d-609 and Neuroblastoma

Methylmercury (MeHg) is a ubiquitous environmental toxicant to which humans can be exposed by ingestion of contaminated food. MeHg has been suggested to exert its toxicity through its high reactivity to thiols, generation of arachidonic acid and reactive oxygen species (ROS), and elevation of free intracellular Ca(2+) levels ([Ca(2+)](i)). However, the precise mechanism has not been fully defined. Here we show that phosphatidylcholine-specific phospholipase C (PC-PLC) is a critical pathway for MeHg-induced toxicity in MDCK cells. D609, an inhibitor of PC-PLC, significantly reversed the toxicity in a time- and dose-dependent manner with concomitant inhibition of the diacylglycerol (DAG) generation and the phosphatidylcholine (PC)-breakdown. MeHg activated the group IV cytosolic phospholipase A(2) (cPLA(2)) and acidic form of sphingomyelinase (A-SMase) downstream of PC-PLC, but these enzymes as well as protein kinase C (PKC) were not linked to the toxicity by MeHg. Furthermore, MeHg produced ROS, which did not affect the toxicity. Addition of EGTA to culture media resulted in partial decrease of [Ca(2+)](i) and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca(2+) in the culture media, D609 completely prevented cell death with parallel decrease in [Ca(2+)](i). Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca(2+)](i) through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA(2), A-SMase, and PKC, or to the generation of ROS.

Topics: Animals; Bridged-Ring Compounds; Calcium; Cell Line, Tumor; Cell Survival; Chelating Agents; Diglycerides; Dogs; Dose-Response Relationship, Drug; Egtazic Acid; Environmental Pollutants; Enzyme Activation; Humans; Kidney; Methylmercury Compounds; Neuroblastoma; Norbornanes; Phosphatidylcholines; Phosphodiesterase Inhibitors; Phospholipases A; Reactive Oxygen Species; Sphingomyelin Phosphodiesterase; Thiocarbamates; Thiones; Type C Phospholipases

In our previous studies, TPA treatment of LA-N-1 cells stimulated the production of diacylglycerol in nuclei, probably through the activation of a phospholipase C. Stimulation of the synthesis of nuclear phosphatidylcholine by the activation of CTP:phosphocholine cytidylyltransferase was also observed. The present data show that both effects were inhibited by the pretreatment of the cells with D609, a selective phosphatidylcholine-phospholipase C inhibitor, indicating that the diacylglycerol produced through the hydrolysis of phosphatidylcholine in the nuclei is reutilized for the synthesis of nuclear phosphatidylcholine and is required for the activation of CTP:phosphocholine cytidylyltransferase.

Topics: Antioxidants; Bridged-Ring Compounds; Cell Division; Cell Nucleus; Choline-Phosphate Cytidylyltransferase; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrolysis; Lipid Metabolism; Neuroblastoma; Norbornanes; Phosphatidylcholines; Phosphodiesterase Inhibitors; Thiocarbamates; Thiones; Time Factors; Tumor Cells, Cultured; Type C Phospholipases