| Polybrominated diphenyl ethers(PBDEs)are widely used brominated flame retardants with semi-volatility,high lipophilicity and refractory degradation.2,2’,4,4’-tetrabromodiphenyl ether(BDE47)is the most abundant PBDEs homologue detected in blood and breast milk,it has been attracted much attention because of its significant increase in environmental and organism.Large number of epidemiological studies have show that the concentration of BDE47 in plasma is negatively correlated with the level of Thyroid hormones(THs),suggesting that BDE47 has the effect of thyroid hormone homeostasis.Some studies have shown that the concentration of BDE47 in umbilical cord blood of neonates is negatively correlated with their birth weight,suggesting that exposure to BDE47 at early stage may be deleterious to embryonic development.However,the relationship between thyroid hormone homeostasis caused by BDE47 and developmental toxicity remains unclear.Besides,most studies regarding the underlying mechanism by which PBDEs interfer with thyroid hormone homeostasis mainly focused on the hypothalamus-pituitary-thyroid axis(HPT axis)or thyroid hormone receptors(TRs),while the study on the regulation of thyroid hormone deiodinase(DIO)in peripheral tissues is relatively rare.Biotransformation is an important process in which exogenous chemicals exert toxicity in organism,mainly mediated by Cytochrome P450(CYP450).Previous studies have confirmed that BDE47 can be metabolized to a series of metabolites by CYP3A,resulting in stronger toxicity in reproduction and development.However,the role of CYP3A-mediated biotransformation in the disturbance of thyroid hormone homeostasis by BDE47 and its relationship with developmental toxicity needs to be clarified.In this study,wild-type(WT)and cyp3a65 knockout(KO)zebrafish were exposed to BDE47(1μM)to investigate the role of CYP3A65(homologous to human CYP3A4)in early developmental toxicity induced by BDE47 and the mechanism of interfering thyroid hormone homeostasis.PartⅠ:Effects of CYP3A65 on BDE47-induced thyroid hormone homeostasis disruption and developmental dysplasia in ZebrafishObiective:To clarify the role of CYP3A65-mediated biotransformation in thyroid hormone homeostasis by BDE47 and its relationship with the developmental toxicity in zebrafish.Methods:The cyp3a65 knockout zebrafish model constructed by TALEN technology was validated by Real-time PCR and Western Blot.And then,wildtype(WT)and knockout(KO)embryos were both treated with BDE47(1μM)in the same way.At0-144 hpf,the toxicology endpoints of zebrafish were assessed,including hatching rate,deformity rate,body length,eye size and swim bladder inflation.By comparing the phenotypic differences between WT and KO groups,we aim to explore the function of cyp3a65 in BDE47-induced toxicity.At 144 hpf,thyroid hormone were detected by ELISA kit,including T3,T4,rT3 and TSH.Besides,the expression of deiodinase(dio)regulating thyroid hormone metabolism was detected by Real-time PCR to preliminarily explore the possible causes of thyroid hormone disorder in zebrafish.Results:In contrast to controls,BDE47 resulted in delayed hatching of embryos during 24-58 hpf;At 96 hpf,BDE47 had no significant effect on embryo body length,but it reduced eye size to 7×10~4μm~2(control group was 8×10~4μm~2);At 120 hpf,the inflation rate of swim bladder in control group reached 100%,while zebrafish in BDE47 group was only 86%;At 144 hpf,the body length decreased significantly to3.6×10~3μm(control group was 3.8×10~3μm)and the deformity rate in BDE47-exposed group was as high as 70%,and the main abnormality was spinal curvature,while none of controls was malformated.However,in cyp3a65-knockout group exposed to BDE47,the hatching rate of embryos dramastically increased,the body length increased to 3.7×10~3μm and deformity rate at 144 hpf decreased to 58%.Besides,the eye size at 96 hpf(7.7×10~4μm~2)and swim bladder inflation rate at 120hpf(94%)were also significantly improved.In addition,compared with the control group,the expression of type I deiodinase(dio1)and type II deiodinase(dio2)of zebrafish in BDE47/WT group did not change significantly,while the expression of type III deiodinase(dio3a,dio3b)increased significantly,with the level of T3significantly decreased and rT3 dramastically increased.However T4 and TSH didn’t display any kind of alteration.Further analysis showed that the ratio of T3 to T4decreased significantly.After deleting cyp3a65,the expression of three types of deiodinase in zebrafish was similar to that in control group,and the levels of T3,rT3and T3/T4 ratio were also significantly improved.PartⅡ:The mechanisms of CYP3A65-mediated bioactivation of BDE47 in the thyroid hormone homeostasis disruption in ZebrafishObiective:To analyze the metabolism of BDE47 in zebrafish,and to explore the role of CYP3A65 on BDE47 biotransformation and the molecular mechanism on imbalance of thyroid hormone homeostasis.Methods:The UPLC-MS/MS and GC-MS/MS were applied to quantitatively detect the concentration of BDE47 metabolites in wild-type(WT)and cyp3a65 knockout(KO)zebrafish.The zebrafish embryos were then exposed to BDE47 metabolites till144 hpf to analyze the accumulation level and transformation relationship.In order to identify the main BDE47 metabolites mediated by CYP3A65 that interfere the expression of dio3,we treated WT and KO zebrafish embryos with BDE47metabolites till 144 hpf and then detected their effects on the expression of type III deiodinase(dio3a,dio3b)by Real-time PCR.Results:BDE47 can be metabolized into 6 products in zebrafish embryos,including3-OH-BDE47,5-OH-BDE47,6-OH-BDE47,3-MeO-BDE47,5-MeO-BDE47 and6-MeO-BDE47,and the content of hydroxylated metabolites(OH-BDE47)is higher than those of methoxy metabolites(MeO-BDE47);the content of MeO-BDE47 in the BDE47/WT group is higher than that in the BDE47/KO group,while the content of OH-BDE47 is lower than that in the BDE47/KO group,indicating that CYP3A65might be the dominant enzyme that metabolizes BDE47 to MeO-BDE47.All of metabolites can accumulate in zebrafish,and mutual transformation between3-OH-BDE47 and 3-MeO-BDE47,6-OH-BDE47 and 6-MeO-BDE47 were confirmed while 5-OH-BDE47 was only transformed into 5-MeO-BDE47.Further analysis shows that only 3-MeO-BDE47 could significantly up-regulate the expression of dio3a and dio3b of zebrafish in BDE47/WT and BDE47/KO group,indicating that 3-MeO-BDE47 was the key metabolite of BDE47 mediated by CYP3A65,which interfered the expression of dio3.Based on the above results,we speculated that 3-MeO-BDE47 might be the main metabolite of BDE47 that increased the expression of dio3 mediated by CYP3A65.ConclusionsBased on the model of wild-type(WT)and cyp3a65 knockout(KO)zebrafish,this study elaborated the role of CYP3A65 in disturbing the thyroid hormone homeostasis of zebrafish and its relationship with developmental abnormalities.We also discussed the main metabolites of BDE47 metabolized by CYP3A65 in zebrafish and the mechanism regarding thyroid hormone disorder from the perspective of biotransformation.The following conclusions are obtained:1.BDE47 results in early developmental anomaly of zebrafish,which could be mitigate by knocking down cyp3a65.In addition,BDE47 leads to thyroid hormone disorder in zebrafish,which can be significantly improved by knocking down cyp3a65,suggesting a relationship between thyroid hormone disorder caused by BDE47 and the early development of zebrafish,and CYP3A65 plays an important role in this process.2.CYP3A65 is the main metabolic enzyme of BDE47.All of BDE47 metabolites are accumulative in zebrafish.OH-BDE47 and MeO-BDE47 can be transformed into each other in zebrafish,except that 5-OH-BDE47 is only transformed into5-MeO-BDE47.3.3-MeO-BDE47 is the key metabolites that upregulates the expression of dio3 in zebrafish,suggesting that 3-MeO-BDE47 may be the main metabolite of BDE47leading to thyroid hormone disorder in zebrafish. |
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