Abstract：
Clothianidin, classified as a second-generation neonicotinoid, has achieved extensive application due to its high efficacy against insect pests. This broad-spectrum usage has resulted in its frequent detection in environmental surveys. CYP2C19 and CYP3A4 are crucial for converting clothianidin to desmethyl-clothianidin (dm-clothianidin). The expression of these CYP450s can be significantly influenced by genetic polymorphisms. The objective of our research was to examine the catalytic effects of 27 CYP3A4 variants and 31 CYP2C19 variants on the metabolism of clothianidin within recombinant insect microsomes. These variants were assessed through a well-established incubation procedure. In addition, the concentration of its metabolite dm-clothianidin was quantified by employing an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Lastly, the kinetic parameters of these CYP3A4 and CYP2C19 variants were calculated by applying Michaelis-Menten kinetic analysis to fit the data. The observed changes in enzyme activity were related to the metabolic transformation of clothianidin to dm-clothianidin. In the CYP2C19 metabolic pathway, one variant (CYP2C19.23) showed no notable change in intrinsic clearance (CLint), four variants (CYP2C19.29, .30, .31 and L16F) demonstrated a marked increase in CLint (110.86-183.46 %), and the remaining 25 variants exhibited a considerable decrease in CLint (26.38-89.79 %), with a maximum decrease of 73.62 % (CYP2C19.6). In the CYP3A4 metabolic pathway, 26 variants demonstrated significantly reduced CLint (10.54-52.52 %), with a maximum decrease of 89.46 % (CYP3A4.20). Our results suggested that most variants of CYP3A4 and CYP2C19 significantly altered the enzymatic activities associated with clothianidin metabolism to various degrees. This study provides new insights into assessing the metabolic behavior of pesticides and delivers crucial data that can guide clinical detoxification strategies.
摘要：
Clothianidin,被归类为第二代新烟碱，由于其对害虫的高效能，已取得了广泛的应用。这种广谱的使用导致了它在环境调查中的频繁检测。CYP2C19和CYP3A4对于将噻虫胺转化为去甲基-噻虫胺（dm-噻虫胺）至关重要。这些CYP450的表达可能受到遗传多态性的显着影响。我们研究的目的是检查27种CYP3A4变体和31种CYP2C19变体对重组昆虫微粒体中噻虫胺代谢的催化作用。通过完善的孵育程序评估这些变体。此外,其代谢物dm-clothianidin的浓度通过使用超高效液相色谱串联质谱（UPLC-MS/MS）进行定量。最后,这些CYP3A4和CYP2C19变体的动力学参数是通过应用Michaelis-Menten动力学分析拟合数据来计算的.观察到的酶活性变化与噻虫胺向dm-噻虫胺的代谢转化有关。在CYP2C19代谢途径中，一个变体（CYP2C19.23）在内在清除率（CLint）中没有显着变化，四个变体（CYP2C19.29、.30、.31和L16F）显示出Clint的显着增加（110.86-183.46％），剩下的25个变异体显示出相当大的减少（26.38-89.79%），最大降幅为73.62%(CYP2C19.6)。在CYP3A4代谢途径中，26个变体显示出明显减少的CLint（10.54-52.52％），最大降幅为89.46%(CYP3A4.20)。我们的结果表明，CYP3A4和CYP2C19的大多数变体在不同程度上显着改变了与噻虫胺代谢相关的酶活性。这项研究为评估农药的代谢行为提供了新的见解，并提供了可以指导临床解毒策略的关键数据。
