Intracellular Ceramide Analysis Induced By 50-Hz Magnetic Fields Exposure Using High Performance Liquid Chromatography Tandem Mass Spectrometry

Power frequency (50/60 Hz) magnetic fields (MFs), which belongs to extremely low frequency magnetic fields (ELF-MFs) is produced mainly from electric power system including high voltage overhead power lines (transmission lines),low voltage overhead power lines, residential and occupational electric appliances and so on. With the increase of exposure to power frequency MFs, there is more concern about the related biological effects, both in vitro and in vivo. Epidemiological studies have indicated that power frequency MFs from the vicinity of high-voltage transmission lines increases the incidence of childhood cancer, leukemia, breast cancer and so on. However, there are also opposite results suggested that there was no related between occupational exposure to power frequency MFs and brain tumors or breast cancer. Animal studies showed that exposure to power frequency MFs could produce effects on the hormone levels, fertility, and behavior. None the less there are also negative results. In addition, the results from cellular research in vitro showed that power frequency MFs could induce various biological effects such as enhance cell proliferation, cause DNA damage, and so on. Some other studies showed that ELF-MFs could also produce some non-thermal positive effects, such as retaining calcium in tibias of aged rats, treatment of inflammatory disease, and tendonitis therapy. However, the mechanisms of biological effects of ELF-MFs, especially the loci where ELF-MFs transfers into biological signals and the signal transduction pathways in cells is still unclear.It is well-known that external stimuli, such as CD95, CD40, Fas ligand, ultraviolet andγ-rays could trigger activation and translocation of acid sphingomyelinase (A-SMase) onto the lipid raft of the plasma membrane, hydrolysis of sphingomyelin into ceramide (CER) which can link each other by hydrogen bonds, and induce clustering of lipid raft and the corresponding receptors, amplifying the primary signals, resulting in follow-up biological effects. In the previous studies, we have found that exposure50 Hz MFs at 0.4 mT could induce clustering of epidermal growth factor receptor (EGFR) on cellular membrane, and the clustering was inhibited by disrupting the lipid raft structure. The results suggested that EGFR clustering induced by 50 Hz MFs depended on intact lipid rafts on cellular membrane. We also found that 0.4 mT, 50 Hz MFs exposure could induced clustering of A-SMase from a diffused state to a concentrated state on the cell membrane, which co-localized with lipid raft, and imipramine,an inhibitor of A-SMase activity could block the EGFR clustering induced by 50 Hz MFs exposure, which could be recovered by adding C2-CER in culture medium. It is suggested that A-SMase may participate in the process of EGFR clustering, and CER plays a role in this pathway.Sphingolipids is one of the structural components of the biological membranes of all eukaryotic organisms. The central backbone of CERs is a sphingoid base attached to an acyl chain by an amide bond. The species of CER are defined according the number of carbon atoms attached to the amide-linked fatty acid moiety. Only even-numbered carbon CERs (C2～C28) exists in nature. The long chain CERs (C16～C24) is indicated to be the species generated following cellular stress, such as apoptosis-inducing ligands, DNA damage, and heat shock. In order to investigate the direct effects of 50 Hz MFs exposure on CERs in culture cells, in the present study, reversed-phase high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was applied to analyze the change of CERs in FL cells after 50 Hz MFs exposure for different durations, which has the capacity to selectively target specific analytes from a vast array of compounds in biological samples. Briefly, at first, precursor ion scanning (PIS) mode was performed to obtain the spectrogram of CERs and its derivatives, search lipid maps Structure Database (LMSD) based on the characteristic ion peaks, and make a preliminary recognition of CERs. Then the CERs standard were used to make a confirmation depends on retention time. Next, CER were detected in the multiple reaction mode (MRM) by tandem mass spectrometry in the positive ion mode, and all extracted ion peaks were integrated for quantitative analysis in different 50 Hz MFs, the C17-CER was used as inner standard because it is not a naturally occurring species.Validation studiesC16,C17,C18,C24-CER standards were employed to make experimental parameters optimization in multiple reaction monitoring(MRM) mode. Calibration curves for a mixture of CERs concentrations were made, and the resulting lines of identity were linear with regression coefficients close to 1. Results from the accuracy study for the detection limits of C16-CER, C17-CER, C18-CER and C24-CER were 3.50,2.18,9.32 and 1.02μg/ml respectively; the quantification limits were 11.67, 7.26,31.06 and 3.40μg/ml respectively. Recovery for the CER isolation from cells is 86.67±5.64%.Profile CERs in cells FL cells exposured in different 50 Hz MFs at 0.4 mT for Omin,5min,15min or 30min respectively were mixed in one sample. The total lipids was extracted using Folch method. Analyses were performed using electrospray ionization in the positive ion mode with PIS to select both parent ions (m/z 300-800) and characteristic daughter ions (m/z 264). We exported each single peak for their parent ions on LMSD to screen the probable CERs. The results showed there may be C16,C18 and C24-CER in lipid samples.Confirmation of probable CERsThe CERs standards were employed to exclude the interference of overlapped molecular mass using electrospray ionization in the positive ion mode with MRM to select both parent and characteristic daughter ions to each CER. The results that the retention time of the probable C24-CER in cells samples was in the rention time window (5%) of extracted C24-CER standard confirmed C24-CER exist in cells samples but riot C16,C18-CER. It was found the retention time of extracted CERs standards is shorter than pure CERs standards, indicated that the extraction procedure have an effect on the retention time of CERs, which partly explain the difference between retention time of C24-CER in cells lipid sample and in pure standard.QuantificationBased on the previous results, in the part experiment, the effects of 50 Hz MFs exposure at 0.4mT for different durations on intracellular C24-CER level were explored. C17-CER which is not a naturally occurring species was employed as an inner standard, analyses were performed with MRM mode. The cells were divided into 4 groups:sham exposure,5min exposure,15min exposure and 30min exposure. The results showed that the background value of C24-CER in FL cells was 1.62μg/mg protein. Compared to sham group, exposure to 50 Hz,0.4mT MFs for 5,15 or 30min respectively could enhance the C24-CER level in FL cells (p<0.05). The C24-CER level reached the peak at 15 min exposure time. Compared with 15min exposure, the C24-CER level decreased after 30 min exposure. However, there is no significant statistical difference (p>0.05),and it was still higher than sham and 5min exposure groups (p<0.05)Conclusion Based on the present studies, we got the following conclusions:1) We found C24-CER does exist in FL cells, and the background level is about 1.62μg/mg protein, no other CERs was found.2) Exposure to 50Hz,0.4mT MFs for 5min,15min or 30min could increase the C24-CER level in FL cells.