A Sphingolipids Metabolism Study In The Mechanism Of 50 Hz ELF MF-induced Receptor Clustering

Power frequency magnetic fields, which belong to extremely low frequency magnetic field (ELF-MF), are generated by 50 or 60 Hz alternating current, and usually exists around industry and agriculture electric appliances, household electric appliances, electric power transmission and distribution lines. In recent decades, with rapid development of electric power and widely application of electric appliances, power frequency electromagnetic fields is becoming a kind of potential pollution followed with water, air and solid garbage.50 Hz MF, as a physical stimulus, has been shown its wide biological deleterious effects based on a number of studies. But it is still unclear how cells or organisms transform a physical stimuli signal to a biological signal, and then transduce and induce its final effects. In the previous study, we have found that cells induce to 50 Hz MF could induce clustering of epidermal growth factor receptor (EGFR) and tumor necrosis factor receptor (TNFR) on cellular membrane. Some kinases which usually connect tightly with receptors and mediate intracellular signaling transduction, including SAPK and MAPK, could be phosphorylated and activated by 50 Hz MF. So we supposed that the cellular membrane, especially receptors, could possibly be one of the target sites which ELL-MF interacts with cells or organisms.Several researches showed that a detergent-insoluble glycolipid-enriched complex on cellular membrane called lipid rafts and a kind of hydratase named acid sphingomyelinase (ASM) may possibly be involved in triggering receptor's clustering. Ligand-induced receptor clustering is essential for the activation of many receptors as well as the downstream signaling pathway. The fundamental mechanism for the ligand-induced receptor's clustering has been elucidated. The possible processes includ: receptor ligation, ASM translocation and activation, ceramide generation, fusion of lipid rafts, then the receptors clustering on signaling platforms. The molecular ordering of the early events of certain receptor clustering provides a new tool for study in the mechanism of receptor clustering induced by many non-ligand stimuli. Like the ligand, Some kinds of extracellular factors such as UV-C,γradiation, could also induced receptor clustering. And they almost shared the same mechanism which has been elucidated above. The clustering mostly occurs at the lipid rafts, and sphingolipids play an important role in this process.Sphingolipids which has a long chain sphingoid backbone, not only are cellular structural components, but also act as important role in many intracellular signal transduction pathways. Thus, it is one reason that why kinds of lipids were often involved in many studies related to mechanism of cell surface receptor clustering.In the present study, human amnion FL cells were chosen to investigate the relationship among a 50 Hz MF-induced epidermal growth factor receptor (EGFR) clustering, lipid rafts and acid sphingomyelinase (ASM). We explore the possible mechanism of receptor clustering induced by 50 Hz MF, and study the effects of 0.4 mT 50 Hz ELF-MF on cellular sphingolipids metabolism using the lipidomic highthrough-approach.The role of lipid rafts in EGFR clustering induced by 50Hz magnetic fieldsConfocal microscopy was used to observe EGFR clustering with immunofluorescence method. Cells were divided into two groups, including Nystatin pretreatment group which cells were pretreated with Nystatin, the inhibitor of lipid rafts for 1 h and non-pretreatment group. Every group was set up four subgroups for following treatment respectively: a) sham exposure, b) MF exposure, cells were exposed to 50 Hz, 0.4 mT MF for 15 min, c) positive control, cells were treated with 100 ng/ml EGF for 15 min, and d) resolvent control, cells were treated with DMSO for 15 min. The result showed that both EGF treatment and 50 Hz MF exposure could induce a formation of bright-green spots or bands around the cells, which indicate that both two treatments could induce EGFR clustering. Neither sham exposure nor DMSO treatment could induce the effects. However, Nystatin pretreatment could eliminate or decrease the effect. It is suggested that the clustering of EGFR induced by 50 Hz MF occurred primarily at lipid rafts. 50Hz magnetic fields induces ASM redistribution and co-localization with lipid raftsIn this part of study, cells were divided into three groups for following treatment: a) sham exposure, b) MF exposure, cells were exposure to 50 Hz MF at 0.4 mT for 15 min, and c) positive control, cells were treated with EGF (100 ng/ml) for 15 min. After treatment, the ASM and lipid rafts were labelled respectively with Cy3 and FITC using immunofluorescence method. Confocal microscopy was also employed for observing distribution of ASM and lipid rafts. The results showed that MF exposure could change cellular ASM (labeled with Cy3) from a diffused state in the sham exposure group to a concentrated state on the cell membrane, and co-localized with lipid rafts (labeled with FITC). The statistic difference was significant (P<0.01, students't test) compared to sham exposure group. It is suggested that exposure to 50 Hz MF at 0.4 mT for 15 min could trigger ASM translocated to a concentrated area where the lipid rafts located.The effects of 50Hz magnetic fields on sphingolipids metabolismWe studied the effects of 0.4 mT 50 Hz ELF-MF exposure on cellular sphingolipids metabolism using a lipidomic highthroughput-approach. FL cells were exposed to 50 Hz MF at 0.4 mT for 0 (sham exposure), 5, 15, and 30 min. Cellular sphingolipids were extracted after magnetic fields exposure. Matrix assisted laser desorption/ionization-time of flight MS (MALDI-TOF MS) was used to analyze changes of cellular sphingolipids. The results showed that exposure cells to 50 Hz MF for 30 min could induce a new peak of m/z 704 which corresponded to a kind of ceramide, and the disappearance of peak of m/z 727 which corresponded to a certain kind of sphingomyelin. There appeared one kind of ceramide-monohexoside (CMH) after 15 and 30 min exposure, respectively. The other spectrum peaks, including the peak of m/z 509 (p<0.01, p<0.05 ), m/z 611 (p<0.05, p<0.05 ) and m/z 705 (p<0.01, p<0.05, p<0.01), which corresponde to ceramide, were appeared in both exposure and sham exposure group. However, it is higher than sham exposure group and the statistic difference is significant. It is suggested that 50 Hz MF exposure could affect the ceramide level in cells. Unlike the ceramide, the change of sphingomyelin metabolism was irregular. ConclusionBased on the present experiment, we got the following conclusions:1. EGFR clustering induced by 50 Hz MF depended on intact lipid rafts on cellular membrane. The clustering occurred primarily at lipid rafts.2. Exposure to 50 Hz MF at 0.4 mT could trigger ASM translocate to a concentrated area of cell, and co-localize with lipid rafts.3. Exposure to 50 Hz MF at 0.4 mT could induce ceramide a change of chemical leves. However, the change of sphingomyelin metabolism was irregular.