title: Protective efficacy of Anopheles minimus CYP6P7 and CYP6AA3 against cytotoxicity of pyrethroid insecticides in Spodoptera frugiperda (Sf9) insect cells.
abstract: 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 λ-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.
authors: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand 10400.
authors: P
authors: P
authors: Duangkaew
authors: D
authors: D
authors: Kaewpa
authors: P
authors: P
authors: Rongnoparut
mesh_terms: D000818:Animals; D000852:Anopheles / Q000201:enzymology* / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D003584:Cytological Techniques / Q000379:methods; D015870:Gene Expression; D007306:Insecticides / Q000378:metabolism* / Q000494:pharmacology* / Q000633:toxicity; D011722:Pyrethrins / Q000378:metabolism* / Q000494:pharmacology* / Q000633:toxicity; D018411:Spodoptera; D013194:Staining and Labeling / Q000379:methods; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D007306:Insecticides; D011722:Pyrethrins; D013778:Tetrazolium Salts; D013844:Thiazoles; D003577:Cytochrome P-450 Enzyme System; C022616:thiazolyl blue