Facilitation of Acetylcholine Signaling by the Dithiocarbamate

Azim Ziyaei HalimehjaniSahar ShokrgozarPetr Beier ... Azim Ziyaei Halimehjani , Mojtaba Hajiloo Shayegan , Mohammad Mahmoodi Hashemi , and Behrooz ...
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Chem. Res. Toxicol. 2002, 15, 26-32

Facilitation of Acetylcholine Signaling by the Dithiocarbamate Fungicide Propineb Marina Marinovich,* Barbara Viviani, Valerie Capra,† Emanuela Corsini, Laura Anselmi,‡ Gianluigi D’Agostino,§ Amalia Di Nucci,‡ Marco Binaglia, Marcello Tonini,‡ and Corrado L. Galli Laboratory of Toxicology and Section of Theoretical Pharmacology and Receptor Modeling, Department Pharmacological Sciences, University of Milan, Department of Internal Medicine and Therapeutics, Section of Clinical and Experimental Pharmacology, and Department of Experimental and Applied Pharmacology, University of Pavia, Italy Received July 5, 2001

Dithiocarbamates (DTCs) are used mainly in agriculture as pesticides and as alcohol deterrent drugs. Neurological complications as well as movement disorders characterized by plastic rigidity, muscle twitch and paralysis are the prevailing symptoms in chronically exposed animals and humans. We investigated whether propineb interfered with peripheral cholinergic transmission in various isolated model systems. In electrically stimulated longitudinal musclemyenteric plexus preparations (LMMPs), propineb (0.01-1000 nM) concentration-dependently enhanced the amplitude of both neurogenic twitch contractions and tritiated acetylcholine ([3H]ACh) release. The maximum percent increase was achieved by 10 nM propineb and was 19% and 14%, respectively. The effect on twitch contractions was partially antagonized by hexamethonium, a ganglionic nicotinic receptor blocker. In unstimulated LMMPs, propineb (10 pM, 10 nM, 10 µM) did not affect contractions to applied acetylcholine (ACh; 1 nM-10 µM), a finding indicating that propineb has no anticholinesterase activity. In human neuroblastoma cells (SH-SY5Y), propineb facilitated ACh release evoked by KCl depolarization. The increase in ACh release was not associated with detectable alterations of intracellular Ca2+ ([Ca2+]i) homeostasis. Binding studies carried out with R-bungarotoxin in striated muscle cells (L6) failed to demonstrate any influence of propineb on both affinity and capacity of skeletal muscle nicotinic receptors. In conclusion, propineb was found to interfere with cholinergic transmission in LMMPs and SH-SY5Y cells. In LMMPs, the potentiation of cholinergic transmission is partly dependent on the activation of ganglionic nicotinic receptors. Other targets relevant to cholinergic transmission seem not to be affected by propineb.

Introduction Dithiocarbamates (DTCs) are synthetic organic chemicals used in agriculture as pesticides with fungicide, insecticide and herbicide properties (1) and, in the clinical setting, as alcohol deterrent drugs (e.g., disulfiram). Occupational exposure or acute intoxication to various DTCs have been associated with several adverse effects. Neurological complications, as well as movement disorders characterized by plastic rigidity with cogwheeling and muscle twitch are the prevailing symptoms in exposed individuals (1-3). In long-term experiments, exposed animals showed muscle weakness, ataxia, paralysis and histological changes in neuronal and nonneuronal tissues (calcification in the brain stem and cerebellum and dystrophic changes in leg muscle) (1). Several in vivo and in vitro studies have been carried out to identify the mode of action of DTCs, including their mechanisms of toxicity. From these studies emerged an impairment of central neurotransmitter pathways as a relevant event of DTC toxicity. Indeed, DTCs have been * To whom correspondence should be addressed. Phone: +39-0258358316. Fax: +39-02-58358260. E-mail: [email protected]. † Section of Theoretical Pharmacology and Receptor Modeling. ‡ Department of Internal Medicine and Therapeutics. § Department of Experimental and Applied Pharmacology.

reported to deplete noradrenaline stores (4) and to increase acetylcholine (ACh) levels in the rat brain (5), to induce dopamine, DOPAC and glutamate 6, 7) from striatum, and to inhibit dopamine (6) and glutamate uptake and vesicular transport (7-9). It has been hypothesized that DTC-induced alterations of central adrenergic and cholinergic systems may underlie memory deficits (5, 10), while disruption of dopamine balance has been regarded as the primary cause of movement impairment. This is partly due to the fact that the symptomathology associated with DTC intoxication resembles a permanent extrapyramidal syndrome similar to parkinsonism (6). Although the central cholinergic system should be considered as a target of DTC toxicity (11), no attention has been paid so far on the effects of DTCs on peripheral cholinergic transmission, despite the evidence that these compounds may adversely affect the function of peripheral excitable tissues (1). Propineb [polymeric zinc propylenebis(dithiocarbamate)] is widely used in Europe, mainly in the Mediterranean area, as fungicide and due to its large spectrum of action on fungi an expansion of the market is expected. This study was designed to investigate whether propineb interferes with the peripheral cholinergic system. For this purpose, several aspects of the cholinergic function were

10.1021/tx015538c CCC: $22.00 © 2002 American Chemical Society Published on Web 12/22/2001

Facilitation of Acetylcholine Signaling

evaluated by means of distinct experimental approaches. Strips of longitudinal muscle + myenteric plexus preparations (LMMPs) from the guinea pig ileum allowed us to assess the influence of propineb on electrically induced [3H]ACh release and on acetylcholinesterase activity. Propineb was also assessed on K+-evoked ACh release in human cholinergic neuroblastoma cells (SH-SY5Y), whereas the potential interaction with nicotinic receptors was determined by radioreceptor binding assay with R-bungarotoxin in a rat skeletal muscle cell line (L6).

Experimental Procedures Animals. Male Dunkin-Hartley guinea-pigs (250-400 g) were used. Animals were housed in standard animal facilities, providing constant temperature (21 ( 1 °C), relative humidity (50-55%), and alternating 12 h light and dark cycles. They were provided with standard laboratory chow and tap water available ad libitum. The animals were killed by CO2 inhalation and bled. All animal care procedures were in accordance with the local Animal Care Committee, and no weight loss or decease was observed after receipt of rats in our animal facility. All efforts were made to minimize the suffering of animals. Longitudinal Muscle-Myenteric Plexus Preparations (LMMPs). Segments of guinea-pig distal ileum were taken 10 cm proximal to the ileo-caecal junction, cleared of their intraluminal contents and trasferred to a Petri dish containing oxygenated (95% O2 + 5% CO2) Tyrode solution. A 10 cm long segment of ileum was stretched over a glass rod. The longitudinal muscle layer with attached myenteric plexus was separated from the underlying circular muscle by stroking tangentially away from the mesenteric attachment with a cotton wad. LMMPs of about 30 mm in length were mounted in a 5 mL organ bath containing Tyrode solution at 37 °C and bubbled with a mixture of O2 (95%) and CO2 (5%). The strips were mounted isometrically under a tension of 5 mN (12). Isometric contractions were recorded by means of a force-displacement trasducer. All experiments were started after at least 45 min equilibration with changing of the bathing solution every 15 min. Neurogenic Contractions in Electrically Stimulated LMMPs. Electrical field stimulation (EFS) was delivered with a stimulator by means of two platinum electrodes placed at the top and the bottom of the chamber. Maximal contractions were evoked by rectangular pulses with the following parameters: 0.1 Hz, 40-60 V, 0.5 ms pulse duration. Under these conditions, electrically evoked contractions are nerve-mediated and cholinergic in nature (13). After at least 10 min of reproducible twich contractions, noncumulative concentration-response curves to propineb (range 0.1 pM-100 nM) were constructed. In a separate set of experiments, the effect of propineb (10 nM) was investigated in the presence of the nicotinic receptor blocker hexamethonium (10, 30 µM). Any drug-induced change in the amplitude of contractions was expressed as percentage of the predrug (control) twich amplitude. ACh-Induced Contractions in Unstimulated LMMPs. In resting preparations, cumulative concentration-response curves to the contractile effects of ACh (1 nM to 10 µM) were constructed in the absence and in the presence of propineb (10 pM, 10 nM, and 10 µM). Propineb was allowed to equilibrate for at least 30 min prior to the ACh administration. Submaximal contractions to ACh in the absence and in the presence of propineb were expressed as a percentage of maximal response induced by 10 µM ACh in control conditions. [3H]ACh Release in Electrically Stimulated LMMPs. For tritiated acetylcholine ([3H]ACh) release studies, neuronal ACh stores were labeled according to the procedure previously described (14). Briefly, LMMPs were incubated with [3H]choline (2.5 µCi mL-1; 32 nM) for 30 min, during which time they were continuously stimulated at 0.1 Hz (0.5 ms pulse duration, 40 V/cm). At the end of the labeling period, the preparations were superfused with Tyrode’s solution containing 10 µM hemicho-

Chem. Res. Toxicol., Vol. 15, No. 1, 2002 27 linium to prevent uptake of choline, at a constant rate of 2 mL min-1. After a 120 min washout (time zero), superfusion fluid was collected continuously for 3 min periods (6 mL samples). LMMPs were stimulated two times for 3 min, beginning at 9 (S1) and 54 (S2) min after time zero. The release was evoked with 18 pulses at a frequency of 0.1 Hz, 0.5 ms pulse duration, 40 V cm-1. Aliquots (1 mL) of the superfusate were added to 5 mL of Ultima Gold, and the tritium content was measured by liquid scintillation spectrometry. Quench correction curves were established and external standardization was used for counting efficiency. Both resting and stimulation-evoked outflow of radioactivity were expressed in disintegration per second Bequerels per gram (Bq g-1) of tissue. The increase in the release caused by stimulation was obtained by subtracting the calculated spontaneous outflow from the total tritium outflow during 3 min stimulation plus the following 12 min (stimulation outflow period). The decline of the spontaneous outflow was calculated by fitting a linear regression line to the values (expressed in Bq) of three 3-min samples before and after the stimulation outflow period. The effects of 10 nM propineb exposed 10 or 30 min before S2 were evaluated by using the ratio S2/S1 as a percentage of the equivalent ratio obtained in control experiments. Cell Cultures. Human neuroblastoma cell line (SH-SY5Y) and rat skeletal muscle myoblasts (L6) were used. SH-SY5Y cells were cultured in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), streptomycin (0.1 mg mL-1) and penicillin (100 UI mL-1). In all experiments, the cells were seeded at a density of 12 500 cells/mL in 12 wells cell culture dishes. L6 cells were cultured in Dulbecco’s modified MEM (DMEM) supplemented with 10% inactivated horse serum (HS), 2% chicken embryo extract (CEE), streptomycin (0.1 mg mL-1), and penicillin (100 UI mL-1). To obtain myoblast fusion and differentiation into striated muscular fibers, cells were seeded at a density of 5 × 103 cells mL-1 in 12 well cell culture dishes in DMEM supplemented with 5% HS and 1% CEE. After 24 h, plating medium was replaced, and L6 cells were cultured for 2 weeks in DMEM + 1% HS + insulin 0.3 mM. ACh Release in SH-SY5Y Cells. ACh release from SHSY5Y cells was detected using the chemiluminescent method described by Israel and Lesbats (15). Briefly, SH-SY5Y cells were incubated for 5 min at room temperature with propineb (10 nM) and then stimulated for 1 min with KCl 50 mM. An aliquot of the buffer was collected at the end of each treatment. ACh present in the sample was hydrolyzed by 1.25 units of acetylcholinesterase and the choline produced was transformed by 2.5 units of choline oxidase. H2O2 produced reacted with luminol and horseradish peroxidase, and the light emitted was recorded by a luminometer. ACh concentration in the sample was calculated from a standard curve based on known amount of ACh. Results were expressed as picolmoles per milligram of protein. The protein content of cell monolayers was measured according to the method of Lowry (16) by direct solubilization in NaOH 1 N. Determination of Intracellular Free Ca2+ Concentration. SH-SY5Y cells were loaded in MEM supplemented with 2% fetal calf serum (FBS) and 5 mM Fura-2/AM for 30 min at room temperature and then resuspended in HEPES buffer. The Fura 2 fluorescence ratio signal was measured in a doublewavenlength fluorometer and calibrated in terms of [Ca2+]i as described by Grynkiewicz et al. (17). Ligand Binding Assays. Binding studies were performed at equilibrium on confluent adherent L6 at 25 °C for 16 h in 24-well dishes pretreated with 0.2% bovine serum albumine (BSA). A mixed type protocol (18) was performed with increasing concentration of [3H]propionylated-R-bungarotoxin (0.03-3 nM, 53 Ci mmol-1) (19) and 0.01-1 µM unlabeled homologous ligand in 250 mL of MEM containing 1% HS, 0.3 mM insulin, 0.2% BSA, and protease inhibitor cocktail. Cells were exposed to 10 nM propineb during a 5 min preincubation before binding with [3H]-R-bungarotoxin, for the entire 16 h incubation period with

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Figure 1. Typical tracing representative of the effect of propineb (1-100 nM) on contractions induced by electrical stimulation (0.1 Hz every 10 s, 0.5 ms pulse duration 60 V) in guinea pig longitudinal muscle + myenteric plexus strips. [3H]-R-bungarotoxin or during the last 5 min of the incubation period. Control and propineb-treated cells were washed once with ice-cold PBS containing 0.2% BSA and lysed in 0.25 N NaOH. Radioactivity was then measured by liquid scintillation and protein content was determined by Bradford dye-binding procedure. Statistical analysis of ligand-binding data was performed within the LIGAND program (20) using the statistical principle of the “extra sum of squares” to compare different models. Parameter errors are always expressed in % coefficient of variation (% CV). Nonspecific binding, calculated as one of the unknown parameters of the model, was 15-30% of the total binding. Binding is expressed as the ratio of bound ligand concentration over total ligand concentration, (B/T, dimensionless), vs the logarithm of total ligand concentration (log T). B (in M) is the sum of “hot”, “cold”, and nonspecific binding bound; T (in M) is the sum of “hot” and “cold” ligand incubated. All the curves shown are computer generated. Each experiment was performed at least three times in duplicates. Statistical Analysis. Data are expressed as mean ( standard error of the mean (SEM). Differences between means were analyzed by Student’s t-test for paired and unpaired data and with one-way analysis of variance (ANOVA) when applicable. Values of P < 0.05 were taken as statistically significant. Solutions and Chemicals. The Tyrode solution (pH 7.3) had the following composition (mM): NaCl 136.9, KCl 2.7, CaCl2 1.8, MgCl2 1.04, NaHCO3 11.9, NaH2PO4 0.4, glucose 5.5. The composition of HEPES buffer was (mM) NaCl 150, KCl 5, MgSO4 1.2, CaCl2 1.8, HEPES 25, d-Glucose 10, Na2HPO4 1.2. Rat skeletal muscle myoblasts (L6) and human neuroblastoma cells (SH-SY5Y) were from ATCC (Rockford, MD. [3H]Choline and [3H]propionylated-R-bungarotoxin were from Amersham (Life Science, Little Chalfont, Buckinghamshire, U.K.). Propineb (89% pure) was kindly provided by Bayer (Leverkusen, Germany).

Results Neurogenic Contractions in Electrically Stimulated LMMPs. Repetitive electrical stimulation of LMMPs evoked twitch-like contractions of reproducible amplitude. These responses are neurogenic and cholinergic in nature (14). Administration of propineb (1-100 nM) caused a concentration-dependent increase in the amplitude of twitch contractions (Figure 1). The maximum percent increase (19 ( 3.8%, n ) 7) was achieved within 3 min exposure by 10 nM propineb (Figure 2). The potentiating effect of 10 nM propineb was partially (p < 0.05) antagonized by 10, 30 µM hexamethonium (Figure 3). ACh-Induced Contractions in Unstimulated LMMPs. In unstimulated LMMPs, propineb (10 pM, 10 nM, and 10 µM) did not affect the amplitude of contractions caused by exogenous ACh (1 nM-10 µM) (Figure 4).

Figure 2. A quantitation of the effects of propineb (10 pM-1 µM) on electrically induced twitch contractions. Values are means ( SEM of four to eight experiments. With the exception of the effect caused by 1 µM propineb, all the other drug-induced potentiations are significant for p < 0.03 or better.

Figure 3. Potentiating effect of 10 nM propineb on electrically induced twitch contractions in guinea pig longitudinal muscle myenteric plexus preparations in the absence and in the presence of 10 or 30 µM (p < 0.05 compared to control) hexamethonium. Values are means ( SEM, n ) 4; (*) p < 0.05 vs group treated only with propineb.

[3H]ACh Release in Electrically Stimulated LMMPs. EFSs at 0.1 Hz (18 pulses) applied 45 min apart (S1 and S2) caused an increase in [3H]ACh release (Figure 5). Propineb (10 nM) applied 10 min before S2 potentiated [3H]ACh release by 13.8% (n ) 3) as observed by the enhancement of the ratio S2/S1 (0.92 ( 0.02 vs 0.81 ( 0.01 obtained in control conditions, Figure 5). Conversely, propineb (10 nM) applied 30 min before S2 did not affect [3H]ACh release (S2/S1 ratio: 0.77 ( 0.01 vs 0.79 ( 0.02, n ) 4). The 3H-resting outflow (SB) was not affected by propineb (Figure 5). ACh Release in SH-SY5Y Cells. In a human cell line of neuroblastoma (SH-SY5Y), incubation for 5 min with propineb (10 nM) did not affect basal ACh release (2.5 pmol mg-1 protein). The administration of KCl (50 mM) increased ACh release up to 7.5 pmol mg-1 protein.

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Figure 6. ACh-release stimulated by 50 mM KCl in SH-5YSY cells. White column, control vehicle, gray column, 10 nM propineb. Values are means ( SEM of three independent samples. (*) p < 0.05 vs the control (unstimulated cells); (**) p < 0.01 vs control cells stimulated with KCl. Table 1. [Ca2+]i (nM) in Resting and Stimulated SY5Y Cells resting cells

Figure 4. Concentration-response curves for ACh in inducing contractile responses in guinea pig longitudinal muscle myenteric plexus preparations in the absence (O) or in the presence of various propineb [(]) 10 pM; (b) 10 nM; (2) 10 µM] concentrations. Values are means ( SEM, n ) 4.

control, vehicle propineb 10 nM

0 min

5 min

KCl (50 mM)

92.5 ( 93.4 ( 1.5

95.2 ( 7.8 96.3 ( 7.7

205 ( 25.8 219 ( 26.4

8.9a

a Values are the mean ( SE of four different detections. SHSY5Y cells loaded with Fura-2 were exposed to DMSO or 10 nM Propineb for 5 min and then stimulated with 50 mM KCl. Recording of Ca2+ traces stopped 3 min after KCl addition when the signal reached a steady level.

Figure 5. Potentiating effects of 10 nM propineb on [3H]ACh release evoked by EFS in guinea pig longitudinal muscle myenteric plexus preparations. SB is the 3H resting outflow, whereas S1 and S2 (delivered 45 min apart) represent [3H]ACh release induced at 0.1 Hz for 3 min (18 pulses), 0.5 ms, 40 V/cm. S2/S1 ratio in control condition is 0.81. In the presence of 10 nM propineb (applied 3 min before S2), the S2/S1 ratio is 0.92 with a significant increase (11.4%; p < 0.05) compared to control. Values are means ( SEM, n ) 4.

Propineb enhanced by approximately 2-fold ACh release elicited by KCl (Figure 6). In Fura-2 loaded SH-SY5Y cells, the same concentration of KCl significantly enhanced (p < 0.01) intracellular calcium concentration [Ca2+]i (Table 1). Propineb (10 nM) did not affect basal [Ca2+]i content, or the enhancement of [Ca2+]i induced by KCl (Table 1). Ligand Binding Assays. To investigate a potential interaction of propineb with nicotinic receptors, the binding of [3H]-R-bungarotoxin to R1 nicotinic/skeletal muscle-type ACh receptors was assessed on insulindifferentiated L6 skeletal muscle cells in the absence and presence of 10 nM propineb (Figure 7). Both control and propineb-treated cells showed monophasic binding curves resolved by LIGAND program in a single site model with the binding parameters reported in Figure 5. [3H]-RBungarotoxin bound in a specific and saturable manner

Figure 7. Binding of [3H]-R-bungarotoxin to nicotinic/muscletype ACh receptors assessed on insulin-differentiated L6 skeletal muscular cells in the absence and presence of 10 nM propineb at the indicated times. (b) 3H-R-bungarotoxin control curve; (O) 5 min prior incubation with propineb; (9) 16 h coincubation with propineb; (0) last 5 min co-incubation with propineb. (Inset) Binding parameters for [3H]-R-bungarotoxin in L6 cells in the absence and presence of 10 nM propineb; Kds are expressed as nanomolar. Bmaxs are expressed as femtomole per milligram of protein. Parameters are expressed means ( % Coefficient of Variation (C. V.).

with an affinity value (Kd 0.19 nM ( 35% C. V.) in agreement with affinities previously obtained in other tissues or cells (19), and a maximum number of binding sites (Bmax) equal to 5.13 fmol mg-1 protein (29% C. V. Computer analysis of binding data obtained from propineb-

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treated cells demonstrated that the affinities for the agonist [3H]-R-bungarotoxin and the binding capacities of the nicotinic receptor were not statistically different from those of the control cells.

Discussion In animals, exposure to DTC is followed by movement impairment, muscle weakness and hind legs paralysis, whereas in humans the development of a parkinsonianlike extrapyramidal syndrome characterized by rigidity and, less frequently, tremor and bradykinesia has been described (21-25). The concentrations causing neurotoxic and behavioral effects in animals ranged from about 30 to 3000 mg kg-1 b.w (1, 11), depending on the type of DTC and on the duration of the exposure; a change in catecholamine metabolism and disorders of central nervous system were observed in workers exposed for at least 1 year to concentrations of zineb never exceeding 1 mg/m3 (1). On the basis of the symptoms observed, attention has been mainly focused on DTC-induced alterations of central dopaminergic system (6, 11). Nevertheless, many of the symptoms related to dithiocarbamate intoxication in vivo could involve an alteration of cholinergic transmission, which may generate an unbalance between cholinergic and dopaminergic systems. The results reported here using different biological models concur in proving this assumption. In longitudinal muscle + myenteric plexus preparations from the guinea pig ileum, short term (3-10 min) exposure to subnanomolar/nanomolar concentrations of propineb amplified electrically induced neurogenic cholinergic contractions and tritiated ACh release. The potentiating effect of propineb on neurogenic contractions was counteracted by hexamethonium indicating a partial involvement of nicotinic ganglionic transmission. This effect may result from a direct interaction of propineb with ganglionic nicotinic receptors or indirectly via an action of released ACh on nicotinic receptors present on nerve terminals of excitatory motoneurons, which exert a positive feedback mechanism on ACh release. The latter mechanism has been elegantly demonstrated in the myenteric plexus of guinea pig intestine recently (26, 27). The use of resting LMMPs allowed the exclusion of the influence of propineb on smooth muscle contractility due to activation of muscarinic receptors by exogenous ACh or on tissue cholinesterases. In fact, propineb failed to modify ACh-induced contractions indicating that the drug does not interfere with muscarinic transmission and is devoid of anticholinesterase properties. On the other hand, the lack of anticholinesterase activity was confirmed in [3H]ACh release experiments with prolonged (30 min) exposure to the drug. Under these conditions, propineb failed to modify ACh release at variance with the effect produced by compounds possessing anticholinesterase properties (e.g., eserine) in the same preparation (14). In our hands, propineb was also found to potentiate ACh release in response to a depolarizing stimulus (i.e., KCl administration) in SH-SY5Y neuroblastoma cells. Neurosecretory responses have generally a strict requirement for Ca2+ and drug-induced increase in the intracellular free Ca2+ concentration [Ca2+]i, including that evoked by KCl, triggers neurotransmitter release (2831). In the effort to identify the molecular mechanisms

Marinovich et al.

involved in propineb-stimulated increase of Ach release, we monitored [Ca2+]i levels in a neuronal cell line (SHSY5Y cells) exposed to propineb and the possible interference with the signal elicited by a depolarizing stimuli. KCl induced a reversible peak of [Ca2+]i that was only slightly affected by propineb, suggesting that the facilitated ACh release could be independent of an amplification of Ca2+ response. Nevertheless, it is not possible to exclude that the increase of Ca2+ caused by KCl could have eventually masked an additive effect of propineb on the intracellular ion concentration. Apart from the partial involvement of ganglionic nicotinic receptors in propineb-induced facilitation of Ach release, as observed in electrically stimulated LMMPs, other mechanisms related to neuronal membrane permeability may underlie the effect of the drug on both LMMPs and SH-SY5Y cells. For example, a blockade of voltage-gated potassium channels might also lead to a potentiation of ACh release in both model systems. Voltage-gated potassium channels are known to regulate the amount of neurotransmitter released from presynaptic nerve terminals by modulating the duration of the presynaptic action potential and their blockade by selective toxins has been shown to increase ACh release at the neuromuscolar junction (32). CS2 or zinc ions, which are known to possess a neurotoxic potential (33, 34), are usually considered as potential mediators of propineb toxicity. However, due to the lack of both morphological changes and intermolecolar cross-linking characteristic of CS2 (35) and the difference of toxic concentrations, we can assume that propineb effect is not mediated by CS2. By contrast, the effects of zinc ions on neuronal function deserve a special consideration. Zn2+ is present in several regions of the brain, where it is stored in synaptic vesicles of nerve terminals and released upon stimulation (36, 37). The effect of Zn2+ on spontaneous or stimulus evoked excitatory postsynaptic potentials (EPSPs) and frequency of micro end-plate potentials (MEPP), however, was detectable above 10 µM concentration (38-40) and it is thus conceivable that the effects observed at subnanomolar/nanomolar concentrations under our experimental conditions are due to propineb itself and not to a leakage of zinc. This consideration is further supported by the fact that disulfiram, a structure-related compound not containing zinc, causes the same in vivo symptoms of propineb. Alternatively, other mechanisms could be involved in propineb-induced facilitation of ACh release. DTCs are known to easily interact with proteins due to their affinity for -SH groups (41). Most synaptic vesicles in the presynaptic terminal are associated with cytoskeletal elements near the active zone. During the early phase of exocytosis, there is a rapid formation of a fusion pore, a narrow cytoplasmic bridge that unites the vesicle membrane with the plasma membrane at the active zone. It is possible to assume that a propineb-induced modification of the proteins involved in neurosecretion could facilitate ACh release. Propineb could also induce release of Ach as a result of an interaction with the carrier for this neurotransmitter on the synaptic vesicles or by increasing the permeability of the vesicular membrane through an aspecific mechanism. Both these possibilities are suggested by disulfiram, that disrupts dopamine balance with similar mechanisms (7).

Facilitation of Acetylcholine Signaling

Nevertheless, whatever is the underlying mechanism, the propineb-induced enhancement of ACh release may contribute to alter motor function in animals and humans chronically exposed to low doses of propineb. In fact, a low/moderate increase in ACh release (by approximately 15%, such as that observed in LMMPs experiments) might not be relevant in acute conditions. However, the persistence of enhanced levels of ACh resulting from propineb chronic exposure may produce important changes in the physiology of central cholinergic neurons (contributing to the unbalance between cholinergic and dopaminergic transmission) leading to tremors and muscle twitches. By contrast, a marked increase in ACh release (such as that caused by propineb in SH-5YSY cells) may produce paresis due to nicotinic receptor desensitization or blockade of cholinergic endplate transmission caused by an excess of nerve terminal depolarization. Finally, we checked the possibility that propineb could interact with muscular-type nicotinic receptors. Binding studies carried out on striated muscle fibers of L6 differentiated cells using the specific nicotinic receptor ligand R-bungarotoxin failed to demonstrate any influence of propineb on both affinity (Kd) and capacity (Bmax) of nicotinic receptors. As it can be easily inferred from the binding parameters presented in Figure 7 all the Kds and Bmaxs are within a 2-fold range, a value below the limit of resolution of this technique (42). This implies that propineb may recognize, at least in part, only the neuronal-type nicotinic receptor and that the neurological complications such as muscle twitches and tremors may involve central nicotinic receptors rather then peripheral (muscular-type) nicotinic receptors. Propineb is currently used as a fungicide in a large number of crops (apples, grapes, tomatoes, onions, etc.), and international regulatory agencies estimated for humans a theoretical maximum daily intake of 0.35 mg/ person/day (0.00585 mg/kg body weight/day) through the diet (3). Even if we observed an effect at very low doses of propineb, it is unlikely that propineb content in the diet might represent a real risk for human beings; this consideration may be, however, different when other scenarios of exposure are considered (for example, plant or agricolture workers). In conclusion, in LMMPs and SH-SY5Y cells, propineb was found to facilitate ACh release evoked by electrical stimulation or KCl depolarization, through a mechanism partially involving neuronal-type nicotinic receptors. At the concentrations tested (nanomolar range), no other targets relevant to cholinergic transmission (i.e., muscarinic receptors, muscular-type nicotinic receptors, anticholinesterase activity) seem to be affected by propineb. We can thus hypothesize that some of the effects observed following propineb exposure (i.e., tremors and muscle twitches) may result from low/moderate activation of central and peripheral parasympathetic pathways, whereas muscle weakness, atrophy, and paresis occurring after long-term exposure may be due, at least in part, to desensitization of neuronal-type nicotinic receptors or, alternatively, to pronounced nerve terminal depolarization caused by an excess of ACh release, as observed in SH-SY5Y cells.

References (1) WHO (1988) Dithiocarbamate pesticides, ethylenthiourea and propylenthyourea: a general introduction. Environmental Health Criteria, Vol. 78, World Health Organization.

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