Role Of Mitochondria In The Toxication Of The Rat Lymphocyte Induced By Sulfur Mustard

The suppression of the immune system induced by Sulfur mustard,can affect sufferers living quality.Lymphocyte is the major element in the immune system, whose quality and quantity response to the function of the immune system and haematopoietic system in vivio.lt has been found that the cell toxicity induced by sulfur mustard depends on the concentration of mustard gas and post-exposure time.However,the mechanism of sulfur mustard-treated lymphocyte toxicity has not been clarified completely and no successful prevention and cure measure has yet been found.Objiective: Apoptosis and necrosis are two different types of cell death induced by sulfur mustard.Using mitochondria as a main target,we studied the apoptotic toxicity of lymphocyte in vitro and the effect on the immune organ and lymphocyte in vivio induced by sulfur mustard.The relationship between the function of the mitochondria and the toxicity of lymphocyte treated with sulfur mustard has been studied from the change of oxidative/deoxidative state,MPP,Cyt-c and the mitochondria stuctur.The aim of this experiment is to offer more understanding on the route of the toxicity of lymphocyte induced by sulfur mustard,and more theory about the diagnosis,prevention and cure measure.Method: (1)Lymphocyte in vitro intoxication:lymphocytes separated from rats spleen were cultured in vitro.HE stained cell and DNA fragment were detected to make them as the markers of cell damage (apoptosis) induced by sulfur mustard;The change of GSH and GSSG were detected by fluorescence spectrophotometry to reflect the oxidative/deoxidative state; MTT assay was used to measure cell viability(mitochondria function),and fluorescent probe labled with Rhodamine 123 was used to study mitochondria potential;Cyt-c releasing was detected by Western blot and IC blot;the ultrastructure of mitochondria was detected by Transmission electron microscope. (2) Animal in vivo intoxication:Wistar rats were randomly divided into control group and experimental group.The dose of sulfur mustardinjecting to experimental rats was 0.6LD50.The control rat was injected physiological saline solution.We observed and noted the clinic intoxication symptom.The spleen and thymus in the experimental group were collected afer 2h,6h,12h,24h intoxication.The level of ROS,SOD,MDA,and Cyt-c in cell or in mitochondria was measured by ultraviolet spectrophotometry;the change of GSH and GSSG were detected by fluorescence spectrophotometry;the morphology of spleen was investigated with HE staining. (3) Intervention of the GSH on the mitochondria function in vitro: We assay the level of GSH, mitochondria function, and DNA fragment in lymphocyte treated with thee dose of sulfur mustard and sulfur mustard with GSH respectively.Results: (1) The morphological changes as apoptosis and the DNA ladder of the lymphocyte were observed after intoxication.(2)The mitochondria potential decreased at 4h.There was a liner correlation between the mitochondria potential and the level of GSH in lymphocyte.Mitochondria function also decreaed at 4h and there was a liner correlation between the mitochondria function and the level of GSH in lymphocyte.There was a liner correlation between the mitochondria potential and cell viability in lymphocyte. (3)The content of the band of the Cyt-c was increased significantly at 4h compared with control group in western blot,and the same results in the IC.(4)There were no distinct clinic intoxication symptom and organ changes after intoxication compared with control group.When the morphology of spleen were changing,the oxidative/deoxidative state were changing too.And the cytochome (c,cl,aa3,b) of the mitochongdria respiration chain were losing in diferrent content.(5)GSH can maintain the mitochondria function,and alleviate the apoptosis.Conclusions: (1)Apoptosis and necrosis are involed in lymphocyte cytotoxication induced by sulfur mustard,and the mitochondria play the key role in these process.(2) 100mol/L sulfur mustard can induce lymphocyte apoptosis, and the ultrastructure of the mitochongdria chan...