Chronic Toxicity And Neurotoxic Mechanism Studies Of KCN

Part One: Chronic Toxicity Studies of KCNObjective: To study the chronic toxicity, the toxicity severity, the main target organand the reversibility of damage in Wistar rats with successive KCN intragastricadministration, so as to find the effective dosage of chronic toxicity, and provide data forchronic cyanide poisoning. Methods: According to the weight,60healthy Wistar rats ofeither gender were randomly divided into control group, low-dose group and high-dosegroup, with20Wistar rats in each group. The drug was administrated by intragastrically atthe daily dosage of0,5,10mg/kg for the three groups respectively for14daysconsecutively with7days of recovery period. General food intake was monitored daily,and the body weight was measured at the7thday,14thday and21stday of administration.At the14thday (withdrawal period) and21thday (recovery period) after administration,half rates of each group were selected to collect blood samples to measure thehematological indexes of red blood cell, hemoglobin, hematocrit, mean cell volume, meancorpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet, whiteblood corpuscle, neutrophils, lymphocytes, monocytes, eosinophils, basophils,granulocytes, large unstained cells, reticulocyte, prothrombin time, activated partialthromboplastin time, thrombin time and plasma fibrinogen and blood biochemical indexesof serum alanine transarninase, aspartate aminotransferase, alkaline phosphatase, lactatedehydrogenase, total bilirubin, blood urea nitrogen, creatinine, total protein, albumin,glucose, total cholesterol, triglyceride, creatine phosphate kinase, Ca, P, K, Naand Cl. Atthe14thday and21thday after administration, half of the experimental animals hadundergone necropsy to make the histopathological examination and bone marrow cellcount and classification. Results:(1)After administration, KCN-treated rats showed upsevere toxicity such as Respiratory depression, tremors, especially the high-dose group, butall returned to normal in20-30minutes.(2)14days after consecutive gastricadministration, body weight of the high dose group male rats decreased.(3) Food intake ofeach group was less affected.(4) No significant effect of KCN was observed onhematological indices of the rats.(5) There was no effect on blood biochemical parametersof KCN-treated rats.(6) The results of bone marrow slides showed no significant effect.(7)During withdrawal period, the absolute and relative brain weight of female rats in the high dose group were significantly decreased; In some rats of high dose group, the gittercells in cerebral cortex showed focal hyperplasia and constituted gliomesenchymal nodule.In the bound of gray matter and white mater, a little neurons degenerated and was filledwith glial cells in focal sites. Some rats even showed vacuolar degeneration in cerebralwhite matter and myelinated fibres decreased significantly. Part of rats in high dose groupshowed focal demyelination at basilar part of the cerebral white matter. In some rats ofhigh dose group neurocytes in gyrus hippocampi decreased significantly, and evenappeared focal elimination in large quantity in some areas, where was filled with glia cells.no obvious abnormality was detected during the recovery period. Conclusions: After theconsecutive gastric administration for14days, KCN-treated rats showed severe toxicity,the target organ is the central nervous system, but the toxic effects are reversible. Part two: Neurotoxic Mechanism Studies of KCNObjective: To study the whole genome changes of Wistar rat’s brain and find thepossible mechanism of neurotoxicity based on KCN14-days gavage test; and to carry outblood and urine metabolomics analysis to identify the impact of metabolites afteradministration. Methods:(1) Preparation of gene chip：Twelve brain tissues of Wistar rat(4from0mg/kg dose group during withdraw period,4from10mg/kg dose group duringwithdraw period and4from10mg/kg dose group during recovery period) were extractedto collect the RNA. After labeling and amplification, the RNA was hybridized with genechips. The chips were scanned and differentially expressed genes were selected.(2)Metabolomics analysis：Urine of24rats at the14days of administration (8rats eachgroup), and serum samples were collected to make metabolomics analysis and the KCNtoxicity biomarkers were screened. Results:(1) the signal of hybridized chip was clearwith low background and high signal to noise ratio. Genechip detection and clusteranalysis achieved effective results.(2) The differentially expressed genes (n=289) werescreened with significant up-expression (signal ratio≧2) or significant down-expression(signal ratio≦0.5) with T-tests.(3) The results of Gene Ontology showed that thesedifferentially expressed genes reflected a variety of biological functions, including positiveregulation of neuroblast proliferation, cerebral cortex development, negative regulation ofneuron apoptosis, positive regulation of cell proliferation, response to calcium ion,regulation of action potential, regulation of cell differentiation, etc.(4) The possiblesignaling pathways of KCN neurotoxicity included focal adhesion s, dopaminergicsynaptic signaling pathway, glutamatergic synaptic signaling pathway, ECM receptorinteraction signaling pathway, neuroactive ligand-receptor interaction, Neurotrophinsignaling pathway, calcium signaling pathway.（5）Metabolomics analysis: In the serummetabolites of high-dose group and low-dose group of KCN, the levels of Phosphocholine,Glycerolphosphocholine, Glutamine, and Methionine increased significantly, while thelevel of lysine reduced significantly. In the Urine metabolites of high-dose group andlow-dose group of KCN, the levels of TMAO, p-Hydroxyphenylacetate, Nicotinate,Hippurate, Dimethylamine increased significantly, while the levels of Succinate, Lactate,Creatinine, Citrulline, Acetate reduced significantly；Conclusions:(1) The central nervoussystem toxicity of KCN may be related to the following effects: the neurotoxicity of KCNis related with calcium homeostasis disorders and Ca2+influx in the nerve cells; byregulating genes of focal adhesion pathway and ECM receptor pathway such as ITGB6, the cell-cell interaction is affected; by regulating genes of dopaminergic synaptic signalingpathway and glutamatergic synaptic signaling pathway such as GRIN2A, synaptictransmission and signaling between nerve cells are regulated; by regulating genes ofNeurotrophin signaling pathway such as NTRK2, the function of nerve cells is affected byNeurotrophin combining with tyrosine kinase receptor Trk.(2) Therefore, the neurotoxicityof KCN may be caused through multiple signaling pathways.The most importantmechanism is due to calcium homeostasis imbalance caused by Ca2+influx, resulting inlipid peroxidation, which therefore further affects the synaptic transmission and signaltransduction in the nerve cells and generate CNS toxicity.(3) Metabolomics analysissuggests that the levels of serum glutamine and Lysine, and the levels of urinary Acetate,Succinate and Citrate can be used as biomarkers of neurotoxicity in rats caused by KCN.However, all these results need further studies to confirm.