Neurotoxicity And Molecular Mechanism Of Tetrabromobisphenol A Bis (2-Hydroxyethyl) Ether In Zebrafish At Different Life Stages

New pollutants may have the characteristics of biological toxicity,environmental persistence and bioaccumulation.Even if the concentration in the environment is low,it may pose a great risk to the ecological environment or human health.At present,"New Pollutant Problems and Challenges" has been listed as one of the major frontier scientific issues in China.Tetrabromobisphenol A bis(2-hydroxyethyl)ether A [TBBPA-DHEE] is one of the derivatives of tetrabromobisphenol A(TBBPA).Because of its wide use,it is released into the environment through many ways and accumulates in various media and organisms.Preliminary studies shows that TBBPA-DHEE is a new pollutant with endocrine disrupting effect,its biological toxicity,ecological and health risks have attracted great attention.In addition,there are few studies on its neurotoxicity to aquatic organisms,especially neurodevelopmental toxicity.The neurotoxicity and their mechanisms of TBBPA-DHEE on aquatic organisms need to be studied urgently.Therefore,this paper focuses on the possible neurotoxicity caused by TBBPA-DHEE exposure and its mechanism of action,especially neurodevelopmental toxicity,and establishes zebrafish exposure models at different life stages(embryonic stage,larval stage,juvenile stage,sexual development stage and adult stage),and systematically studies the neurotoxicity caused by TBBPA-DHEE exposure by behavioral,histological,biochemical and molecular biology methods.Transcriptomics was used to explore the molecular mechanism of its toxicity,so as to reveal the harm and potential health risks of TBBPA-DHEE exposure to aquatic ecosystems,and provide scientific basis for the environmental management of this new pollutant.The main research contents are as follows:(1)Neurodevelopmental toxicity and molecular mechanisms of TBBPA-DHEE exposure in zebrafish embryo.At the embryonic stage,zebrafish was exposed from 0 to 96 hpf,and the exposure concentrations of TBBPA-DHEE were 0.05,0.5 and 5 mg/L.The results showed that after TBBPA-DHEE exposure,the mortality,deformity rate and heart rate of zebrafish embryos increased significantly,while the hatching rate and body length decreased significantly.The levels of thyroid stimulating hormone(TSH)and growth hormone(GH)in the body decreased significantly,showing a dose-dependent relationship.Behavioral studies show that the autonomous movement ability of embryos under light/dark conditions is significantly reduced.Histological study showed that the number of neurons in zebrafish embryonic brain decreased significantly,the nucleus of neurons and the relaxation of nerve fibers were induced.Transcriptional studies showed that TBBPA-DHEE could significantly affect the gene expression of zebrafish embryos,and 579 differentially expressed genes(459 up-regulated,120 downregulated)were screened.KEGG and GO enrichment analysis showed that TBBPA-DHEE was mainly enriched in phototransduction pathway,complement system,JAK-STAT signaling pathway and cytokine-cytokine interaction signaling pathway.RT-q PCR results showed that TBBPA-DHEE could significantly reduce the expression of neurodevelopmental related genes(gnat2,opn1mw1,c5,f5,lepa,lepb,stat3,jak1,ghra,clu,tnfrsf1 b and tgfb1a)in zebrafish.Therefore,TBBPA-DHEE exposure has neurodevelopmental toxicity on zebrafish embryos,its possible molecular mechanism is that it interferes with embryo growth and neurobehavior,and affects the developmental endpoints,hormone levels and the expression of genes involved in neurodevelopmental pathways.(2)Neurodevelopmental toxicity and molecular mechanism of TBBPA-DHEE exposure to zebrafish larval.At the larval stage,zebrafish were exposed from 4 to 11 dpf,and the exposure concentrations of TBBPA-DHEE were 0.05,0.3 and 1.5 mg/L.The results showed that after TBBPA-DHEE exposure,the mortality and deformity rate of larvae increased significantly,the body length decreased significantly,all of which were dose-dependent.Behavioral studies show that the motor ability of larvae was significantly reduced.The activities of acetylcholinesterase(ACh E),superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)in vivo decreased significantly.Histopathological and AO staining experiments showed that TBBPA-DHEE could induce apoptosis of brain cells and damage brain tissues of larvae.Transcriptional studies showed that TBBPA-DHEE significantly affected the gene expression of larvae,and 691 differentially expressed genes(539 up-regulated and 152 down-regulated)were screened.KEGG and GO enrichment analysis showed that differentially expressed genes were mainly enriched in complement and coagulation cascade,estrogen signaling pathway,TNF and IL-17 signaling pathway.Therefore,TBBPA-DHEE exposure caused neurodevelopmental toxicity to zebrafish larvae,and its possible molecular mechanism was that it decreased enzyme activity in zebrafish larvae,and affected the expression of genes in neurodevelopment and endocrine hormone secretion signal pathway,leading to apoptosis of nervous system cells and damage of brain tissue.(3)Neurodevelopmental toxicity and molecular mechanism of TBBPA-DHEE exposure to juvenile zebrafish.At the juvenile stage,zebrafish were exposed from 11 to 35 dpf,and the exposure concentrations of TBBPA-DHEE were 0.05,0.3 and 1.5 mg/L.The results showed that after TBBPA-DHEE exposure,the swimming distance and speed of juvenile fish in high concentration exposure group increased significantly,while that in median concentration exposure group decreased significantly,while that in low concentration exposure group had no significant effect.The content of excitatory neurotransmitters(dopamine,norepinephrine and epinephrine)in the body increased significantly,and showed a dose-dependent relationship,which indicated that TBBPA-DHEE could significantly affect its neurotransmitter secretion and produce endocrine disrupting effect.Transcriptional studies showed that TBBPA-DHEE could significantly affect the gene expression of juvenile fish,and 286 differentially expressed genes(176 up-regulated and 110down-regulated)were screened.KEGG and GO enrichment analysis showed that differentially expressed genes were mainly enriched in PPAR signaling pathway,fatty acid degradation,fatty acid metabolism and cholesterol metabolism signaling pathway.Therefore,TBBPA-DHEE exposure has neurodevelopmental toxicity to zebrafish in juvenile stage,and its possible molecular mechanism is to cause neurotransmitter disorder,activate PPAR signaling pathway and affect gene expression in fatty acid metabolism and other signaling pathways.(4)Neurodevelopmental toxicity of TBBPA-DHEE exposure to zebrafish during sexual development and its molecular mechanism.At the stage of sexual development,zebrafish was exposed from 35 to 75 dpf,and the exposure concentrations of TBBPA-DHEE were 0.05,0.2 and0.3 mg/L.The results showed that after TBBPA-DHEE exposure,the swimming distance and speed of zebrafish during sexual development decreased significantly,and their locomotor ability decreased significantly.The levels of gonadotropin-releasing hormone(Gn RH),luteinizing hormone(LH)and follicle-stimulating hormone(FSH)in vivo decreased significantly.Transcriptional studies showed that TBBPA-DHEE could significantly affect the gene expression of zebrafish during sexual development,and 216 differentially expressed genes(5 up-regulated and 211 down-regulated)were screened.KEGG and GO enrichment analysis showed that the differentially expressed genes were mainly enriched in cell cycle,oocyte meiosis,progesteronemediated oocyte maturation and ovarian steroidogenesis signal pathway.Therefore,TBBPADHEE exposure has neurodevelopmental toxicity on zebrafish during sexual development,and its molecular mechanism may be that its exposure affects gene expression in cell cycle,progestin- mediated oocyte maturation and ovarian steroidogenesis of zebrafish during sexual development,resulting in neuroendocrine disruption.(5)Neurotoxicity and molecular mechanism of long-term TBBPA-DHEE exposure to male and female zebrafish.In this study,zebrafish was exposed from 35 to 135 dpf,and the exposure concentrations of TBBPA-DHEE were 0.05,0.2 and 0.3 mg/L.The results showed that after long-term exposure to TBBPA-DHEE,the locomotor behavior and ability of male and female zebrafish decreased significantly.The level of steroid hormone in vivo decreased significantly,and the level of vitellogenin(VTG)increased significantly.Transcriptional studies showed that 1568 genes were up-regulated and 542 genes were down-regulated in male zebrafish exposure group,and 1265 genes were up-regulated and 535 genes were down-regulated in female zebrafish exposure group.GO and KEGG enrichment analysis showed that differentially expressed genes were mainly enriched in cell cycle and p53 signaling pathway,which were all potential pathways of neuroendocrine disruptors.Therefore,long-term exposure of TBBPA-DHEE is neurotoxic to adult zebrafish,and its possible mechanism is that it affects the neural behavior of male and female zebrafish,affects the gene expression of neuroendocrine system-related pathways such as cell cycle and p53 signal transduction,and finally interferes with the neuroendocrine system function.In summary,TBBPA-DHEE exposure was neurotoxic to zebrafish at different life stages,including embryonic,larval,juvenile and sexual development stages.TBBPA-DHEE showed similar neurodevelopmental toxic effects.However,the neurotoxicity mechanism of pathways to zebrafish at different life stages is different.