Miah Mohammad Asaduzzaman, Elzaki Mohammed Esmail Abdalla, Han Zhaojun
Key Laboratory of Integrated Crop Pest Management in Eastern China (Agricultural Ministry of China) College of Plant Protection Nanjing Agricultural University Nanjing 210095 Jiangsu China.
Ecol Evol. 2017 Jun 2;7(14):5032-5040. doi: 10.1002/ece3.3047. eCollection 2017 Jul.
Cytochrome P450 monooxygenases (CYPs) usually overexpressed in resistant strain were found involved in oxidative detoxification of insecticides. In this study, an investigation was conducted to confirm if resistance irrelevant CYPs which were not overexpressed in resistant strain before, were capable of degrading insecticides. Three resistance irrelevant CYPs viz. CYP417A2v2, CYP425A1v2, and CYP4DJ1 from CYP4 family of were randomly selected for experiments. CYP417A2v2 and CYP425A1v2 were found expressed successfully in Sf9 cell line while CYP4DJ1 was not expressed successfully and out of two expressed CYPs, only CYP417A2v2 showed its efficient catalytic activity. For catalytic activity, three traditional model probe substrates and five insecticides were assayed. For the probe substrates screened, -nitroanisole and ethoxycoumarin were preferentially metabolized by CYP417A2v2 (specific activity 3.76 ± 1.22 and 1.63 ± 0.37 nmol min mg protein, respectively) and they may be potential diagnostic probes for this enzyme. Among insecticides, only imidacloprid was efficiently degraded by CYP417A2v2. Incubation of imidacloprid with CYP417A2v2 of and subsequent HPLC, LC-MS, and MS/MS analysis revealed the formation of imidacloprid metabolites, that is, 4' or 5'hydroxy-imidacloprid by hydroxylation. This result implies the exemption of CYPs character that it is not always, all the CYPs degrading insecticides being selected and overexpressed in resistant strains and the degrading CYPs without mutations to upregulate could be candidates during insecticide resistance evolution. This characterization of individual insect CYPs in insecticide degradation can provide insight for better understand of insecticide resistance development.
细胞色素P450单加氧酶（CYPs）通常在抗性品系中过表达，被发现参与杀虫剂的氧化解毒过程。在本研究中，进行了一项调查，以确认之前在抗性品系中未过表达的与抗性无关的CYPs是否能够降解杀虫剂。从CYP4家族中随机选择了三个与抗性无关的CYPs，即CYP417A2v2、CYP425A1v2和CYP4DJ1进行实验。发现CYP417A2v2和CYP425A1v2在Sf9细胞系中成功表达，而CYP4DJ1未成功表达，在两个成功表达的CYPs中，只有CYP417A2v2显示出其高效的催化活性。对于催化活性，测定了三种传统模型探针底物和五种杀虫剂。对于筛选的探针底物，对硝基苯甲醚和乙氧基香豆素优先被CYP417A2v2代谢（比活性分别为3.76±1.22和1.63±0.37 nmol min mg蛋白），它们可能是该酶的潜在诊断探针。在杀虫剂中，只有吡虫啉被CYP417A2v2有效降解。吡虫啉与CYP417A2v2孵育并随后进行HPLC、LC-MS和MS/MS分析，揭示了吡虫啉代谢物的形成，即通过羟基化形成4'或5'-羟基吡虫啉。这一结果意味着CYPs的特性并非总是如此，并非所有降解杀虫剂的CYPs都在抗性品系中被选择和过表达，并且在杀虫剂抗性进化过程中，未发生上调突变的降解CYPs可能是候选者。单个昆虫CYPs在杀虫剂降解中的这种特性可为更好地理解杀虫剂抗性发展提供见解。
