The function of two P450s, CYP9M10 and CYP6AA7, in the permethrin resistance of Culex quinquefasciatus

Abstract

Cytochrome P450 monooxygenases play a critical role in insecticide resistance by allowing resistant insects to metabolize insecticides. Previous studies revealed that two P450 genes, CYP9M10 and CYP6AA7, are not only up-regulated but also induced in resistant Culex mosquitoes. In this study, CYP9M10 and CYP6AA7 were separately co-expressed with cytochrome P450 reductase (CPR) in insect Spodoptera frugiperda (Sf9) cells using a baculovirus-mediated expression system and the enzymatic activity and metabolic ability of CYP9M10/CPR and CYP6AA7/CPR to permethrin and its metabolites, including 3-phenoxybenzoic alcohol (PBOH) and 3-phenoxybenzaldehyde (PBCHO), characterized. PBOH and PBCHO, both of which are toxic to Culex mosquito larvae, can be further metabolized by CYP9M10/CPR and CYP6AA7/CPR, with the ultimate metabolite identified here as PBCOOH, which is considerably less toxic to mosquito larvae. A cell-based MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assay revealed that Sf9 cells expressing CYP9M10/CPR or CYP6AA7/CPR increased the cell line's tolerance to permethrin, PBOH, and PBCHO. This study confirms the important role played by CYP9M10 and CYP6AA7 in the detoxification of permethrin and its metabolites PBOH and PBCHO.

Figure 1

Biochemical characterization of P450 content and CPR activity of co-expressed P450/CPR. (A) P450 content detected in microsomal protein of Sf9 cells expressing CYP9M10; (B) P450 content detected in microsomal protein of Sf9 cells expressing CYP6AA7; (C) CPR activity measured in microsomal protein of Sf9 cells co-expressing CYP9M10/CPR; (D) CPR activity measured in microsomal protein of 6AA7/CPR expressing Sf9 cells.

Figure 2

The role of P450/CPR in the detoxification of permethrin in Sf9 insect cells. (A) Viability of Sf9 cells co-expressing CYP9M10/CPR, the cells expressing chloramphenicol acetyltransferase (CAT) protein [Invitrogen], and parental Sf9 cells treated with 12.5, 25, 50, 100, 200, and 400 µM of permethrin; (B) Effects of permethrin and PBO on viability of co-expressing CYP9M10/CPR Sf9 cells and Sf9 cells alone, treated with 0.1, 1, and 10 µM of PBO; (C) Viability of CYP6AA7/CPR co-expressing Sf9 cells and Sf9 cells alone, treated with 12.5, 25, 50, 100, 200, and 400 µM of permethrin. Student’s t-test was used for the statistical significance analysis. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

The role of CYP9M10/CPR and CYP6AA7/CPR in the detoxification of PBOH or PBCHO in Sf9 insect cells. (A) Viability of CYP9M10 or CYP6AA7 and CPR co-expressing Sf9 cells and Sf9 cells alone, treated with 31.25, 62.5, 125, 250, 400, 500, and 700 µM of PBOH; (B) Viability of CYP9M10 or CYP6AA7 and CPR co-expressing Sf9 cells and Sf9 cells alone, treated with 31.25, 62.5, 125, 250, 400, 500, and 700 µM of PBCHO. Student’s t-test was used for the statistical significance analysis. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Permethrin metabolism by microsomes of CYP/CPR. (A) Control: permethrin (20 µM) incubated with CYP9M10/CPR or CYP6AA7/CPR microsomes (100 pmol P450 in 0.1 M Tris pH = 7.5) for 2 h at 30 °C without NADPH. The black arrows indicate the peaks for PBOH, trans-permethrin, and cis-permethrin; (B) Permethrin metabolized by microsomes of CYP9M10/CPR expressing Sf9 cells with NADPH; (C) Permethrin metabolized by microsomes of CYP6AA7/CPR expressing Sf9 cells with NADPH; (D) Permethrin turnover rate in the CYP/CPR co-expressed microsomal protein with and without NADPH, and the rate of formation of the metabolite PBOH in microsomes of CYP9M10/CPR or CYP6AA7/CPR expressing Sf9 cells.

Figure 5

Metabolism profile of PBOH by CYP6AA7/CPR or CYP9M10/CPR microsomes. (A) Control: PBOH (20 µM) incubated with CYP9M10/CPR or CYP6AA7/CPR microsomes (100 pmol P450 in 0.1 M Tris pH = 7.5) for 2 h at 30 °C without NADPH. The black arrows indicate the peaks for PBOH and its metabolite PBCOOH; (B) PBOH metabolized by microsomes of CYP9M10/CPR expressing Sf9 cells with NADPH; (C) PBOH metabolized by microsomes of CYP6AA7/CPR expressing Sf9 cells with NADPH; D. Metabolism rate of PBOH in different CYP/CPR microsomes. The results are shown as the mean ± S.E (n ≥ 3). Statistical significance is represented by P ≤ 0.05 using one-way ANOVA.

Figure 6

Metabolism profile of PBCHO by CYP6AA7/CPR and CYP9M10/CPR microsomes. (A) Control: PBCHO (10 µM) treated with CYP/CPR microsomes (100 pmol P450 in 0.1 M Tris pH = 7.5) for 15 min at 30 °C without NADPH. The black arrows indicate the peaks for PBCHO and PBCOOH; (B) PBCHO metabolized by CYP9M10/CPR microsomes. The red arrows indicate the PBCOOH and the reduced amount of PBCHO; (C) PBCHO metabolized by CYP6AA7/CPR microsomes. The red arrows indicate the PBCOOH and the reduced amount of PBCHO; (D) Metabolism rate of PBCHO with different CYP/CPR microsomes. Statistical significance is represented by P ≤ 0.05, with the letters using one-way ANOVA.