Calcium Signaling in Neuronal Cells Exposed to the Munitions Compound Cyclotrimethylenetrinitramine (RDX)

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Abstract
Cyclotrimethylenetrinitramine (RDX) has been used extensively as an explosive in military munitions. Mechanisms for seizure production, seen in past animal studies, have not been described. Increased calcium levels contribute to excitotoxicity, so in this study neuroblastoma cells are loaded with calcium-indicating dye before application of 1.5 µM to 7.5 mM RDX, with fluorescence recorded for 30 cycles of 11 seconds each. The lowest concentration of RDX increases calcium fluorescence significantly above baseline for cycles 2 to 8; millimolar concentrations increase calcium fluorescence significantly above baseline for cycles 2 to 30. Increases in calcium, like those of 200 nM carbachol, are prevented with 10 mM of calcium chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N tetra-acetic acid (EGTA, tetrasodium salt). Calcium channel blocker verapamil (20 μM), Ca2+-ATPase inhibitor thapsigargin (5 μM), and general membrane stabilizer lidocaine (10 mM) partially attenuate carbachol- and RDX-induced increases in calcium, suggesting that RDX transiently increases intracellular calcium by multiple mechanisms.

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