| Flame retardants are a class of compounds that are widely used in plastics,paints and textile fabrics to prevent fire.In recent years,with phase-out ofpolybrominated diphenyl ethers?PBDEs?,organophosphate flame retardants?OPFRs?and novel brominated flame retardants?NBFRs?,as major alternatives,were continued to increase,especially the increase growth of OPFRs usage leads to the metabolites ubiquity in the environmental media even human urine.However,the endocrine disrupting potency of these chemicals were still unclear.This study focused on NBFRs and OPFRs metabolites,by constructing estrogen receptor??ER??,glucocorticoid receptor?GR?and mineralocorticoid receptor?MR?mediated dual-luciferase reporter system using CHO cells,combined with human adrenocortical cancer cell?H295R?steroid experiments and molecular docking experiments,to evaluate the endocrine disrupting effects of four NBFRs with high detection rates in the environment and three primary metabolites from the most widely used OPFRs.On the basis of in vitro experiments,zebrafish was used as a model organism,and the endocrine disrupting effects of OPFRs metabolites was further evaluated in vivo through acute toxicity experiments and real-time fluorescent quantitative reverse transcription polymerase chain reaction?q RT-PCR?experimental analysis methods.The major work and achievements are as follows:?1?The endocrine disrupting effects of four NBFRs:In this study,we adopted two in vitro models?reporter gene assays and H295R cell line?to investigate the endocrine-disrupting effects of four NBFRs?2,4,4,6-Tetrabromocyclohexa-2,5-dienone??/?-TBCO?,2,3,4,5,6-pentabromoethylbenzene?PBEB?,2-Ethylhexyl2,3,4,5-tetrabromobenzoate?EHTBB?and Bis?2-ethylhexyl?3,4,5,6-tetrabromophthalate?BEHTBP??.The molecular docking platform was adopted to further confirm the results of the in vitro assessment.Our results revealed that PBEB and EHTBB exhibited ER?antagonistic activity at the concentration of 10-5 M and 10-6 M.?/?-TBCO exhibited ER?antagonistic activity only at a concentration of 10-5 M or above.The RIC20 values of?/?-TBCO,PBEB and EHTBB were 8.0×10-6 M,7.9×10-7 M and 9.2×10-7 M,respectively.Notably,none of the four NBFRs produced ER?agonistic activity in the range of tested concentration.For GR activity,?/?-TBCO,PBEB,BEHTBP,and EHTBB did not show significant GR agonistic effects.Only PBEB showed GR antagonistic activity at 10-5 M,and the RIC20 value was 8.6×10-6 M.When co-exposed with 5.0×10-10 M aldosterone?AD?,none of the four NBFRs showed the MR agonistic and antagonistic activities in the range of tested concentration.The results of molecular docking were consistent with the reporter gene assay.For H295R cell line assay,all?/?-TBCO,PBEB and EHTBB induced17?HSD,CYP11B1 and CYP17 upregulation.?2?The endocrine disrupting effects of three OPFRs metabolites:In this study,three primary metabolites from the most widely used OPFRs including?bis?1-chloro-2-propyl?phosphate?BCIPP?,bis?1,3-dichloro-2-propyl?phosphate?BDCIPP?and diphenyl phosphate?DPHP??were chosen to investigate the potential endocrine disruption effects by in vitro,in vivo and in silico assays.The luciferase reporter assay revealed that all of the three metabolites exhibited a dose-response agonistic activity to estrogen receptor alpha?ER??with the REC20 value of BCIPP,BDCIPP and DPHP at 6.5?10-8 M,4.9?10-7 M and 1.2?10-6 M,respectively.Neither of them showed anti-oestrogenic activities.In contrary,BCIPP,BDCIPP and DPHP acted as an antagonism rather than agonist to mineralocorticoid receptor?MR?with the RIC20 values of 8.1?10-7 M,1.1?10-8 M and4.2?10-7 M,respectively.For GR activity,only BCIPP and BDCIPP exhibited GR antagonistic activity and their RIC20 values were 4.7?10-8 M and 6.9?10-6 M,respectively.As the same one,none of the three OPFRs metabolites showed GR agonistic activity.The results of molecular docking were consistent with the reporter gene assay.In H295R cell line,both BCIPP and BDCIPP upregulated the genes encoded for estrogenic synthesis enzymes such as 17?HSD and CYP19.All of the three compounds stimulated the transcription of CYP11B1 that encoded for cortisol synthesis,while only BCIPP and DPHP also triggered CYP11B2 that regulation on corticosterone synthesis.BDCIPP may inhibit the synthesis of progesterone since CYP11A1,STAR,and 3-?HSD were all downregulated.?3?Zebrafish embryo toxicity of three OPFRs metabolites:In this study,zebrafish was used as a model organism to further evaluate three metabolites of OPFRs in vivo.First,the results of acute toxicity experiments showed that BCIPP,BDCIPP and DPHP caused the death of zebrafish embryos at 12 hpf,and the maximum induction can be found under BCIPP exposure,the mortality of zebrafish embryos reached 58.3%at 10-5 M.In general,all the three OPFRs metabolites induced mortality and delaying hatch in zebrafish embryos.At 96 hpf,the malformation and mortality of zebrafish larvae were significantly induced by three OPFRs metabolites.The mortality of zebrafish larvae was37.5%at 10-5 M of BCIPP,which was higher than BDCIPP and DPHP(13.3%and 6.25%at 10-5 M).No apparently changes were observed at 10-6 M or below.The malformation of zebrafish were significantly induced by BCIPP,BDCIPP and DPHP at all tested concentrations.The observed malformation types including pericardial edema,hypopigmentation,yolk sac swelling,short tail and curved spine.And BCIPP reached a maximum deformity rate of 62.5%at 10-5 M.Both of the HPA axis marked as ER,VTG,CYP17 and CYP19,and HPG axis marked as GR,MR and CRH were disrupted by BCIPP,BDCIPP and DPHP in zebrafish larval. |
PDF Full Text Request