Expression Of Cyp3a65 And Its Influence On BDE47-induced The Dysplasia Of Zebrafish (Danio Rerio)

Polybrominated diphenyl ethers(PBDEs)are a class of brominated flame retardant chemicals(BFRs),which have been applied in various polymer-based consumer productions.Due to their physicochemical properties and adverse effects on human health,PBDEs are worldwide concerned as persistent organic pollutants(POPs).2,2’,4,4’-tetrabromodiphenyl ether(BDE47)is the dominant PBDE congener in human blood and milk.It was reported that BDE47 could disrupt endocrine and exert negative effects on human development.Previous studies demonstrated that the concertation of BDE47 in cord blood was related to lower birth weight,indicating that exposure to BDE47 is harmful to embryo development.However,the underlying mechanism of BDE47-induced development toxicity is still unclear.Biotransformation is critical in the process that xenobiotics produce toxic effects on creatures in which Cytochrome P450,a class of phase I metabolic enzymes,plays an important role.Our previous studies suggested that BDE47 could be metabolized to OH-BDE47 via CYP3A and exert toxic defects by inducing oxidative stress and apoptosis.However,the role of CYP3A-mediated biotransformation in BDE47-induced developmental toxicity is still unclear.In this study,we employed zebrafish embryo to investigate the developmental toxicity.To explore the role of CYP3A in BDE47-induced toxicity,cyp3a65,the homologous gene of human CYP3A4 in zebrafish,was knocked down via morpholino antisense oligos.Because CYP450-mediated biotransformation is a redox reaction,embryos were co-exposed to BDE47 and antioxidant chemical N-acetyl-L-cysteine(NAC)and isoliquiritigenin(ISL)to further explore whether oxidative stress is included in BDE47-induced developmental toxicity.Part I:Distribution and induction of cyp3a65 in zebrafish embryos by BDE47Objective:To determine where cyp3a65 locates in zebrafish,when cyp3a65 starts to express,how much the expression of cyp3a65 is and whether BDE47 induces the expression of cyp3a65.Methods:0-168 hpf zebrafish embryos were collected at 24 hour intervals.The distribution of cyp3a65 was identified via whole-mount in situ hybridization and the expression via Real-time PCR.After embryos exposed to BDE47,the diatribution and expression of cyp3a65 were determined by the same way.Results:cyp3a65 mainly distributes in the liver and intestines of zebrafish embryo.Its expression occurs from 72 hpf and significantly increases during 96-144 hpf in a time-dependent manner.However,the transcription level of cyp3a65 barely changes after 144 hpf.Besides,BDE47 apparently induces the expression of cyp3a65 during 72-120 hpf whereas this effect disappears during 144-168 hpf.Part II:The role of cyp3a65 in BDE47-induced developmental toxicity of zebrafishObjective:To explore the effects of BDE47 on developmental embryos and the function of cyp3a65 in BDE47-induced toxicity.Methods:cyp3a65in embryos was knocked down via morpholino antisense oligos.And then,wildtype and knockdown embryos were both treated with BDE47 in the same way.At 0-168 hpf,the toxicology endpoints of zebrafish were assessed,including spontaneous movement,hatching,mortality and malformation.Besides,the behavior of 120 hpf larvae was analysed in order to evaluate the neurodevelopment of zebrafish.Results:In contrast to controls,BDE47 results in contemporary delayed hatching of embryos during 48-62 hpf and increases the incidence of the malformation in a dose-dependent manner.At 168 hpf,the abnormality rate of zebrafish exposed to 0,5 and 1 pM BDE47 is 27.8%and 68%while none of controls is malformated.Also,0.5 and 1μM BDE47 induced about 6.4%and 16.8%of larval dying at 168 hpf.However,the abnormality rate of cyp3a65-knockdown zebrafish,which is also exposed to BDE47,dramastically decreased to 18%whereras the rate in wildtype larvae is 100%at 120 hpf.According to behavior analysis,120 hpf larvae in control group swim under the light and dark at 0.25 mm/s and 2 mm/s respectively,while BDE47-exposed larvae move at 0.8 mm/s both in the light and dark.However,the velocity of cyp3a65-knockdown larvae becomes 0.4 mm/s in the light and 1.6 mm/s in the dark with the same treatment of BDE47.The activity and total move distance of larvae appear similar change to velocity.The activity and total move distance in controls are altered with the change of light while BDE47-exposed larvae don’t display the kind of alteration.Knokdowning cyp3a65 results in the alteration along with illumination changes occurring again.Part Ⅲ:The role of oxidative stress in BDE47-induced developmental toxicityObjective:To determine whether BDE47 could induce oxidative stress and apoptosis in zebrafish embryos and further result in developmental toxicity by this way.Methods:After merrely exposed to BDE47 or co-exposed to BDE47 and antioxidative chemicals(NAC or ISL)for 120 h,oxidative stress was evaluated by determining the production of ROS,activity of antioxidant enzymes and changes of related genes encoding antioxidant proteins.And then,changes of genes related to apoptosis were determined via Real-time PCR and apoptotic cell in larval were detected via acridine orange staining.Finally,the behavior of larvae in all groups was analysed at 120 hpf in order to evaluate the effects of BDE47 and antioxidative chemicals exerted on the neurodevelopment of larvae.Results:BDE47 induces excessive ROS production in larval and alters transcript levels of genes encoding antioxidant proteins,including Cu/zn-sod,Cat and Mn-sod.Subsquently,the activity of antioxidant enzymes SOD and CAT was descreased by BDE47.In addition,BDE47 results in cell apoptosis in larval via upregulating p53,caspase3,caspase9 and downregulating Bcl-2/Bax.Futhermore,NAC and ISL could significantly reduce BDE47-induced malformation.At 120 hpf,the abnormality rate of BDE47-exposed larvae is 90%while 30%and 28%in NAC/BDE47 and ISL/BDE47 respectively.NAC and ISL reduce oxidative stress and cell apoptosis by increasing the activity of SOD and CAT,down-regulating the transcript level of genes related to apoptosis.In addition,according to behavior analysis,120 hpf larvae in control group swim under the light and dark at 0.45 mm/s and 1.5 mm/s respectively,while BDE47-exposed larvae move at 0.8 mm/s both in the light and dark.However,the velocity of NAC/BDE47 and ISL/BDE47 larvae becomes 0.7mm/s,0.5 mm/s in the light and 1.0 mm/s,1.2 mm/s in the dark with the same treatment of BDE47.The activity and total move distance of larvae appear similar change to velocity.The activity and move distance in controls are altered with the change of light while BDE47-exposed larvae don’t display the kind of alteration.NAC and ISL bring the alteration along with illumination changes back to BDE47-exposed zebrafish.ConclusionIn the present study,we explore the role of cyp3a65 and oxidative stress in BDE47-induced developmental toxicity.Major findings are depicted as follows:1.cyp3a65 is mostly expressed in liver and intestines of zebrafish during 0-168 hpf.Its expression starts from 72 hpf and increased time-dependently until 144 hpf.BDE47 could increase the expression of cyp3a65 during 72-120 hpf while it doesn’t work after 144 hpf.2.BDE47 results in growth retardation,deformation and abnormal behaviors of zebrafish,which could be mitigate by knocking down cyp3a65,indicating that cyp3a65-mediated biotransformation plays a critical role in BDE47-induced developmental toxicity.3.BDE47 could lead to the abnormal development of zebrafish by inducing oxidative stress and apoptosis.Besides,antioxidant chemical NAC and ISL have the ability of alleviating BDE47-induced developmental toxicity via inhibiting p53/caspase pathway.