Calcium And The Endoplasmic Reticulum Associated-pathway Participated In The Paraoxon-induced Apoptosis In EL4 Cells.

Organophosphorus compounds (OPCs) are frequently utilized in agriculture, industry and medicine all over the world. Because of their wide uses, OPCs are also widespread environmental pollutants and potent toxicants to many kinds of organisms, including human beings. Therefore, until the present time, toxic effects and toxicological mechanisms of OPCs have been attended to and been studied in vitro or in vivo by many researchers. In recent years, more and more studies have indicated that induction of apoptosis is a new toxic effect of OPCs, in vitro or in vivo, and that apoptotic cell death may play an important role in OPC-mediated impairing effects. It is necessary to elucidate molecular mechanisms involved in OPC-induced apoptosis, and some researchers have investigated signaling pathways in apoptosis induced by OPCs.According to results obtained now, intracellular calcium signaling has been strongly implicated in induction of apoptosis and regulation of the apoptotic signaling pathways and the endoplasmic reticulum (ER)-associated pathway is one of the three major apoptotic pathways which have been found to date. Until the present time, no one has investigated whether intracellular calcium signals were involved in OPC-induced apoptosis at the early stage of apoptotic process, and whether the ER-associated pathway participated in apoptosis induced by OPCs. In the present study, we used the experimental model in which paraoxon (POX) induced apoptosis in murine EL4 T-lymphocytic leukemia cells. The objective of this study is to investigate whether intracellular calcium signals were involved in POX-induced apoptosis in EL4 cells at the early stage of apoptotic process, and whether the ER-associated pathway participated in this apoptotic process.We used real-time laser scanning confocal microscopy (LSCM) to examine POX-induced changes of intracellular calcium signals at the early stage (0-2 h) of POX-induced apoptosis in EL4 cells, and we investigated apoptotic rates and morphological changes of EL4 cells after treatment with POX using fluorescent microscopy. Caspase-12 is one of key elements in the ER-pathway. We also evaluated changes of caspase-12 expression in POX-induced apoptosis in EL4 cells, using immunohistochemistry (IHC). In the present research, EGTA was used to chelate Ca2+in extracellular medium; heparin and procaine were used as the specific antagonists to IP3 receptor (IP3 R)-associated Ca2+channels and ryanodine receptor (Ry R)-associated Ca2+channels respectively, to inhibit Ca2+efflux from the ER.POX (1-10 nM) increased intracellular calcium signals of EL4 cells in a dose-dependent manner at the early stage (0-2 h) of POX-induced apoptosis, and apoptotic rates of EL4 cells after treatment with POX for 16h also increased in a dose-dependent manner. Pre-incubation with EGTA, heparin or procaine could partly inhibit POX-induced intracellular calcium elevation and apoptosis in EL4 cells. Additionally, POX could up-regulate caspase-12 expression in a dose-dependent manner, and pre-incubation with EGTA, heparin or procaine could significantly inhibit POX-induced increase of caspase-12 expression.Our results suggest that POX induced intracellular calcium increases in EL4 cells at the early stage (0-2 h) of POX-induced apoptotic process, which included the mobilization of Ca2+stored in the ER and Ca2+influx from extracellular medium. Ca2+release from the ER was via IP3 R-associated Ca2+channels and Ry R-associated Ca2+channels. The present study firstly proved that calcium signaling was an important upstream messenger in POX-induced apoptotic process in EL4 cell, and the ER-associated pathway was also involved in this apoptosis.