The Effects Of 50-Hz Magnetic Fields Exposure On Ceramide Metabolism And Related Signaling Pathways In FL Cells

Objective:To analyze the effects of 50-Hz magnetic fields (MFs) exposure on ceramide metabolism and related signaling pathways in human amnion (FL) cells.Methods:FL cells were seeded at a density of 2×105 cells/ml on a 60-mm petri dishes and cultured for 24 h, then exposed to a 50-Hz,0.4 mT MF for 60 min. Total lipid was extracted and analyzed with a HPLC-MS/MS method for sphingomyelin (SM), sphinganine, sphingosine, and sphingosine-1-phosphate (SIP) quantitatively. FL cells were pretreated with imipramine, an inhibitor of A-SMase, and then exposed to a 50-Hz, 0.4 mT MF for 60 min to investigate the mechanism of SM hydrolysis. Cathepsin D activity was assayed using a fluorometric kit. Phosphorylation status of the catalytic subunit of PP2A and ERK1/2 was evaluated after exposure to a 50-Hz,0.4 mT MF for 60 min. Then FL were pretreated with SKI Ⅱ, an inhibitor of SphK, and the relationship between ERK1/2 phosphorylation and SIP was investigated after exposure to a 50-Hz, 0.4 mT MF for 60 min. After exposure to MF for 60 min, FL cells were cultured sequentially for indicated durations (0 h,3 h,6 h,12 h,24 h,36 h), and the proliferation of cells was detected by Cell Counting Kit. In addition, FL cells were pretreated with U0126 (an inhibitor of ERK kinases) and SKI Ⅱ respectively, and exposed to a 50-Hz, 0.4 mT MF for 60 min with indicated culture durations (24 h and 36 h) sequentially, and the proliferation of FL cells was investigated.Results:Compared with sham group, MF exposure for 60 min decreased the levels of SMs. Especially, the differences in the C24-SM, C24:1-SM and C16-SM groups were statistically significant (P<0.05). The decrease of SMs induced by MF could be inhibited by pretreatment with imipramine. In addition, the levels of sphinganine and SIP were increased after MF exposure for 60 min (P<0.05). It is suggested that MF exposure could activate the de novo pathway of ceramide. MF exposure for 60 min could not affect the cathepsin D activity and phosphorylation of PP2Ac on 307 tyrosine residue. But MF exposure for 60 min enhanced ERK1/2 phosphorylation and promoted cell proliferation (P<0.05), which could be inhibited by SKI Ⅱ and U0126 respectively. In addition, cell proliferation induced by MF exposure could be also inhibited by SKI II.Conclusions:1. Exposure to a 50-Hz,0.4 mT MF could trigger acid sphingomyelinase (A-SMase) activation, leading to the hydrolysis of sphingomyelin (SM) to ceramide, and increase the generation of sphinganine in FL cells, which may affect the synthesis of ceramide from the de novo pathway.2. Exposure to a 50-Hz,0.4 mT MF had no effect on cathepsin D activity or PP2Ac tyrosine307 phosphorylation in FL cells.3. A 50-Hz,0.4 mT MF exposure elevated levels of p-ERKl/2 by increasing the generation of SIP, and promoted FL cell proliferation.