Sensitive Period And Mechanism Of Bisphenol A On Neurotoxicity In Zebrafish Embryo

Objective:In this study,we compared the differences of development toxicity induced by BPA in the embryo of zebrafish in order to investigate the sensitive period and its possible mechanism.Method:1.Detection of development toxicity sensitive period of zebrafish embryos induced by BPA.Embryos from Cleavage Period to Segmentation Period(1-24 hpf),Pharyngula Period(24-48 hpf)and Hatching Period(48-72 hpf)were exposed to 25 ?M BPA,and then cultured to 96 hpf in system water.Mortality,malformation rate,hatching rate,heart rate and alternation of behavior were observed to screen the sensitive period of BPA induced zebrafish development toxicity.2.Transcriptomic analysis by RNA-sequencing(RNA-seq)were used to screen the target gene.Zebrafish larvae were collected at 96 hpf,and then were using for transcriptomic analysis by RNA-sequencing(RNA-seq).After the screen of differentially expressed genes and functional analysis,the target gene of BPA on the neurotoxicity sensitive period of zebrafish was identified,and then verified by fluorescence quantitative PCR.3.Gene rescue experiment of zebrafish.The rescue experiment was carried out by injecting synthetic cypin gene into zebrafish embryos at 1-cell stage,and then the embryos were immediately exposed to BPA to 24 hpf.Mortality and alternation of behavior of zebrafish after overexpression of cypin gene were further observed.Results:1.Embryos from Cleavage Period to Segmentation Period(1-24 hpf)exposed in BPA shown the increased motility and the decreased locomotor activity,which showed that the speed of free swimming in 10 minutes was 18.97%lower than that of the control group;the distance of movement decreased significantly under light-dark cycle stimulation,that is decreased 29.7%in light cycles,respectively,decreased 18.4%respectively in three dark cycles.The heart rate of zebrafish decreased in Pharyngula Period(24-48 hpf)and Hatching Period(48-72 hpf),especial in Pharyngula Period(24-48 hpf)2.Transcriptome analysis showed that there were 131 differentially expressed genes with 39 being up-regulated and 92 being down-regulated in from Cleavage Period to Segmentation Period(1-24 hpf)exposure group.Subsequently,we screened 89 specifically expressed genes with 31 being up-regulated and 58 being down-regulated in from Cleavage Period to Segmentation Period(1-24 hpf)exposure group.Finally,through GO enrichment analysis,gene function analysis and fluorescence quantitative PCR,we found that the gene guanine deaminase(cypin)mRNA level in the group mentioned above was significantly decreased.Therefore,cypin was used as the target gene for further study.3.The results of Rescue experiment showed that cypin mRNA injection could restore the ability of motion of zebrafish,but the mortality had no significant change.Conclusions:1.The sensitivity of BPA to zebrafish embryo neurotoxicity and lethality is from cleavage stage to somatic stage(1-24 hpf),which is manifested by inhibition of zebrafish's motor behavior and increased mortality;BPA is sensitive to cardiotoxicity of zebrafish embryos.The period is pharyngeal(24-48hpf)and hatching(48-72hpf),which shows that the heart rate of zebrafish is reduced,and the pharyngeal period(24-48hpf)is more sensitive.2.The mechanism of zebrafish embryos sensitive to BPA neurotoxicity is:BPA mediates neurotoxicity by down-regulating the expression of cypin mRNA from the cleavage stage to the somatic stage(1-24hpf)and affects the zebrafish's motor behavior.