Study On The Role And Mechanisms Of The Biotransformation Of CYP3A65 In BDE47-induced The Developmental Abnormalities In Zebrafish

Polybrominated diphenyl ethers（PBDEs）are one of the most widely used additive brominated flame retardants.2,2’4,4’-tetrabromodiphenyl ether（BDE47）is one of the most important PBDEs and it has the highest content in blood and breast milk,accounting for more than 50%of the total PBDEs.Studies have shown that BDE47 has developmental toxicity,reproductive toxicity,neurotoxicity,and endocrine disruptive toxicity.It can also affect adult susceptibility through direct action on genes or epigenetic effects in early developmental stages.Multiple birth cohort studies have shown that prenatal exposure to BDE47 is associated with mental retardation,hyperactivity disorder,and neurological impairment,suggesting that exposure to BDE47 may impair normal development in children.Biotransformation has always been an important way for environmental chemicals to exert their biological effects.The process requires the participation of specific metabolic enzymes and is metabolized to some active metabolites.Our previous studies showed that BDE47 induced the expression of CYP450（especially CYP3A）and was metabolized in vitro as a metabolizing substrate for CYP3A4,we concluded the mechanism of CYP3A4 induced toxicity of BDE47.Development is closely related to human reproductive health,zebrafish is the most suitable research model for developmental toxicity.CYP3A65 gene has 54%homology with human CYP3A4,but its role and mechanism in the developmental toxicity of zebrafish caused by BDE47 are not yet clear.In this study,BDE47 was applied to zebrafish by establishing in vitro BDE47exposure model and using emerging models of zebrafish as a developmental toxicity model by mapping the basal expression profile of CYP3A65 in zebrafish.We verified developmental toxic effect and detected active major metabolites in vivo.These three aspects are inseparable and interlocking.The effects of BDE47 on the spatial and temporal expression profiles of zebrafish CYP3A65 and its regulatory mechanisms were highlighted.In addition,the molecular mechanism of CYP3A65-mediated biotransformation in the dysplasia of zebrafish caused by BDE47 was elucidated.The results of the study will help to thoroughly understand the role of CYP3A65 in in situ metabolism of BDE-47 and its developmental anomalies effects,we will provide an important basis for controlling the developmental toxicity of PBDEs.PartⅠ:The basic expression profile of CYP3A65 and its regulation mechanism induced by BDE47 in zebrafishObiective:To investigate the expression of CYP3A65 and its molecular regulation mechanism induction by BDE47 in zebrafish.Methods:We employed zebrafish embryo to investigate the developmental toxicity.We detected the expression of CYP3A65 at different developmental stages and different organs by q RT-PCR,in situ hybridization and western blotting.We designed the situ hybridization probes in hybridization experiments.The lack of commercial zebrafish CYP3A65 antibody in western blotting experiments,we prepare the polyclonal specific antibodies.By analyzing the zebrafish CYP3A65expression profile and the occurrence of developmental abnormalities,it is determined that the potential window period of BDE47 metabolically activated by CYP3A65.In addition,the miRNAs（dre-mir-142a-3p）that may be involved in the regulation were screened by the Targetscan database and validated by dual luciferase reporter assays,which systematically explored the basic expression profile of CYP3A65 and its role in BDE47-induced zebrafish malformations.Results:The zebrafish CYP3A65 was mainly expressed in the liver and intestinal tissues of fish zebrafish.CYP3A65 began to express from 72hpf and reached the peak at 144hpf after birth.The teratogenic window was mainly found at 6-72hpf after zebrafish birth.BDE47 Can significantly induce the expression of CYP3A65 in zebrafish,and the expression level increases with the increase with the dose.In addition,dre-mir-142a-3p may be involved in the regulation of CYP3A65 expression through bioinformatics analysis and database prediction.It confirmed that dre-mir-142a-3p can target the 3’UTR region of CYP3A65 by dual luciferase reporter assays.The expression of CYP3A65 was settled.The expression of dre-mir-142a-3p of BDE47-treated zebrafish was down-regulated and CYP3A65 was up-regulated.Therefore,BDE47 could induce the expression of CYP3A65 through dre-mir-142a-3p.PartⅡ:The effect and molecular mechanism of BDE47 on zebrafish developmental abnormalitiesObiective:To screen the target organs of BDE47-induced zebrafish developmental abnormalities,we carefully study the toxic effect of BDE47,and clarify the role of CYP3A65 in BDE47-induced zebrafish developmental abnormalities.We investigate the role of CYP3A65 in developmental toxicity of zebrafish through high-throughput sequencing and bioinformatics analysis.Methods:The zebrafish embryo and zebrafish adult development model with BDE47exposure were established.Malformations of zebrafish juveniles and adult larvae and their target organ pathological features were assessed using toxicological methods.Systematic evaluation of zebrafish（F0 and F1 generation）generation nervous system developmental damage.Zebrafish embryos were knocked down by CYP3A65microinjection with antisense morpholino-oligonucleotide（MO）.Talen technique was used to construct a CYP3A65 knockout zebrafish model.The role of CYP3A65 in developmental malformation of zebrafish caused by BDE47 was clarified through the analysis of indicators related to developmental abnormalities.We identified the molecular mechanism of CYP3A65 in the BDE47-treated zebrafish using deep-sequencing technology.Results:BDE47 caused dorsolateral flexion of zebrafish embryos and adult spines,abnormal angiogenesis development,loosely arranged zebrafish retinal bipolar cell layers,photoreceptor layer damage,dark adaptation damage,impaired motor function and limited dark cycle activity.In addition,it was found that the offspring also had spinal flexion and limited movement.About 75%knockdown efficiency of CYP3A65,The zebrafish developmental malformation of BDE47-induced was significantly reduced（p<0.001）,and neuromotor function returned to normal.This finding was also validated in the CYP3A65 knockout zebrafish model.Zebrafish behavioral experiments showed that CYP3A65 knockout significantly improved BDE47-induced neurodevelopmental toxicity in zebrafish.The results of deep-sequencing of transcriptome showed that BDE47 damages the visual development pathway of zebrafish.On one hand,BDE47 can induce the up-expression of CYP3A65.On the other hand,it can inhibit the expression of retinol saturase（RETSAT）.CYP3A65 can accelerate metabolism to remove retinol and cause zebrafish synthetic disorders of metabolism of retinol,which may lead to visual impairment of zebrafish.Visual damage may cause zebrafish behavioral abnormalities.BDE47-induced dorsal curvature of zebrafish may be associated with thyroid hormone metabolism and synthesis.These results need to be further verified.PartⅢ:The relationship between CYP3A65 in situ metabolism of BDE47 and dysplasia of zebrafishObiective:We aim to analyze the distribution and metabolism of BDE47 and its influence on zebrafish developmental abnormalities,and to explore the role of CYP3A65 during in-situ metabolism of BDE47 leads to abnormal development of zebrafish.Methods:Firstly,it was confirmed that the effect of CYP3A65-mediated BDE47biotransformation was metabolic activation.Lentivirus protein expression system（PLJM）was used to construct a cell line that highly expressed CYP3A65,and cytotoxicity was used to determine the type of effect.Secondly,we expressed CYP3A65 protein in vitro using the Baculovirus/sf9 expression system.In order to obtain the enzyme activity of CYP3A65 and we established a system of POR and CYP3A65 protein co-expression in baculovirus/sf9.pFast Bac-dual-POR-CYP3A65plasmid was constructed by molecular cloning,and then DH10Bac was used to transform it into baculovirus DNA.Then the baculovirus DNA was transformed into sf9 cells to produce CYP3A65-POR virus solution.The sf9 cells co-express metabolically active CYP3A65 and POR proteins were generated.In vitro metabolic assay was used to analyze the metabolic features of CYP3A65 on BDE47.Finally,in wild-type and CYP3A65 knockout zebrafish models,BDE47 and its active metabolites were detected by GC-MS/MS,and their metabolic profiles were analyzed.Results:The highly expressed CYP3A65 cells（Beas-2B-CYP3A65）were successfully constructed.The cytotoxicity results confirmed that BDE47 could be metabolized by CYP3A65.The plasmid pFast Bac-dual-POR-CYP3A65 was successfully constructed and the zebrafish CYP3A65 protein was successfully expressed in vitro.And the 6-OH-BDE47 was the active metabolite of BDE47,and which can also be detected in a zebrafish exposure model.ConclusionThis study,firstly,confirme that CYP3A65 in zebrafish activates BDE47 by in situ metabolic transformation and 6-OH-BDE47 is a main active metabolite,resulting in back bending,impaired retinal photoreceptor cells,and neurodevelopmental impairment,and there is a sub-generational effect.The molecular mechanism exists direct and indirect ways.On the one hand,BDE47 can induce the high expression of CYP3A65 through dre-mir-142a-3p,and BDE47 can be metabolized by CYP3A65 to form more toxic metabolites 6-OH-BDE47.On the other hand,the metabolic process of BDE47 can trigger oxidative stress,interfere with thyroid hormone metabolism and retinol metabolism,which responsible for zebrafish developmental toxicity.In this study,we firstly constructed a CYP3A65 knockout zebrafish,prepared zebrafish CYP3A65 polyclonal antibody,expressed zebrafish CYP3A65 enzymes in vitro,prepared high-expressing cell lines,constructed a lifetime exposure model.From the perspective of chemical in situ metabolism,the effects of BDE47 on the development of zebrafish and its molecular mechanism were explored,and some new findings were obtained,these results provide an important theoretical basis for systematically evaluating the reproductive development effects of brominated flame retardants in the environment,and provide important technical support for the health risk assessment.1.CYP3A65 is mainly expressed in the zebrafish liver and intestine tissues.It is expressed from zebrafish 72hpf and reaches the expression peak at 144hpf.BDE47can significantly induce the expression of CYP3A65,and dre-mir-142a-3p participates in regulatory process of the expression of CYP3A65.2.The metabolic activation of BDE47 by CYP3A65 leads to spine bending back,retinal photoreceptor cell damage and neurodevelopmental damage in zebrafish,and there is a sub-generational effect relationship.The knock-out of CYP3A65 can significantly improve the development malformations caused by BDE47 in zebrafish.3.CYP3A65 is the main metabolic enzyme of BDE47 in zebrafish,the active metabolite is 6-OH-BDE47,suggesting that the abnormal development of zebrafish caused by BDE47 is related to the activated metabolites produced by CYP3A65-mediated biotransformation.4.The biotransformation of BDE47 mediated by CYP3A65 is a metabolic activation.BDE47 can interfere with the synthesis of retinol metabolism,then induce zebrafish developmental toxicity.In addition to exogenous metabolism,the endogenous metabolism disorders also play an important role in zebrafish abnormalities.5.CYP3A65 is highly homologous to human CYP3A4,and CYP3A is the main metabolic enzyme for drug metabolism and environmental pollutants.The constructed CYP3A65^-/-zebrafish model will provide an important model for clinical drug screening and environmental risk assessment.