The Mechanism Of Sphingosine-1-Phosphate Regulating Cell Proliferation Induced By 50 Hz Magnetic Field And Co-exposure With DEHP

The extremely low-frequency electromagnetic fields（ELF-EMFs）,with a frequency range of 0-300 Hz,are mainly generated from the use of power transmission,domestic appliances and industrial electronic equipment.Specifically,magnetic fields（MFs）with a frequency of 50 or 60 Hz,which are also called power frequency magnetic fields（PFMFs）,exists widely in public and occupational environments.With the rapid development of science and technology,the intensity of ELF-EMFs increased gradually in the environment,which raised public concerns about the potential adverse effects thereof on human health.Epidemiological investigations have reported that ELF-EMFs exposure were associated with cancers,adverse pregnancy outcomes,neurodegenerative diseases,etc.The International Agency for Research on Cancer（IARC）even classified ELF-EMFs as a possibly carcinogen to humans（Group 2B）in 2002.However,the biological mechanism underlying the effects of ELF-EMFs is still not sufficiently understood.The uncertainty over the potential adverse health effects of ELF-EMFs urgently requires clarification.On the other hand,the ELF-EMFs usually exits with many chemical pollutants in our environment.Thus,it also becomes a great concern whether there is a synergistic effect with the combined exposure to the weak ELF-EMFs and low-dose environmental chemicals.Studies have reported that PFMF exposure could influence cell proliferation,differentiation,migration and apoptosis.However,the mechanism of these cellular effects induced by PFMF has not been elucidated clearly.Our recent study found that a 0.4 m T50 Hz magnetic field（MF）exposure could promote Sphingosine-1-phosphate（S1P）production and mediate cell proliferation.In this study,the mechanism of S1P-related signal pathway on regulating the PFMF-induced cell proliferation,as well as the potential synergistic effects of PFMF and low-dose of di-（2-ethylhexyl）phthalate（DEHP）,was explored in human amniotic epithelial（FL）cells,which are sensitive to the magnetic fields.The results showed that the exposure to a PFMF at 0.4 m T for 1 h or 24 h could induce the FL cell proliferation and increase the percentage of G2/M phase.Although PFMF exposure had no significant effect on apoptosis,it could promote the anti-apoptotic effect.After inhibiting S1P synthetase with SphK inhibitor（SKI II）,we found that the PFMF-induced cell proliferation was inhibited,and the anti-apoptosis effect was also weakened.It is suggested that S1P was involved in the process of PFMF-induced cell proliferation and anti-apoptosis.In order to clarify the specific role of S1P-related pathways in PFMF-induced cell proliferation,the metabolism,transport and related downstream signal molecules of S1P were explored.Results found that PFMF exposure could activate SphK1,but had no effect on SphK2,S1P lyase（SPL）and S1P phosphorylase（SPP）,indicating that PFMF exposure increase the S1P production mainly via promoting the synthesis of S1P.The study showed that the expression of S1P transporter（sphingolipid transporter 2,SPNS2）was up-regulated by PFMF exposure;knocking down SPNS2 could inhibit PFMF-induced cell proliferation;exogenous S1P rescued the decrease of cell proliferation caused by inhibition of S1P synthesis;and antagonists of S1PR1/3 attenuated PFMF-induced cell proliferation.These findings suggested that the PFMF-increased S1P could promote cell proliferation through activating S1PR1 and S1PR3 in the"inside-out"way.In order to reveal the downstream signal molecules activated by SphK1-S1P-S1PR1/3,we detected the phosphorylation level of proliferation-related signal molecules exposed to PFMF.It was found that Akt and Erk-related signal molecules could be activated by PFMF.After treated with SKI II,si SphK and S1PRs antagonist respectively,the results showed that inhibition or knockdown of SphK1 and antagonism of S1PR1/3 could significantly inhibit Erk phosphorylation as well as cell proliferation induced by PFMF exposure,but did not affect Akt phosphorylation.These results indicated that PFMF promoted cell proliferation via SphK1-S1P-S1PR1/3-activated Erk pathway.Further studies found that PFMF exposure raised the level of ROS through increasing intracellular Ca²⁺,and cascadedly activated Akt-SphK1 which triggered SphK1-S1P-S1PR1/3-Erk cascade signal pathway and promoted the FL cell proliferation finally.Additionally,we found that exposure to low-dose DEHP also could promote FL cell proliferation,while high dose of DEHP had an inhibitive effect.In order to investigate the possible synergistic effects,the under-threshold concentration/intensity of DEHP（0.1μM）and PFMF（0.2 m T）were chosen in the followed experiments.Results showed when FL cells were treated jointly by a 0.2 m T PFMF and 0.1μM DEHP,the proliferation rate of cells was significantly higher than that of DEHP group and MF group,suggesting that the co-exposure of PFMF and DEHP had a synergistic effect on the cell proliferation.Inhibition the activity of Akt,SphK1 and Erk with specific inhibitor could effectively attenuate cell proliferation induced by co-exposure,indicating that co-exposure of PFMF and DEHP could cooperatively activate the same pathway to promote cell proliferation.To further explore the effects on embryonic development exposed to single or combined factors of PFMF and DEHP,zebrafish embryos were exposed to different concentrations/intensities of DEHP and PFMF,or co-exposed to 0.25 mg/L DEHP and0.2 m T PFMF.Results showed that higher-concentrations DEHP or 0.4 m T PFMF exposure alone could increase the heart rate of zebrafish embryos,meanwhile,co-exposure could synergistically increase the heart rate and the activation of Akt and Sphk1 of zebrafish embryos.Based on the present experiments,we concluded:1)PFMF exposure could promote FL cell proliferation,change the distribution of cell cycle and enhance anti-apoptosis;2)PFMF exposure increased the level of ROS mediated by Ca²⁺to stimulate the activation of Akt,and then trigger SphK1-S1P-S1PR1/3-ERK cascade signal pathway to promote FL cell proliferation;3)Co-exposure to under-threshold DEHP and PFMF could synergistically promote the FL cell proliferation via activating Akt-SphK1-Erk signal pathway;4)Exposure to PFMF and DEHP alone or in combination could increase the heart rate of zebrafish embryos.