| With the development of power industry,environmental electromagnetic fields have raised public concerns for the possible hazard health effects.The biological effects of extremely low frequency electromagnetic fields(ELF-EMF)(0-300 Hz),especially the power frequency electromagnetic fields(PF-EMF)(50 Hz/ 60 Hz),have been widely investigated.Based on limited epidemiological evidence,the International Agency for Research on Cancer(IARC)has classified the extremely low frequency magnetic fields(ELF-MF)as a possibly carcinogen to humans(Group 2B)in 2002,however,the results of laboratory studies were not consistent on the biological effect by ELF-MF exposure.Furthermore,the mechanism of ELF-MF exposure acting on organism remains unclear.Thus,further studies are necessary to explore ELF-MF induced biological effects and its underling mechanism.Our laboratory has investigated ELF-MF induced biological effects for many years,and previous studies have found that JAR and JEG-3 respond differently to ELF-MF exposure under the same experimental conditions.In JAR cells,50 Hz MF at 3.0 mT and 2.5 ?M of chloride cadmium combined exposure(co-exposure)can temporarily reduce the inhibition of 2.5 ?M of cadmium exposure alone on cell viability,while the effects of the co-exposure on cellular behaviors remain to be evaluated in JEG-3 cells.To further investigate the ELF-MF induced biological effects and underlying mechanism,this study used JEG-3 cells as the research cellular model.After single or co-exposure to 50 Hz MF at 3.0 mT and 5?M of cadmium for 6 hours,proteomics and metabolomics approaches were applied to analyze the changes of protein expression profile and metabolite expression profile.Then,the changes of cell viability and cell cycle were investigated by cell counting kit-8(CCK-8)assay and flow cytometry at different time points under the same exposure condition.The results of proteomics showed that,when the JEG-3 cells were exposed to MF and/or cadmium for 6 hours,differential expressed proteins were screened under the cut-off of 1.2 fold changes.Compared with the sham group,MF exposure resulted in 8proteins up regulated and 2 proteins down regulated,which play roles in intracellular trafficking,secretion,vesicular transport,posttranslational modification,nucleotide transport and metabolism;cadmium exposure resulted in 52 proteins up regulated and32 down regulated,which play roles in signal transduction,translation and ribosome structure;MF and cadmium co-exposure resulted in 60 proteins up regulated and 47 proteins down regulated,which were involved in transcription and posttranslational modification.Compared with the cadmium exposure group,MF and cadmium co-exposure resulted in 8 proteins up regulated and 21 proteins down regulated,which were involved in intracellular trafficking,secretion,vesicular transport,posttranslational modification and protein turnover.The results of Western Blot showed that compared with the sham group,single or co-exposure to MF and cadmium could significantly increase the expression level of HSP70,single or co-exposure to MF and cadmium could significantly increase the expression level of Clusterin.We firstly applied ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC-QTOF/MS)to screen the differential expressed metabolites.841 known metabolites were identified in the positive ion mode and 727 in the negative ion mode.Differential expressed metabolites were screened under cut-off 2 fold changes.In the positive ion mode,compared with the sham group,MF exposure resulted in 18 metabolites up regulated and 12 metabolite down regulated;cadmium exposure resulted in 23 metabolites up regulated and 11 metabolites down regulated;MF and cadmium co-exposure resulted in 21 metabolites up regulated and 8 metabolites down regulated.Compared with the cadmium exposure group,MF and cadmium co-exposure resulted in 19 metabolite up regulated and 14 metabolite down regulated.Differential expressed metabolites were mainly enriched in the pathways of D-arginine and D-ornithine metabolism,glycerophospholipid metabolism and arginine and proline metabolism.In the negative ion mode,compared with the sham group,MF exposure resulted in 52 metabolites up regulated and 17 metabolite down regulated;cadmium exposure resulted in 45 metabolites up regulated and 14 metabolites down regulated;MF and cadmium co-exposure resulted in 37 metabolites up regulated and 18 metabolites down regulated.Compared with the cadmium exposure group,MF and cadmium co-exposure resulted in 16 metabolite up regulated and 26 metabolite down regulated.Differential expressed metabolites were mainly enriched in the pathways of D-arginine and D-ornithine metabolism,one carbon pool by folate and purine metabolism.Another metabolomics approach,gas chromatography time-of-flight mass spectrometry(GC-TOF/MS)was applied to verify the contents of differential expressed metabolites.Compared with the sham group,MF exposure could increase the expression level of L-Phenylalanine,L-Histidine and Pyroglutamic acid,and decrease the expression level of L-Cystine;cadmium exposure could increase the expression level of L-Phenylalanine and D-Ribose,and decrease the expression level of L-Cystine;MF and cadmium co-exposure could increase the expression level of L-Phenylalanine and D-Ribose;Compared with the cadmium exposure group,MF and cadmium co-exposure could increase the expression level of L-Cystine.As to cellular behaviors analysis,the CCK-8 assay showed that,compared with the sham exposure group,the cell viability of JEG-3 cells was significantly increased at 24 hours in MF exposure group;the cell viability was significantly decreased at 6 hours,24 hours and 48 hours in cadmium exposure,or MF and cadmium co-exposure group.The flow cytometry showed that,the cell cycle synchronization could be achieved by treated with 20 ng/ml nocodazole for 18 hours,then cells were exposed to MF and/or cadmium.Compared with the sham group,MF exposure for 1 hour had no significant effect on cell cycle;cadmium exposure significantly increased the percentage of S phase cells;MF and cadmium co-exposure significantly increased the percentage of G1 and S phase cells and significantly decreased the percentage of G2 phase cells.However,there was no significant difference in cell cycle between each two groups after single exposure or co-exposure for 2 hours.In conclusion,under 50Hz MF at 3.0 mT and/or 5?M of cadmium exposure conditions,1)both single exposure to MF or cadmium and co-exposure to MF and cadmium,can change protein and metabolite profiles in JEG-3 cells.Single or co-exposure to MF and cadmium could significantly increase the expression level of HSP70 and Clusterin.Single or co-exposure to MF and cadmium could change the expression levels of L-Phenylalanine,L-Histidine,L-Cystine,Pyroglutamic acid and D-Ribose;2)MF exposure for certain duration could significantly increase cell viability in JEG-3 cells,while cadmium exposure alone,or MF and cadmium co-exposure significantly decreased cell viability;3)MF exposure did not change cell cycle while cadmium exposure alone,or MF and cadmium co-exposure significantly increased the percentage of S phase cells. |
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