Natural and synthetic allatotoxins: suicide substrates for juvenile hormone biosynthesis.

Cytotoxic agents with antijuvenile hormone activity in insects have been discovered. Their mechanism of action may involve an oxidative bioactivation into a reactive quinone methide.

The V410M mutation associated with pyrethroid resistance in Heliothis virescens reduces the pyrethroid sensitivity of house fly sodium channels expressed in Xenopus oocytes.

Some strains of Heliothis virescens carry a novel sodium channel mutation, corresponding to the replacement of Val410 by Met (designated V410M) in the house fly Vssc1 sodium channel, that is genetically and physiologically associated with pyrethroid resistance. To test the functional significance of this mutation, we created a house fly Vssc1 sodium channel containing the V410M mutation by site-directed mutagenesis, expressed wildtype and specifically mutated sodium channels in Xenopus laevis oocytes, and evaluated the effects of the V410M mutation on the functional and pharmacological properties of the expressed channels by two-electrode voltage clamp. The V410M mutation caused depolarizing shifts of approximately 9mV and approximately 5mV in the voltage dependence of activation and steady-state inactivation, respectively, of Vssc1 sodium channels. The V410M mutation also reduced the sensitivity of Vssc1 sodium channels to the pyrethroid cismethrin at least 10-fold and accelerated the decay of cismethrin-induced sodium tail currents. The degree of resistance conferred by the V410M mutation in the present study is sufficient to account for the degree of pyrethroid resistance in H. virescens that is associated with this mutation. Although Val410 is located in a sodium channel segment identified as part of the binding site for batrachotoxin, the V410M mutation did not alter the sensitivity of house fly sodium channels to batrachotoxin. The effects of the V410M mutation on the voltage dependence and cismethrin sensitivity of Vssc1 sodium channels were indistinguishable from those caused by another sodium channel point mutation, replacement of Leu1014 by Phe (L1014F), that is the cause of knockdown resistance to pyrethroids in the house fly. The positions of the V410M and L1014F mutations in models of the tertiary structure of sodium channels suggest that the pyrethroid binding site on the sodium channel alpha subunit is located at the interface between sodium channel domains I and II.

The molecular biology of knockdown resistance to pyrethroid insecticides.

The term "knockdown resistance" is used to describe cases of resistance to diphenylethane (e.g. DDT) and pyrethroid insecticides in insects and other arthropods that result from reduced sensitivity of the nervous system. Knockdown resistance, first identified and characterized in the house fly (Musca domestica) in the 1950's, remains a threat to the continued usefulness of pyrethroids in the control of many pest species. Research since 1990 has provided a wealth of new information on the molecular basis of knockdown resistance. This paper reviews these recent developments with emphasis on the results of genetic linkage analyses, the identification of gene mutations associated with knockdown resistance, and the functional characterization of resistance-associated mutations. Results of these studies identify voltage-sensitive sodium channel genes orthologous to the para gene of Drosophila melanogaster as the site of multiple knockdown resistance mutations and define the molecular mechanisms by which these mutations cause pyrethroid resistance. These results also provide new insight into the mechanisms by which pyrethroids modify the function of voltage-sensitive sodium channels.

PCR-based homology probing reveals a family of GABA receptor-like genes in Drosophila melanogaster.

A polymerase chain reaction (PCR)-based homology probing strategy was employed to screen Drosophila melanogaster genomic DNA for sequences encoding a conserved amino acid 'signature motif' known to be present in vertebrate GABA receptor and glycine receptor subunit genes. This approach yielded three discrete amplified sequence elements (designated LCCH1, LCCH2, and LCCH3) that contained open reading frames and > 40% amino acid sequence identity to the corresponding regions of vertebrate ligand-gated chloride channel genes. Genomic DNA clones corresponding to each element were isolated and sequenced, and predicted amino acid sequences corresponding to the second (M2) and third (M3) transmembrane domains of vertebrate genes were analyzed for identity or similarity to known sequences. LCCH1 was identical to the Rdl gene, a known GABA receptor subunit gene from D. melanogaster, whereas LCCH2 and LCCH3 were novel D. melanogaster sequences that exhibited structural similarity to other members of the ligand-gated chloride channel gene family. LCCH2 was equally divergent in M2 and M3 (46-49% amino acid identity) from all other known members of this family and may therefore represent a new subunit or receptor class within this family. LCCH2 was localized by in situ hybridization to cytogenetic region 75A on the left arm of chromosome 3. LCCH3 was closely related to mammalian (79% amino acid identity) and snail (96% amino acid identity) GABA receptor beta subunits and may therefore be the homologue in D. melanogaster of this subunit class. LCCH3 was localized by in situ hybridization to cytogenetic region 13F on the X chromosome.(ABSTRACT TRUNCATED AT 250 WORDS)

The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids.

Voltage-sensitive sodium channels encoded by a full-length cDNA corresponding to the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes either alone or in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1 cRNA alone produced very small (50-150 nA) sodium currents, whereas the combination of Vssc1 and tipE cRNAs produced robust (0.5-3 microA), rapidly inactivating sodium currents. The pyrethroid insecticide cismethrin prolonged the sodium current carried by Vssc1/tipE sodium channels during a depolarizing pulse and induced a tail current after repolarization. The Vssc1 cDNA was specifically mutated to substitute phenylalanine for leucine at position 1014 of the inferred amino acid sequence (L1014F), a polymorphism shown previously to be associated with the kdr (knockdown resistance) trait of the house fly. The L1014F substitution reduced the sensitivity of expressed house fly sodium channels to cismethrin at least 10-fold and increased the rate of decay of pyrethroid-induced sodium tail currents. These results demonstrate that the resistance-associated L1014F mutation confers a reduction in the sensitivity of house fly sodium channels to pyrethroids that is sufficient to account for the kdr resistance trait.

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica).

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.

Characterization of voltage-sensitive sodium channel gene coding sequences from insecticide-susceptible and knockdown-resistant house fly strains.

The kdr insecticide resistance trait of the house fly (Musca domestica .L.), which confers reduced neuronal sensitivity to DDT and pyrethroid insecticides, was previously shown to exhibit tight genetic linkage to restriction fragment length polymorphism markers lying within a voltage-sensitive sodium channel gene that is homologous to the para gene of Drosophila melanogaster. In the present study, the 6315 nucleotide coding sequences of this voltage-sensitive sodium channel gene from insecticide-susceptible (NAIDM strain) and kdr (538ge strain) house flies were determined by automated direct DNA sequencing of PCR fragments obtained by amplification on first strand cDNA from adult heads. The deduced 2105-residue amino acid sequence from each strain exhibited overall structure and organization typical of sodium channel alpha subunit genes and was 90.0% identical to that of the D. melanogaster para gene product. We did not detect any splice variants among voltage-sensitive sodium channel cDNAs obtained from adult house fly head preparations. Comparison of the coding sequence of the voltage-sensitive sodium channel gene of the kdr house fly strain to that of the NAIDM strain revealed 12 amino acid differences in the 538ge strain. The significance of these polymorphisms as candidate resistance-conferring mutations is discussed.

Mutations in the house fly Vssc1 sodium channel gene associated with super-kdr resistance abolish the pyrethroid sensitivity of Vssc1/tipE sodium channels expressed in Xenopus oocytes.

The super-kdr insecticide resistance trait of the house fly confers resistance to pyrethroids and DDT by reducing the sensitivity of the fly nervous system. The super-kdr genetic locus is tightly linked to the Vssc1 gene, which encodes a voltage-sensitive sodium channel alpha subunit that is the principal site of pyrethroid action. DNA sequence analysis of Vssc1 alleles from several independent super-kdr fly strains identified two amino acid substitutions associated with the super-kdr trait: replacement of leucine at position 1014 with phenylalanine (L1014F), which has been shown to cause the kdr resistance trait in this species, and replacement of methionine at position 918 with threonine (M918T). We examined the functional significance of these mutations by expressing house fly sodium channels containing them in Xenopus laevis oocytes and by characterizing the biophysical properties and pyrethroid sensitivities of the expressed channels using two-electrode voltage clamp. House fly sodium channels that were specifically modified by site-directed mutagenesis to contain the M918T/L1014F double mutation gave reduced levels of sodium current expression in oocytes but otherwise exhibited functional properties similar to those of wildtype channels and channels containing the L1014F substitution. However, M918T/L1014F channels were completely insensitive to high concentrations of the pyrethroids cismethrin and cypermethrin. House fly sodium channels specifically modified to contain the M918T single mutation, which is not known to exist in nature except in association with the L1014F mutation, gave very small sodium currents in oocytes. Assays of these currents in the presence of high concentrations of cismethrin suggest that this mutation alone is sufficient to abolish the pyrethroid sensitivity of house fly sodium channels. These results define the functional significance of the Vssc1 mutations associated with the super-kdr trait of the house fly and are consistent with the hypothesis that the super-kdr trait arose by selection of a second-site mutation (M918T) that confers to flies possessing it even greater resistance than the kdr allele containing the L1014F mutation.

Pharmacological characterization of the voltage-dependent sodium channels of rainbow trout brain synaptosomes.

Batrachotoxin, aconitine, and veratridine, alkaloid activators of voltage-dependent sodium channels, stimulated 22Na+ uptake by rainbow trout brain synaptosomes. The potency and efficacy of activation by these compounds decreased in the following order: batrachotoxin greater than aconitine much greater than veratridine. Aconitine-stimulated sodium uptake was completely inhibited by tetrodotoxin, a specific blocker of voltage-dependent sodium channels. Polypeptide toxins in the venom of the scorpion, Leiurus quinquestriatus, and the insecticide DDT enhanced veratridine-dependent sodium uptake but had no effect on non-specific uptake. These studies identify appropriate conditions for measuring sodium channel-dependent 22Na+ uptake in trout brain synaptosomes and characterize some of the pharmacological properties of trout brain sodium channels. Trout sodium channels differed from those in rat and mouse brain in their responses to batrachotoxin, aconitine, veratridine, and DDT but not to tetrodotoxin and Leiurus venom toxins. These results suggest that the specificity of some of the neurotoxin-binding domains of the trout brain sodium channel may differ from those of sodium channels in mammalian brain.

Mechanisms of action of ibogaine and harmaline congeners based on radioligand binding studies.

Assays using radioligands were used to assess the actions of ibogaine and harmaline on various receptor types. Ibogaine congeners showed affinity for opiate receptors whereas harmaline and harmine did not. The Ki for coronaridine was 2.0 microM at mu-opiate receptors. The Kis for coronaridine and tabernanthine at the delta-opiate receptors were 8.1 and 3.1 microM, respectively. Ibogaine, ibogamine, coronaridine and tabernanthine had Ki values of 2.08, 2.6, 4.3 and 0.15 microM, respectively, for kappa-opiate receptors. Long-lasting, dose-dependent behavioral effects of ibogaine have been reported. The possibility that these effects were due to irreversible binding properties of ibogaine at kappa-receptors was considered; however, radioligand wash experiments showed a rapid recovery of radioligand binding after one wash. A voltage-dependent sodium channel radioligand demonstrated Ki values in the microM range for all drugs tested. Using radioligand binding assays and/or 36Cl- uptake studies, no interaction of ibogaine or harmaline with the GABA receptor-ionophore was found. The kappa-activity of ibogaine (or an active metabolite) may be responsible for its putative anti-addictive properties whereas the tremorigenic properties of ibogaine and harmaline may be due to their effects on sodium channels.

Action of the pyrethroid insecticide cypermethrin on rat brain IIa sodium channels expressed in xenopus oocytes.

Pyrethroid insecticides bind to a unique site on voltage-dependent sodium channels and prolong sodium currents, leading to repetitive bursts of action potentials or use-dependent nerve block. To further characterize the site and mode of action of pyrethroids on sodium channels, we injected synthetic mRNA encoding the rat brain IIa sodium channel alpha subunit, either alone or in combination with synthetic mRNA encoding the rat sodium channel beta1 subunit, into oocytes of the frog Xenopus laevis and assessed the actions of the pyrethroid insecticide [1R,cis,alphaS]-cypermethrin on expressed sodium currents by two-electrode voltage clamp. In oocytes expressing only the rat brain IIa alpha subunit, cypermethrin produced a slowly-decaying sodium tail current following a depolarizing pulse. In parallel experiments using oocytes expressing the rat brain IIa alpha subunit in combination with the rat beta1 subunit, cypermethrin produced qualitatively similar tail currents following a depolarizing pulse and also induced a sustained component of the sodium current measured during a step depolarization of the oocyte membrane. The voltage dependence of activation and steady-state inactivation of the cypermethrin-dependent sustained current were identical to those of the peak transient sodium current measured in the absence of cypermethrin. Concentration-response curves obtained using normalized tail current amplitude as an index of the extent of sodium channel modification by cypermethrin revealed that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit increased the apparent affinity of the sodium channel binding site for cypermethrin by more than 20-fold. These results confirm that the pyrethroid binding site is intrinsic to the sodium channel alpha subunit and demonstrate that coexpression of the rat brain IIa alpha subunit with the rat beta1 subunit alters the apparent affinity of this site for pyrethroids.

Receptor-like stereospecific binding of a pyrethroid insecticide to mouse brain membranes.

A heterogeneous particulate fraction of mouse brain homogenates binds NRDC 157 (3-phenoxybenzyl [1R, cis]-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropanecarboxylate), a potent pyrethroid insecticide, stereospecifically and with high affinity. Stereospecific binding is a minor component of total binding (2.8%); the remainder of observed binding is predominantly nonspecific and unsaturable. Stereospecific binding is half-saturated at 4 X 10(-8)M and fully saturated at concentrations in excess of 1 X 10(-7)M. The stereospecific binding capacity of this preparation was 200-250 pmoles of NRDC 157 per gram equivalent of brain tissue (2.3-2.8 pmol/mg protein). This binding site may represent the neural receptor involved in the stereospecific toxic action of pyrethroids.

Inhibition of gamma-aminobutyric acid-stimulated chloride flux in mouse brain vesicles by polychlorocycloalkane and pyrethroid insecticides.

Selected polychlorocycloalkane and pyrethroid insecticides were evaluated as inhibitors of gamma-aminobutyric acid (GABA)-dependent chloride flux into mouse brain vesicles. The inhibitory potencies of the polychlorocycloalkane insecticides, measured as concentrations producing 50% inhibition, spanned a 1200-fold concentration range in the following order of decreasing potency: 12-ketoendrin; isobenzan; endrin; dieldrin; heptachlor epoxide; aldrin; heptachlor; and lindane. For the cyclodienes, inhibition of chloride uptake was closely correlated with both mammalian toxicity and the ability to displace the binding of [35S]t-butylbicyclophosphorothionate (TBPS). However, lindane was much less potent as an inhibitor of GABA-dependent chloride uptake than would be expected from its acute toxicity or potency as an inhibitor of [35S]TBPS binding. Mirex and chlordecone were poor inhibitors of GABA-dependent chloride uptake, indicating that other sites are likely to be involved in their toxic action. The pyrethroid insecticide deltamethrin gave 50% inhibition of GABA-dependent chloride uptake at 25 microM, but the extent of inhibition was not increased at higher concentrations. In addition, the nontoxic enantiomer of deltamethrin produced dose-dependent inhibition in the chloride flux assay with a potency about 10-fold less than deltamethrin. These results demonstrate the utility of this functional assay to identify compounds that act at the GABAA receptor-ionophore complex and implicate this complex as the principal site of neurotoxic action for cyclodiene insecticides. Although lindane and deltamethrin also altered GABAA receptor-ionophore function, their low potencies and the incomplete stereospecificity of deltamethrin inhibition suggest that this complex is not involved in the neurotoxic action of lindane and alpha-cyano-substituted pyrethroids.

Differential sensitivity of sodium channel isoforms and sequence variants to pyrethroid insecticides.

Pyrethroids are commonly regarded as safe insecticides. However, some widely used pyrethroids, particularly single neurotoxic isomers of potent Type II compounds, have acute oral toxicities comparable to many organophosphorus insecticides. The majority of studies of the action of pyrethroids on voltage-sensitive sodium channels, the principal target sites for these compounds, have not considered differences in sodium channel structure as determinants of sensitivity. In mammals, voltage-sensitive sodium channels are encoded by a multi-gene family and exhibit both anatomical and developmental regulation of expression. Studies in this laboratory using cloned rat sodium channel isoforms expressed in Xenopus oocytes have documented profound differences in pyrethroid sensitivity between isoforms. Although the role of sodium channel gene mutations in altering pyethroid sensitivity has not been addressed in the case of the mammalian sodium channel gene family, the potential significance of allelic variation is illustrated in studies of point mutations in a sodium channel gene of the house fly that confer resistance to the lethal actions of pyrethroids and modify the sensitivity of house fly sodium channels expressed in Xenopus oocytes to these compounds. It is of particular interest that some of these resistance-associated mutations in the fly sodium channel occur at amino acid residues that are also the sites of mutations in human skeletal muscle sodium channels that are associated with inherited paralytic disorders. These findings document the pharmacological significance of structural differences between sodium channel isoforms and between genetic variants of an individual isoform as determinants of pyrethroid sensitivity.

Metabolic considerations in pyrethroid design.

1. Synthetic pyrethroids, based on the naturally-occurring insecticidal components of pyrethrum extract, emerged in the 1970s as the fourth major chemical class of synthetic insecticides. They are widely used today in the control of agriculture and household pests and disease vectors. 2. Early efforts in the design of synthetic analogues focused on the need to identify novel structural moieties that preserved or enhanced intrinsic insecticidal activity while eliminating known sites of metabolic and photolytic attack in the natural compounds. Subsequent efforts focused on achieving high levels of insecticidal activity while minimizing costs of synthesis and retaining desirable levels of selective toxicity. 3. The synthetic compounds obtained in these efforts constitute a group of insecticides having unprecedented biological activity against target species with low acute toxicity to mammals. 4. The evolutionary development of the pyrethroids illustrates how knowledge of metabolic fate can contribute to the design of novel insecticides with improved insecticidal activity and selective toxicity.

Effects of non-neural mechanisms on pyrethroid structure-activity relationships.

Structural requirements for high insecticidal activity in pyrethroid insecticides are very stringent. Observed structure-activity relationships may arise either from specificity at the site of pyrethroid action in the nervous system, from selectivity in the pharmacokinetic processes governing the appearance and persistence of compounds at that site, or from a combination of these mechanisms. Recent studies of the metabolism of trans and cis isomers of pyrethroids in insect tissue preparations in vitro and of their pharmacokinetic behavior in insects in vivo permit an assessment of the impact of non-neural mechanisms on the toxicity differences observed between these isomers.

Neurotoxic actions of pyrethroid insecticides.

Pyrethroid insecticides interact with a variety of neurochemical processes, but not all of these actions are likely to be involved in the disruption of nerve function. Several lines of evidence suggest that the voltage-sensitive sodium channel is the single principal molecular target site for all pyrethroids and DDT analogs in both insects and mammals. The alterations of sodium channel functions identified in both biophysical and biochemical studies are directly related to the effects of these compounds on intact nerves. The pyrethroid recognition site of the sodium channel exhibits the stringent stereospecificity predicted by in vivo estimates of intrinsic neurotoxicity in both insects and mammals. Type I and Type II compounds produce qualitatively different effects on sodium channel tail currents, divergent actions on intact nerves, and different effects on the excitability of vertebrate skeletal muscle. Moreover, compounds that are defined as intermediate in the Type I/Type II classification scheme are also intermediate in their effects on sodium channel kinetics. The range of different actions on sensory and motor nerve pathways arising from these qualitatively different effects at the level of the sodium channel appear to be sufficient to explain the distinct poisoning syndromes that have been identified in both insects and mammals. Thus, it does not appear necessary to invoke different primary target sites for Type I and Type II compounds to explain their actions in whole animals. Although the voltage-sensitive sodium channel is likely to be the principal site of pyrethroid action, it is probably not the only site involved in intoxication. Insect neurosecretory neurons are sensitive to very low concentrations of pyrethroids, and disruption of the neuroendocrine system has been implicated as a factor contributing to the irreversible effects of pyrethroid intoxication in insects. Since action potentials in these nerves are carried by calcium ions through TTX-insensitive voltage-gated cation channels, these findings provide evidence that pyrethroids can alter neuronal excitability through an action on voltage-sensitive channels other than the sodium channel. Actions on voltage-sensitive calcium channels may also be involved in the effects of pyrethroids on neurotransmitter release in mammals. The proconvulsant actions of pyrethroids mediated through the peripheral-type benzodiazepine receptor may also contribute to pyrethroid intoxication. Both Type I and Type II compounds are potent proconvulsants in vivo at doses well below those required to produce pyrethroid-dependent intoxication.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrolysis of pyrethroid insecticides by soluble mouse brain esterases.

trans-Permethrin, a pyrethroid insecticide, was hydrolyzed by one or more carboxylesterases located in the soluble fraction of mouse brain homogenates. The apparent affinity of this activity for trans-permethrin was greater than that reported for mouse hepatic carboxylesterase activity, but the apparent maximum velocity was considerably lower than that of the hepatic activity. Soluble brain esterases also hydrolyzed several other pyrethroid esters with a substrate specificity different from that of the hepatic esterases. In particular, alpha-cyano-3-phenoxybenzyl esters of noncyclopropane acids (e.g., fenvalerate and fluvalinate) were hydrolyzed by brain esterases at rates equal to or greater than that measured for trans-permethrin. These results suggest that hydrolysis in the brain may contribute to the detoxication of some pyrethroids in mammals.

Neurotoxic insecticides inhibit GABA-dependent chloride uptake by mouse brain vesicles.

The neurotoxic insecticides endrin, dieldrin, aldrin, lindane (gamma-1,2,3,4,5,6-hexachlorocyclohexane) and deltamethrin inhibited gamma-aminobutyric acid-dependent 36Cl- uptake by mouse brain vesicles. Of the insecticides examined, the chlorinated cyclodienes endrin and dieldrin were the most potent, producing 50% inhibition at 2.8 and 13.9 microM, respectively. Lindane and deltamethrin were less effective, and with deltamethrin the effect was incompletely stereospecific. These results demonstrate the disruption of gamma-aminobutyric acid receptor-chloride ionophore function in mammalian brain by neurotoxic insecticides and provide evidence that this complex is the principal site of cyclodiene action.