Characterization of 11 commercial pyrethroids on the functional attributes of rat brain synaptosomes

The action of 11 commercial pyrethroids on Ca²⁺ influx and glutamate release was assessed using high-throughput functional assays with rat brain synaptosomes to better understand the mechanistic nature of pyrethroid-induced neurotoxicity and aid in the reassessment of pyrethroids in vivo. Concentration-dependent response curves for each of the non-cyano and α-cyano containing pyrethroids were determined and the data used in a cluster analysis. The previously characterized α-cyano pyrethroids that induce the CS-syndrome (cypermethrin, deltamethrin, and esfenvalerate) increased Ca²⁺ influx and glutamate release, and clustered with two other α-cyano pyrethroids (β-cyfluthrin and λ-cyhalothrin) that shared these same actions. Previously characterized T-syndrome pyrethroids (bioallethrin, cismethrin, and fenpropathrin) did not share these actions and clustered with two other non-cyano pyrethroids (tefluthrin and bifenthrin) that likewise did not elicit these actions. Our current findings indicate that pyrethroids that have an α-cyano group (with the exception of fenpropathrin) were more potent enhancers of Ca²⁺ influx and glutamate release under depolarizing conditions than pyrethroids that did not possess this functional group. The collective data set does not support the hypothesis that pyrethroids, as a class, act in a similar fashion at presynaptic nerve terminals.     

Action of cismethrin and deltamethrin on functional attributes of isolated presynaptic nerve terminals from rat brain

Isolated presynaptic nerve terminals (synaptosomes) prepared from rat brain were used to evaluate the actions of a tremor (T)-syndrome (cismethrin) and a choreoathetosis-salivation (CS)-syndrome (deltamethrin) pyrethroid on the functional attributes of synaptosomes by measuring calcium influx and endogenous neurotransmitter (l-glutamate) release with fluorescent assays. Both cismethrin and deltamethrin stimulated calcium influx, however, only deltamethrin enhanced Ca²⁺-dependent neurotransmitter release and its action was stereospecific, concentration-dependent, stimulated by depolarization, unaltered by tetrodotoxin, and blocked by ω-conotoxin GVIA. Our results delineate a separate action of deltamethrin on presynaptic nerve terminals from that elicited by cismethrin and implicate Ca_v2.2 calcium channels as target sites for deltamethrin that is consistent with the observed in vivo release of neurotransmitter at the onset of convulsive symptom caused by CS-syndrome pyrethroids. This information will allow a more complete understanding of the molecular and cellular nature of pyrethroid-induced neurotoxicity and expands our knowledge of the structure–activity relationships of pyrethroids in regards to their action on voltage-sensitive calcium channels.     

PKC-dependent phosphorylations modify the action of deltamethrin on rat brain N-type (CaV2.2) voltage-sensitive calcium channel

Voltage clamp electrophysiological studies using wild type Ca_V2.2 and its β₃ subunit coexpressed in Xenopus oocytes revealed that deltamethrin increased the rate of activation, prolonged inactivation and reduced peak current. Site-directed mutagenesis of threonine 422 to glutamic acid (T422E, one of five protein kinase C (PKC)-dependent phosphorylation sites) resulted in a channel that acted as if it were permanently phosphorylated. This resulted in an increased probability of opening during depolarization and a reduced inhibition by the βγ subunit of heterotrimeric G-protein. Deltamethrin treatment of T422E Ca_V2.2 enhanced peak current ∼50% over ethanol-treated controls with an EC₅₀ of 9.8 × 10⁻¹¹ M.Phosphorylation of wild type Ca_V2.2 is evoked by the phorbol ester, phorbol 12-myristrate, 13 acetate (PMA), by activating endogenous protein kinase C (PKC) in oocytes. PKC-dependent phosphorylation activated by PMA of wild type Ca_V2.2 has been previously shown to slow channel deactivation and increased Ca²⁺ influx and subsequent neurotransmitter release. Following PMA-activated phosphorylation, deltamethrin significantly increased peak current and slowed deactivation of the phosphorylated channel, which would be consistent with slower channel closure, greater Ca²⁺ influx and enhanced neurotransmitter release seen in vivo. Deltamethrin treatment in the absence of PMA-activated phosphorylation resulted in no effect on the deactivation kinetics of unphosphorylated Ca_V2.2 or the T422E mutant. Thus, Ca_V2.2 is modified by deltamethrin but the resulting perturbations are dependent upon its PKC-dependent phosphorylation state.     

Mutation of threonine 422 to glutamic acid mimics the phosphorylation state and alters the action of deltamethrin on Cav2.2

Effects of deltamethrin on voltage-sensitive calcium channels (VSCC) from rat brain (Ca_v2.2) expressed in Xenopus oocytes were assessed electrophysiologically. Deltamethrin reduced peak current of wild-type Ca_v2.2 in a stereospecific and concentration-dependent manner with an EC₅₀ of 1 × 10⁻⁹ M. Phosphorylation of threonine 422 enhances voltage-sensitive calcium current, increases the probability that Ca_v2.2 will open under depolarizing conditions and antagonizes the inhibition of the channel by the betagamma subunit of heterotrimeric G-protein (Gβγ). Site-directed mutagenesis of threonine 422 to glutamic acid (T422E) results in a channel that acts as if it were permanently phosphorylated. Deltamethrin (10⁻⁷ M) significantly enhanced peak current via the T422E channel (1.5-fold) compared to the nontreated control and the increase was significantly greater than for either the wild-type (T422) or T422A (permanently unphosphorylated mutant) channels. The effect of deltamethrin on T422E Ca_v2.2 was stereospecific and concentration-dependent with an EC₅₀ of 9.8 × 10⁻¹¹ M. Thus, Ca_v2.2 is modified by deltamethrin but the resulting perturbation is dependent upon the phosphorylation state of threonine 422.     

Binary mixtures of pyrethroids produce differential effects on Ca influx and glutamate release at isolated presynaptic nerve terminals from rat brain

Isolated rat brain synaptosomes were used to evaluate the action of pyrethroid mixtures on Ca²⁺ influx and subsequent glutamate release under depolarizing conditions. In equipotent binary mixtures at their respective and/or estimated EC₅₀s with deltamethrin always as one of the two components, cismethrin, λ-cyhalothrin, cypermethrin, esfenvalerate and permethrin were additive and S-bioallethrin, fenpropathrin and tefluthrin were less-than-additive on Ca²⁺ influx. In binary mixtures with deltamethrin always as one of the two components, esfenvalerate, permethrin and tefluthrin were additive and λ-cyhalothrin was less-than-additive on glutamate release. Binary mixture of S-bioallethrin and cismethrin was additive for both Ca²⁺ influx and glutamate release. Only a subset of pyrethroids (S-bioallethrin, cismethrin, cypermethrin, and fenpropathrin) in binary mixtures with deltamethrin caused a more-than-additive effect on glutamate release. These binary mixtures were, however, only additive (cismethrin and cypermethrin) or less-than-additive (S-bioallethrin and fenpropathrin) on Ca²⁺ influx. Therefore, increased glutamate release evoked by this subset of pyrethroids in binary mixture with deltamethrin is not entirely occurring by Ca²⁺-dependent mechanisms via their action at voltage-sensitive calcium channels. These results suggest that pyrethroids do not share a common mode of toxicity at presynaptic nerve terminals from rat brain and appear to affect multiple target sites, including voltage-sensitive calcium, chloride and sodium channels.     

Action of deltamethrin on N-type (Cav2.2) voltage-sensitive calcium channels in rat brain

Isolated presynaptic nerve terminals prepared from whole rat brain were used to evaluate the action of deltamethrin on voltage-sensitive calcium channels by measuring calcium influx and endogenous glutamate release. Deltamethrin-enhanced K⁺-stimulated calcium influx and subsequent Ca²⁺-dependent glutamate release. The effect of deltamethrin was concentration-dependent, stereospecific, blocked by ω-conotoxin MVIIC but unaltered in the presence of tetrodotoxin. These results suggest that N-type voltage-sensitive calcium channels are a site of action at the presynaptic nerve terminal. Electrophysiological studies were carried out using rat brain Ca_v2.2 and β₃ subunits coexpressed in Xenopus oocytes to validate such action. Deltamethrin reduced barium peak current in a concentraion-dependent and stereospecific manner, increased the rate of activation, and prolonged the inactivation rate of this channel. These experiments support the conclusion that N-type voltage-sensitive calcium channel operation is altered by deltamethrin.     

Electrophysiological identification of site-insensitive mechanisms in knockdown-resistant strains (kdr,super-kdr) of the housefly larva (Musca domestica)

The effects of pyrethroids were studied upon isolated segmental nerves and neuromuscular junctions in both susceptible (Cooper) and knockdown-resistant (kdr; super-kdr) strains of housefly larvae (Musca domestica L.). Isolated segmental nerves contained neither cell bodies nor synaptic contacts; thus, any effects of pyrethroids were attributed solely to their actions upon voltage-dependent Na⁺ channels. Threshold concentrations of the type II pyrethroid, deltamethrin, required to elevate the spontaneous firing rate of these nerves were determined. Both resistant strains were about ten times less sensitive to deltamethrin than the susceptible strain, but insensitivity of super-kdr nerves was no greater than in the less resistant kdr strain. At neuromuscular junctions, the minimum concentrations of pyrethroids needed to trigger massive increases in the frequency of miniature excitatory postsynaptic potentials (mEPSPs) were determined for deltamethrin and the type I pyrethroid, fenfluthrin. With fenfluthrin there was no detectable difference between the junctions of kdr and super-kdr strains, which were both about ten-fold less sensitive than Cooper junctions. With deltamethrin, kdr junctions were about 30 times less sensitive than those of Cooper; super-kdr junctions were dramatically insensitive to deltamethrin, being some 10000- and 300-fold less sensitive than those of Cooper and kdr respectively. Thus, in the synaptic assay, super-kdr conferred an extension in resistance over kdr only against the type II pyrethroid, it being ineffective against fenfluthrin. We suggest that kdr resistance comprises at least two site-insensitive areas within the nervous system. One involves insensitivity of the Na⁺ channel and has similar efficacy in both kdr and super-kdr strains against type I and II pyrethroids; the other is associated with the presynaptic terminal and is particularly effective in super-kdr resistance against type II pyrethroids. The latter could be associated with Ca²⁺-activated phosphorylation of proteins involved with neurotransmitter release. Such phosphorylation reactions are known to be perturbed by pyrethroids, especially by type II compounds.     

Deltamethrin: A neurophysiological study of the sites of action

Recent experiments on the mode of action of pyrethroids have indicated that those pyrethroids containing an α-cyano phenoxybenzyl group may act on GABA-mediated chloride channels. The crayfish stretch receptor neuron provides a useful preparation for examining the effects of pyrethroids on these channels and on sodium channels. The lowest concentration of deltamethrin to have an effect on sodium channels was 10⁻¹² M, but the response of the preparation to GABA appeared to be unaffected by concentrations of deltamethrin up to 10⁻⁷ M. Although 10⁻⁶ M deltamethrin had a slight effect on the GABA response of the dactyl abductor muscle, it appears that the majority of the effects of cyano pyrethroids in invertebrates could be accounted for solely by their action on sodium channels.     

Actions of pyrethroid insecticides on insect axonal sodium channels

The actions of pyrethroid insecticides were tested on isolated giant axons of the cockroach Periplaneta americana, using oil-gap, single-fibre recording techniques. Current-clamp and voltage-clamp experiments were used to determine the actions of pyrethroids on axonal membrane potentials and ionic currents. Treatment with deltamethrin at micromolar concentrations caused gradual depolarisation of the axon accompanied by a reduction in amplitude of the action potential. This depolarisation was enhanced by an increase in stimulation frequency. Other synthetic pyrethroids: 3,4,5,6-tetrahydrophthalimidomethyl (1RS)-cis-3-[(RS)-2,2-dimethylcyclopropyl]-2,2-dimethylcyclopropanecarboxylate, biopermethrin and its (1S)-enantiomer, (1R)-tetramethrin, S-bioallethrin, bioresmethrin and its (1S)-enantiomer, cismethrin, and 5-benzyl-3-furylmethyl (E)-(1R)-cis-2,2-dimethyl-3-(2-oxothiolan-3-ylidenemethyl)cyclopropanecarboxylate (RU-15525, ‘Kadethrin’) were investigated. The (1S)-enantiomers were inactive, but all the other pyrethroids tested, apart from deltamethrin, induced prolonged negative (depolarising) after-potentials. All the treatments with the active pyrethroids resulted in the appearance of a voltage and time-dependent ‘maintained’ sodium conductance. The duration of this ‘slow’ conductance varied considerably depending on the pyrethroid under test. Clearly, the effectiveness of pyrethroids on whole insects is not determined only by the degree to which they directly modify the properties of sodium channels. Nevertheless, voltage-clamp experiments on isolated axons readily permit direct comparison of the actions of different pyrethroids on the sodium channels of insect neurones.     

Enhancement of neurotransmitter release from invertebrate synaptosomes by pyrethroids during pulsed-depolarization: A functional assay for effects on repolarization

The release of [³H]neurotransmitters was used as a functional assay to assess the actions of selected neurotoxins on the synaptosomal membranes prepared from the invertebrate nervous systems of squid and house fly. A reproducible release of [³]neurotransmitter was evoked by pulsed-depolarization in the presence of elevated K⁺ or of veratridine. Pretreatment with deltamethrin resulted in a substantial enhancement of [³H]neuro-transmitter release during pulsed-depolarization. This enhanced neurotransmitter release was greatly reduced or absent when synaptosomes of knockdown-resistant house flies were examined. No enhanced neurotransmitter release due to deltamethrin pretreatment was apparent from any synaptosomal preparation under non-depolarizing conditions. Under similar conditions, collaborative experiments demonstrated that deltamethrin causes a significant change in protein phosphorylation activities which follow depolarization. The most significant change caused by deltamethrin was the prolonged elevation of the level of phosphorylation on a number of key synaptic proteins beyond the normal time of their recovery to the dephosphorylated state. The most notable protein reacting to deltamethrin in this manner was calcium-cadlmodulin-dependent protein kinase.     

Effects of pyrethroids on neuronal excitability of adult honeybees Apis mellifera

Pyrethroids act on the nervous system as a primary target organ and exert their neurotoxic effects primarily by altering the conductance of sodium channel, leading to hyperexcitation. However, few studies investigated the effects of pyrethroids on neuronal excitability of honeybee brain neurons. In this study, a whole-cell patch-clamp technique was used to record current threshold, the minimum current to induce an action potential, and peak sodium current in the dissociated honeybee brain neurons treated with bifenthrin, deltamethrin and fluvalinate in vitro & in vivo. The study showed that these pyrethroids greatly suppressed the neuronal excitability as revealed by increasing current injected and inhibited the peak sodium current in honeybees. The three pyrethroids also inhibited steady-state inactivation in addition to reduction of sodium peak current.     

Effects of deltamethrin and its commercial formulation DECIS on different cell types in vitro: Cytotoxicity,cellular binding,and intracellular localization

To investigate the effect of deltamethrin and other pyrethroids on nonexcitable cells, we tested these molecules on animal (fibroblasts) and plant (algae) cells in culture. The effects of pyrethroids on cellular proliferation vary according to their structure. Deltamethrin (from 5 × 10⁻⁵ M) affected the growth of fibroblasts, but was inactive on other cells. Commercial formulations (DECIS EC and DECIS FLO) were more active to cells, but this effect was due mostly to the matrix. However, these matrices facilitated the penetration of deltamethrin into cells. Studies of [³H]deltamethrin uptake indicated the labeling of macromolecules, but labeling was reduced significantly after treatment with organic solvents. Autoradiographic studies with [³H]deltamethrin showed labeling of cells at the cytpolasmic level.     

不同生境中稻飞虱种群动态研究

菊酯类农药对水稻螟虫有优异的防治效果,但引起稻飞虱的更大发生。作者用溴氰菊酯、氰戊菊酯及对稻飞虱高效的噻嗪酮等杀虫剂和对稻飞虱不同抗感水平的水稻品种组成不同的化学、品种环境,系统观察稻飞虱种群动态差异。结果表明,同一水稻品种,不同农药,稻飞虱种群增长量不一样。同一农药,不同水稻品种,稻飞虱的发生亦不同。溴氰菊酯或氰戊菊酯用到感虫品种上,有利因子重叠,稻飞虱的发生量最大;用到中抗品种上,有利因子与不利因子重叠,有效地抑制稻飞虱种群的增长。先将溴氰菊酯喷洒到感虫品种上,10天后再用噻嗪酮,两个有利因子重叠后再加一不利因子,仍可控制稻飞虱的为害。     

Mechanisms for multiple resistances in field populations of common cutworm, Spodoptera litura (Fabricius) in China

The mechanisms for multiple resistances had been studied with two field resistant strains and the selected susceptible and resistant strains of Spodoptera litura (Fabricius). Bioassay revealed that the two field strains were both with high resistance to pyrethroids (RR: 63-530), low to medium resistance to organophosphates and carbamates, AChE targeted insecticides (RR: 5.7-26), and no resistance to fipronil (RR: 2.0-2.2). Selection with deltamethrin in laboratory could obviously enhance the resistance of this pest to both pyrethroids and AChE targeted insecticides. Synergism test, enzyme analysis and target comparison proved that the pyrethroid resistance in this pest associated only with the enhanced activity of cytochrome P450 monooxygenase (MFO) and esterase. However the resistance to the AChE targeted insecticides depended on the target insensitivity and also the enhanced activity of MFO and esterase. Thus, the cross-resistance between pyrethroids and the AChE targeted insecticides was thought to be resulted from the enhanced activity of MFO and esterase.     

The action of pyrethroids on the voltage-sensitive calcium channel of Paramecium tetraurelia

Calcium regulation is an important event in synaptic transmission and neuronal function, which is governed by a very intricate signal transduction system which is not completely understood. Using a variety of pharmacological assays, we have characterized the action of deltamethrin on the ciliary voltage-sensitive calcium channel and on phospholipase C activity of Paramecium tetraurelia Sonneborn, an organism that does not possess a voltage-sensitive sodium channel. In fura-2 fluorometric assays, which examined whole cells and ciliary membrane vesicles enriched with calcium channels, deltamethrin stimulated Ca²⁺ uptake. We also determined that the phospholipase C activity of the ciliary membrane vesicles is regulated by the βγ-subunit from heterotrimeric G-proteins. Subsequent treatment with deltamethrin resulted in a substantial and highly significant increase in phospholipase C activity. These results provide evidence that the molecular mode of action of pyrethroids on the voltage-sensitive calcium channel is distinct from the action of this insecticide on the voltage-sensitive sodium channel and may be dependent, in part, upon an interaction with the βγ-subunit of heterotrimeric G-protein.     

Differential susceptibilities to pyrethroids in field populations of Chilo suppressalis (Lepidoptera: Pyralidae)

To assess the feasibility of pyrethroids for rice insect control, we examined susceptibilities of six field populations of rice stem borer Chilo suppressalis (Walker) to 10 pyrethroids using the topical application method in laboratory in 2004 and 2005. Our results showed that the seven pyrethroids with high fish-toxicity (i.e., β-cyfluthrin, λ-cyhalothrin, β-cypermethrin, deltamethrin, S-fenvalerate, α-cypermethrin, and fenpropathrin) were more effective against C. suppressalis than the three compounds with low fish-toxicity (i.e., cycloprothrin, etofenprox, and silafluofen). The results also showed that all 10 of the pyrethroids were much more effective than methamidophos and monosultap for C. suppressalis control. In addition, we found that susceptibilities of some field populations of C. suppressalis to some high fish-toxicity pyrethroids were significantly reduced, and our results indicated that a Ruian (RA) field population showed a year-to-year variation in susceptibility to most tested pyrethroids between 2004 and 2005. Our data indicated that the tolerance levels increased dramatically in RA population, especially to β-cyfluthrin and deltamethrin. This study provided the first assessment of resistance to pyrethroids in field populations of C. suppressalis. In addition, a close correlation between resistance ratios to the 10 compounds and differences of the structures of these compounds was established in the RA05 population, which was resistant to most of the pyrethroids tested while it was still very susceptible to fenvalerate with no cross resistance. Finally, the feasibility and precaution were discussed in selecting pyrethroids as alternatives to replace high toxicity organophosphates for C. suppressalis control and insecticide resistance management.     

Stability of pyrethroids on wheat in storage

The four pyrethroids, permethrin, phenothrin, fenvalerate and deltamethrin were applied to wheat which was stored for 52 weeks at 25 or 35°C, and either 12 or 15% moisture content. Rates of loss were calculated from residue analyses of the wheat at five intervals during storage. Calculated half-lives (weeks) for the pyrethroids at 25°C (12% moisture) and 35°C (15% moisture) were: permethrin 252 and 44, phenothrin 72 and 29, fenvalerate 210 and 74, and deltamethrin 114 and 35, respectively.     

两种挥发性拟除虫菊酯杀虫剂对伊蚊的驱避作用研究

拟除虫菊酯杀虫剂广泛应用于蚊香及其他驱蚊产品,不仅对蚊子有杀虫作用,而且具有驱避效应,然而拟除虫菊酯杀虫剂对蚊子驱避作用的机理尚不清楚.本研究发现两种挥发性拟除虫菊酯杀虫剂四氟甲醚菊酯和氯氟醚菊酯对白纹伊蚊和埃及伊蚊具有非接触驱避作用.触角电位(EAG)试验表明除四氟甲醚菊酯有微弱EAG响应外,其他拟除虫菊酯杀虫剂均没...     

Effects of the pyrethroids bioallethrin,deltamethrin and RU-15525 on the electrical activity of a cuticular mechanoreceptor of the cockroach Periplaneta americana

A study has been made of the effects of bioallethrin, RU-15525 [5-benzyl-3-furylmethyl (1R)-cis-2,2-dimethyl-3-(tetrahydro-2-oxo-3-thienylidenemethyl)-cyclopropanecarboxylate, ‘Kadethrin’], and deltamethrin on the electrical activity, measured in vivo, of a cuticular mechanoreceptor of Periplaneta americana. The modifications induced by these pyrethroids on the membrane excitability can be classified into two groups: Type I effects (bioallethrin) are characterised by a substantial increase in the number of action potentials triggered at the initiation site by a given mechanical stimulation, by an electrical activity persisting after mechanical stimulus has been stopped (repetitive activity), and possibly, by an inhibition of excitability of the cell membrane. Type II effects (RU-15525 and deltamethrin), are characterised by an inhibition of the excitability of the initiation site. In the case of RU-15525, there was a transient spontaneous electrical activity. Both types of effects have been linked to an action on the sodium channel, particularly at the initiation site. The preparation studied, which possessed no synapses, was shown to be more sensitive to deltamethrin (which is also the most insecticidal of the three pyrethroids) than to either allethrin or RU-15525. These results suggest that it is unnecessary to envisage a main target (sodium channel) that is different for the two types of pyrethroid.     

Neuropharmacological studies of an insecticidal trihaloimidazole derivative in the crayfish neuromuscular preparation

A trihaloimidazole derivative (S-377; 1-(4-chlorobutoxymethyl)-2,4,5-trichloroimidazole) caused giant excitatory junctional potentials by a single electrical stimulus to the excitatory nerves. Repetitive discharges were simultaneously induced in the nerve fibres. S-377 also caused repetitive firing in the inhibitory nerve fibres. Permethrin showed quite similar actions on both the junction and nerve fibres. It was supposed that S-377 inhibited the inactivating system of the sodium channel, like pyrethroids. However, S-377 reduced the number of spikes of the permethrin-induced repetitive discharges on the excitatory nerve fibres. These observations suggest that binding sites of S-377 on sodium channels are different from those of pyrethroids.