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首页> 外文期刊>Tropical biomedicine. >Protective efficacy of Anopheles minimus CYP6P7 and CYP6AA3 against cytotoxicity of pyrethroid insecticides in Spodoptera frugiperda (Sf9) insect cells.
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Protective efficacy of Anopheles minimus CYP6P7 and CYP6AA3 against cytotoxicity of pyrethroid insecticides in Spodoptera frugiperda (Sf9) insect cells.

机译：微小按蚊CYP6P7和CYP6AA3对拟除虫菊酯（Sf9）昆虫细胞中拟除虫菊酯类杀虫剂的细胞毒性的保护作用。

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摘要

Cytochrome P450 monooxygenases (P450s) are enzymes known to metabolize a wide variety of compounds including insecticides. Their overexpression leading to enhanced insecticide detoxification could result in insecticide resistance in insects. The increased mRNA expression of two P450 genes, CYP6P7 and CYP6AA3, has been previously observed in laboratory-selected deltamethrin-resistant Anopheles minimus, a major malaria vector in Southeast Asia, suggesting their role in detoxification of pyrethroids. In this study CYP6P7 and CYP6AA3 were expressed in insect Spodoptera frugiperda (Sf9) cells via baculovirusdirected expression system. Insecticide detoxification capabilities of Sf9 cells with and without expression of CYP6P7 or CYP6AA3 were evaluated using 3-(4,5-dimethyl-thiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The results revealed that CYP6P7- or CYP6AA3-expressing cells showed significantly higher cytoprotective capability than parental Sf9 cells against cytotoxicity of pyrethroids including permethrin, cypermethrin and deltamethrin. Such cytoprotective effect was not observed for bioallethrin (pyrethroid), chlorpyrifos (organophosphate) and propoxur (carbamate). Moreover, expression of CYP6AA3, but not CYP6P7, could protect cells against lambda-cyhalothrin cytotoxicity. In MTT assays upon co-incubation with piperonyl butoxide (P450 inhibitor), cytoprotective ability of CYP6P7 and CYP6AA3 against deltamethrin was diminished, implying that pyrethroid detoxification was due to activities of P450 enzymes. Insecticide detoxification capabilities of CYP6P7 and CYP6AA3 observed from MTT assays were correlated to their pyrethroid metabolizing activities observed from in vitro reconstitution enzymatic assays. Thus MTT assays using cells expressing P450 enzymes of interest could be primarily used to determine detoxification activities of enzymes against cytotoxic insecticides.
机译：细胞色素P450单加氧酶（P450）是已知可代谢多种化合物（包括杀虫剂）的酶。它们的过表达导致增强的杀虫剂排毒作用，可能导致昆虫对杀虫剂产生抗药性。 CYP6P7和CYP6AA3这两个P450基因的mRNA表达增加，以前在实验室选择的耐溴氰菊酯的东南亚最小疟原虫中检测到，表明它们在拟除虫菊酯的解毒中起作用。在这项研究中，CYP6P7和CYP6AA3通过杆状病毒定向表达系统在昆虫贪食夜蛾（Sf9）细胞中表达。使用3-（4,5-二甲基-噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）分析评估有或没有CYP6P7或CYP6AA3的Sf9细胞的杀虫剂解毒能力。结果表明，表达CYP6P7-或CYP6AA3的细胞对拟除虫菊酯包括氯菊酯，氯氰菊酯和溴氰菊酯的细胞毒性显示出比亲代Sf9细胞更高的细胞保护能力。对于生物Allethethrin（拟除虫菊酯），毒死rif（有机磷酸酯）和Prooxur（氨基甲酸酯）未观察到这种细胞保护作用。此外，CYP6AA3而非CYP6P7的表达可以保护细胞免受λ-氯氟氰菊酯的细胞毒性。在与胡椒基丁醚（P450抑制剂）共孵育的MTT分析中，CYP6P7和CYP6AA3对溴氰菊酯的细胞保护能力减弱，这表明拟除虫菊酯的解毒归因于P450酶的活性。从MTT分析中观察到的CYP6P7和CYP6AA3的杀虫剂解毒能力与从体外重组酶分析中观察到的拟除虫菊酯的代谢活性相关。因此，使用表达目的P450酶的细胞进行的MTT分析可主要用于确定酶对细胞毒性杀虫剂的解毒活性。

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