Study On The Neurotoxicity And Its Toxicokinetics Of Colistin In Mice

Colistin is an antibiotic polypeptide produced in culture broth of an aerobic spore-forming rod Bacillus Polymyxa Var. colistinus. Although the effectiveness of colistin against multidrug-resistance gram-negative bacteria has not been questioned, early colistin was restricted with its nephrotoxicity and neurotoxicity. In recent year, scholars have been forced to reappraise the clinical value of colistin due to multidrug-resistance gram-negative superbugs. So it is important significance of toxicity mechanism of colistin. In this paper, the mechanism of neurotoxicity induced by colistin was incestigated in vitro and in vivo in mice.In vivo test, the LD50of colistin after i.v.administration in Kunming mice with weight in18-22g were investigated in acute test. Sixty Kunming mice i.v. administrated with maximal tolerance dose of colistin were killed (0.17,0.33,0.5,1.0,2.0,3.0,4.0,6.0,12and24h after injection of colistin) in toxicokinetic test. Blood sample and tissues were collected and concentrations of colistin in plasma and tissues were determined by UPLC/MS/MS. The other18mice were used to determine chemistry parameters in plasma. In subacute test, forty eight mice were divided into four groups randomly, and i.v.administered colistin dosages of0,2.5,5.0,7.5mg-kg-1,respectively. Observed clinical symptoms and determined content of N-Acetyl-Neuraminic and AChE activity in plasma, brain, spinal cord of mice. The other forty eight mice were used to neurobehavioral test (gait, grid walking, heat sensation conduction) in the1d,3d,7d,14d after injection of colistin dosages of0,2.5,5.0,7.5mg·kg-1The results showed as follows:(1) The LD50of colistin after i.v.administration in mice were12.5mg·kg-1, which showed higher toxicity by i.v.administration. Obvious toxic symptoms were observed in mice.(2) In toxicokinetic test, a simple UPLC/MS/MS method had been developed for the quantification of colistin in plasma and tissues. Mice i.v. administrated with maximal tolerance dose of colistin could reflect clinical symptoms of myasthenial of limbs, ataxic, convulsions and dyspnea. It is showed mice were sensitive to colisitn and could be as a animal model. AUC0-∞was25.5μg·h·mL-1and t1/2λz was9.8h, Vz was5.5L·kg-1in plasma. All data showed colistin easily penetrated all tissues. The half life was longest in brain which showed colistin might be accumulated in brain. AUC0-∞is the highest in kidney which showed kidney might be target organ.(3) In subacute test, the dosages of colistin with5mg·kg-1and7.5mg·kg-1could increase content of N-Acetyl-Neuraminic in plasma and AChE activity in plasma, brain and decrease content of N-Acetyl-Neuraminic in spinal cord and brain of mice, which showed colistin could cause neurons damage and apoptosis in mice. The result of neurobehavioral test showed the high dosages of colistin group could cause neurobehavioral change in mice.In vitro test, the dorsal root ganglion (DRG) neurons of mice was selected to study the mechanism of colistin neurotoxicity. The DRG neurons was treated for24h with colistin at dosages of0,62.5,125,250and500μg·mL-1in DMEM/F12. In order to study the mechanism of colistin neurotoxicity, the items about morphology, biochemical, DNA fragmentation, oxygen stress,[Ca2+]i were detected. The results showed as follows:(1) The dosages of colistin with125μg·mL-1could cause DRG neurons cell pathologic change and no apoptosis; the dosages of colistin with250and500μg·mL-1could cause DRG neurons cell pathologic change and apoptosis from the results of morphology, AO/EB, Hoechst33258, DNA Ladder, Caspase-3activity. The cytotoxicity of colistin increased with the increase of concentrations.(2) With the increase of the concentration of colisitn, the activity of ROS increased and the content of GSH decreased in DRG neurons cell. Colistin could lead to the oxidative stress and damage DRG neurons. The results showed that oxidative stress could be one of the mechanisms of colistin neurotoxicity.(3) With the increase of the concentration of colisitn, the content of Ach in DRG neurons cell culture medium decreased. The results showed colistin could affect DRG neurons release Ach.(4) With the increase of the concentration of colisitn, the calcium ion concentration ([Ca2+]i) increased in DRG neurons cell and decreased in DRG neurons cell culture medium. The results showed the change of [Ca2+]i might be one of the mechanism of colistin-induced neurons apoptosis.In sum, high concentration colistin could cause neurons pathologic change, lead to the oxidative stress and induce apoptosis by many ways, finally, damage nervous system.