Effects And Mechanisms Of Extremely Low Frequency Magnetic Field On Embryo Development Of Zebrafish Danio Rerio

Extremely low frequency magnetic field(ELF-MF)is a kind of ubiquitous physical environment factor.With the development of science and society,organisms,such as human,mammals and fish,are continuously exposure to ELF-MF,especially to power frequency electromagnetic field(50/60 Hz)of low intensity.Nowadays,more and more attention has been paid to the biological effects of ELF-MF exposure,however little is known about the exact biological effects as well as the mechanism.In this study,zebrafish(Danio rerio)embryos were used to identify the biological effects of ELF-MF exposure with different magnetic induction intensities and different exposure mode.And the molecular mechanism of such effect was also studied by fluorescent quantitative PCR and gene chip analysis.In the present study,fertilized embryos were divided into seven groups as control,sham,and five experimental groups.Embryos of experimental groups were continuously exposed to 50 Hz ELF-MF with intensities of 30,100,200,400,and 800 μT.The sham group was treated as the experimental groups,but without any ELF-MF exposure.The control group was not subjected to anything.The mortality,abnormality rates,hatching rate,and heart rate of zebrafish were detected at 24,36,48,60,72,and 96 hpf(hours post fertilization),respectively.The results showed that no significant differences in embryo mortality and abnormality(P>0.05)were found in all the experimental groups.It has been found that the hatching rate was significantly decreased in relatively high intensity ELF-MF-exposed groups(50 Hz,200,400,and 800 μT)at 48-60 hpf of zebrafish embryos(P<0.05),showing as delayed hatching,whereas such effect was not observed in the low intensity ELF-MF-exposed groups(50 Hz,30,100 μT).In addition,the heart rate of zebrafish embryos was significantly decreased in relatively high intensity ELF-MF-exposed groups(50 Hz,200,400,and 800 μT)at 36-60 hpf(P<0.05),whereas no significant differences was found in relatively low intensity ELF-MF-exposed groups(50 Hz,30,100 μT).Apoptosis of zebrafish were qualitatively detected using acridine orange(AO)staining assays at 96 h post exposure to ELF-MF,and the expression of the apoptosis-related genes caspase-3,caspase-8,caspase-9,and p53 were quantitatively tested by real-time quantitative PCR.It showed that notable signals of apoptosis mainly in the ventral fin and spinal column at high intensity ELF-MF-exposed groups(50 Hz,200,400,and 800μT).At the same time,the transcription of apoptosis-related genes(caspase-3,caspase-9)was significantly upregulated in ELF-MF-exposed embryos(P<0.05).In conclusion,the overall results demonstrated that ELF-MF exposure has detrimental effects on the embryonic development of zebrafish by inducing apoptosis.To comprehensively and systematically analyze the possible way of how ELF-MF affects fish embryonic development,gene chip technology was applied to study zebrafish which has been exposed to ELF-MF for 96 h.GO(Gene Ontology)analysis and pathway analysis were used to screen differentially expressed genes.There are 36232 transcripts in the transcriptome,and the differentially expressed gene was selected while the ratio of the gene expression level between experimental group and control group is ≥2 or ≤0.5.It was found that there are 4268(11.78%),3494(9.64%)and 3859(10.55%)differentially expressed genes in 30 μT,100 μT and 800 μT ELF-MF exposure group,respectively.In which up-regulated genes were 2688,1995,and 1736,and down-regulated genes were 1580,1499,and 2123,respectively.GO analysis and Pathway analysis are carried out based on the function of genes,and the results showed that the up-regulated genes exposed to 30 μT and 100 μT mainly related to metabolism,membrane function and ionic bonding,such factors primarily induce the transformation of signal transduction pathway,like calcium channel.Interestingly,the up-regulated genes of 800 μT ELF-MF exposure group were involved in immune process,leading to changes of immune channel,such as Cytokine-cytokine receptor interaction,Jak-STAT signaling pathway and Toll-like receptor signaling pathway.The results obtained from GO analysis and Pathway analysis showed that ELF-MF in low intensity as 30 μT or 100 μT and in high intensity like 800 μT might have different action mode on embryonic development of zebrafish.The weaker one mainly affect the signaling transduction of zebrafish embryos while the stronger one impact its immune function.Moreover,the effects of ELF-MF with different exposure models on the development of zebrafish embryos was also studied in the present study.Fertilized eggs of zebrafish were exposed to 50 Hz magnetic field with the induction intensity of 100 and 200 μT,respectively.Each exposure group with the same ELF-MF intensity was divided into "1 h on/5 h off","2 h on/4 h off","3 h on/3 h off" and "all-day exposure" groups based on the model of exposure time.The developmental-related indicators,such as mortality,abnormality rates,hatching rate,and heart rate of zebrafish embryos were detected until 96 hpf.The results showed that no significant differences were found in embryonic development of zebrafish in the different model of intermittently exposure.In summary,this study analyzed the effects of ELF-MF exposure on embryonic development of zebrafish with different intensities,and discussed the molecular mechanisms of the biological effects generated by ELF-MF exposure.In addition,the biological effects of ELF-MF with different exposure model was also discussed.The present study provide new evidence on the biological effects as well as its mechanism of ELF-MF,which throw new light on the health risk assessments of ELF-MF in the future study.