| Polybrominated biphenyls?PBBs?were once widely used as brominated flame retardants for the production of thermoplastics,fibers,and various electronic products.Their residues have been detected in water,soil and air,and their derivatives are present in various biological samples,posing a potential threat on human health.The exposure to PBBs is reported to affect thyroid hormone homeostasis in mammals,however,the specific molecular mechanism remains largely unkown.How the metabolism of PBBs affects its thyroid disrupting effect is unclear.Since such information is extremely important for the health risk assessment of PBBs,it is necessary to investigate the molecular mechanism of thyroid disruption by PBBs and evaluate the effect of biotransformation on endocrine disrupting effect of PBBs.The tetrabromobiphenyl 3,3',5,5'-tetrabromobiphenyl?BB80?was chosen as the target PBB,and its thyroid disrupting effects were evaluated using the human recombinant TR?gene yeast via two-hybrid yeast technique.The metabolite of BB80?OH-BB80?is revealed to have significantly enhanced TR?disrupting effect.The underlying molecular mechanism of the TR?disrupting effect of OH-BB80 was studied by the investigation of the molecular interactions between OH-BB80 and the human TR?ligand binding domain?LBD?using the fluorescence spectroscopy and molecular docking.Using zebrafish as a model,the effects of the exposure to BB80and OH-BB80 on the developmental toxicity,thyroid hormone level and gene expression at 6 dpf,14 dpf and 21 dpf in the transition stage from zebrafish larvae to adult,residues of BB80 and OH-BB80,thyroid histology and motor behavior in zebrafish larvae were further evaluated.The thyroid endocrine disrupting effect in zebrafish was further evaluated by short-term elimination of BB80 and OH-BB80 in zebrafish larvae.There may be difference for the thyroid disruption between BB80and OH-BB80 by the comparison of thyroid disrupting effects of the exposure to BB80 and OH-BB80 at the early and late stage of zebrafish.The main conclusions are as follows:?1?The in vitro metabolism of BB80 by human liver microsome?HLM?was performed and BB80 can be in vitro metabolized.The 2,2'-dihydroxy-3,3',5,5'-tetrabromobiphenyl?OH-BB80?was identified as the metabolite using ultra-high performance liquid chromatography coupled quadrupole-time of flight mass spectrometer?UPLC-Q-TOF-MS/MS?.The metabolite was further synthesized and confirmed by mass spectrometry.?2?The agonistic/antagonistic effects of BB80 and OH-BB80 toward TR?were evaluated by recombinant human TR?two-hybrid yeast bioassay.BB80 shows no TR agonistic or antagonistic effects,while OH-BB80 has enhanced TR antagonistic effect in a concentration-dependent manner with IC20 at 2?mol/L,indicating the metabolic activation of BB80 toward TR?disruption.?3?The effect of OH-BB80 on the conformational changes of TR?LBD was in vitro analyzed by the fluorescence spectrum assay.OH-BB80 induces static quenching of TR?LBD,causing red shift of fluorescence emission peaks of tryptophan residues,causing conformational changes of TR?LBD.OH-BB80interacts with TR?LBD mainly through hydrophobic interaction and electrostatic interaction.The measured binding energy is-30 KJ·mol-1.The molecular docking further revealed that OH-BB80 interacts mainly with surrounding hydrophobic amino acids and has one hydrogen bond with Phe272.?4?Using zebrafish as the model,the effect of the exposure to BB80 and OH-BB80 on the thyroid endocrine system at the early development stage of zebrafish was evaluated.The transcription levels of genes related to HPT axis-related genes at 6dpf and 14 dpf were measured using quantitavie PCR.The contents of thyroid hormones?T3 and T4?and thyroid stimulating hormone?TSH?at 14 dpf were measured using ELISA assay.The expression of tsh?,tg and tpo is significantly up-regulates and T3 and T4 levels in zebrafish larvae are increased at the early stage of exposure to OH-BB80.However,BB80 up-regulates the expression of crh,tsh?and tpo and induces the increase of T4 in zebrafish at the late stage of exposure.This indicates the difference of the thyroid disruption between BB80 and OH-BB80.The morphology of zebrafish thyroid follicles and follicular cells at 14 dpf was further observed and BB80 causes thyroid damage in zebrafish.?5?We compared the hormone levels and gene expression levels in the zebrafish with the exposure to BB80 and OH-BB80 at 2 hpf to 14 dpf and without the exposure to BB80 and OH-BB80 at the elimination stage?14 dpf-21 dpf?.The hormones level recovers after the elimination stage,and tg and tpo are down-regulated.However,the gene expression of HPT axis negative feedback regulation has no significant difference.These results indicate the recovery mechanism of zebrafish after the disruption by BB80 and OH-BB80.The direct regulation of thyroid hormone synthesis may be the main way to restore hormone homeostasis in zebrafish.?6?After the short-term elimination,the disruption induced by BB80 and OH-BB80 still exerts effect on thyroid endocrine system of zebrafish.The contents of BB80 and OH-BB80 in zebrafish before and after the elimination stage were quantified by GC-MS/MS and LC-MS/MS.The total body burden in the larvae decreases by 9.7%,15.0%and 5.0%after the elimination phase at the initial exposure groups for BB80 with the concentration of 2,10 and 50?g/L,and decreases by 82.2%,92.6%and 90.4%for OH-BB80.The neurotoxicity is induced by BB80 and OH-BB80 as revealed by the evaluation of the motor behavior of zebrafish.The accumulation of BB80 in zebrafish causes sustained neurotoxicity.We show the metabolic activation of BB80 on human TR?disrupting effects mediated by HLM and elucidate the molecular mechanism of OH-BB80 on TR?disruption at the molecular level.We further evaluate the effect on thyroid endocrine disruption of BB80 and OH-BB80 to zebrafish at early development,and investigate the mechanism of the disruption and the mechanism in the recovery of the disruption.This research provides scientific data for comprehensive human health risk assessment of BB80 and its metabolites. |
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