| The bromobenzene flame retardants are widely used in polystyrene,resin,textile,c oating and electronic appliances.Due to the extensive usage,the residues of bromobenz ene flame retardants have been continuously detected in various environmental matrices such as air,water,sediments and soil.Their potential toxicities to human have caused i ncreasing public concern.However,whether the exposure to bromobenzene flame retard ants can result in potential thyroid receptor?TR?disrupting effect still remained largely unknown.The molecular mechanism of these chemicals also needs to be elucidated.W e investigated the TR disrupting effect of six emerging bromobenzene flame retardants i ncluding BTBPE,DBDPE,HBB,PBEB,pTBX,TBPhA.To probe the possible involve d molecular initiating event?MIE?,the molecular interactions between thyroid receptor 1 igand binding domain?TR? LBD?protein and these contaminants were investigated.Th e main results are listed as follows:?1?Thyroid disrupting effects of six contaminants were evaluated by dual lucifera se reporter gene system using CV-1 cells transfected by pGal4-L-TR plasmid.There wa s no significant TR agonistic effect for six contaminants at the concentrations of 1×10-10 M,1×10-9 M,1×10-8 M,1×10-7 M and 1×10-6 M.BTBPE,HBB and PBEB showed anti-thyroid disrupting effect in concentration-dependent mode.The maximal anti-thyroid activity for BTBPE,HBB and PBEB were 60%,62%and 66%,respectively.?2?The effect of Human liver microsomes?HLM?and human CYP3A4-mediated biotransformation to the anti-thyroid activity of the six flame retardants was investigated.BTBPE and PBEB exhibited metabolic attenuation effects on TR antagnonistic activity after the HLM-mediated metabolism.Prior to and after the metabolism,the antagonisti c rate of BTBPE and PBEB decreased from 22.69%to 12.19%.27.68%to 13.27%,r espectively.BTBPE and PBEB exhibited metabolic attenuation effects on TR antagnonis tic activity after the CYP3A4-mediated metabolism.Prior to and after the metabolism,t he antagonistic rate of BTBPE and PBEB decreased from 22.69%to 13.59%,27.68%t o 14.64%,respectively.The anti-thyroid activity of HBB showed no obvious changes af ter the biotransformation.Our result indicated that CYP3A4 enzyme plays an important role in mediating the attenuation effect of BTBPE and PBEB on TR,suggesting that t he biotransformation should be taken into full consideration in the toxicity evaluation of contaminants.?3?The influence of BTBPE,HBB and PBEB on the enzymatic activity of hum an CYP3A4 was studied using the P450-GloTM CYP3A4?Luciferin-IPA?bioassay.BTB PE,HBB and PBEB can inhibit the activity of CYP3A4 enzyme with the half maxima 1 inhibitory concentration?IC50?values of 0.33 ?M,0.52 ?M and 0.65 ?M,respectivel y,which may disrupt the regular process of CYP3A4-mediated biotransformation.?4?The molecular interaction between TR? LBD and BTBPE,HBB,PBEB was probed by the fluorescence spectroscopy.The binding of three flame retardants induced the static quenching of TR? LBD.The binding ability ranks as PBEB>HBB>BTBPE,in line with the ranking order with their anti-thyroid activity,indicating that the intera ctions between the contaminants and TR? LBD may be one of the important factors co ntributing to the anti-thyroid activity.The interactions between TR? LBD and the conta minants were mainly caused by the electrostatic interaction and hydrophobic interaction. |
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