain antioxidant enzymes and the expression of heat shock protein (HSP-70) in different organs of rats. Rats were treated with 0.50 LD(50) (7.0 mg/kg) of potassium cyanide (KCN; oral) and/or alpha-ketoglutarate (A-KG; 1.0 g/kg; oral) daily for 14 days, and various biochemical variables were measured in brain, liver, and kidney after 7 and 14 days of treatments and a 7-day recovery period. Cyanide significantly reduced the activity of glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CA) in all the organs after 7 days, while the activity of GPx in brain, liver, and kidney, GR in liver, and CA in brain remained diminished up to 14 days. The gene-expression profile of corresponding enzymes did not show any difference between the control and treatment groups. Elevated levels of malondialdehyde were observed in brain and kidney 7 and 14 days after cyanide. Cyanide also increased the expression of HSP-70 activity in brain after 7 days alone. Regression of toxicity was observed after the withdrawal of KCN. Treatment of A-KG was found to prevent all the biochemical alterations caused by cyanide. This study reveals that oxidative stress caused by cyanide was independent of the expression of antioxidant enzyme activity at the gene level, and all changes responded favorably to A-KG, indicating its therapeutic potential.

The principle mitochondrial target where the respiratory inhibitors CO, CN(-), and NO act in the execution of their acute toxic effects is complex IV of the electron-transport chain, cytochrome c oxidase. However, there is a paucity of studies in the literature regarding the concerted effects of such poisons. Accordingly, the combined inhibitory effects of CO + CN(-), NO + CN(-), and NO + CO on the activity of cytochrome c oxidase preparations are reported. Only in the case of CO + CN(-) do the effects of the two inhibitors seem to be additive as expected. NO appears to be antagonistic toward the effects of the other two inhibitors; that is, the effects of both CO an CN(-) on enzyme activity are ameliorated by NO when present. To further clarify these observations, the ligand substitutions of heme-bound CN(-) by NO in cytochrome c oxidase and hemoglobin have also been briefly investigated. These results suggest that displacement of CN(-) from the ferric hemoproteins by NO is rate-limited by heme reduction-and in the case of the enzyme, the presence of nonligand-binding electron-transfer centers facilitates the reaction. The findings are discussed in relation to the idea that NO does not behave as a classic reversible (by dissociation) inhibitor.

During cerebral hypoxia or ischaemia, mitochondrial dysfunction is induced which can lead to free radical production and cell death. This phenomenon is mimicked by the acute administration of mitochondrial poisons such as 3-nitropropionic acid (3-NPA) and potassium cyanide (KCN), with the production of reactive molecular species secondary to the activation of glutamate receptors. Also during ischaemia, the kynurenine pathway of tryptophan metabolism is activated, leading to the production of quinolinic acid and kynurenic acid which can modulate N-methyl-D-aspartate (NMDA) receptors as agonist and antagonist respectively. Since kynurenic acid is known to be neuroprotective, we have now examined its ability to prevent the neurotoxic effects of mitochondrial dysfunction in primary cultures of postnatal rat cerebellar granule neurons. Viability was quantified using the Alamar Blue (AB) assay and by direct morphological examination. Both 3-NPA and KCN (10 microM-1 mM) reduced neuronal viability in a concentration-dependent manner. The NMDA receptor antagonists 2-amino-5-phosphonopentanoic acid (D-AP5) at a concentration of 50 microM, and a 10 microM dose of (+)-5-Methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine hydrogen maleate (MK-801) prevented cell death, although the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) at a concentration of 10 microM did not. The antioxidant enzymes catalase and superoxide dismutase, and the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) afforded partial protection. Kynurenic acid, a glutamate antagonist with preference for the glycine site of the NMDA receptors, had no protective effect at all against 3-NPA or KCN toxicity at concentrations up to 1 mM. Although these data confirm a major role for NMDA receptors and oxidative stress in the neurotoxic effects of mitochondrial inhibitors, they reveal a resistance to kynurenic acid which suggests a non-classical activation of NMDA receptors by mitochondrial inhibitors that is independent of the glycine site or which occurs distal to the site of action of kynurenic acid.