Interaction of tetramethrin and deltamethrin at the single sodium channel in rat hippocampal neurons.

Type I and type II pyrethroids are known to modulate the sodium channel to cause persistent openings during depolarization and upon repolarization. Although there are some similarities between the two types of pyrethroids in their actions on sodium channels, the pattern of modification of sodium currents is different between the two types of pyrethroids. In the present study, interactions of the type I pyrethroid tetramethrin and the type II pyrethroid deltamethrin at rat hippocampal neuron sodium channels were investigated using the inside-out single-channel patch clamp technique. Deltamethrin-modified sodium channels opened much longer than tetramethrin-modified sodium channels. When 10 microM tetramethrin was applied to membrane patches that had been exposed to 10 microM deltamethrin, deltamethrin-modified prolonged single sodium currents disappeared and were replaced by shorter openings which were characteristic of tetramethrin-modified channel openings. These single-channel data are compatible with previous whole-cell competition study between type I and type II pyrethroids. These results are interpreted as being due to the displacement of the type II pyrethroid molecule by the type I pyrethroid molecule from the same binding site or to the allosteric interaction of the two pyrethroid molecules at separate sodium channel sites.     

Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids

Voltage-gated sodium channels (Na_v channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Na_v channel, Para (also called DmNa_v), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNa_v (para^ts) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNa_v and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNa_v/DSC1 double mutant line by introducing a para^ts1 allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNa_v variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNa_v channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para^ts1 mutant flies was almost completely abolished in para^ts1;DSC1^−/− double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w^1118A), and the para^ts1;DSC1^−/− double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para^ts1 mutant. Our findings revealed distinct roles of the DmNa_v and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance.     

Elucidation of pyrethroid and DDT receptor sites in the voltage-gated sodium channel

DDT and pyrethroid insecticides were among the earliest neurotoxins identified to act on voltage-gated sodium channels. In the 1960s, equipped with, at the time, new voltage-clamp techniques, Professor Narahashi and associates provided the initial evidence that DDT and allethrin (the first commercial pyrethroid insecticide) caused prolonged flow of sodium currents in lobster and squid giant axons. Over the next several decades, continued efforts by Prof. Narahashi’s group as well as other laboratories led to a comprehensive understanding of the mechanism of action of DDT and pyrethroids on sodium channels. Fast forward to the 1990s, genetic, pharmacological and toxicological data all further confirmed voltage-gated sodium channels as the primary targets of DDT and pyrethroid insecticides. Modifications of the gating kinetics of sodium channels by these insecticides result in repetitive firing and/or membrane depolarization in the nervous system. This mini-review focuses on studies from Prof. Narahashi’s pioneer work and more recent mutational and computational modeling analyses which collectively elucidated the elusive pyrethroid receptor sites as well as the molecular basis of differential sensitivities of insect and mammalian sodium channels to pyrethroids.     

Pyrethroids: involvement of sodium channels in effects on inositol phosphate formation in guinea pig synaptoneurosomes

The effects of pyrethroids were studied on phosphoinositide breakdown in guinea pig synaptoneurosomes. Similar to other agents that activate voltage-dependent sodium channels, type I and type II pyrethroids stimulated phosphoinositide breakdown. Type II pyrethroids, like deltamethrin and fenvalerate, were more potent and, at least for deltamethrin, more efficacious than type I pyrethroids, like allethrin, resmethrin and permethrin. The effects of type II pyrethroids could be partially inhibited by the sodium channel blocker tetrodotoxin. The effects of allethrin and resmethrin were not affected by 5 microM tetrodotoxin. Stimulation of phosphoinositide breakdown by fenvalerate was additive to the stimulation elicited by the receptor agonists carbamylcholine and norepinephrine, but not to the stimulation elicited by sodium channel agents (batrachotoxin, scorpion venom and pumiliotoxin B). Stimulation by allethrin was not additive to the stimulation elicited either by receptor agonists or sodium channel agents. A submaximal concentration of allethrin, a type I pyrethroid, did not greatly affect the dose-dependent stimulation elicited by a type II pyrethroid, deltamethrin, while a higher concentration of allethrin prevented further stimulation by type II pyrethroids. A local anesthetic, dibucaine, which inhibits sodium channel activation, inhibited phosphoinositide breakdown induced by type II, but not by type I pyrethroids, except at higher concentrations. Thus, type II pyrethroids appear to stimulate phosphoinositide breakdown in synaptoneurosomes in a manner analogous to other sodium channel agents, while type I pyrethroids elicit phosphoinositide breakdown by a different mechanism, probably not involving sodium channels.     

Pyrethroids and enhanced inhibition in the hippocampus of the rat

The pyrethroid insecticides have been divided into two classes on the basis of their biochemical actions and behavioral indices of toxicity. Both types of pyrethroids have effects on sodium conductance, and Type II pyrethroids have been reported to antagonize gamma-aminobutyric acid (GABA) by interacting with the t-butyl-bicyclophosphorothionate (TBPS)/picrotoxinin binding site. The dentate gyrus of the hippocampus is equipped with GABAergic recurrent inhibitory circuits. The present experiment was designed to demonstrate dissociation in the biochemistry of pyrethroids by activating the perforant path with pairs of stimulus pulses and monitoring the recurrent inhibition in this circuit. Antagonism of GABA leads to a reduction in inhibition, measured as an increase in the size of the population spike in response to the second pulse of the pair. The GABAergic properties of the pyrethroids were assessed by examining paired pulse inhibition before and after oral treatment with 20 mg/kg of cismethrin (Type I), 20 mg/kg of fenvalerate, or 10 mg/kg of deltamethrin (Type IIs). Input/output (I/O) functions revealed a reduction in excitatory postsynaptic potential (EPSP) following cismethrin and deltamethrin. Population spike height was unaffected. Fenvalerate had no effect on I/O functions. In contrast to the prediction of reduced inhibition following treatment with Type II pyrethroids, deltamethrin and fenvalerate increased inhibition up to 500 and 150 ms interpulse intervals, respectively. Cismethrin was without effect on paired pulse inhibition. These findings fail to provide evidence of GABA antagonistic properties of Type II pyrethroids and may be best explained by a differential effect of these three pyrethroids on sodium channel kinetics.     

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.     

Increase of sodium current after pyrethroid insecticides in mouse neuroblastoma cells

The effects of 4 different pyrethroid insecticides on sodium channel gating in internally perfused, cultured mouse neuroblastoma cells (N1E-115) were studied using the suction pipette, voltage clamp technique. Pyrethroids increased the amplitude of the sodium current, sometimes by more than 200%. Activation of the sodium current occurred at more hyperpolarized potentials than under control conditions. The declining phase of the sodium current during depolarization was markedly slowed down and after repolarization of the membrane a large, slowly decaying sodium tail current developed. Pyrethroids did not affect the sodium current reversal potential, steady-state sodium inactivation or recovery from sodium channel inactivation. The amplitude of the pyrethroid-induced slow tail current was always proportional to the sodium current at the end of the preceding depolarizing pulse. The rate of decay of the slow tail current strongly depended on pyrethroid structure and increased in the order deltamethrin, cyphenothrin, fenfluthrin and phenothrin. The rate of decay further depended on membrane potential and temperature. Below -85 m V the instantaneous current-voltage relationship of the slow tail current showed a negative slope conductance. The tail current decayed more slowly at low temperatures. Arrhenius plots indicated that the relaxation of open sodium channels to a closed state involved a higher energy barrier for pyrethroid-affected than for normal channels. The energy barrier was higher after deltamethrin than after the non-cyano pyrethroid fenfluthrin. It is concluded that in mammalian neuronal membrane pyrethroids selectively reduce the rate of closing of sodium channels both during depolarization and after repolarization of the nerve membrane.     

Point Mutations in Homology Domain II Modify the Sensitivity of Rat Nav1.8 Sodium Channels to the Pyrethroid Insecticide Cismethrin

Two point mutations in homology domain II of the house fly Vssc1 voltage-sensitive sodium channel α subunit, M918T and L1014F, are associated with resistance to pyrethroid insecticides and reduce the pyrethroid sensitivity of Vssc1 sodium channels expressed in Xenopus laevis oocytes. To assess the impact of these residues as determinants of pyrethroid sensitivity in another sequence context, we mutated the corresponding positions of the rat pyrethroid-sensitive, TTX-resistant peripheral nerve sodium channel (rNa_v1.8; also called SNS or PN3) and determined the sensitivity of native and mutated channels expressed in Xenopus oocytes to the pyrethroid insecticide cismethrin. The rNa_v1.8 channel, like other vertebrate sodium channel isoforms, contains a conserved isoleucine residue at sequence position 780 that aligns with the conserved methionine at position 918 of Vssc1 and other insect sodium channels. Channels mutated to contain methionine at position 780 (I780M) exhibited enhanced sensitivity to cismethrin and larger decay constants for pyrethroid-modified channel states. In contrast, the mutation corresponding to M918T in the Vssc1 channel (I780T) profoundly decreased the cismethrin sensitivity of expressed channels. Insertion of the mutation corresponding to L1014F (L879F in rNa_v1.8) reduced the cismethrin sensitivity of channels having either isoleucine or methionine at position 780, whereas channels containing the I780T/L879F double mutation were insensitive to this insecticide. Mutations at Ile780 and Leu879 also modified the voltage dependence of rNa_v1.8 channels, but these effects were not related to changes in pyrethroid sensitivity. These results confirm the importance of residues in homology domain II as fundamental determinants of the pyrethroid sensitivity of sodium channels.     

Interactions of tetramethrin, fenvalerate and DDT at the sodium channel in rat dorsal root ganglion neurons

Type I and type II pyrethroids and dichlorodiphenyltrichloroethane (DDT) are known to modulate the sodium channel to cause the hyperexcitatory symptoms of poisoning in animals. However, since the degrees to which neuronal sodium channel parameters are altered differ, a question is raised as to whether these insecticides bind to the same site in the sodium channel. Competition patch-clamp experiments were performed using rat dorsal root ganglion neurons which are endowed with tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels.d-trans-Tetramethrin,S,S-fenvalerate andp,p′-DDT caused a slowly rising and slowly falling tail current o to be developed in tetrodotoxin-sensitive sodium channels. In tetrodotoxin-resistant sodium channels, these insecticides, particularly tetramethrin and fenvalerate, generated a large and prolonged tail current upon repolarization. The effects of tetramethrin were reversible after washing with drug-free solution, whereas the effects of fenvalerate and DDT were irreversible. When fenvalerate application was followed by tetramethrin application, the characteristic changes in current by fenvalerate disappeared and the characteristic changes by tetramethrin appeared. After washout, the characteristic current pattern of fenvalerate reappeared. These results can be explained by assuming that the tetramethrin molecule displaces the fenvalerate molecule from the same binding site in the sodium channel protein, or that tetramethrin and fenvalerate bind to separate sodium channel sites which interact allosterically with each other. DDT interacted with fenvalerate and tetramethrin in the same manner.     

钠通道在昆虫拟除虫菊酯抗药性中的重要作用

电压门控的钠通道是拟除虫菊酯 (Pyrethroid)和滴滴涕 (dichloro diphenyl trichloroethane)的共同靶变点 ,是它们产生交互抗性 (击倒抗性 ,Knockdownresistance)的基础。由于拟除虫菊酯具有对昆虫和其他无脊椎动物呈高毒性 ,而对哺乳动物呈低毒性等特点 ,研究拟除虫菊酯的杀虫和抗药性机理 ,无论对人畜中毒的防治 ,还是对农业害虫的治理都有极为重大的意义     

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 α-cyano pyrethroids (β-cyfluthrin, cypermethrin, deltamethrin, esfenvalerate, fenpropathrin and λ-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_v1.8 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 α-cyano pyrethroids, deltamethrin, λ-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 α-cyano pyrethroids (λ-cyfluthrin, cypermethrin, deltamethrin and fenpropathrin) reduced open chloride channel probability. The R-isomers of λ-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, λ-cyhalothrin, cyfluthrin and cypermethrin belong to a second. Fenpropathrin and esfenvalerate occupy an intermediate position between these two groups.     

An examination of the proconvulsant actions of pyrethroid insecticides using pentylenetetrazol and amygdala kindling seizure models

Pyrethroid insecticides have recently been purported to possess strong proconvulsant potential. The seizure-inducing properties of two pyrethroids were assessed by pentylenetetrazol (PTZ) seizure models (repeated ip, suprathreshold ip, and iv), and electrical kindling of the amygdala. The efficacy of po versus ip routes of deltamethrin administration was compared using iv-PTZ administration and tests of locomotor activity in a figure-eight maze. Both po and ip deltamethrin produced comparable decreases in motor activity indicating the effectiveness of both of these exposure routes on biological activity. The Type I pyrethroid, cismethrin (15 mg/kg, po), produced a 17% reduction in the threshold dosage of ip-PTZ required to induce a seizure, while delaying the onset of generalized seizure activity. The Type II pyrethroid, deltamethrin (10 mg/kg, po), failed to alter threshold or latency to seizure onset, but did increase seizure duration. No differences were revealed between po (0, 10, 15 mg/kg) or ip (0, 1, 10 mg/kg) administered deltamethrin on seizure thresholds or durations following iv-PTZ. Seizure severity, however, was enhanced by pyrethroids administered po in the iv-PTZ and suprathreshold-ip PTZ tests, ip deltamethrin was without effect. Cismethrin (0, 8, 15 mg/kg) and deltamethrin (0, 6, 10 mg/kg) administered po daily, 2 hours prior to electrical kindling stimulation facilitated amygdala kindling to a minimal but equivalent degree at the highest dosage. This dosage also evoked strong behavioral signs of toxicity. Deltamethrin also induced spontaneous seizures in partially kindled animals in the absence of stimulation. Thus strong evidence of proconvulsant activity of pyrethroids was not evident. The primary effects were limited to an enhancement of seizure severity in response to PTZ (tonic seizures) and the provocation of spontaneous seizures in partially kindled animals.     

Interaction of insecticides of the pyrethroid family with specific binding sites on the voltage-dependent sodium channel from mammalian brain

Measurement of neurotoxin binding in rat brain membranes and neurotoxin-activated 22Na+ influx in neuroblastoma cells were used to define the site and mechanism of action of pyrethroids and DDT on sodium channels. A highly potent pyrethroid, RU 39568, alone enhanced the binding of [3H]batrachotoxinin A 20-alpha-benzoate up to 30 times. This effect was amplified by the action of neurotoxins such as sea anemone toxins and brevetoxin acting at different sites of the sodium channel protein in brain membranes. The ability of various pyrethroids and DDT to enhance batrachotoxin binding was related to their capacity to activate tetrodotoxin sensitive 22Na+ uptake. These results point to an allosteric mechanism of pyrethroids and DDT action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels. Pyrethroids do not block [3H]tetrodotoxin binding, 125I-Anemonia sulcata toxin 2 binding, 125I-Tityus serrulatus toxin gamma binding at neurotoxin receptor sites 1, 3 and 4 respectively. Pyrethroids appear to act at a new neurotoxin receptor site on the sodium channel. The distribution of pyrethroid binding sites in rat brain was determined by quantitative autoradiographic procedures using the property of pyrethroids to reveal binding sites for [3H]batrachotoxinin A 20-alpha-benzoate.     

Differential state-dependent modification of inactivation-deficient Nav1.6 sodium channels by the pyrethroid insecticides S-bioallethrin, tefluthrin and deltamethrin

Pyrethroid insecticides disrupt nerve function by modifying the gating kinetics of transitions between the conducting and nonconducting states of voltage-gated sodium channels. Pyrethroids modify rat Na(v)1.6+β1+β2 channels expressed in Xenopus oocytes in both the resting state and in one or more states that require channel activation by repeated depolarization. The state dependence of modification depends on the pyrethroid examined: deltamethrin modification requires repeated channel activation, tefluthrin modification is significantly enhanced by repeated channel activation, and S-bioallethrin modification is unaffected by repeated activation. Use-dependent modification by deltamethrin and tefluthrin implies that these compounds bind preferentially to open channels. We constructed the rat Na(v)1.6Q3 cDNA, which contained the IFM/QQQ mutation in the inactivation gate domain that prevents fast inactivation and results in a persistently open channel. We expressed Na(v)1.6Q3+β1+β2 sodium channels in Xenopus oocytes and assessed the modification of open channels by pyrethroids by determining the effect of depolarizing pulse length on the normalized conductance of the pyrethroid-induced sodium tail current. Deltamethrin caused little modification of Na(v)1.6Q3 following short (10ms) depolarizations, but prolonged depolarizations (up to 150ms) caused a progressive increase in channel modification measured as an increase in the conductance of the pyrethroid-induced sodium tail current. Modification by tefluthrin was clearly detectable following short depolarizations and was increased by long depolarizations. By contrast modification by S-bioallethrin following short depolarizations was not altered by prolonged depolarization. These studies provide direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Na(v)1.6Q3 channels in the open state and imply that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure-activity relationships.     

Differential sensitivity of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels to the insecticide allethrin in rat dorsal root ganglion neurons

The pyrethroid insecticides are known to modify neuronal sodium channels to cause a prolongation of whole cell current. The sodium channels expressed in the dorsal root ganglion neurons of the rat are of two types, one highly sensitive to tetrodotoxin and the other highly resistant to tetrodotoxin. The pyrethroid allethrin exerted profound effects on tetrodotoxin-resistant sodium channels while causing minimal effects on tetrodotoxin-sensitive sodium channels. Currents derived from tetrodotoxin-resistant sodium channels were greatly prolonged during a step depolarization; the tail currents upon repolarization were also augmented and prolonged. In the tetrodotoxin-sensitive sodium channel currents, these changes caused by allethrin were much smaller or negligible. The activation and inactivation voltages of tetrodotoxin-resistant peak sodium currents were not significantly altered by allethrin. The differential action of allethrin on the two types of sodium channels would be important not only in identifying the target molecular structure but also in interpreting the symptoms of poisoning in mammals.     

The action of pyrethroids on sodium channels in myelinated nerve fibres and spinal ganglion cells of the frog

The interaction of pyrethroids with the voltage-dependent sodium channel was studied in voltage-clamped nodes of Ranvier and isolated spinal ganglion neurons of the clawed frog, Xenopus laevis. In the node, pyrethroids prolonged the sodium tail current associated with a step repolarization of the membrane. It was found that the amplitude of the slow, pyrethroid-induced, sodium tail current (PIT) first increased and then decreased as a function of the duration of membrane depolarization (to -5 mV). This decrease of the PIT amplitude was absent when depolarizations to the sodium equilibrium potential (+40 mV) were used. Measurements of changes in sodium reversal potential indicated that sodium ion depletion in the perinodal space is largely responsible for the inactivation of the pyrethroid-modified sodium current. Inactivation is not completely abolished by pyrethroid treatment since the probability of channel opening, measured in membrane patches excised from spinal ganglion cells, decreased slowly during prolonged depolarization. Analysis of unitary currents indicated that both activation and inactivation are retarded by pyrethroids. The arrival of sodium channels in the pyrethroid-modified open state followed a time course that was slower than both activation and inactivation of unmodified sodium channels. Our findings indicate that sodium channels are modified when in the closed resting state and that both opening and closing kinetics are delayed by pyrethroids.     

Effects of an environmentally-relevant mixture of pyrethroid insecticides on spontaneous activity in primary cortical networks on microelectrode arrays

Pyrethroid insecticides exert their insecticidal and toxicological effects primarily by disrupting voltage-gated sodium channel (VGSC) function, resulting in altered neuronal excitability. Numerous studies of individual pyrethroids have characterized effects on mammalian VGSC function and neuronal excitability, yet studies examining effects of complex pyrethroid mixtures in mammalian neurons, especially in environmentally relevant mixture ratios, are limited. In the present study, concentration-response functions were characterized for five pyrethroids (permethrin, deltamethrin, cypermethrin, β-cyfluthrin and esfenvalerate) in an in vitro preparation containing cortical neurons and glia. As a metric of neuronal network activity, spontaneous mean network firing rates (MFR) were measured using microelectorde arrays (MEAs). In addition, the effect of a complex and exposure relevant mixture of the five pyrethroids (containing 52% permethrin, 28.8% cypermethrin, 12.9% β-cyfluthrin, 3.4% deltamethrin and 2.7% esfenvalerate) was also measured. Data were modeled to determine whether effects of the pyrethroid mixture were predicted by dose-addition. At concentrations up to 10 μM, all compounds except permethrin reduced MFR. Deltamethrin and β-cyfluthrin were the most potent and reduced MFR by as much as 60 and 50%, respectively, while cypermethrin and esfenvalerate were of approximately equal potency and reduced MFR by only ∼20% at the highest concentration. Permethrin caused small (∼24% maximum), concentration-dependent increases in MFR. Effects of the environmentally relevant mixture did not depart from the prediction of dose-addition. These data demonstrate that an environmentally relevant mixture caused dose-additive effects on spontaneous neuronal network activity in vitro, and is consistent with other in vitro and in vivo assessments of pyrethroid mixtures.     

Interactions of the pyrethroid fenvalerate with nerve membrane sodium channels: temperature dependence and mechanism of depolarization

Depolarization of nerve membranes is an important component of the mode of action of pyrethroids, and its negative temperature dependence parallels that of insecticidal activity. We studied the mechanism and temperature dependence of depolarization of crayfish giant axons by pyrethroids, using intracellular microelectrode and voltage clamp techniques. Membrane depolarization caused by tetramethrin and fenvalerate was greater at 10 degrees C than at 21 degrees C, and was reversible upon changing the temperature. Short-duration depolarizing pulses in voltage-clamped fenvalerate-treated axons induced prolonged sodium currents that are typical of other pyrethroids, but the decay of the tail current following repolarization was extremely slow, lasting several minutes at the large negative holding potential of -120 mV. At the normal resting potential, the tail current did not decay completely, and even without stimulation, a steady-state sodium current developed, which could account for the depolarization. The steady-state current induced by fenvalerate at the resting potential was much larger at 8 degrees C than at 21 degrees C, accounting for the negative temperature dependence of the depolarization. The negative temperature dependence of the steady-state current seems to be due ultimately to the great stabilizing effect of low temperature on the open-modified channel. When the steady-state current was induced at the resting potential, hyperpolarization to more negative potentials caused it to decay with exactly the same time course as tail currents induced by short-duration depolarizing pulses, indicating that both types of currents are carried by identically-modified channels. The modified channels were shown to be inactivated very slowly at potentials more positive than - 100 mV, accounting for the limited depolarization observed in micro-electrode experiments. Even when applied directly to the internal face of the membrane, the effect of fenvalerate on the sodium channel developed slowly, taking more than 90 min to reach its final level. Fenvalerate did not significantly affect potassium currents.     

Modulation of nerve membrane sodium channel activation by deltamethrin.

Deltamethrin is a highly potent pyrethroid insecticide that causes hypersensitivity, choreoathetosis, tremors, and paralysis in mammals. It is known to modify the sodium channel in such a way as to prolong the tail current associated with step repolarization following a depolarizing pulse. Using the axial-wire voltage-clamp technique with the giant axon of the squid Loligo pealei, we have demonstrated that deltamethrin also greatly slows the opening of the sodium channel. This was first observed as a decrease, by as much as 80%, in the peak sodium current flowing during a short, 10 ms depolarization. Current flowing through these slowly opening deltamethrin modified sodium channels was observed during the first depolarizing pulse after deltamethrin exposure and developed with a time constant of 320 ms. This supports the idea that deltamethrin can modify sodium channels when they are in the closed or resting state. Further, evidence of this hypothesis was provided by experiments using 0.1 and 10 microM deltamethrin and measuring the tail current amplitude after depolarizing pulses of varying duration (1-1200 ms). The mean time constant for the increase in tail current amplitude was almost concentration independent; 253 ms at 0.1 microM and 193 ms at 10 microM. We conclude that deltamethrin modifies the activation kinetics of sodium channels in such a way as to slow opening and that this modification occurs predominantly when channels are in the closed or resting state.     

Functional reconstitution of rat Nav1.6 sodium channels in vitro for studies of pyrethroid action

The ability to reconstitute sodium channel function and pharmacology in vitro using cloned subunits of known structure has greatly enhanced our understanding of the action of pyrethroid insecticides at this target and the structural determinants of resistance and interspecies selectivity. However, the use of reconstituted channels raises three critical questions: (1) Which subunits and subunit combinations should be used? (2) Which heterologous expression system is preferred? (3) Which combination of subunits and expression system best represents the function of native neuronal channels in the organism of interest? This review considers these questions from the perspective of recent research in this laboratory on the action of pyrethroid insecticides on rat Na_v1.6 sodium channels by comparing the effects of heteroligomeric complex composition on channel function and insecticide response when channels are expressed in either Xenopus oocytes or stably-transformed HEK293 cells. These comparisons provide new insight into the influence of cellular context on the functional and pharmacological properties of expressed channels, the modulatory effects of sodium channel auxiliary subunits on the action of pyrethroids, and the relative fidelity of the Xenopus oocyte and HEK293 cell expression systems as model systems for studying of channel function and pyrethroid action.