David M. Soderlund

Molecular Mechanisms of Insecticide Resistance

Four decades of intensive use of synthetic organic insecticides to control arthropod pests and disease vectors have led to the selection of insecticide or acaricide resistance in approximately 450 arthropod species (Georghiou 1986). In the most extreme cases, such as the Colorado potato beetle (Leptinotarsa decemlineata) in parts of the eastern United States, populations are resistant to virtually all chemicals available for control (Forgash 1984). The deleterious consequences of pesticide resistance in arthropods include increased levels of environmental contamination and risks of applicator and agricultural worker exposure from higher rates of pesticide application; increases in pest control costs; disruption of ecologically sound pest control strategies; increased incidence of human, animal, and plant diseases in which transmission depends on insect vectors; and, in the most extreme case, the complete destruction of agricultural production systems on a local or regional basis.

Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances

Synthetic pyrethroid insecticides were introduced into widespread use for the control of insect pests and disease vectors more than three decades ago. In addition to their value in controlling agricultural pests, pyrethroids are at the forefront of efforts to combat malaria and other mosquito-borne diseases and are also common ingredients of household insecticide and companion animal ectoparasite control products. The abundance and variety of pyrethroid uses contribute to the risk of exposure and adverse effects in the general population. The insecticidal actions of pyrethroids depend on their ability to bind to and disrupt voltage-gated sodium channels of insect nerves. Sodium channels are also important targets for the neurotoxic effects of pyrethroids in mammals but other targets, particularly voltage-gated calcium and chloride channels, have been implicated as alternative or secondary sites of action for a subset of pyrethroids. This review summarizes information published during the past decade on the action of pyrethroids on voltage-gated sodium channels as well as on voltage-gated calcium and chloride channels and provides a critical re-evaluation of the role of these three targets in pyrethroid neurotoxicity based on this information.

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.

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.

Evidence for a separate mechanism of toxicity for the Type I and the Type II pyrethroid insecticides

Neurotoxicity and mechanistic data were collected for six alpha-cyano pyrethroids (beta-cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin and lambda-cyhalothrin) and up to six non-cyano containing pyrethroids (bifenthrin, S-bioallethrin [or allethrin], permethrin, pyrethrins, resmethrin [or its cis-isomer, cismethrin] and tefluthrin under standard conditions. Factor analysis and multivariate dissimilarity analysis were employed to evaluate four independent data sets comprised of (1) fifty-six behavioral and physiological parameters from an acute neurotoxicity functional observatory battery (FOB), (2) eight electrophysiological parameters from voltage clamp experiments conducted on the Na(v)1.8 sodium channel expressed in Xenopus oocytes, (3) indices of efficacy, potency and binding calculated for calcium ion influx across neuronal membranes, membrane depolarization and glutamate released from rat brain synaptosomes and (4) changes in chloride channel open state probability using a patch voltage clamp technique for membranes isolated from mouse neuroblastoma cells. The pyrethroids segregated into Type I (T--syndrome-tremors) and Type II (CS syndrome--choreoathetosis with salivation) groups based on FOB data. Of the alpha-cyano pyrethroids, deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin arrayed themselves strongly in a dose-dependent manner along two factors that characterize the CS syndrome. Esfenvalerate and fenpropathrin displayed weaker response profiles compared to the non-cyano pyrethroids. Visual clustering on multidimensional scaling (MDS) maps based upon sodium ion channel and calcium influx and glutamate release dissimilarities gave similar groupings. The non-cyano containing pyrethroids were arrayed in a dose-dependent manner along two different factors that characterize the T-syndrome. Bifenthrin was an outlier when MDS maps of the non-cyano pyrethroids were based on sodium ion channel characteristics and permethrin was an outlier when the MDS maps were based on calcium influx/glutamate release potency. Four of six alpha-cyano pyrethroids (lambda-cyfluthrin, cypermethrin, deltamethrin and fenpropathrin) reduced open chloride channel probability. The R-isomers of lambda-l-cyhalothrin reduced open channel probability whereas the S-isomers, antagonized the action of the R-isomers. None of the non-cyano pyrethroids reduced open channel probability, except bioallethrin, which gave a weak response. Overall, based upon neurotoxicity data and the effect of pyrethroids on sodium, calcium and chloride ion channels, it is proposed that bioallethrin, cismethrin, tefluthrin, bifenthrin and permethrin belong to one common mechanism group and deltamethrin, lambda-cyhalothrin, cyfluthrin and cypermethrin belong to a second. Fenpropathrin and esfenvalerate occupy an intermediate position between these two groups.

Pyrethroids, knockdown resistance and sodium channels†

Knockdown resistance to DDT and the pyrethrins was first described in 1951 in the housefly (Musca domestica L.). This trait, which confers reduced neuronal sensitivity to these insecticides, was subsequently shown to confer cross-resistance to all synthetic pyrethroid insecticides that have been examined to date. As a consequence, the worldwide commercial development of pyrethroids as a major insecticide class over the past three decades has required constant awareness that pyrethroid overuse has the potential to reselect this powerful resistance mechanism in populations that previously were resistant to DDT. Demonstration of tight genetic linkage between knockdown resistance and the housefly gene encoding voltage-sensitive sodium channels spurred efforts to identify gene mutations associated with knockdown resistance and understand how these mutations confer a reduction in the sensitivity of the pyrethroid target site. This paper summarizes progress in understanding pyrethroid resistance at the molecular level, with particular emphasis on studies in the housefly.

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.

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.

Pyrethroid insecticides: potent, stereospecific enhancers of mouse brain sodium channel activation

Abstract Deltamethrin and NRDC 157, pyrethroid insecticides that produce different poisoning syndromes in mammals, enhanced veratridine-dependent, sodium channel-mediated 22Na+ uptake in mouse brain synaptosomes. Concentrations producing half-maximal enhancement were 2.5 × 10−8 M (deltamethrin) and 2.2 × 10−7 M (NRDC 157). This effect was stereospecific: The nontoxic 1S enantiomers had no significant effect on veratridine-dependent activation. At high deltamethrin concentrations, enhancement was maximal at 5 × 10−5−1 × 10−4 M veratridine. Pyrethroid enhancement was completely blocked by 5 × 10−6 M tetrodotoxin, and neither pyrethroid affected 22Na+ uptake in the absence of veratridine at concentrations up to 1 × 10−5 M. The relative potencies of deltamethrin and NRDC 157 in the synaptosomal sodium channel assay agree well with their relative acute toxicities to mice when administered by intracerebral injection. These findings demonstrate that pyrethroids exemplifying both characteristic poisoning syndromes are potent, stereospecific modifiers of sodium channel function in mammalian brain.

Pyrethroid-hydrolyzing esterases in southern armyworm larvae: Tissue distribution, kinetic properties, and selective inhibition

Abstract Esterase activity hydrolyzing both [1 RS,trans ]- and [1 RS,cis ]-permethrin was detected in crude homogenates of the following southern armyworm ( Spodoptera eridania Cramer) larval tissues: cuticle, gut, fat body, head capsule, Malpighian tubules, and silk gland. Neither substrate was detectably hydrolyzed by hemolymph. The highest esterase activities per insect equivalent of tissue were found in cuticle, gut, and fat body for the trans isomer and in cuticle and gut for the cis isomer. Each preparation hydrolyzed the trans isomer more rapidly, but the degree of specificity varied greatly between tissues. Differences in apparent K m and V max values between the three most active tissues were threefold or less for trans isomer hydrolysis, but differences between tissues of up to 100-fold were found for K m and V max values for cis isomer hydrolysis. Hydrolysis of the trans isomer in cuticle, gut, and fat body homogenates was only partially inhibited by α-naphthyl N -propylcarbamate (NPC). Concentrations of NPC giving maximal inhibition of trans isomer hydrolysis had little effect on the hydrolysis of the cis isomer. These results demonstrate that pyrethroid-hydrolyzing activity is broadly distributed in insect tissues and results from the combined activity of several esterases with different properties. It is likely that the trans and cis isomers of permethrin are hydrolyzed by separate enzymes in this insect.