DEVELOPMENT OF EVALUATION SYSTEM FOR IMMUNOTOXICITY BY DIOXIN COMPOUNDS

点击翻译按钮获取中文摘要

Our aim of this project is a development of assay system for immunotoxicity by dioxin compounds. We tried to find a method for a large number of samples, high sensitivity and low background for the detection of T cell apoptosis. We tested caspase 3 assay system as a possible candidate method. In the process of apoptosis by TCDD, caspase 3 was activated in L-MAT cells. Therefore, we utilized fluorescence dye conjugated to peptide of PARP as a substrate to improve the sensitivity of apoptosis assay. The apoptosis of L-MAT cells by TCDD can be detected as low as 10^5 cells by this method. Using fluorometric-measuring equipment with 96 well culture dish, we developed a mass-screening system.Furthermore, we analyzed the pathway of apoptotic signal to caspase 3. A specific inhibitor of Protein kinase C-theta (PKC-θ) blocked the apoptosis of L-MAT cells by TCDD. 1,2-Diacylglycerol (DAG) is known to activate PKC-θ, therefore, DAG producing enzyme, phospholipase Cγ(PLCγ), was blocked by a specific inhibitor, U73122. The blockade of PLCγ suppressed the apoptosis of L-MAT cells by TCDD. We concluded that TCDD activates PLCγ by some mechanism.