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1.  Effects of Anticholinesterases on Catalysis and Induced Conformational Change of the Peripheral Anionic Site of Murine Acetylcholinesterase 
Conventional insecticides targeting acetylcholinesterase (AChE) typically show high mammalian toxicities and because there is resistance to these compounds in many insect species, alternatives to established AChE inhibitors used for pest control are needed. Here we used a fluorescence method to monitor interactions between various AChE inhibitors and the AChE peripheral anionic site, which is a novel target for new insecticides acting on this enzyme. The assay uses thioflavin-T as a probe, which binds to the peripheral anionic site of AChE and yields an increase in fluorescent signal. Three types of AChE inhibitors were studied: catalytic site inhibitors (carbamate insecticides, edrophonium, and benzylpiperidine), peripheral site inhibitors (tubocurarine, ethidium bromide, and propidium iodide), and bivalent inhibitors (donepezil, BW284C51, and a series of bis(n)-tacrines). All were screened on murine AChE to compare and contrast changes of peripheral site conformation in the TFT assay with catalytic inhibition. All the inhibitors reduced thioflavin-T fluorescence in a concentration-dependent manner with potencies (IC50) ranging from 8 nM for bis(6)-tacrine to 159 μM for benzylpiperidine. Potencies in the fluorescence assay were correlated well with their potencies for enzyme inhibition (R2 = 0.884). Efficacies for reducing thioflavin-T fluorescence ranged from 23–36% for catalytic site inhibitors and tubocurarine to near 100% for ethidium bromide and propidium iodide. Maximal efficacies could be reconciled with known mechanisms of interaction of the inhibitors with AChE. When extended to pest species, we anticipate these findings will assist in the discovery and development of novel, selective bivalent insecticides acting on AChE.
doi:10.1016/j.pestbp.2013.04.001
PMCID: PMC3758491  PMID: 24003261
catalytic site; carbamate; donepezil; edrophonium; ethidium; fluorescence; propidium; tacrine; tacrine dimer; thioflavin-T
2.  Diversity and Convergence of Sodium Channel Mutations Involved in Resistance to Pyrethroids 
Pyrethroid insecticides target voltage-gated sodium channels, which are critical for electrical signaling in the nervous system. The intensive use of pyrethroids in controlling arthropod pests and disease vectors has led to many instances of pyrethroid resistance around the globe. In the past two decades, studies have identified a large number of sodium channel mutations that are associated with resistance to pyrethroids. The purpose of this review is to summarize both common and unique sodium channel mutations that have been identified in arthropod pests of importance to agriculture or human health. Identification of these mutations provides valuable molecular markers for resistance monitoring in the field and helped the discovery of the elusive pyrethroid receptor site(s) on the sodium channel.
doi:10.1016/j.pestbp.2013.02.007
PMCID: PMC3765034  PMID: 24019556
3.  Indoxacarb, Metaflumizone, and Other Sodium Channel Inhibitor Insecticides: Mechanism and Site of Action on Mammalian Voltage-Gated Sodium Channels 
Sodium channel inhibitor (SCI) insecticides were discovered almost four decades ago but have only recently yielded important commercial products (eg., indoxacarb and metaflumizone). SCI insecticides inhibit sodium channel function by binding selectively to slow-inactivated (non-conducting) sodium channel states. Characterization of the action of SCI insecticides on mammalian sodium channels using both biochemical and electrophysiological approaches demonstrates that they bind at or near a drug receptor site, the "local anesthetic (LA) receptor." This mechanism and site of action on sodium channels differentiates SCI insecticides from other insecticidal agents that act on sodium channels. However, SCI insecticides share a common mode of action with drugs currently under investigation as anticonvulsants and treatments for neuropathic pain. In this paper we summarize the development of the SCI insecticide class and the evidence that this structurally diverse group of compounds have a common mode of action on sodium channels. We then review research that has used site-directed mutagenesis and heterologous expression of cloned mammalian sodium channels in Xenopus laevis oocytes to further elucidate the site and mechanism of action of SCI insecticides. The results of these studies provide new insight into the mechanism of action of SCI insecticides on voltage-gated sodium channels, the location of the SCI insecticide receptor, and its relationship to the LA receptor that binds therapeutic SCI agents.
doi:10.1016/j.pestbp.2013.03.004
PMCID: PMC3780446  PMID: 24072940
indoxacarb; DCJW; metaflumizone; RH3421; sodium channel; local anesthetic receptor
4.  Inhibitor profile of bis(n)-tacrines and N-methylcarbamates on acetylcholinesterase from Rhipicephalus (Boophilus) microplus and Phlebotomus papatasi 
Pesticide biochemistry and physiology  2013;106(3):10.1016/j.pestbp.2013.03.005.
The cattle tick, Rhipicephalus (Boophilus) microplus (Bm), and the sand fly, Phlebotomus papatasi (Pp), are disease vectors to cattle and humans, respectively. The purpose of this study was to characterize the inhibitor profile of acetylcholinesterases from Bm (BmAChE1) and Pp (PpAChE) compared to human and bovine AChE, in order to identify divergent pharmacology that might lead to selective inhibitors. Results indicate that BmAChE has low sensitivity (IC50 = 200 μM) toward tacrine, a monovalent catalytic site inhibitor with sub micromolar blocking potency in all previous species tested. Similarly, a series of bis(n)-tacrine dimer series, bivalent inhibitors and peripheral site AChE inhibitors possess poor potency toward BmAChE. Molecular homology models suggest the rBmAChE enzyme possesses a W384F orthologous substitution near the catalytic site, where the larger tryptophan side chain obstructs the access of larger ligands to the active site, but functional analysis of this mutation suggests it only partially explains the low sensitivity to tacrine. In addition, BmAChE1 and PpAChE have low nanomolar sensitivity to some experimental carbamate anticholinesterases originally designed for control of the malaria mosquito, Anopheles gambiae. One experimental compound, 2-((2-ethylbutyl)thio)phenyl methylcarbamate, possesses >300-fold selectivity for BmAChE1 and PpAChE over human AChE, and a mouse oral LD50 of >1500 mg/kg, thus providing an excellent new lead for vector control.
doi:10.1016/j.pestbp.2013.03.005
PMCID: PMC3811934  PMID: 24187393
Leishmaniasis; sandfly; cattle fever; southern cattle tick; insecticides; selectivity
5.  Mechanism of action of sodium channel blocker insecticides (SCBIs) on insect sodium channels 
Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides, with a mechanism of action different from those of other classes of insecticides that target voltage-gated sodium channels. These compounds have no effect at hyperpolarized membrane potentials, but cause a voltage-dependent, nearly irreversible block as the membrane potential is depolarized. The mechanism of action of SCBIs is similar to that of local anesthetics (LAs), class I anticonvulsants and class I antiarrhythmics. In this article, we review the physiological actions of these compounds on the whole animal, the nervous system and sodium channels, and also present the results from recent studies that elucidate the receptor site of SCBIs.
doi:10.1016/j.pestbp.2009.09.001
PMCID: PMC3765036  PMID: 24013950
6.  Coexpression with Auxiliary β Subunits Modulates the Action of Tefluthrin on Rat Nav1.6 and Nav1.3 Sodium Channels 
We expressed the rat Nav1.3 and Nav1.6 sodium channel α subunit isoforms in Xenopus oocytes either alone or with the rat β1 and β2 auxiliary subunits in various combinations and assessed the sensitivity of the expressed channels to resting and use-dependent modification by the pyrethroid insecticide tefluthrin using the two-electrode voltage clamp technique. Coexpression with the β1 and β2 subunits, either individually or in combination, did not affecting the resting sensitivity of Nav1.6 channels to tefluthrin. Modification by tefluthrin of Nav1.6 channels in the absence of β subunits was not altered by the application of trains of high-frequency depolarizing prepulses. By contrast, coexpression of the Nav1.6 channel with the β1 subunit enhanced the extent of channel modification twofold following repeated depolarization. Coexpression of Nav1.6 with the β2 subunit also slightly enhanced modification following repeated depolarization, but coexpression of Nav1.6 with both β subunits caused enhanced modification following repeated depolarization that was indistinguishable from that found with Nav1.6+β1 channels. In contrast to Nav1.6, the resting modification by tefluthrin of Nav1.3 channels expressed in the absence of β subunits was reduced by repeated depolarization. However, tefluthrin modification of the Nav1.3 α subunit expressed with both β subunits was enhanced 1.7-fold by repeated depolarization, thereby confirming that β subunit modulation of use-dependent effects was not confined to the Nav1.6 isoform. These results show that the actions of pyrethroids on mammalian sodium channels in the Xenopus oocyte expression system are determined in part by the interactions of the sodium channel α subunit with the auxiliary β subunits that are part of the heteromultimeric sodium channel complexes found in neurons and other excitable cells.
doi:10.1016/j.pestbp.2011.10.003
PMCID: PMC3346283  PMID: 22577241
voltage-gated sodium channels; Nav1.6 isoform; Nav1.3 isoform; β subunit; voltage clamp; tefluthrin
7.  Actions of Tefluthrin on Rat Nav1.7 Voltage-Gated Sodium Channels Expressed in Xenopus Oocytes 
In rats expression of the Nav1.7 voltage-gated sodium channel isoform is restricted to the peripheral nervous system and is abundant in the sensory neurons of the dorsal root ganglion. We expressed the rat Nav1.7 sodium channel α subunit together with the rat auxiliary β1 and β2 subunits in Xenopus laevis oocytes and assessed the effects of the pyrethroid insecticide tefluthrin on the expressed currents using the two-electrode voltage clamp method. Tefluthrin at 100 µM modified of Nav1.7 channels to prolong inactivation of the peak current during a depolarizing pulse, resulting in a marked "late current" at the end of a 40-ms depolarization, and induced a sodium tail current following repolarization. Tefluthrin modification was enhanced up to two-fold by the application of a train of up to 100 5-ms depolarizing prepulses. These effects of tefluthrin on Nav1.7 channels were qualitatively similar to its effects on rat Nav1.2, Nav1.3 and Nav1.6 channels assayed previously under identical conditions. However, Nav1.7 sodium channels were distinguished by their low sensitivity to modification by tefluthrin, especially compared to Nav1.3 and Nav1.6 channels. It is likely that Nav1.7 channels contribute significantly to the tetrodotoxin-sensitive, pyrethroid-resistant current found in cultured dorsal root ganglion neurons. We aligned the complete amino acid sequences of four pyrethroid-sensitive isoforms (house fly Vssc1; rat Nav1.3, Nav1.6 and Nav1.8) and two pyrethroid-resistant isoforms (rat Nav1.2 and Nav1.7) and found only a single site, located in transmembrane segment 6 of homology domain I, at which the amino acid sequence was conserved among all four sensitive isoform sequences but differed in the two resistant isoform sequences. This position, corresponding to Val410 of the house fly Vssc1 sequence, also aligns with sites of multiple amino acid substitutions identified in the sodium channel sequences of pyrethroid-resistant insect populations. These results implicate this single amino acid polymorphism in transmembrane segment 6 of sodium channel homology domain I as a determinant of the differential pyrethroid sensitivity of rat sodium channel isoforms.
doi:10.1016/j.pestbp.2011.06.001
PMCID: PMC3181098  PMID: 21966053
voltage-gated sodium channel; Nav1.7 isoform; pyrethroid; tefluthrin; peripheral nervous system; dorsal root ganglion
8.  Investigations of the constitutive overexpression of CYP6D1 in the permethrin resistantLPR strain of house fly (Musca domestica) 
House fly (Musca domestica) CYP6D1 is a cytochrome P450 involved in metabolism of xenobiotics. CYP6D1 is located on chromosome 1 and its expression is inducible in response to the prototypical P450 inducer phenobarbital (PB) in insecticide susceptible strains. Increased transcription of CYP6D1 confers resistance to permethrin in the LPR strain, and this trait maps to chromosomes 1 and 2. However, the constitutive overexpression of CYP6D1 in LPR is not further increased by PB and the non-responsiveness to PB maps to chromosome 2. It has been suggested that a single factor on chromosome 2 could be responsible for both the constitutive overexpression and lack of PB induction of CYP6D1 in LPR. We examined the PB inducibility of CYP6D1v1 promoter from LPR using dual luciferase reporter assays in Drosophila S2 cells and found the CYP6D1v1 promoter was able to mediate PB induction, similar to the CYP6D1v2 promoter from the insecticide susceptible CS strain. Therefore, variation in promoter sequences of CYP6D1v1 and v2 does not appear responsible for the lack of PB induction of CYP6D1v1 in LPR; this suggests an unidentified trans acting factor is responsible. HR96 has been implicated in having a role in PB induction in Drosophila melanogaster and M. domestica. Therefore, house fly HR96 cDNA was cloned and sequenced to examine if this trans acting factor is responsible for constitutive overexpression of CYP6D1v1 in LPR. Multiple HR96 alleles (v1–v10) were identified, but none were associated with resistance. Expression levels of HR96 were not different between LPR and CS. Thus, HR96 is not the trans acting factor responsible for the constitutive overexpression of CYP6D1 in LPR. The identity of this trans acting factor remains elusive.
doi:10.1016/j.pestbp.2011.02.012
PMCID: PMC3134379  PMID: 21765560
Pyrethroid resistance; CYP6D1 transcription; Phenobarbital induction; HR96; Musca domestica; S2 cells
9.  Possible connection between imidacloprid-induced changes in rice gene transcription profiles and susceptibility to the brown plant hopper Nilaparvatalugens Stål (Hemiptera: Delphacidae) 
Pesticide Biochemistry and Physiology  2012;102-531(3):213-219.
Graphical abstract
Insecticide imidacloprid application induced the susceptibility of rice to the brown planthopper and changes of gene expression levels of rice.
Highlights
► Microarray was used to identify changes in imidacloprid-induced rice gene expression. ► A total of 225 genes were differentially expressed following imidacloprid treatment. ► These differentially expressed genes were mainly classified into eight functional groups.
The chemical pesticide, imidacloprid (IMI) has long-lasting effectiveness against Hemiptera. IMI is commonly used to control the brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae). Some chemical pesticides, however, can induce the susceptibility of rice to BPH, which has indirectly led to the resurgence of BPH. The mechanism of the chemical induction of the susceptibility of rice to BPH was not previously understood. Here, a 44 K Agilent Rice Expression Microarray was used to identify changes in gene expression that accompany IMI-induced rice susceptibility to BPH. The results showed that 225 genes were differentially expressed, of which 117 were upregulated, and 108 were downregulated. Gene ontology annotation and pathway analysis revealed that differentially expressed genes were mainly classified into the eight functional groups: oxidation reduction, regulation of cellular process, response to stress, electron carrier activity, metabolic process, transport, signal transducer, and organismal development. The genes encoding plant lipid transfer protein, lignin peroxidase, and flavonol-3-O-methyltransferenase may be important responses to the IMI-induced susceptibility of rice to BPH. The reliability of the microarray data was verified by performing quantitative real-time PCR and the data provide valuable information for further study of the molecular mechanism of IMI-induced susceptibility of rice.
doi:10.1016/j.pestbp.2012.01.003
PMCID: PMC3334832  PMID: 22544984
Imidacloprid; Rice brown planthopper; Susceptibility; Microarray
10.  Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals 
Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABAA receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC50s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABAA receptors. Moreover, GluCls of cockroach neurons had low IC50s for fipronil. Two types of glutamate-induced chloride current were obswerved: desensitizing and non-desensitizing, with fipronil IC50s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides.
doi:10.1016/j.pestbp.2009.07.008
PMCID: PMC2885805  PMID: 20563240
11.  Regulation of cytochrome P450 expression in Drosophila: Genomic insights 
Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers / induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action.
doi:10.1016/j.pestbp.2009.06.009
PMCID: PMC2890303  PMID: 20582327
12.  State-Dependent Modification of Voltage-Gated Sodium Channels by Pyrethroids 
Pyrethroids disrupt nerve function by altering the rapid kinetic transitions between conducting and nonconducting states of voltage-gated sodium channels that underlie the generation of nerve action potentials. Recent studies of pyrethroid action on cloned insect and mammalian sodium channel isoforms expressed in Xenopus laevis oocytes show that in some cases pyrethroid modification is either absolutely dependent on or significantly enhanced by repeated channel activation. These use-dependent effects have been interpreted as evidence of preferential binding of at least some pyrethroids to the open, rather than resting, state of the sodium channel. This paper reviews the evidence for state-dependent modification of insect and mammalian sodium channels expressed in oocytes by pyrethroids and considers the implications of state-dependent effects for understanding the molecular mechanism of pyrethroid action and the development and testing of models of the pyrethroid receptor.
doi:10.1016/j.pestbp.2009.06.010
PMCID: PMC2905833  PMID: 20652092
pyrethroid; cismethrin; cypermethrin; deltamethrin; permethrin; tefluthrin; sodium channel; insect; rat; human
13.  The effects of triazophos on the trehalose content, trehalase activity and their gene expression in the brown planthopper Nilaparvata lugens (Stål) (Hemiptera: Delphacidae) 
Graphical abstract
A previous study demonstrated that the flight capacity of Nilaparvata lugens adults treated with triazophos was enhanced significantly. However, the physiological and regulative mechanisms of the flight enhancement are not well understood. Trehalose is a primary blood sugar in insects, and the enzyme trehalase is involved in energy metabolism. The present study investigated the effects of triazophos on the trehalose content, trehalase activity (soluble trehalase and membrane-bound trehalase) and the mRNA transcript levels of their corresponding genes (NlTre-1 and NlTre-2) in fifth instar nymphs, as well as in the brachypterous and macropterous N. lugens adult females. Our findings showed that the trehalose content in fifth instar nymphs as well as in the brachypterous and the macropterous adults significantly decreased following triazophos treatment. However, the glucose content, soluble trehalase activity and expression level of NlTre-1 mRNA increased significantly compared to the controls. No significant enhancement of NlTre-2 expression was found, indicating that regulation of energy metabolism of triazophos-induced flight capacity in N. lugens was not associated with NlTre-2 expression. In addition, soluble trehalase activity and the expression level of NlTre-1 mRNA in the macropterous females was significantly higher than that in the brachypterous females. The present findings provide valuable information on the molecular and regulative mechanisms of the increased flight capacity found in adult N. lugens after treatment with triazophos.
Highlights
► The physiological and regulative mechanisms of insecticide-induced the flight enhancement are not well understood. ► The trehalose content in fifth instar nymphs, and in the brachypterous and the macropterous adults of the insect decreased following triazophos treatment. ► The glucose content, soluble trehalase activity and expression level of NlTre-1 mRNA increased significantly compared to the controls. ► Soluble trehalase activity and the expression level of NlTre-1 mRNA in the macropterous females was higher than that in the brachypterous females.
A previous study demonstrated that the flight capacity of Nilaparvata lugens adults treated with triazophos was enhanced significantly. However, the physiological and regulative mechanisms of the flight enhancement are not well understood. Trehalose is a primary blood sugar in insects, and the enzyme trehalase is involved in energy metabolism. The present study investigated the effects of triazophos on the trehalose content, trehalase activity (soluble trehalase and membrane-bound trehalase) and the mRNA transcript levels of their corresponding genes (NlTre-1 and NlTre-2) in fifth instar nymphs, as well as in the brachypterous and macropterous N. lugens adult females. Our findings showed that the trehalose content in fifth instar nymphs as well as in the brachypterous and the macropterous adults significantly decreased following triazophos treatment. However, the glucose content, soluble trehalase activity and expression level of NlTre-1 mRNA increased significantly compared to the controls. No significant enhancement of NlTre-2 expression was found, indicating that regulation of energy metabolism of triazophos-induced flight capacity in N. lugens was not associated with NlTre-2 expression. In addition, soluble trehalase activity and the expression level of NlTre-1 mRNA in the macropterous females was significantly higher than that in the brachypterous females. The present findings provide valuable information on the molecular and regulative mechanisms of the increased flight capacity found in adult N. lugens after treatment with triazophos.
doi:10.1016/j.pestbp.2011.03.007
PMCID: PMC3102831  PMID: 21760647
Nilaparvata lugens; Trehalose; Glucose; Soluble trehalase (NlTre-1); Membrane-bound trehalase (NlTre-2); Triazophos

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