Therapeutic effects of some cholinolytics in organophosphate intoxications

The therapeutic effects of pipethanate (sycotrol) and two newly synthetized cholinolytics, DPX-8 and ANC-51, were compared with atropine in mice poisoned by DDVP, fluostigmine, phospholine, and paraoxon. The antagonistic activity of tested drugs against acetylcholine-induced contraction of rat ileum and oxotremorine-induced salivation and tremor in the mouse was also studied. The anticholinergic activity of pipethanate, DPX-8, and ANC-51 was weaker than that of atropine. However, the therapeutic effect of pipethanate was higher than that of atropine in mice poisoned by the organophosphates. DPX-8 and ANC-51 afforded a better antidotal effect than atropine only in DDVP-poisoned mice.     

Determination of Organophosphorus Acid Anhydrase in Blood

In an attempt to develop a new conceptual approach to paraoxon antagonism by encapsulating organophosphorus acid anhydrase (OPA anhydrase) into carrier erythrocytes, it necessitated the spectral determination of OPA anhydrase activity in blood. OPA anhydrase determination usually is conducted in serum or plasma. This presents a problem, because blood contains various substances that interfere with the determination of OPA anhydrase. This enzyme used paraoxon as the substrate, liberating p-nitrophenol and diethylphosphate. OPA anhydrase has a broad substrate specificity and is capable of hydrolyzing other organophosphorus compounds, i.e., pesticides, nerve gases, etc. Blood OPA anhydrase activity was determined with paraoxon as the substrate. This reaction was terminated by adding hydrochloric acid to the reaction mixture. The p-nitrophenol then was extracted with dichloromethane, and p-nitrophenol was determined at 400 nm. The sensitivity of this method can be greatly enhanced by increasing the sample size or by using a longer reaction time.     

Immunomodulation by poly-YE reduces organophosphate-induced brain damage

Accidental organophosphate poisoning resulting from environmental or occupational exposure, as well as the deliberate use of nerve agents on the battlefield or by terrorists, remain major threats for multi-casualty events, with no effective therapies yet available. Even transient exposure to organophosphorous compounds may lead to brain damage associated with microglial activation and to long-lasting neurological and psychological deficits. Regulation of the microglial response by adaptive immunity was previously shown to reduce the consequences of acute insult to the central nervous system (CNS). Here, we tested whether an immunization-based treatment that affects the properties of T regulatory cells (Tregs) can reduce brain damage following organophosphate intoxication, as a supplement to the standard antidotal protocol. Rats were intoxicated by acute exposure to the nerve agent soman, or the organophosphate pesticide, paraoxon, and after 24 h were treated with the immunomodulator, poly-YE. A single injection of poly-YE resulted in a significant increase in neuronal survival and tissue preservation. The beneficial effect of poly-YE treatment was associated with specific recruitment of CD4⁺ T cells into the brain, reduced microglial activation, and an increase in the levels of brain derived neurotrophic factor (BDNF) in the piriform cortex. These results suggest therapeutic intervention with poly-YE as an immunomodulatory supplementary approach against consequences of organophosphate-induced brain damage.     

Evaluation of nicotinic receptors agonists and antagonists against paraoxon exposed PC12 cells

Chronic and acute exposure to organophosphate pesticides may lead to persistent neurological and neurobehavioral effects, which cannot be explained by acetylcholinesterase (AChE) inhibition alone. In an attempt to elucidate the mechanism by which paraoxon affects the nicotinic receptors gene expression, the effects of exposure of PC12 cells to 100 μM concentrations of paraoxon for 48 h in the presence and the absence of nicotinic acetylcholine receptors (nAChRs) agonists and antagonists were characterized. Paraoxon at 100 μM significantly inhibited AChE activity. On the mRNA level, the ₄ and β₂ subunits of nAChR mRNA were significantly decreased in the cells exposed to paraoxon. On the protein level, ₄ and β₂ subunits of nAChR protein were also significantly reduced. Mecamylamine (10 μM), dihydro-β-erythroidine (DHβE) (5 μM) and nicotine (10 μM) efficiently prevented the decrease of ₄ and β₂ nAChR mRNA and protein in PC12 cells, but carbamaylcholine a weak agonist of nAChR was not efficient. These observations suggest that ₄β₂ nAChRs are involved in paraoxon related toxicity and nicotinic receptors antagonists could play some protective role against organophosphate related damages.     

Ventilatory effects of low-dose paraoxon result from central muscarinic effects

Paraoxon induces respiratory toxicity. Atropine completely reversed parathion- and paraoxon-induced respiratory toxicity. The aim of this study was to assess the peripheral or central origin of ventilatory effects of low-dose paraoxon. Male Sprague-Dawley rats were given paraoxon 0.215 mg/kg subcutaneously and treated with either atropine (10 mg/kg sc) or ascending doses of methylatropine of 5.42 (equimolar to that of atropine), 54.2, and 542 mg/kg administered subcutaneously 30 min after paraoxon. Ventilation at rest was assessed using whole-body plethysmography and rat temperature using infra-red telemetry. Results are expressed as mean ± SE. Statistical analysis used two-way ANOVA for repeated measurements. Paraoxon induced a significant decrease in temperature 30 min after injection lasting the 90 min of the study period. This effect was partially corrected by atropine, but not by methylatropine whatever the dose. Paraoxon induced a decrease in respiratory rate resulting from an increase in expiratory time associated with an increase in tidal volume. Atropine completely reversed the ventilatory effects of low-dose paraoxon while the equimolar dose of methylatropine had no significant effects. The 54.2 and 542 mg/kg doses of methylatropine had no significant effects. Atropine crosses the blood-brain barrier and reverses peripheral and central muscarinic effects. In contrast, methylatropine does not cross the blood-brain barrier. Atropine completely reversed the ventilatory effects of low-dose paraoxon, while methylatropine had no significant effects at doses up to 100-fold the equimolar dose of atropine. We conclude that the ventilatory effects of low-dose paraoxon are mediated by disrupted muscarinic signaling in the central nervous system.     

The central effects of paraoxon on blood pressure of rabbits after intravenous administration or infusion via a vertebral artery

In the present study paraoxon (28–825 g/kg, i.v.) was administered into rabbits treated with N-methylatropine (1 mg/kg). In urethane-anaesthetized rabbits pressor and depressor effects were observed. In pentobarbitone-anaesthetized rabbits, however, only depressor effects were noticed. Dexetimide (0.25–0.5 mg/kg) prevented the pressor and depressor action of paraoxon. Furthermore, paraoxon (83–825 g) was infused into a vertebral artery of pentobarbitone-anaesthetized animals. After administration via this route the depressor effect was similar to i.v. administration. 30 min after administration of paraoxon acetylcholinesterase activity was determined in the medulla oblongata, the pons and the hypothalamus. The enzyme has to be inhibited almost completely to induce an effect on blood pressure.It is concluded that the vascular effects of paraoxon seem to be mediated by a mechanism involving stimulation of central muscarinic receptors, probably by accumulated acetylcholine, which in turn induces a decrease in blood pressure.     

Paraoxon suppresses Ca(2+) spike and afterhyperpolarization in snail neurons: Relevance to the hyperexcitability induction

The effects of organophosphate (OP) paraoxon, active metabolite of parathion, were studied on the Ca(2+) and Ba(2+) spikes and on the excitability of the neuronal soma membranes of land snail (Caucasotachea atrolabiata). Paraoxon (0.3 muM) reversibly decreased the duration and amplitude of Ca(2+) and Ba(2+) spikes. It also reduced the duration and the amplitude of the afterhyperpolarization (AHP) that follows spikes, leading to a significant increase in the frequency of Ca(2+) spikes. Pretreatment with atropine and hexamethonium, selective blockers of muscarinic and nicotinic receptors, respectively, did not prevent the effects of paraoxon on Ca(2+) spikes. Intracellular injection of the calcium chelator BAPTA dramatically decreased the duration and amplitude of AHP and increased the duration and frequency of Ca(2+) spikes. In the presence of BAPTA, paraoxon decreased the duration of the Ca(2+) spikes without affecting their frequency. Apamin, a neurotoxin from bee venom, known to selectively block small conductance of calcium-activated potassium channels (SK), significantly decreased the duration and amplitude of the AHP, an effect that was associated with an increase in spike frequency. In the presence of apamin, bath application of paraoxon reduced the duration of Ca(2+) spike and AHP and increased the firing frequency of nerve cells. In summary, these data suggest that exposure to submicromolar concentration of paraoxon may directly affect membrane excitability. Suppression of Ca(2+) entry during the action potential would down regulate Ca(2+)-activated K(+) channels leading to a reduction of the AHP and an increase in cell firing.     

Forskolin potentiates the paraoxon-induced hyperexcitability in snail neurons by blocking afterhyperpolarization

One characteristic of organophosphate poisoning is the ability to increase excitability or induce epileptiform activity in nerve cells, but underlying mechanisms are not fully understood. We have previously reported that paraoxon, an organophosphate compound, at submicromolar concentrations effectively suppress Ca(2+) spikes and modulate the activity of snail neurons. This effect was unrelated to acetylcholinesterase (AChE) inhibition but was found to involve the direct or indirect modulation of ion channels [Vatanparast J, Janahmadi M, Asgari AR, Sepehri H, Haeri-Rohani A. Paraoxon suppresses Ca(2+) spike and afterhyperpolarization in snail neurons: relevance to the hyperexcitability induction. Brain Res 2006a;1083(1):110-7]. In the present study, the interaction of paraoxon with cAMP formation on the modulation of Ca(2+) spikes and neuronal excitability was examined. Forskolin, the activators of adenylate cyclase, suppressed afterhyperpolarization (AHP) and increased the activity of snail neurons without any significant effect on the Ca(2+) spike duration. Pretreatment with forskolin, although attenuated the suppressing effect of paraoxon on the duration of Ca(2+) spikes but also potentiated the paraoxon-induced hyperexcitability by enhancing the suppressive effects of paraoxon on AHP. Our findings support the possible involvement of cAMP formation in the paraoxon-induced AHP suppression and neuronal hyperexcitability, although activation of cAMP pathway may attenuates some effects of paraoxon.     

Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways

The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.     

Acute effects of the organophosphate paraoxon on schedule-controlled behavior and esterase activity in rats: Dose-response relationships

The effects of acute intraperitoneal administration of paraoxon on behavioral and biochemical parameters were studied in male rats. Rats were trained to press a lever under an FR10 schedule of reinforcement. Rats were injected with 3 sublethal doses of paraoxon (0.5, 0.75, and 1.0 mg/kg) and performance was monitored for four days after exposure. Response rates were depressed significantly for days 1 and 2 with 0.75 and 1.0 mg/kg, but not 0.5 mg/kg, even though there was inhibition of brain and plasma cholinesterases at all doses. Performance recovered prior to brain AChE recovery. There was no clear-cut threshold of brain AChE inhibition required to yield performance deficits, nor was there a direct correlation between significant inhibition in peripheral enzymes which could serve as markers (plasma aliesterases, butyrylcholinesterase, non-iso-OMPA-sensitive cholinesterase, and hepatic aliesterases) and performance deficits, suggesting that other noncholinergic targets may play a role in OP-induced behavioral deficits.