Inhibiting Cytochrome C Oxidase Leads to Alleviated Ischemia Reperfusion Injury

BACKGROUND AND OBJECTIVES: The overall purpose of this study was to investigate the role of cytochrome C oxidase (CcO) in preventing ischemia reperfusion-induced cardiac injury through gaseous signaling molecule pathways. MATERIALS AND METHODS: We used CcO inhibitor, potassium cyanide (KCN) to mimic the pre-treatment of gaseous signaling molecules in a global ischemia/reperfusion (IR) injury model in rats. Intracellular reactive oxygen species (ROS) was determined by measuring mitochondrial H2O2 and mitochondrial complex activity. RESULTS: KCN pre-treatment led to decreased infarction area after IR injury and improved cardiac function. KCN pre-treated group challenged with IR injury was associated with reduced ROS production through inhibition of activity and not downregulation of CcO expression. In addition, KCN pre-treatment was associated with enhanced expression and activity of mitochondrial antioxidase, suggesting the role of CcO in regulating IR injury through oxidative stress. CONCLUSION: KCN pre-treatment reduced the severity of IR injury. The potential mechanism could be increased endogenous anti-oxidase activity and consequently, the enhanced clearance of ROS.

Cyanide Poisoning Deaths Detected at the National Forensic Service Headquarters in Seoul of Korea: A Six Year Survey (2005~2010)

The records of 255 cyanide poisoning deaths obtained from National Forensic Service (NFS) headquarters, located in Seoul of Korea, from 2005 to 2010 were retrospectively reviewed. The mean age was 41.88 +/- 13.09 and range was 6~80 years (unknown in seven cases). The number of deaths of males and females were 200 and 53, respectively (unknown in two cases). The largest number of cases occurred in people aged 40-49 years (81 cases, 31.8%), followed by the age groups 30~39 years (51 cases, 20%), 50~59 years (44 cases, 17.2%) and 20~29 years (43 cases, 16.9%). The total number of deaths among other age groups (below 10, 10~19, 60~69, 70~79, over 80 years and unknown) were 36, representing only 14.1%. Of all cyanide poisoning deaths, 97.3% were due to suicide, and 14.5% of the total number who died received medical treatment. The most frequent site for ingestion was the person's own residence (120 cases, 47.1%) and the route of administration was mainly oral (252, 98.8%). From the total of 255 cyanide poisoning cases, white powders were submitted for analysis in 92 cases. Potassium cyanide and sodium cyanide occupied 51 and 41 cases, respectively. This study showed that poisoning deaths due to cyanide are one of the continuously reported public health problems in Korea. Enforcement of regulations and safety education to prevent cyanide poisoning should be carried out by the government.

Alterations in Membrane Transport Function and Cell Viability Induced by ATP Depletion in Primary Cultured Rabbit Renal Proximal Tubular Cells

This study was undertaken to elucidate the underlying mechanisms of ATP depletion-induced membrane transport dysfunction and cell death in renal proximal tubular cells. ATP depletion was induced by incubating cells with 2.5 mM potassium cyanide (KCN)/0.1 mM iodoacetic acid (IAA), and membrane transport function and cell viability were evaluated by measuring Na+-dependent phosphate uptake and trypan blue exclusion, respectively. ATP depletion resulted in a decrease in Na+-dependent phosphate uptake and cell viability in a time-dependent manner. ATP depletion inhibited Na+-dependent phosphate uptake in cells, when treated with 2 mM ouabain, a Na+ pump-specific inhibitor, suggesting that ATP depletion impairs membrane transport functional integrity. Alterations in Na+-dependent phosphate uptake and cell viability induced by ATP depletion were prevented by the hydrogen peroxide scavenger such as catalase and the hydroxyl radical scavengers (dimethylthiourea and thiourea), and amino acids (glycine and alanine). ATP depletion caused arachidonic acid release and increased mRNA levels of cytosolic phospholipase A2 (cPLA2). The ATP depletion-dependent arachidonic acid release was inhibited by cPLA2 specific inhibitor AACOCF3. ATP depletion-induced alterations in Na+-dependent phosphate uptake and cell viability were prevented by AACOCF3. Inhibition of Na+-dependent phosphate uptake by ATP depletion was prevented by antipain and leupetin, serine/cysteine protease inhibitors, whereas ATP depletion-induced cell death was not altered by these agents. These results indicate that ATP depletion-induced alterations in membrane transport function and cell viability are due to reactive oxygen species generation and cPLA2 activation in renal proximal tubular cells. In addition, the present data suggest that serine/cysteine proteases play an important role in membrane transport dysfunction, but not cell death, induced by ATP depletion.

Effect of Scutellaria flavonoids on KCN-induced damages in rat pheochromocytoma PC12 cells.

BACKGROUND & OBJECTIVE: Cerebral hypoxia is known to be involved in many neurodegenerative diseases such as Alzheimer's and cerebrovascular dementia. The present study was designed to investigate the effects of flavonoids from aerial part of Scutellaria baicalensis Georgi (SSF) on potassium cyanide (KCN) -induced hypoxic cytotoxicity in rat pheochromocytoma cell line PC12, and to understand the probable mechanism. METHODS: The rat pheochromocytoma cell line PC12 was subjected to hypoxia by 200 microM KCN for 30 min. The cytotoxicity of KCN was assessed by cell viability assay, morphological observation, lactate dehydrogenase (LDH) release, malondialdehyde (MDA) production, and the activities of superoxide dismutase (SOD) and Na+-K+-ATPase measurements. The effects of SSF on the changes induced by KCN in PC12 cells were detected. RESULTS: Treatment of PC12 cells with 200 micriM KCN for 30 min increased cell death when compared with control, as assayed by MTT reduction, morphological observation and lactate dehydrogenase release measurement. These cell lesions were accompanied by disorders in SOD and Na+-K+-ATPase activities as well as MDA production. In contrast, the PC12 cells pre-treated with SSF for 24 h prior to 200 microM KCN exposure have shown protection against hypoxic toxicity. The KCN - induced decreased cell viability and activities of SOD and Na+-K+-ATPase, as well as increased MDA production were reversed by SSF pre-treatment. INTERPRETATION & CONCLUSION: SSF exerted neuroprotections against KCN - induced hypoxic cytotoxicity in PC12 cells and the probable mechanisms involved free radicals and energy metabolism. Our findings may have implications in future in the treatment of neurodegenerative diseases.

Effect of pre-treatment of alpha-ketoglutarate on cyanide-induced toxicity and alterations in various physiological variables in rodents / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the effects of pre-treatment of alpha-ketoglutarate (alpha-KG) on cyanide-induced lethality and changes in various physiological parameters in rodents.</p><p><b>METHODS</b>The LD50 of potassium cyanide (KCN) given orally (po), intraperitoneally (ip), subcutaneously (sc) or intravenously (iv) was determined in male mice, in the presence or absence alpha-KG given po, ip or iv. alpha-KG was administered 10, 20 or 40 min prior to KCN at 0.50, 1.0 or 2.0 g/kg by po or ip route, and at 0.10, 0.20 or 0.40 g/kg by iv route. Protection index (PI) was calculated as the ratio of LD50 of KCN in the presence of alpha-KG (protected animals) and LD50 of KCN in the absence of alpha-KG (unprotected animals). In a separate experiment, several physiological variables viz. mean arterial pressure (MAP), heart rate (HR), respiratory rate (RR), neuromuscular transmission (NMT) and rectal temperature (RT) were measured in anesthetized female rats pre-treated (-10 min) with po (2.0 g/kg) or iv (0.125 g/kg) alpha-KG and then administered sub-lethal (0.75 LD50) or lethal (2.0, 4.0 or 8.0 LD50) doses of KCN (po).</p><p><b>RESULTS</b>PI of 4.52, 6.40 and 7.60 at -10 min, 3.20, 5.40 and 6.40 at -20 min, and 1.40, 3.20 and 5.40 at -40 min of po administration with a-KG was observed for 0.50, 1.0 and 2.0 g/kg doses, respectively, against KCN given by po route. When KCN was given ip, a PI of 3.38, 4.79 and 5.70 was observed for 0.50, 1.0 and 2.0 g/kg alpha-KG given ip (-10 min), respectively. A lower PI of 3.37, 2.83 and 2.38 was observed when KCN given sc was challenged by 2.0 g/kg alpha-KG given ip at -10, -20 or -40 min, respectively. Similarly, a PI of 3.37, 2.83 and 2.0 was noted when KCN given sc was antagonized by 2.0 g/kg alpha-KG given po at -10, -20 or -40 min, respectively. No appreciable protection was observed when lower doses of alpha-KG (ip or po) challenged KCN given by sc route. Pre-treatment of iv or po administration of alpha-KG did not afford any protection against KCN given po or iv route. Oral treatment of 0.75 LD50 KCN caused significant decrease in MAP and HR after 15 min, RR after 30 min and NMT after 60 min. There was no effect on RT. No reduction in MAP, HR, RR and RT was observed when rats received 2.0 or 4.0 LD50 KCN after pre-treatment of alpha-KG (po; 2.0 g/kg). However, no protection was observed on NMT. Protective efficacy of alpha-KG was not observed on MAP, HR, RR, and NMT decreased by 8.0 LD50 KCN. Decrease in MAP and NMT caused by 2.0 LD50 KCN (po) was resolved by iv administration of alpha-KG.</p><p><b>CONCLUSIONS</b>Cyanide antagonism by alpha-KG is best exhibited when both alpha-KG and KCN are given by po route. The protective effect of a-KG on cyanide-induced changes in several physiological parameters also indicates a promising role of alpha-KG as an alternative cyanide antidote.</p>

Effect of alpha-ketoglutarate on cyanide-induced biochemical alterations in rat brain and liver / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate the biochemical changes in rat brain and liver following acute exposure to a lethal dose of cyanide, and its response to treatment of alpha-ketoglutarate (alpha-KG) in the absence or presence of sodium thiosulfate (STS).</p><p><b>METHODS</b>Female rats were administered 2.0 LD50 potassium cyanide (KCN; oral) in the absence or presence of pre-treatment (-10 min), simultaneous treatment (0 min) or post-treatment (+2-3 min) of alpha-KG (2.0 g/kg, oral) and/or STS (1.0 g/kg, intraperitoneal, -15 min, 0 min or + 2-3 min). At the time of onset of signs and symptoms of KCN toxicity (2-4 min) and at the time of death (5-15 min), various parameters particularly akin to oxidative stress viz. cytochrome oxidase (CYTOX), superoxide dismutase (SOD), glutathione peroxidase (GPx), reduced glutathione (GSH) and oxidized glutathione (GSSG) in brain, and CYTOX, sorbitol dehydrogenase (SDH), alkaline phosphatase (ALP), GSH and GSSG in liver homogenate were measured.</p><p><b>RESULTS</b>At both time intervals brain CYTOX, SOD, GPx, and GSH significantly reduced (percent inhibition compared to control) to 24%, 56%, 77%, and 65%, and 44%, 46%, 78%, and 57%, respectively. At the corresponding time points liver CYTOX and GSH reduced to 74% and 63%, and 44% and 68%, respectively. The levels of GSSG in the brain and liver, and hepatic ALP and SDH were unchanged. Pre-treatment and simultaneous treatment of a-KG alone or with STS conferred significant protection on above variables. Post-treatment was effective in restoring the changes in liver but failed to normalize the changes in the brain.</p><p><b>CONCLUSIONS</b>Oral treatment with alpha-KG alone or in combination with STS has protective effects on cyanide-induced biochemical alterations in rat brain and liver.</p>

Modulation of Muscarinic K+ Channel by Protein Kinase C in Ischemic Rat Atrial Myocytes

BACKGROUND AND OBJECTIVES: Recent studies have shown that many kinds of K+ channels, including the muscarinic K+ channel (KACh), are activated in the ischemic heart. It is known that these channels can be modulated by phosphorylation. However, little is known about the function of the KACh in ischemic hearts. In this study, we examined whether the KACh channel is mediated by protein kinase C (PKC) activation in rat atrial myocytes under ischemic conditions. MATERIALS AND METHODS: Single atrial cells of adult rat heart were prepared by collagenase digestion. Channel activity of KACh was recorded by cell-attached configuration from single atrial cells under ischemic conditions, using a patch clamp technique. To simulate ischemia, adenosine or potassium cyanide (KCN) was applied to atrial myocytes, and Western blot was performed to specify PKC isoforms. RESULTS: Adenosine and KCN markedly increased KACh channel activity. The responses to adenosine and KCN were increased 3-fold at mean open time from that observed with control. Channel activity of KACh was blocked by pretreatment with PKC antagonists such as sphingosine, Go 6976, and rottlerin. PKC alpha and PKC betaI isoform levels were increased in the membrane fraction of ischemic heart, indicating that ischemic stress might trigger translocation of cytosolic PKC to the cell membrane. CONCLUSION: These results show that KACh channels are modulated by PKC activation under ischemic conditions induced by adenosine or KCN. Therefore, the channels can protect the heart from ischemic stress by increasing channel activity.

Neural reflex regulation of arterial pressure in pathophysiological conditions: interplay among the baroreflex, the cardiopulmonary reflexes and the chemoreflex

The maintenance of arterial pressure at levels adequate to perfuse the tissues is a basic requirement for the constancy of the internal environment and survival.The objective of the present review was to provide information about the basic relfex mechanisms that are responsible for the moment-to-moment regulation of the cardiovascular system. We demonstrate that this control is largely provided by the action of arterial and non-arterial reflexes that detect and correct changes in arterial pressure (baroreflex), blood volume or chemical composition (mechano-and chemosensitive cardiopulmonary reflexes), and changes in bloodgas composition (chemoreceptor reflex). The importance of the integration of these cardiovascular reflexes is well understood and it is clear that processing mainly occurs in the nucleus tractus solitarii, although the mechanism is poorly understood.There are several indications that the interactions of baroreflex, chemoreflex and Bezold-Jarisch reflex inputs, and the central nervous system control the activity of autonomic preganglionic neurons through parallel afferent and efferent pathways to achieve cardiovascular homeostasis. It is surprising that so little appears in the literature about the integration of these neural reflexes in cardiovascular function. Thus, our purpose was to review the interplay between peripheral neural reflex mechanisms of arterial blood pressure and blood volume regulation in physiological and pathophysiological states. Special emphasis is placed on the experimental model or arterial hypertension induced by N-nitro-L-arginine methyl ester (L-NAME) in which the interplay of these three reflexes is demonstrable.

Autonomic processing of the cardiovascular reflexes in the nucleus tractus solitarii

The nucleus tractus solitarii (NTS) receives afferent projections from the arterial baroreceptors, carotid chemoreceptors and cardiopulmonary receptors and as a function of this information produces autonomic adjustments in order to maintain arterial blood pressure within a narrow range of variation.The activation of each of these cardiovascular afferents produces a specific autonomic response by the excitation of neuronal projections from the NTS to the ventrolateral areas of the medulla (nucleus ambiguus, caudal and rostral ventrolateral medulla). The neurotransmitters at the NTS level as well as the excitatory amino acid (EAA) receptors involved in the processing of the autonomic responses in the NTS, although extensively studied, remain to be completely elucidated. In the present review we discuss the role of the EAA L-glutamate and its different receptor subtypes in the processing of the cardiovascular reflexes in the NTS. The data presented in this review related to the neurotransmission in the NTS are based on experimental evidence obtained in our laboratory in unanesthetized rats. The two major conclusions of the present review are that a) the excitation of the cardiovagal component by cardiovascular relfex activation (chemo- and Bezold-Jarisch reflexes) or by L-glutamatae microinjection into the NTS is mediated by N-methyl-D-aspartate (NMDA) receptors, and b) the sympatho-excitatory componente of the chemoreflex and the pressor response to L-glutamate microinjected into the NTS are not affected by an NMDA receptor antagonist, suggesting that the sympatho-excitatory component of these responses is mediated by non-NMDA receptors.

Studies on electron donation to photosystem I sites by exogenous donors in Spirulina thylakoids.

The kinetic parameters of different sites of electron donation to photosystem I (PS I) were evaluated in Spirulina platensis thylakoids. Reduced 2,6-dichlorophenolindophenol (DCIPH2) exhibited two sites of electron donation, with apparent K(m) values of 8 and 40 microM each. The corresponding value for reduced N-tetramethyl-p-phenylenediamine (TMPDH2) and diaminodurene (DADH2) which donate electrons at a single site to PS I were 103 and 48 microM, respectively. The electron donation by these three exogenous donors were differentially inhibited by KCN (70 mM) affecting the apparent K(m) and Rmax values to varying extent. This cyanide inhibition of PS I catalyzed electron transport suggests the presence of plastocyanin in the photosynthetic electron transport chain of Spirulina platensis.

Cyanide induced changes in dynamic pulmonary mechanics in rats.

Effect of subcutaneously (s.c.) administered potassium cyanide (0.5 and 1.0 LD50) and inhalation of hydrogen cyanide (55 ppm or 60.6 mg/m3) for 30 minutes was studied on various physiological parameters related to dynamic pulmonary mechanics in anaesthetized rats. Total pulmonary phospholipid with its fractions were also estimated. Both s.c. (1.0 LD50) and inhalation exposures increased air flow, transthoracic pressure and tidal volume accompanied by significant decrease in pulmonary phospholipids. Inhalation of hydrogen cyanide also exhibited direct effect on the pulmonary cells as evidenced by decreased compliance. The study suggests that inhalation of cyanide is more injurious compared to parenteral route.

Effect of potassium cyanide [KCN] and sodium azide NaN3 on the growth of streptococcus pyogenes Gr A

In order to show that the growth of S. pyogenes Group A was independent of both Fe and O2 as a terminal electron acceptor, KCN and NaN3 were added to the growth medium at levels which completely inhibited growth of aerobic bacteria. These agents have no effect on the growth of S. pyogenes at concentration below 100 mM, proving that the growth was independent of cyt aa3 oxidoreductase and O2

Potentiation of radiation effects in plateau phase human glioma cells by combination of metabolic inhibitors.

Effects of glycolytic inhibitor 2-deoxy-D-glucose (2-DG) on radiation damage were studied in a human glioma cell line (BMG-1), grown to confluence in monolayer. After irradiation (60Co-gamma-rays, 2 Gy) and incubation with low concentrations of 2-DG (0.5, 1.25 mM; 2-DG/glucose = 0.1, 0.25; 2 hr), in the absence or presence of respiratory inhibitor KCN (0.5-2 mM), cells were trypsinized and plated to assay radiation induced cytogenetic damage (micronuclei formation). The observations made were: (1) 2-DG and/or KCN treatments did not induce damage in unirradiated cells. (2) Either of these treatments did not increase radiation induced micronuclei formation. (3) Presence of 2-DG along with KCN (1,2 mM) significantly enhanced the radiation induced micronuclei formation. (4) Preliminary experiments by macrocolony assay showed that radiation induced cell death was also significantly increased by the combined treatment. These observations suggest that presence of clinically feasible, low concentrations of 2-DG (2-DG/glucose < 0.5) for short intervals of time after radiation could increase radiation damage in non-cycling, hypoxic tumour cells with impaired oxidative and increased glycolytic energy metabolism.

A simple technique for hemoglobin estimation to screen for anaemia.

Drabkins method has been modified enabling detection of anaemia in a large population. 132 samples of EDTA blood were subjected to hemoglobin estimation by (1) Direct Drabkin (DD) (2), Filter Drabkin (FD) and (3) Special Filter Drabkin (SFD). Hemoglobin estimations by DD and FD compared well on statistical analysis. SFD with a punch diameter of 10.6 to 10.7 mm compared well with DD and is ideal for screening anaemia in field studies.

Acute cyanide poisoning: a case report with toxicokinetic study.

Cyanide poisoning is a life threatening condition. But specific antidotes exist and can be easily prepared from available substances in hospital. Administration of antidotes will produce methemoglobin, which itself causes hypoxia. Nitrite induced methemoglobin can be extremely dangerous and even lethal. Before administering the antidotes, the diagnosis should be confirmed. Nitrite should not be given if the poisoning is mild or diagnosis is uncertain, to avoid excessive methemoglobin, dosage of sodium nitrite must be adjusted according to hemoglobin level (Table 1). Usage of sodium nitrite and sodium thiosulfate in the recommended doses are safe and effective for cyanide poisoning.

Intermediary metabolic changes in rabbits administered linamarin or potassium cyanide.

Since cassava (Manihot esculenta Crantz) is a staple food item of several million people in the tropics, its toxicity cannot be underestimated. Therefore an attempt has been made to understand the metabolic changes caused by the chronic administration of sublethal doses linamarin, the principal cyanoglucoside of cassava, to rabbits. A significant rise in lactic acid and total cholesterol in liver and brain and a highly significant depletion of phospholipids of brain tissue was observed. There were also significant variations in the lactate dehydrogenase isoenzyme pattern of treated animals as compared with the control rabbits. The findings suggest that some of the biological effects of linamarin are similar to those of free cyanide.

Pattern of enzyme changes in rabbits administered linamarin or potassium cyanide.

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

CT Brain Scan in Case of Cyanide Intoxication

Eating the meat of pheasants which was killed by potassium cyanide, a 27 year old man developed several symptoms of minor intoxication. A few days later, the patient became mute, apathy, somnelent and indifferent but without any parkinsonia features or dementia. On CT brain scan, bilateral symmetrical and non-enhancing focal low densities are noticed in basal ganglia, similar to the carbon monoxide intoxication.