Structure-activity relationship and in vitro inhibition of human cytochrome CYP2A6 and CYP2A13 by flavonoids.

Cigarette smoking is one of the major risk factors of various diseases including respiratory diseases and lung cancer. While the liver-specific CYP2A6 is associated with the nicotine clearance and smoking addiction, the metabolic activation of the tobacco-specific nitrosamine by lung-specific CYP2A13 can lead to lung tumorigenesis.It has been reported that inhibition of CYP2A6 and CYP2A13 enzymes by flavonoids constituents could be an aids in smoking cessation. This study demonstrates the inhibition activity of kaempferol and myricetin and the structure-function relationship of these two flavonoids and previously isolated flavonoids from Vernonia cinerea and Pluchea indica against both enzymes.Kaempferol could inhibit CYP2A6 with Kic value of 1.77 ± 0.47 µM while inhibit CYP2A13 with Kic value of 0.12 ± 0.01 µM. Myricetin could inhibit CYP2A6 with Kic value of 4.06 ± 0.52 µM while inhibit CYP2A13 with Kic value of 1.88 ± 0.03 µM.Molecular docking indicated that CYP2A13 enzyme has strong hydrophobic interaction with ring B of flavonoids compared to CYP2A6 enzyme. The presence of the hydroxyl group at C3 position of ring C and the hydroxyl group at C5' of ring B affected inhibitory activity on both enzymes.

Structural and Functional Studies of the Membrane-Binding Domain of NADPH-Cytochrome P450 Oxidoreductase.

NADPH-cytochrome P450 oxidoreductase (CYPOR), the essential flavoprotein of the microsomal cytochrome P450 monooxygenase system, is anchored in the phospholipid bilayer by its amino-terminal membrane-binding domain (MBD), which is necessary for efficient electron transfer to cytochrome P450. Although crystallographic and kinetic studies have established the structure of the soluble catalytic domain and the role of conformational motions in the control of electron transfer, the role of the MBD is largely unknown. We examined the role of the MBD in P450 catalysis through studies of amino-terminal deletion mutants and site-directed spin labeling. We show that the MBD spans the membrane and present a model for the orientation of CYPOR on the membrane capable of forming a complex with cytochrome P450. EPR power saturation measurements of CYPOR mutants in liposomes containing a lipid/Ni(II) chelate identified a region of the soluble domain interacting with the membrane. The deletion of more than 29 residues from the N-terminus of CYPOR decreases cytochrome P450 activity concomitant with alterations in electrophoretic mobility and an increased resistance to protease digestion. The altered kinetic properties of these mutants are consistent with electron transfer through random collisions rather than via formation of a stable CYPOR-P450 complex. Purified MBD binds weakly to cytochrome P450, suggesting that other interactions are also required for CYPOR-P450 complex formation. We propose that the MBD and flexible tether region of CYPOR, residues 51-63, play an important role in facilitating the movement of the soluble domain relative to the membrane and in promoting multiple orientations that permit specific interactions of CYPOR with its varied partners.

Anti-proliferative effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) isolated from Vernonia cinerea on oral squamous cell carcinoma through inhibition of STAT3 and STAT2 phosphorylation.

BACKGROUND: The high mortality rate of oral cancers has stimulated the search for effective herbal medicines and their pharmacological targets. Vernonia cinerea, a perennial tropical herb, is wildly used as a traditional folk medicine for treatment of intestinal diseases and various skin diseases in addition to possessing anti-cancer activity. However, the effect of 8α-tigloyloxyhirsutinolide-13-O-acetate (8αTGH) as a major sesquiterpene lactone compound found in V. cinerea and the underlying mechanism of its action on oral cancer cells remains unknown. PURPOSE: To investigate the anti-cancer activity of 8αTGH extracted from V. cinerea and the underlying mechanism of its action in oral cancer cells. METHODS: The anti-proliferative effect of 8αTGH on oral squamous cell carcinoma (HSC4) and lung carcinoma (A549) was determined using the SRB colorimetric method. The molecular mechanism of 8αTGH was explored using kinase inhibitors, followed by Western blotting or RT-qPCR. Flow cytometry and Western blotting were used to assess cell cycle arrest. RESULTS: 8αTGH inhibited cancer cell growth more effectively on HSC4 than A549 and was much less effective on tested normal oral cells. 8αTGH inhibited STAT3 phosphorylation on both cancer cells. Notably, 8αTGH was able to suppress the constantly activated STAT2 found only in HSC4. The STAT2 inhibition by 8αTGH consequently caused down-regulation of ISG15 and ISG15 conjugates. As a result, decreased expression of CDK1/2 and Cyclin B1 was detected leading to G2/M cell cycle arrest. CONCLUSION: 8αTGH isolated from V. cinerea preferentially inhibits the proliferation of oral cancer cells by causing G2/M cell cycle arrest via inhibition of both STAT3 and STAT2 phosphorylation. The results provide molecular bases for developing 8αTGH as a drug candidate or a complementary treatment of oral cancer.

Effects of Vernonia cinerea Compounds on Drug-metabolizing Cytochrome P450s in Human Liver Microsomes.

Vernonia cinerea has been widely used in traditional medicines for various diseases and shown to aid in smoking abstinence and has anticancer properties. V. cinerea bioactive compounds, including flavonoids and hirsutinolide-type sesquiterpene lactones, have shown an inhibition effect on the nicotine-metabolizing cytochrome P450 2A6 (CYP2A6) enzyme and hirsutinolides reported suppressing cancer growth. In this study, V. cinerea ethanol extract and its bioactive compounds, including four flavonoids and four hirsutinolides, were investigated for an inhibitory effect on human liver microsomal CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 using cocktail inhibition assays combined with LC-MS/MS analysis. Among tested flavonoids, chrysoeriol was more potent in inhibition on CYP2A6 and CYP1A2 than other liver CYPs, with better binding efficiency toward CYP2A6 than CYP1A2 (Ki values in competitive mode of 1.93 ± 0.05 versus 3.39 ± 0.21 µM, respectively). Hirsutinolides were prominent inhibitors of CYP2A6 and CYP2D6, with IC50 values of 12-23 and 15-41 µM, respectively. These hirsutinolides demonstrated time-dependent inhibition, an indication of mechanism-based inactivation, toward CYP2A6. Quantitative prediction of microsomal metabolism of these flavonoids and hirsutinolides, including half-lives and hepatic clearance rate, was examined. These findings may have implications for further in vivo studies of V. cinerea. Copyright © 2017 John Wiley & Sons, Ltd.

Inhibition of human cytochromes P450 2A6 and 2A13 by flavonoids, acetylenic thiophenes and sesquiterpene lactones from Pluchea indica and Vernonia cinerea.

The human liver cytochrome P450 (CYP) 2A6 and the respiratory CYP2A13 enzymes play role in nicotine metabolism and activation of tobacco-specific nitrosamine carcinogens. Inhibition of both enzymes could offer a strategy for smoking abstinence and decreased risks of respiratory diseases and lung cancer. In this study, activity-guided isolation identified four flavonoids 1-4 (apigenin, luteolin, chrysoeriol, quercetin) from Vernonia cinerea and Pluchea indica, four hirsutinolide-type sesquiterpene lactones 5-8 from V. cinerea, and acetylenic thiophenes 9-11 from P. indica that inhibited CYP2A6- and CYP2A13-mediated coumarin 7-hydroxylation. Flavonoids were most effective in inhibition against CYP2A6 and CYP2A13, followed by thiophenes, and hirsutinolides. Hirsutinolides and thiophenes exhibited mechanism-based inhibition and in irreversible mode against both enzymes. The inactivation kinetic KI values of hirsutinolides against CYP2A6 and CYP2A13 were 5.32-15.4 and 0.92-8.67 µM, respectively, while those of thiophenes were 0.11-1.01 and 0.67-0.97 µM, respectively.

Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V. cinerea that comprise inhibitory activity toward CYP2A6 and MAO-A and MAO-B enzymes using activity-guided assays, with coumarin as substrate of CYP2A6 and kynuramine of MAOs. These compounds were three flavones (apigenin, chrysoeriol, luteolin), one flavonol (quercetin), and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate, 8α-tigloyloxyhirsutinolide-13-O-acetate, and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate). Modes and kinetics of inhibition against the three enzymes were determined. Flavonoids possessed strong inhibitory effect on CYP2A6 in reversible mode, while inhibition by hirsutinolides was mechanism-based (NADPH-, concentration-, and time-dependence) and irreversible. Inhibition by hirsutinolides could not be reversed by dialysis and by addition of trapping agents or potassium ferricyanide. Flavonoids inhibited MAOs with variable degrees and were more prominent in inhibition toward MAO-A than hirsutinolides, while two of hirsutinolides inhibited MAO-B approximately comparable to two flavonoids. These results could have implications in combination of drug therapy for smoking cessation.

Synergy between rhinacanthins from Rhinacanthus nasutus in inhibition against mosquito cytochrome P450 enzymes.

The cytochrome P450 monooxygenases play a major role in insecticide detoxification and become a target for development of insecticide synergists. In this study, a collection of rhinacanthins (rhinacanthin-D, -E, -G, -N, -Q, and -H/I) purified from Rhinacanthus nasutus, in addition to previously purified rhinacanthin-B and -C, were isolated. These compounds displayed various degrees of inhibition against benzyloxyresorufin-O-debenzylation mediated by CYP6AA3 and CYP6P7 which were implicated in pyrethroid resistance in Anopheles minimus malaria vector. Inhibition modes and kinetics were determined for each of rhinacanthins. Cell-based inhibition assays by rhinacanthins employing 3-(4, 5-dimethylthiazol-2-y-l)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity test were explored their synergistic effects with cypermethrin toxicity on CYP6AA3- and CYP6P7-expressing Spodoptera frugiperda (Sf9) cells. Rhinacanthin-B, -D, -E, -G, and -N exhibited mechanism-based inhibition against CYP6AA3, an indication of irreversible inhibition, while rhinacanthin-B, -D, -G, and -N were mechanism-based inhibitors of CYP6P7. There was structure-function relationship of these rhinacanthins in inhibition effects against both enzymes. In vitro enzymatic inhibition assays revealed that there were synergistic interactions among rhinacanthins, except rhinacanthin-B and -Q, in inhibition against both enzymes. These rhinacanthins exerted synergism with cypermethrin toxicity on Sf9 cells expressing each of the two P450 enzymes via P450 inhibition and in addition could interact in synergy to further increase cypermethrin toxicity. The inhibition potentials, synergy among rhinacanthins in inhibition against the P450 detoxification enzymes, and synergism with cypermethrin toxicity of the R. nasutus constituents of reported herein could be beneficial to implement effective resistance management of mosquito vector control.

Structure­function relationships of inhibition of mosquito cytochrome P450 enzymes by flavonoids of Andrographis paniculata.

The cytochrome P450 monooxygenases are known to play a major role in pyrethroid resistance, by means of increased rate of insecticide detoxification as a result of their overexpression. Inhibition of detoxification enzymes may help disrupting insect detoxifying defense system. The Anopheles minimus CYP6AA3 and CYP6P7 have shown pyrethroid degradation activity and been implicated in pyrethroid resistance. In this study inhibition of the extracts and constituents of Andrographis paniculata Nees. leaves and roots was examined against benzyloxyresorufin O-debenzylation (BROD) of CYP6AA3 and CYP6P7. Four purified flavones (5,7,4'-trihydroxyflavone, 5-hydroxy-7,8-dimethoxyflavone, 5-hydroxy-7,8,2',3'-tetramethoxyflavone, and 5,4'-dihydroxy-7,8,2',3'-tetramethoxyflavone), one flavanone (5-hydroxy-7,8-dimethoxyflavanone) and a diterpenoid (14-deoxy-11,12-didehydroandrographolide) containing inhibitory effects toward both enzymes were isolated from A. paniculata. Structure­function relationships were observed for modes and kinetics of inhibition among flavones, while diterpenoid and flavanone were inferior to flavones. Docking of flavones onto enzyme homology models reinforced relationships on flavone structures and inhibition modes. Cell-based inhibition assays employing 3-(4,5-dimethylthiazol-2-y-l)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assays revealed that these flavonoids efficiently increased susceptibility of CYP6AA3- and CYP6P7-expressing Spodoptera frugiperda (Sf9) cells to cypermethrin toxicity, due to inhibition effects on mosquito enzymes. Thus synergistic effects on cypermethrin toxicity of A. paniculata compounds as a result of enzyme inhibition could be useful for mosquito vector control and insecticide resistance management in the future.

Mechanism-based inactivation of cytochrome P450 2A6 and 2A13 by Rhinacanthus nasutus constituents.

  Human cytochrome P450 CYP2A6 and CYP2A13 catalyze nicotine metabolisms and mediate activation of tobacco-specific carcinogens including 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). In this study, we found rhinacanthins A, B, and C isolated from Rhinacanthus nasutus potentially inhibited coumarin 7-hydroxylation mediated by reconstituted purified recombinant CYP2A6 and CYP2A13. Rhinacanthins A-C are mechanism-based inactivators of CYP2A6 and CYP2A13 as they cause concentration, time and NADPH-dependent inhibition. Among the three rhinacanthins, rhinacanthin-B possessed highest inhibitory potency against CYP2A13 with apparent KI and kinact of 0.16 µM and 0.1 min(-1), respectively, while values of 0.44 µM and 0.12 min(-1) were found against CYP2A6. Rhinacanthin-C had least inhibition potency, with apparent KI and kinact of 0.97 µM and 0.07 min(-1) for CYP2A6, respectively, and values of 1.68 µM and 0.05 min(-1) for CYP2A13. Rhinacanthin-A inhibited CYP2A6 and CYP2A13 with apparent KI values of 0.69 and 0.42 µM, respectively and apparent kinact of 0.18 and 0.06 min(-1), respectively. The inhibition of both enzymes by rhinacanthins A-C could not be prevented by addition of trapping agents or reversed by dialysis or potassium ferricyanide. These findings demonstrated that rhinacanthins A-C, which are 1,4-naphthoquinone derivatives, irreversibly inhibited CYP2A6 and CYP2A13 in a mechanism-based inhibition mode.

Distal effect of amino acid substitutions in CYP2C9 polymorphic variants causes differences in interatomic interactions against (S)-warfarin.

Cytochrome P450 2C9 (CYP2C9) is crucial in excretion of commonly prescribed drugs. However, changes in metabolic activity caused by CYP2C9 polymorphisms inevitably result in adverse drug effects. CYP2C9*2 and *3 are prevalent in Caucasian populations whereas CYP2C9*13 is remarkable in Asian populations. Single amino acid substitutions caused by these mutations are located outside catalytic cavity but affect kinetic activities of mutants compared to wild-type enzyme. To relate distal effects of these mutations and defective drug metabolisms, simulations of CYP2C9 binding to anti-coagulant (S)-warfarin were performed as a system model. Representative (S)-warfarin-bound forms of wild-type and mutants were sorted and assessed through knowledge-based scoring function. Interatomic interactions towards (S)-warfarin were predicted to be less favorable in mutant structures in correlation with larger distance between hydroxylation site of (S)-warfarin and reactive oxyferryl heme than wild-type structure. Using computational approach could delineate complication of CYP polymorphism in management of drug therapy.

Modeling of Anopheles minimus Mosquito NADPH-cytochrome P450 oxidoreductase (CYPOR) and mutagenesis analysis.

Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two- to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs.

Inhibition against mosquito cytochrome P450 enzymes by rhinacanthin-A, -B, and -C elicits synergism on cypermethrin cytotoxicity in Spodoptera frugiperda cells.

Rhinacanthus nasutus (Acanthaceae) is a shrub reported to contain insecticidal activities. The current study was conducted to determine whether R. nasutus constituents could inhibit benzyloxyresorufin O-debenzylation (BROD) mediated by CYP6AA3 and CYP6P7. Both enzymes have shown pyrethroid degradation activity and been implicated to play role in pyrethroid resistance in Anopheles minumus (Theobald) mosquito, a malaria vector. Three compounds, rhinacanthin-A, -B, and -C that exhibited potent inhibitory activity were isolated and purified from aerial part of R. nasutus. Their kinetic parameters and modes of inhibition were determined. Rhinacanthin-B was the most potent inhibitor in in vitro inhibition assay and exhibited mechanism-based inhibition against both CYP6AA3 and CYP6P7. Rhinacanthin-C which is a major compound of R. nasutus reversibly inhibited both enzymes in vitro with 2-4 folds less inhibitory potency than rhinacanthin-B. In contrast, rhinacanthin-A reversibly inhibited CYP6AA3, but inhibition against CYP6P7 was a mechanism-based inhibition type. Where mechanism-based inhibition was found, the inhibition showed characteristic of time-, concentration-dependence, and requirement of NADPH. Using 3-(4, 5-dimethylthiazol-2-y-l)-2, 5-diphenyltetrazolium bromide (MTT) cytotoxicity assay in intact Spodoptera frugiperda (Sf9) cells, the three compounds increased susceptibility of CYP6AA3- and CYP6P7-expressing cells to cypermethrin cytotoxicity because of inhibition effect on mosquito enzymes. The combined inhibition effect on mosquito cytochrome P450 enzyme and synergistic effect on cypermethrin cytotoxicity of the three R. nasutus compounds could be beneficial for resistance management strategies in mosquito vector control.

Homology modeling of mosquito cytochrome P450 enzymes involved in pyrethroid metabolism: insights into differences in substrate selectivity.

BACKGROUND: Cytochrome P450 enzymes (P450s) have been implicated in insecticide resistance. Anopheles minumus mosquito P450 isoforms CYP6AA3 and CYP6P7 are capable of metabolizing pyrethroid insecticides, however CYP6P8 lacks activity against this class of compounds. FINDINGS: Homology models of the three An. minimus P450 enzymes were constructed using the multiple template alignment method. The predicted enzyme model structures were compared and used for molecular docking with insecticides and compared with results of in vitro enzymatic assays. The three model structures comprise common P450 folds but differences in geometry of their active-site cavities and substrate access channels are prominent. The CYP6AA3 model has a large active site allowing it to accommodate multiple conformations of pyrethroids. The predicted CYP6P7 active site is more constrained and less accessible to binding of pyrethroids. Moreover the predicted hydrophobic interface in the active-site cavities of CYP6AA3 and CYP6P7 may contribute to their substrate selectivity. The absence of CYP6P8 activity toward pyrethroids appears to be due to its small substrate access channel and the presence of R114 and R216 that may prevent access of pyrethroids to the enzyme heme center. CONCLUSIONS: Differences in active site topologies among CYPAA3, CYP6P7, and CYP6P8 enzymes may impact substrate binding and selectivity. Information obtained using homology models has the potential to enhance the understanding of pyrethroid metabolism and detoxification mediated by P450 enzymes.

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties.

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus-mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH-cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ-cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ-cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.

Transcription analysis of differentially expressed genes in insecticide-resistant Aedes aegypti mosquitoes after deltamethrin exposure.

Aedes aegypti mosquitoes are resistant to various insecticides, including pyrethroids, throughout Thailand. We previously reported that Ae. aegypti from Mae Wong district, Nakhon Sawan Province in north-central Thailand, were resistant to insecticides, including pyrethroids (deltamethrin and permethrin), organophosphates and carbamates, and that high levels of detoxification enzymes were present. In the present study we used the method of suppression by subtractive hybridization to determine differential expression of genes in Mae Wong Ae. aegypti that survived the exposure to increasing doses (approximately 1.5-2x10(-5) M) of deltamethrin beyond the diagnostic dose compared to unexposed mosquitoes. Screening of 350 cDNA clones from the suppression subtractive library by cDNA array hybridization revealed that 58 clones were over-expressed in the mosquito that survived high dose deltamethrin. The over-expressed cDNA insert sequences corresponded to 11 functional genes, five hypothetical protein genes, and five sequences of unknown function that could be located on the supercontig of the Ae. aegypti genome. The functional genes are those coding for cuticular proteins, muscle proteins, proteins related to controlling the release of synaptic vesicles, and other genes such as heat shock protein and small subunit ribosomal RNA. Over-expression of tomosyn and myosin light chain kinase genes was verified using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), confirming their increased expression in response to deltamethrin exposure in insecticide-resistant Ae. aegypti.

Mosquito NADPH-cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolism.

The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane-bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane-bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR-mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c-reducing activity indicated that the wild-type and mutant flAnCYPORs followed a non-classical two-site Ping-Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH-regenerating system, increased CYP6AA3-mediated deltamethrin degradation compared to the wild-type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3-mediated activity supported by wild-type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3-mediated metabolism.

Genetic structure among Thai populations of Aedes aegypti mosquitoes.

Thirty-one field populations of Aedes aegypti (L.) were compared using isozyme starch gel electrophoresis to characterize genetic variation between populations. Ae. aegypti were collected from seven provinces in Thailand. Thirty-one isozyme encoding loci, including 19 polymorphic loci, were characterized. Only small levels of genetic differentiation were observed among the 31 district populations in the seven provinces. Isolation by distance among populations from the seven provinces showed no correlation between genetic variation and geographical distance.

Characterization of Anopheles minimus CYP6AA3 expressed in a recombinant baculovirus system.

Metabolism by cytochrome P450 monooxygenases is a major mechanism implicated in resistance of insects to insecticides, including pyrethroids. We previously isolated the cytochrome P450 CYP6AA3 from deltamethrin-selected resistant strain of Anopheles minimus mosquito, a major malaria vector in Thailand. In the present study, we further investigated the role of CYP6AA3 enzyme in deltamethrin metabolism in vitro. The CYP6AA3 was expressed in Spodoptera frugiperda (Sf9) insect cells via baculovirus-mediated expression system. The enzymatic activity of CYP6AA3 in deltamethrin metabolism was characterized after being reconstituted with An. minimus NADPH-cytochrome P450 reductase and a NADPH-regenerating system. The contribution of CYP6AA3 responsible for deltamethrin metabolism was determined by measurement of deltamethrin disappearance following the incubation period and deltamethrin-derived compounds were detected using combined gas chromatography mass spectrometry analysis. 3-Phenoxybenzaldehyde was a major product of CYP6AA3-mediated deltamethrin metabolism. Deltamethrin degradation and formation of metabolites were NADPH-dependent and inhibited by piperonyl butoxide. Deltamethrin was catalyzed by CYP6AA3 with an apparent K(m) of 80.0 +/- 2.0 and V(max) of 60.2 +/- 3.6 pmol/min/pmol P450. Furthermore, deltamethrin cytotoxicity assays by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue dye exclusion were examined in Sf9 insect cells, with and without expression of CYP6AA3. Results revealed that CYP6AA3 could play a role in detoxifying deltamethrin in the cells. Thus, the results of this study support the role of CYP6AA3 in deltamethrin metabolism.

NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability.

NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.

Trend of temephos resistance in Aedes (Stegomyia) mosquitoes in Thailand during 2003-2005.

The diagnostic dose for temephos susceptibility test was established based on Aedes aegypti, the susceptible Bora (French Polynesia) strain, for practical and routine use. The diagnostic dose was subsequently used to evaluate the susceptibility/resistance status in F1 progenies of field-collected samples from Bangkok and various parts of Thailand. It appeared that Ae. aegypti mosquitoes of one collection site each in Bangkok, Nakhon Sawan (northcentral), and Nakhon Ratchasrima (northeast) were resistant to temephos, with mortality ranging from 50.5 to 71.4%. Moreover, there was a trend of resistance to temephos among Ae. aegypti populations of all studied districts of Nakorn Ratchasima and most areas of Nakhon Sawan, of which those in one area were susceptible. However, various levels of temephos susceptibility were found in Bangkok populations, including resistance and incipient resistance. In Chonburi Province (eastern), all mosquitoes were susceptible to temephos with an indication of tolerance in one sample. Additionally, mosquitoes from Songkhla (south), Chiang Rai (north), Kanchanaburi (west), and Chanthaburi (east) remained susceptible to temephos during the sample collecting period. Bioassay tests on Aedes albopictus populations collected in this study from Nakhon Sawan, Nakorn Ratchasima, Songkhla, and Kanchanaburi revealed high susceptibility to temephos. Although the use of temephos seems to be potentially effective in many areas of the country, a noticeable trend of resistance indicated that alternative vector control methods should be periodically applied.