CYP2B6对农药p,p’-DDT的代谢活性及代谢产物解析

Metabolic activity of CYP2B6 on pesticide p,p’-DDT and analysis of its metabolites

目的:本研究选择细胞色素P450酶(cytochrome P450,CYP)2B6介导p,p’-滴滴涕(dichloro-diphenyl-trichloroethane,DDT)生物转化过程进行研究,为该类持久性有机污染物的风险评估和防治提供依据。方法:建立气相色谱/质谱联用仪(GCMS/MS)的检测方法以检测生物样本中的p,p’-DDT;利用重组酶CYP2B6构建体外代谢模型,采用米式方程计算代谢动力学参数;采用抑制剂8-MOP验证CYP2B6对p,p’-DDT的代谢能力;构建SD大鼠体内代谢模型(大鼠CYP2B1是人CYP2B6的同源酶),尾静脉注射p,p’-DDT和CYP2B6的特异性抑制剂KR-Ⅱ,收集血清(0、5、10、20、30 min)以及肝脏微粒体,比较对照组以及抑制剂组大鼠肝脏中CYP2B1活性以及肝脏和血清中p,p’-DDT原形及代谢产物p,p’-DDE、p,p’-DDD水平的变化。结果:成功建立了多种DDT及代谢产物的GC-MS/MS检测方法。体外代谢证实CYP2B6可代谢p,p’-DDT产生以p,p’-DDE为主的代谢产物,动力学参数K_(m)为15.12μmol/L,每纳摩尔P450酶的V_(max)为12.8nmol/min;抑制剂8-MOP可显著抑制CYP2B6活性,代谢产物p,p’-DDE的含量显著下降。CYP2B1表达被抑制后,大鼠肝脏中CYP2B1的酶活性显著下降,肝脏和血清中的代谢产物p,p’-DDE和p,p’-DDD含量也明显降低。结论:CYP2B6可以代谢p,p’-DDT生成以p,p’-DDE为主的代谢产物,是DDT的主要优势代谢酶。考虑到人群中CYP2B6的表达和活性存在差异,本研究可为DDT暴露危害的易感人群筛选及风险评估的优化提供依据。

Objective:This study is proposed to clarify the biological conversion process of p,p’-dichloro-diphenyl-trichloroethane(DDT)metabolized by cytochrome P450(CYP)2 B6. The result will provide basis for the risk assessment and prevention of persistent organic pollutant. Methods:The detection method based on gas chromatography-mass spectrometry(GC-MS/MS)was established to detect DDT in biological samples. The metabolic model in vitro was established,using recombinant enzyme CYP2 B6 and p,p’-DDT.The enzyme activity was calculated by Michaelis-Menten equation. The 8-MOP as enzyme inhibitor was used to verify the metabolical ability of CYP2 B6 on p,p’-DDT. Meanwhile,the metabolic model in vivo was established in SD rat(rat CYP2 B1 is homologous enzymes of human CYP2 B6)by tail intravenous injection of p,p’-DDT and CYP2 B6 specific chemical inhibitor KR-Ⅱ. The changes of p,p’-DDT prototypes and metabolites(p,p’-DDE,p,p’-DDD)and the changes of enzyme activity were assessed in the serum(0 min,5 min,10 min,20 min,30 min)and liver microsomes. Results:A detection method for DDT was successfully established and tested. Metabolic experiments in vitro confirmed that p,p’-DDT could metabolized by CYP2 B6,and the main metabolite was p,p’-DDE. Kinetic parameter K_(m)was 15.12 μmol/L,and the V_(max)of per nanomolar P450 is 12.8 nmol/min. Inhibitor 8-MOP significantly inhibited the CYP2 B6 activity and metabolites p,p’-DDE content significantly decreased. After CYP2 B1 was inhibited,the metabolic enzyme activity was significantly decreased and the serum p,p’-DDE and p,p’-DDD product content was lowered. Conclusion:CYP2 B6 can metabolize p,p’-DDT to produce metabolites dominated by p,p’-DDE,and it should be the dominant enzyme of DDT in the human body. Considering that there are differences in the expression and activity of CYP2 B6. This study can provide a basis for screening of susceptible populations exposed to DDT and the optimization of risk assessment.