CYP2B6对农药滴滴涕的代谢活性及代谢产物解析
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1.南京医科大学公共卫生学院,生殖医学国家重点实验室;2.现代毒理学教育部重点实验室;3.生殖医学国家重点实验室

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国家自然科学基金项目(91743205, 81903353, 81803198)


Metabolic activity of CYP2B6 on pesticide p, p’-DDT and analysis of its metabolites
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State Key Lab of Reproductive Medicine,Nanjing Medical University, Longmian Avenue

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    摘要:

    [背景]有机氯农药是一类被禁止使用多年的持久性有机污染物(POPs),可在环境和生物体中长期存在并通过食物链放大作用导致人群的长期低剂量暴露,引起广泛的健康损伤。目前,环境及人体中以滴滴涕(p, p’-DDT)为代表的有机氯农药含量仍较高,其毒性与细胞色素P450酶(CYP)介导的生物转化密切相关,但代谢方式和代谢产物仍不十分清楚。 [目的]本研究拟选择CYP2B6介导p, p’-DDT生物转化过程进行研究,为该类POPs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(GC-MS/MS)的检测方法以检测生物样本中DDTs;利用重组酶CYP2B6(rCYP2B6)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(0min,5min,10min,20min,30min)以及肝脏微粒体,比较对照组以及抑制组大鼠肝脏中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Km为15.12 μM,Vmax为12.8 nmol/min/nmol P450;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,应该是DDTs的主要优势代谢酶。考虑到CYP2B6是高突变性的代谢酶,人群中CYP2B6的表达和活性存在差异,本研究可为DDTs暴露危害的易感人群的筛选及风险评估的优化提供依据。

    Abstract:

    [Background] Organic chlorine pesticides (OCPs) are a class of persistent organic pollutants (POPs) that have been prohibited from many years. It can exist in the environment and organisms for a long time and lead to long-term low-dose exposure of people through food chain amplification, causing several disorders and diseases. At present, the content of OCPs represented by p, p’-DDT in the environment is still high, the toxicity is closely related to cytochrome P450 enzyme (CYP)-mediated biotransformation, but its metabolism in the human body is still not clear. [Objective] This study is proposed to clarify the biological conversion process of p, p’-DDT metabolized by CYP2B6. The result will provide basis for the risk assessment and prevention of DDTs. [Methods] The detection method based on gas chromatography-mass spectrometry (GC-MS/MS) was established to detect DDTS in biological samples. The metabolic model in vitro was established, using recombinant enzyme CYP2B6 and p, p'-DDT. The enzyme activity was calculated by Michaelis-Menten equation and DDT (p, p’-DDT) and metabolites (p, p’-DDE、p, p’-DDD) were analyzed in the samples. Using 8-MOP as enzyme inhibitor to verify the metabolical ability of CYP2B6 on p, p'-DDT. Meanwhile, the metabolic model in vivo was established in SD rat (rat CYP2B1 is homologous enzymes of human CYP2B6), using the tail intravenous injection of p, p’-DDT and CYP2B6 specific chemical inhibitor KR-II. The changes of p, p’-DDT prototypes and metabolites (p, p’-DDE、p, p’-DDD) and the change of enzyme activity were assessed in the serum (0min, 5min, 10min, 20min, 30min) and liver microsomes. [Results] A detection method for DDTs was successfully established and tested. Metabolic experiments in vitro confirmed that p, p’-DDT could metabolized by CYP2B6, and the main metabolite was p, p’-DDE, kinetic parameter Km was 15.12 μM, Vmax was 12.8 nmol/min/nmol P450. Inhibitor 8-MOP significantly inhibited the CYP2B6 activity and metabolites p, p’-DDE content significantly decreased. After CYP2B1 was inhibited, the metabolic enzyme activity was significantly decreased and the serum p, p’-DDE, p, p’-DDD product content was lowered. [Conclusion] CYP2B6 can metabolize p, p’-DDT to produce metabolites dominated by p, p’-DDE, and it should be the dominant enzyme of DDTs in the human body. Considering that CYP2B6 is a high mutant metabolic enzyme. There are differences in the expression and activity of CYP2B6, this study can provide a basis for screening of susceptible populations exposed to DDTs and the optimization of risk assessment.

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  • 收稿日期:2021-07-02
  • 最后修改日期:2021-10-01
  • 录用日期:2021-11-23
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