Crane Alice L, Klein Kathrin, Olson James R
Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, NY, USA.
Xenobiotica. 2012 Dec;42(12):1255-62. doi: 10.3109/00498254.2012.702246. Epub 2012 Jul 10.
Chlorpyrifos (CPF), an organophosphorus (OP) pesticide, is bioactivated by cytochrome P450s (CYPs) to the active metabolite chlorpyrifos oxon (CPF-O). Given that human CYP2B6 has the highest intrinsic clearance (CL(int)) for CPF bioactivation, CYP2B6 polymorphisms may impact human susceptibility to CPF at real world environmental and occupational CPF exposure levels. CYP2B6.4,.5,.7, and .18 were over-expressed in mammalian COS-1 cells to assess the impact of CYP2B6 variants on the K(m) and V(max) for bioactivation of CPF. Cell lysates were incubated with CPF (0-100 μM) and the production of CPF-O was measured via HPLC analysis. CYP2B6 content was determined by western blot. CYP2B6.18 had neither detectable protein nor activity levels. The V(max) value for each remaining variant was significantly higher than wild-type (CYP2B6.1, V(max) 4.13 × 10(4) pmol/min/nmol CYP2B6), with CYP2B6.4,.5, and .7 having V(max) values of 4.52 × 10(5), 1.82 × 10(5), and 9.60 × 10(4) pmol/min/nmol CYP2B6, respectively. The K(m) values for these variants ranged from 0.39 to 1.09 μM and were not significantly different from wild-type. All active variants examined had significantly higher CL(int) than CYP2B6.1. Variants of CYP2B6 have altered capacity to bioactivate CPF and may affect individual susceptibility by altering the V(max) for CPF-O formation.
毒死蜱（CPF）是一种有机磷农药，通过细胞色素P450（CYP）生物活化形成活性代谢产物毒死蜱氧磷（CPF - O）。鉴于人类CYP2B6对CPF生物活化具有最高的内在清除率（CL(int)），CYP2B6基因多态性可能会影响人类在实际环境和职业CPF暴露水平下对CPF的易感性。在哺乳动物COS - 1细胞中过表达CYP2B6.4、.5、.7和.18，以评估CYP2B6变体对CPF生物活化的米氏常数（K(m)）和最大反应速度（V(max)）的影响。将细胞裂解物与CPF（0 - 100μM）孵育，并通过高效液相色谱分析测定CPF - O的生成量。通过蛋白质印迹法测定CYP2B6含量。CYP2B6.18既未检测到蛋白质水平也未检测到活性水平。其余每个变体的V(max)值均显著高于野生型（CYP2B6.1，V(max)为4.13×10(4) pmol/min/nmol CYP2B6），CYP2B6.4、.5和.7的V(max)值分别为4.52×10(5)、1.82×10(5)和9.60×10(4) pmol/min/nmol CYP2B6。这些变体的K(m)值范围为0.39至1.09μM，与野生型无显著差异。所有检测的活性变体的CL(int)均显著高于CYP2B6.1。CYP2B6变体对CPF的生物活化能力有所改变，可能通过改变CPF - O形成的V(max)影响个体易感性。
