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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 Ca2+ levels ([Ca2+]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 A2 (cPLA2) 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 [Ca2+]i and partially blocked the toxicity. In contrast, when the cells were treated with MeHg in the presence of Ca2+ in the culture media, D609 completely prevented cell death with parallel decrease in [Ca2+]i. Our results demonstrated that MeHg-induced toxicity was linked to elevation of [Ca2+]i through activation of PC-PLC, but not attributable to the signaling pathways such as cPLA2, A-SMase, and PKC, or to the generation of ROS.