Henk P.M. Vijverberg

Neurotoxicological Effects And The Mode of Action of Pyrethroid Insecticides

Neuroexcitatory symptoms of acute poisoning of vertebrates by pyrethroids are related to the ability of these insecticides to modify electrical activity in various parts of the nervous system. Repetitive nerve activity, particularly in the sensory nervous system, membrane depolarization, and enhanced neurotransmitter release, eventually followed by block of excitation, result from a prolongation of the sodium current during membrane excitation. This effect is caused by a stereoselective and structure-related interaction with voltage-dependent sodium channels, the primary target site of the pyrethroids. Near-lethal doses of pyrethroids cause sparse axonal damage that is reversed in surviving animals. After prolonged exposure to lower doses of pyrethroids axonal damage is not observed. Occupational exposure to pyrethroids frequently leads to paresthesia and respiratory irritation, which are probably due to repetitive firing of sensory nerve endings. Massive exposure may lead to severe human poisoning symptoms, which are generally treated well by symptomatic and supportive measures.

Hydroxylation Increases the Neurotoxic Potential of BDE-47 to Affect Exocytosis and Calcium Homeostasis in PC12 Cells

Background Oxidative metabolism, resulting in the formation of hydroxylated polybrominated diphenyl ether (PBDE) metabolites, may enhance the neurotoxic potential of brominated flame retardants. Objective Our objective was to investigate the effects of a hydroxylated metabolite of 2,2′,4,4′-tetra-bromodiphenyl ether (BDE-47; 6-OH-BDE-47) on changes in the intracellular Ca2+ concentration ([Ca2+]i) and vesicular catecholamine release in PC12 cells. Methods We measured vesicular catecholamine release and [Ca2+]i using amperometry and imaging of the fluorescent Ca2+-sensitive dye Fura-2, respectively. Results Acute exposure of PC12 cells to 6-OH-BDE-47 (5 μM) induced vesicular catecholamine release. Catecholamine release coincided with a transient increase in [Ca2+]i, which was observed shortly after the onset of exposure to 6-OH-BDE-47 (120 μM). An additional late increase in [Ca2+]i was often observed at ≥1 μM 6-OH-BDE-47. The initial transient increase was absent in cells exposed to the parent compound BDE-47, whereas the late increase was observed only at 20 μM. Using the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and thapsigargin to empty intracellular Ca2+ stores, we found that the initial increase originates from emptying of the endoplasmic reticulum and consequent influx of extracellular Ca2+, whereas the late increase originates primarily from mitochondria. Conclusion The hydroxylated metabolite 6-OH-BDE-47 is more potent in disturbing Ca2+ homeostasis and neurotransmitter release than the parent compound BDE-47. The present findings indicate that bioactivation by oxidative metabolism adds considerably to the neurotoxic potential of PBDEs. Additionally, based on the observed mechanism of action, a cumulative neurotoxic effect of PBDEs and ortho-substituted polychlorinated biphenyls on [Ca2+]i cannot be ruled out.

ACTION OF PYRETHROID INSECTICIDES ON THE VERTEBRATE NERVOUS SYSTEM

Vijverberg H.P.M. & van den Bercken J.1982 Neuropathology and Applied Neurobiology8, 421–440

5-HT3 receptors and neurotransmitter release in the CNS: a nerve ending story?

Serotonin (5-HT) 5-HT(3) receptors are ligand-gated ion channels, which are generally thought to be involved in the presynaptic modulation of neurotransmitter release. However, analysis of published data reveals that most of the evidence for the alleged presynaptic role of 5-HT(3) receptors in modulating CNS neurotransmitter release is not compelling. Nevertheless, 5-HT(3) receptors are present in nerve terminals from some brain regions. The increased basic knowledge of the cellular physiology of central 5-HT(3) receptor ligand-gated ion channels provides opportunities for a detailed characterization of the specific presynaptic effects of 5-HT(3) receptors. Such reconsideration is required for the full appreciation of the functional role of 5-HT(3) receptors in the CNS.

Frequency-dependent effects of the pyrethroid insecticide decamethrin in frog myelinated nerve fibres

The pyrethroid insecticide decamethrin (10(-6) M) caused a frequency-dependent depression of the action potential in frog myelinated nerve fibres which was associated with a progressive membrane depolarisation brought about by summation of depolarising after-potentials. Voltage clamp experiments with single nodes of Ranvier showed that this afterpotential was most probably due to a long-lasting prolongation of the transient increase in sodium permeability of the membrane. The results indicate that decamethrin, like the other pyrethroids, specifically affects the sodium channels of the nerve membrane.

Kinetics of the membrane current mediated by serotonin 5-HT3 receptors in cultured mouse neuroblastoma cells.

1. Ionic currents mediated by serotonin 5‐HT3 receptors were studied in the mouse neuroblastoma cell line N1E‐115, using suction pipettes for intracellular perfusion and voltage clamp recording. The dependence of the kinetics of the membrane current on serotonin concentration was investigated. 2. At a holding potential of ‐70 mV application of 5‐HT (5‐hydroxytryptamine creatinine sulphate) causes a transient inward current. The i‐V curve of the peak amplitude is linear between ‐80 and 60 mV. The reversal potential is 20 +/‐ 4 mV (mean +/‐ S.D.). The kinetics of the transient ionic current are independent of the holding potential. 3. In the presence of 5‐HT the membrane current decays to a small steady‐state level with a single‐exponential time course. The time constant of decay decreases with increasing concentration of the agonist, to a minimum value of 6.5 +/‐ 1.5 s for concentrations of 5‐HT greater than or equal to 3 microM. 4. When the agonist is rapidly removed, single‐exponential decay of the ionic current is observed. The time constant of this decay in the absence of 5‐HT amounts to 6.9 +/‐ 1.5 s and is independent of the membrane potential and of the concentration of 5‐HT used. 5. In the presence of low concentrations of 5‐HT the peak amplitude of the inward current evoked with a high concentration of agonist is gradually reduced. The onset of this desensitization follows the same time course as the decay of the membrane current. In the range from 0.7 to 1.5 microM‐5‐HT both kinetic processes show the same steep concentration dependence. 6. Recovery from desensitization, measured at variable intervals after removal of the agonist, can be fitted by a single‐exponential function with a time constant of 18 +/‐ 4 s. 7. The results show that the kinetic properties of the 5‐HT3 receptor‐mediated ionic current can only be described by a complex, co‐operative model.

Temperature- and structure-dependent interaction of pyrethroids with the sodium channels in frog node of Ranvier

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.

Selection of distinct conformational states of the 5-HT3 receptor by full and partial agonists

1 5‐Hydroxytryptamine 5‐HT3 receptor‐mediated ion currents evoked by 5‐HT, quaternary 5‐HT (5‐HTQ), meta‐chlorophenylbiguanide (mCPBG), dopamine and tryptamine in N1E‐115 mouse neuroblastoma cells have been investigated in whole‐cell voltage clamp and single channel patch clamp experiments. 2 The concentration‐dependent activation and desensitization of the ion currents evoked by the agonists yield the potency order: mCPBG > 5‐HTQ  5‐HT > > tryptamine > dopamine, and the efficacy order: 5‐HT  mCPBG  5‐HTQ > > dopamine  tryptamine. Thus, 5‐HT, 5‐HTQ and mCPBG are full agonists, whereas dopamine and tryptamine are partial agonists at the 5‐HT3 receptor. 3 Full and partial agonists cause complete cross‐desensitization and activate single channels with similar conductances and open lifetimes. This shows that full and partial agonists act on the same population of 5‐HT3 receptors. 4 The time course of recovery from desensitization depends on the agonist used. Recovery from partial agonist‐induced desensitization is single exponential, whereas the desensitization induced by full agonists recovers with sigmoid kinetics, suggesting at least 3 steps between 4 states. 5 During the process of recovery from cross‐desensitization, the full agonists activate a larger fraction of the 5‐HT3 receptors than the partial agonists, irrespective of the agonist used to induce desensitization. 6 It is concluded that full and partial agonists induce distinct desensitized states and, during recovery from desensitization, recognize distinct conformations of unoccupied 5‐HT3 receptors. This conformational selection is likely to account for the different efficacies of full and partial 5‐HT3, receptor agonists.

The dopamine response in mouse neuroblastoma cells is mediated by serotonin 5HT3 receptors

Serotonin (5HT) and dopamine (DA) induce, in neuroblastoma N1E-115 cells, a transient membrane depolarization associated with an inward current. The half-maximum response is obtained with 2 microM 5HT or 200 microM DA. The maximum response to 5HT is 2-3 times that to DA. The selective 5HT3 receptor antagonists ICS 205-930 and MDL 72222 at nanomolar concentrations block both the 5HT- and the DA-induced response. High concentrations (10 microM) of 5HT2 receptor antagonists are without effect. It is concluded that, in N1E-115 cells, 5HT and DA activate a single population of 5HT3 receptors.

Competitive Potentiation of Acetylcholine Effects on Neuronal Nicotinic Receptors by Acetylcholinesterase‐Inhibiting Drugs

Abstract: The effects of the acetylcholinesterase inhibitors physostigmine and tacrine on α4β2 and α4β4 subtypes of neuronal nicotinic acetylcholine (ACh) receptors, expressed in Xenopus laevis oocytes, have been investigated. In voltage‐clamp experiments low concentrations of physostigmine and tacrine potentiate ion currents induced by low concentrations of ACh, whereas at high concentrations they inhibit ACh‐induced ion currents. These dual effects result in bell‐shaped concentration—effect curves. Physostigmine and tacrine, by themselves, do not act as nicotinic receptor againsts. The larger potentiation is observed with 10 μM physostigmine on α4β4 nicotinic receptors and amounts to 70% at 1 μM ACh. The mechanism underlying the effects of physostigmine on α4β4 ACh receptors has been investigated in detail. Potentiation of ACh‐induced ion current by low concentrations of physostigmine is surmounted at elevated concentrations of ACh, indicating that this is a competitive effect. Conversely, inhibition of ACh‐induced ion current by high concentrations of physostigmine is not surmounted at high concentrations of ACh, and this effect appears mainly due to noncompetitive, voltage‐dependent ion channel block. Radioligand binding experiments demonstrating displacement of the nicotinic receptor agonist 125I‐epibatidine from its recognition sites on α4β4 ACh receptors by physostigmine confirm that physostigmine is a competitive ligand at these receptors. A two‐site equilibrium receptor occupation model, combined with noncompetitive ion channel block, accounts for the dual effects of physostigmine and tacrine on ACh‐induced ion currents. It is concluded that these acetylcholinesterase‐inhibiting drugs interact with the ACh recognition sites and are coagonists of ACh on α4‐containing nicotinic ACh receptors.