* Institute for Risk Assessment Sciences, Utrecht University, NL-3508 TD Utrecht, The Netherlands; TNO Prins Maurits Laboratory, NL-2280 AA Rijswijk, The Netherlands; and
CNR Center of Cellular and Molecular Pharmacology, University of Milano, I-20129 Milano, Italy
Received June 24, 2004; accepted August 25, 2004
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ABSTRACT |
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Key Words: organophosphate pesticides; neuronal nicotinic acetylcholine receptor; rat brain acetylcholinesterase; Xenopus oocytes; two-microelectrode voltage clamp.
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INTRODUCTION |
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Studies on repeated low-level exposure to organophosphates of many occupational groups such as sheep dippers, pesticide sprayers, and farmers give equivocal results (for review see Ray and Richards, 2001). Some studies describe no adverse health effects or even indicate improvement of cognitive functions (Ivens et al., 1998
). However, several studies describe a range of neurobehavioral and neuropsychiatric disorders after chronic OP exposure at levels that cause no symptoms of acute poisoning (Salvi et al., 2003
; Stephens et al., 1995
). Some of these OP pesticides, e.g., chlorpyrifos, show different toxicology profiles depending on age and gender of the animals (Levin et al., 2002
; Moser et al., 1998
, 2000
). However, these differences cannot be explained by a different sensitivity of AChE of the animals to these pesticides (Mortensen et al., 1998
). It is clear that exposure to low-level doses of OP pesticides, as well as acute poisoning, can lead to several neurological and neurobehavioral changes, which cannot be accounted for on the basis of AChE inhibition alone. It has been suggested that other, more sensitive brain proteins are involved (Ray and Richards, 2001
).
In addition to inhibition of AChE, several organophosphates also interact directly with receptors of the cholinergic system or modulate the receptor expression levels. A number of organophosphorous compounds, e.g., the warfare agents VX and soman and the pesticide metabolites paraoxon and malaoxon, displace the agonist 3H-cis-methyldioxolane from its binding site, suggesting that these compounds interact directly with rat M2 muscarinic ACh receptors (Bakry et al., 1988; Chaudhuri et al., 1993
; Ward et al., 1993
). Binding to the agonist site of M2 as well as M4 muscarinic ACh receptors is also observed for the pesticide metabolites chlorpyrifos oxon, paraoxon, and methylparaoxon (Howard and Pope, 2002
; van den Beukel et al., 1997
). Apart from direct receptor interactions, the pesticides parathion and chlorpyrifos differentially upregulate and downregulate muscarinic ACh receptor expression in neonatal rats (Betancourt and Carr, 2004
; Chaudhuri et al., 1993
; Richardson and Chambers, 2004
; Tang et al., 2003
). Effects on nicotinic acetylcholine receptors (nAChRs) are also reported. Some organophosphates, e.g., echothiophate, DFP, and VX, block the open channel of muscle type nAChRs and desensitize the receptor (Bakry et al., 1988
; Eldefrawi et al., 1988
; Rao et al., 1987
; Tattersall 1990
). The pesticides chlorpyrifos and parathion and their oxon metabolites were shown to inhibit the carbamylcholine-stimulated binding of 3H-thienyl-cyclohexylpiperidine (TCP) to the open ion channel of Torpedo electric organ nAChRs, but enhanced TCP binding in the absence of agonist. In addition, the OPs enhanced the apparent affinity of the nAChRs to carbamylcholine, suggesting that they induce receptor desensitization (Katz et al., 1997
). Chronic DFP treatment results in loss of hippocampal neuronal nAChRs (Stone et al., 2000
). Although the effects of OP pesticides on muscarinic and muscle type nAChRs have been investigated extensively and are suggested to play a role in OP toxicity, the modulation of neuronal nAChR function by OP pesticides remains to be elucidated.
We have systematically investigated effects of a range of organophosphate pesticides on neuronal 4ß2 nAChRs heterologously expressed in Xenopus laevis oocytes. From 21 organophosphates tested, the six compounds that were the more potent ones to inhibit nAChRs were selected. For these compounds the potencies for inhibition of the nAChRs were compared to the potencies for inhibition of rat brain AChE. In addition, receptor binding data and a kinetic analysis of the effects further elucidate the mechanism of action of OPs on neuronal nAChRs.
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MATERIALS AND METHODS |
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Saline solutions for electrophysiology were prepared with distilled water, and solutions for AChE assays were prepared with Milli-Q filtered water (Millipore SA, Molsheim, France). Stock solutions (0.1 M) of azinphos-methyl, diazinon, dibrom, chlorpyrifos, disulfoton, EPN, fenthion, leptophos, malaoxon, malathion, methamidophos, paraoxon-ethyl, phoxim, tri-p-tolyl-phosphate, and tri-p-tolyl-phosphite were prepared in DMSO. Stock solutions (0.01 M) of acephate, dimefox, dimethoate, and echothiophate iodide were prepared in Milli-Q filtered water. Stock solutions (0.1 M and 1 M) of parathion-ethyl and parathion-methyl were prepared in ethanol. The final ethanol concentration in all experiments was < 0.03% (v/v), at which the ACh-induced ion currents were not affected. cDNAs of nicotinic receptor subunits ligated into the pSM plasmid vector containing the SV40 viral promoter were a kind gift from Dr. J. W. Patrick (Baylor College of Medicine, Houston, TX).
Brain AChE preparation. Male Wistar rats (350 g, n = 5; Harlan, Horst, The Netherlands) were decapitated and the brains were removed. The tissue was homogenized (900 rpm, 10 % w/v homogenate) in ice-cold buffer containing 50 mM Tris, 1 M NaCl, 5 mM EDTA and 1 % (v/v) Triton X-100, pH 7.4 and subsequently centrifuged for 10 min at 36,000 x g in a Ti50 rotor at 4°C (L8-70; Beckman Coulter Inc., Fullerton, CA). The supernatant was kept on ice and used within 3 h. An aliquot was drawn to determine the protein content, which ranged from 30 to 37 mg/ml.
Brain AChE inhibition. The stock solutions of the AChE inhibitors were diluted in a phosphate buffer (8 mM KH2PO4 and 48 mM K2HPO4, pH 7.4) to obtain work standards containing 0.001 µM to 1 mM of the inhibitor. Then 25 µl aliquots of the work standard were added to 225 µl of brain homogenate. After incubation for 1 min at 37°C, 25 µl, samples were drawn, added to 250 µl ice-cold phosphate buffer, mixed, and rapidly frozen in liquid nitrogen. These samples were stored at 70°C until analysis. Thawed samples were appropriately diluted and were assayed in quadruplicate for AChE activity using a 96-well microplate modification of the Ellman method (Bueters et al., 2003). Ethopropazine (10 µM) was used as a specific inhibitor of butyrylcholinesterase.
Receptor expression in oocytes. Mature female Xenopus laevis frogs (AmRep, Breda, The Netherlands) were anesthetized by submersion in 0.2% MS-222, and some ovarian lobes were surgically removed. Experimental procedures involving animals were approved by a local ethical committee and were in accordance with Dutch law. Oocytes were defolliculated manually after treatment with collagenase type I (1.5 mg/ml calcium-free Barth's solution) for 1.5 h at room temperature. Plasmids coding for 4 and ß2 subunits of rat neuronal nAChRs (Boulter et al., 1987
) were dissolved in distilled water. Stock solutions containing
4 or ß2 subunit cDNAs at a 1:1 molar ratio, were injected into the nuclei of stages V and VI oocytes within 8 h after harvesting, using a Drummond microinjector. Approximately 1 ng of each plasmid containing
or ß cDNA was injected in a total injection volume of 18.4 nl/oocyte. After injection, the oocytes were incubated at 19°C in modified Barth's solution containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 15 mM HEPES, and 50 µg/ml neomycin. Experiments were performed on oocytes after 36 days of incubation (Zwart and Vijverberg, 1997
).
Electrophysiology. Oocytes were voltage clamped using two microelectrodes (0.52.5 M) filled with 3 M KCl and a custom-built voltage clamp amplifier with high-voltage output stage (Zwart and Vijverberg, 1997
). The external saline was clamped at ground potential by means of a virtual ground circuit using an Ag/AgCl reference electrode and a platinum black-covered platinum electrode for current passing. Membrane current was measured with a current-to-voltage converter incorporated into the virtual ground circuit. The membrane potential was held at 40 mV and all experiments were performed at room temperature (21°23°C). Oocytes were placed in a piece of tubing (internal diameter 3 mm), which was continuously perfused with saline solution (115 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 10 mM HEPES, pH 7.2 with NaOH) at a rate of approximately 20 ml/min. Aliquots of concentrated stock solutions of ACh in distilled water were added to the saline immediately before the experiments. Compounds were applied by switching between control and compound-containing saline with a servomotor-operated valve. The compounds were applied for 20 s during the 40 s ACh application. Agonist applications were alternated by 5 min of superfusion with agonist-free saline to allow the receptors to recover completely from desensitization. To minimize adsorption of organophosphates in the superfusion system, glass reservoirs and Teflon (PTFE) tubing (4 x 6 mm, Rubber, Hilversum, The Netherlands) were used. Membrane currents were low-pass filtered (eight-pole Bessel; 3 dB at 0.3 kHz), digitized (12 bits, 1,024 samples/record), and stored on disk for off-line computer analysis (Zwart and Vijverberg, 1997
).
Binding studiespreparation of oocyte homogenates. Batches of 5080 frozen oocytes expressing rat 4ß2 nAChRs were thawed and homogenized in a Potter homogenizer in an excess of buffer A (50 mM Tris-HCl pH 7, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2 and 2 mM phenylmethylsulfonyl fluoride), centrifuged for 60 min at 30,000 x g, and rinsed twice. The homogenates were resuspended in the same buffer containing 20 µg/ml of the protease inhibitors leupeptin, bestatin, pepstatin A, and aprotinin. Receptor expression ranged from 172 to 286 fmol/mg protein, which corresponds to an average nAChR density of 41 to 50 fmol/oocyte (44.3 ± 1.6; mean ± SEM, n = 5).
3H-Epibatidine binding. Preliminary time course experiments were performed before saturation and competition analyses to determine the time required for 3H-epibatidine to reach equilibrium with the 4ß2 nAChRs. In the epibatidine saturation experiments, aliquots of oocyte homogenates were incubated overnight at 4°C with concentrations of 3H-epibatidine ranging between 0.005 and 2.5 nM diluted in buffer A. Nonspecific binding was determined in parallel by means of incubation in the presence of 100 nM unlabeled epibatidine. At the end of the incubation, the samples were filtered on GFC filters pre-soaked in polyethylenimine through a Brandell apparatus, and the filters were counted in a ß counter. To test the ability of parathion-ethyl and disulfoton to inhibit 3H-epibatidine binding, parathion-ethyl and disulfoton were dissolved in DMSO and then diluted in buffer A just before use. Serial dilutions were pre-incubated for 30 min at room temperature with homogenates containing
4ß2 nAChRs. Subsequently, a final concentration of 0.05 nM 3H-epibatidine was added for overnight incubation at 4°C.
Data analysis. Amplitudes of ion currents were measured and normalized to the amplitude of ACh-induced control responses (100%) to adjust for differences in receptor expression levels between oocytes and for small variations in response amplitude over time. The percentage of inhibition of the ACh-induced ion current by the organophosphates was calculated from the quotient of the amplitude of the response after 20 s coapplication of the organophosphate and that of the control response at the same time point. Standard concentration-effect curves were fitted to the experimental data according the Hill equation:
![]() | (1) |
Ligand-binding data. The experimental data obtained from the saturation binding experiments were analyzed by means of a nonlinear least-squares procedure using the LIGAND program (Gotti et al., 1998). The binding parameters were calculated by fitting the saturation data. An "extra sum of squares" F-test was performed by the LIGAND program to evaluate the different binding models statistically (i.e., one-site vs. two-site models, comparison of the binding parameters, etc.). The Ki values were determined by means of LIGAND, using the data obtained from three independent competition experiments.
Kinetics and two-step model. The dual rate of inhibition and the apparently concentration-dependent kinetics of reversal of inhibition were interpreted as a two-step sequential chemical equilibrium between organophosphate and nicotinic receptor:
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![]() | (2a) |
![]() | (2b) |
![]() | (2c) |
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RESULTS |
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Chlorpyrifos, disulfoton, EPN, fenthion, parathion-ethyl, and parathion-methyl inhibited the ACh-induced ion current substantially. For all of these compounds, inhibition of the 1 µM ACh-induced ion current was less than the inhibition of the 1 mM ACh-induced ion current (Fig. 1). The concentration-dependent effects of chlorpyrifos, disulfoton, EPN, fenthion, parathion-ethyl, and parathion-methyl at 1 µM ACh and 1 mM ACh were investigated in detail, as shown for the inhibition of the 1 mM ACh-induced ion current by disulfoton in Figure 2. The kinetics of the responses in Figure 2 illustrate that the rate of onset of inhibition increased with the concentration of organophosphate. At wash-out of the organophosphate, the reverse was observed. The rate of recovery of the ACh-induced ion current decreased with the concentration of organophosphate. On the wash-out of high concentrations of organophosphates the ACh-induced ion current did not fully recover before termination of the superfusion with ACh.
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Noncompetitive Mechanisms
The binding experiments demonstrate that the inhibition of the ACh-induced ion current by the organophosphates disulfoton and parathion-ethyl is due to noncompetitive effects. Therefore, various possibilities for noncompetitive inhibition of the neuronal type nAChR by the organophosphates have been investigated. To assess whether the organophosphate itself is the agent causing the inhibitory effect, experiments were performed in which ACh and parathion-ethyl were coapplied and subsequently washed out, each separately or both together. The result shows a rebound tail current indicating reversal of block only when parathion-ethyl is washed out (Fig. 6A). On wash-out of ACh, the remaining inward current rapidly declined to zero irrespective of whether parathion-ethyl remained present or not and rebound tail currents were not observed. These results show that parathion-ethyl blocks the ion current and rules out the possibility that the inhibitory effect is caused by an enhancement of the channel blocking potency of ACh in the presence of parathion-ethyl.
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Onset and Reversal of Inhibition
The kinetics of the effects of parathion-ethyl show that the rate of onset of inhibition increased with organophosphate concentration (Fig. 7A). Values of the apparent fast and slow rate constants of the onset of inhibition were obtained by fitting eq. (3) to the data as shown in the inset of Figure 7A and plotted against parathion-ethyl concentration. An inverse relation between organophosphate concentration and the rate of reversal of inhibition was obtained on wash-out (Fig. 7B). The rate of reversal of inhibition of the ACh-induced ion current decreased with increasing concentration of parathion-ethyl. After administration of high concentrations of organophosphates, the inhibitory effect did not fully reverse on wash-out of the organophosphate within the period until the termination of superfusion with ACh (Fig. 7 inset). The features of inhibition are consistent with a sequential model as shown in equation (2). Regression lines according to eq. (2a) and eq. (2b) in Figure 7A show an approximate compliance of the effects observed with the two-step sequential mechanism of equation (2). The rate constants obtained from the regressions are: kon = 0.013 µM1s1, koff = 1.45 s1 (Kd,1 = 112 µM), and k+1 = 0.60 s1, k1 = 0.057 s1 (Kd,2 = 0.10), and the apparent affinity of the overall process of inhibition by parathion-ethyl, according to eq. (2c), Kd,apparent = 9.8 µM. For disulfoton rate constants obtained are: kon = 0.046 µM1s1, koff = 0.48 s1, (Kd,1 = 10 µM), k+1 = 0.23 s1, and k1 = 0.021 s1, (Kd,2 = 0.09) resulting in an apparent affinity for disulfoton of 0.85 µM. These values are within the same order of magnitude as the IC50 values of the inhibition curves of parathion-ethyl and disulfoton (Table 1).
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DISCUSSION |
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The organophosphates tested have a wide range of inhibitory potencies. For the inhibition of nAChRs, the more potent pesticides, in order of potency, were disulfoton > parathion-ethyl, parathion-methyl > fenthion. The inhibitory effects are more potent at high concentrations of ACh than at low concentrations of the agonist (Fig. 3). For six organophosphates, that were shown to inhibit 4ß2 nAChRs, the potencies for inhibition of rat brain AChE were determined: chlorpyrifos and parathion-ethyl significantly inhibited AChE activity, whereas parathion-methyl, fenthion, EPN, and disulfoton did not. This was expected, because most organophosphates need to be metabolized to their active oxons to cause potent AChE inhibition; e.g., reported values of the IC50 of inhibition of rat brain cholinesterase are 35 nM for paraoxon and 3536 nM for malaoxon (Ward et al., 1993
). Theoretically, the first effect of exposure to an organophosphate may consist of impairment of cholinergic transmission resulting from nAChR inhibition. After formation of the active metabolites, AChE will be inhibited, resulting in an increase of ACh in the synaptic cleft (Soreq and Seidman, 2001
). The elevated concentration of ACh will, at the same time, cause an increase of nAChR activation and enhance the inhibitory effect of the organophosphates on the nAChRs. Thus the stimulating effects of AChE inhibition on nicotinic neurotransmission are counteracted by an enhanced potency of inhibition of nAChRs by the organophosphates. In practice, symptoms related to inhibition of AChE may prevail, even for poisoning by the "thio" OP compounds, because of the much higher potency of the oxon metabolites for inhibition of AChE. Kinetic studies with chlorpyrifos have shown that AChE inhibition in plasma occurs within minutes and changes over hours after oral dosing (Timchalk et al., 2002
), indicating that the oxon metabolite is rapidly formed. Thus, the main effect of the interaction of the parent compound with the nAChR may be to postpone the symptoms of AChE inhibition. A carefully designed study would be required to investigate the various possibilities and to distinguish between symptoms specifically related to effects of OP compounds on AChE and on muscarinic and nicotinic AChR.
Although the mechanisms of AChE inhibition have been described in detail (Soreq and Seidman, 2001), the nature of the inhibitory effect of the organophosphate pesticides on nicotinic ACh receptors is less evident. Competition binding experiments demonstrate that the organophosphates parathion-ethyl and disulfoton at concentrations <10 mM do not bind to the agonist-recognition site of rat
4ß2 nAChRs (Fig. 5). This demonstrates that a noncompetitive mechanism is responsible for the inhibitory effect of the organophosphate on the nAChR-mediated ion current. Because parathion-ethyl can inhibit the
4ß2 nAChRs in the absence of ACh (Fig. 6), it is concluded that channel opening is not required for the inhibitory effect. The biphasic kinetics of onset of inhibition and the concentration-dependence of the kinetics of reversal of inhibition (Fig. 7) suggest that the ion current is inhibited by a two-step mechanism. Fitting a sequential two-step equilibrium, with a rapidly reversible association and dissociation of the organophosphate followed by a slowly reversible transition (Zhao et al., 1999
), to the inhibitory effects of parathion-ethyl and disulfoton (Fig. 7) yielded potencies of the OPs comparable to those obtained from the inhibition curves (Fig. 3). The kinetics of reversal of inhibition are inversely related to OP concentration. This finding is similar to what has been reported before for the inhibition of human muscle type nAChRs in TE-671 cells by the philanthotoxin PhTX-(12). PhTX-(12) is a weak open channel blocker and is supposed to enhance receptor desensitization (Brier et al., 2003
). In addition, it has been suggested that the noncompetitive interaction of chlorpyrifos and parathion and their oxons with muscle nAChRs also leads to receptor desensitization (Katz et al., 1997
). Based on these and the present results, it is more likely that the secondary blocked state in our model (eq. 2) represents a desensitized state rather than a blocked state, because enhancement of desensitization would also account for the enhanced potency of parathion-ethyl and disulfoton at elevated ACh concentration (Fig. 3).
The toxicological relevance of effects of OP pesticides on neuronal nAChRs lies in the fact that a number of neurotoxic symptoms cannot be accounted for on the basis of AChE inhibition alone. Although the effects of desensitization of 4ß2 nAChRs are still unclear, some indication of effects to be expected can be derived from studies on knockout mice. Mice lacking the
4 nAChR subunit display a reduced antinociceptive effect of nicotine (Marubio et al., 1999
) and elevated anxiety (Ross et al., 2000
). Knock-out of the ß2 nAChR subunit demonstrated that the ß2 subunit is involved in the reinforcing properties of nicotine (Picciotto et al., 1998
) and in passive avoidance learning (Cordero-Erausquin et al., 2000
). Thus, chronic or repeated desensitization of
4ß2 nAChRs might result in several neurological and neurobehavioral deficits. In this respect, it is remarkable that in farmers chronically exposed to OP pesticides, increased anxiety is one of the more frequently diagnosed symptoms (Salvi et al., 2003
).
In conclusion, a number of OP pesticides have been demonstrated to interact directly with neuronal 4ß2 nAChRs to inhibit the ACh-induced response in a noncompetitive way. The inhibition is accounted for by a two-step mechanism resulting in receptor desensitization. The results indicate that neuronal AChRs constitute additional targets for the effects of OPs on the nervous system.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Institute for Risk Assessment Sciences, Utrecht University, P.O. Box 80176, NL-3508 TD Utrecht, The Netherlands. Tel + 31 (30) 2535397. Fax + 31 (30) 2535077. E-mail: H.Vijverberg{at}iras.uu.nl.
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