Balanced modulation of neuromuscular synaptic transmission via M1 and M2 muscarinic receptors during inhibition of cholinesterases.

Organophosphorus (OP) compounds that inhibit acetylcholinesterase are a common cause of poisoning worldwide, resulting in several hundred thousand deaths each year. The pathways activated during OP compound poisoning via overstimulation of muscarinic acetylcholine receptors (mAChRs) play a decisive role in toxidrome. The antidotal therapy includes atropine, which is a nonspecific blocker of all mAChR subtypes. Atropine is efficient for mitigating depression in respiratory control centers but does not benefit patients with OP-induced skeletal muscle weakness. By using an ex vivo model of OP-induced muscle weakness, we studied the effects of the M1/M4 mAChR antagonist pirenzepine and the M2/M4 mAChR antagonist methoctramine on the force of mouse diaphragm muscle contraction. It was shown that weakness caused by the application of paraoxon can be significantly prevented by methoctramine (1 µM). However, neither pirenzepine (0.1 µM) nor atropine (1 µM) was able to prevent muscle weakness. Moreover, the application of pirenzepine significantly reduced the positive effect of methoctramine. Thus, balanced modulation of neuromuscular synaptic transmission via M1 and M2 mAChRs contributes to paraoxon-induced muscle weakness. It was shown that methoctramine (10 µmol/kg, i.p.) and atropine (50 µmol/kg, i.p.) were equieffective toward increasing the survival of mice poisoned with a 2xLD50 dose of paraoxon.

Intramuscular atenolol and levetiracetam reduce mortality in a rat model of paraoxon-induced status epilepticus.

Organophosphorus (OP) compounds are chemical threat agents and are irreversible inhibitors of the enzyme acetylcholinesterase that lead to a hypercholinergic response that could include status epilepticus (SE). SE particularly targets the heart and brain and despite existing therapies, it is still associated with significant mortality and morbidity. Here, we investigated the effect of intramuscular (i.m.) adjunct therapy consisting of atenolol (AT) and levetiracetam (LV) when administered after paraoxon (POX)-induced SE. The combination therapy was administered twice daily for 2, 7, or 14 days. POX exposure in rats produced rapid SE onset that was treated with atropine, pralidoxime chloride, and midazolam. Here, AT + LV therapy produced significant reductions in POX SE mortality assessed at 30 days post-SE. AT + LV therapy exhibited muscle pathology inflammation scores that were not significantly different from saline-treated controls. Pharmacokinetic analyses revealed that the i.m. route achieved faster and stabler plasma therapeutic levels for both AT and LV under OP SE conditions compared with oral administrations. Our data provide evidence of the safety and efficacy of i.m. AT + LV therapy for reducing mortality following POX SE.

Mechanism Underlying Organophosphate Paraoxon-Induced Kinetic Tremor.

Organophosphates (OPs) inhibit cholinesterase and hyperactivate the acetylcholinergic nervous system in the brain, causing motor disorders (e.g., tremor and seizures). Here, we performed behavioral and immunohistochemical studies in mice and rats to investigate the tremorgenic mechanism of paraoxon, an active metabolite of parathion. Treating animals with paraoxon (0.15-0.6 mg/kg, i.p.) elicited kinetic tremor in a dose-dependent manner. Expressional analysis of Fos protein, a biomarker of neural excitation, revealed that a tremorgenic dose of paraoxon (0.6 mg/kg) significantly and region-specifically elevated Fos expression in the cerebral cortex (e.g., sensory cortex), hippocampal CA1, globus pallidus, medial habenula, and inferior olive (IO) among 48 brain regions examined. A moderate increase in Fos expression was also observed in the dorsolateral striatum while the change was not statistically significant. Paraoxon-induced tremor was inhibited by the nicotinic acetylcholine (nACh) receptor antagonist mecamylamine (MEC), but not affected by the muscarinic acetylcholine receptor antagonist trihexyphenidyl (THP). In addition, paraoxon-induced Fos expression in the IO was also antagonized by MEC, but not by THP, and lesioning of the IO markedly suppressed tremorgenic action of paraoxon. The present results suggest that OPs elicit kinetic tremor at least partly by activating IO neurons via nACh receptors.

MRS of brain metabolite levels demonstrates the ability of scavenging of excess brain glutamate to protect against nerve agent induced seizures.

This study describes the use of in vivo magnetic resonance spectrocopy (MRS) to monitor brain glutamate and lactate levels in a paraoxon (PO) intoxication model. Our results show that the administration of recombinant glutamate-oxaloacetate transaminase (rGOT) in combination with oxaloacetate (OxAc) significantly reduces the brain-accumulated levels of glutamate. Previously we have shown that the treatment causes a rapid decrease of blood glutamate levels and creates a gradient between the brain and blood glutamate levels which leads to the efflux of excess brain glutamate into the blood stream thereby reducing its potential to cause neurological damage. The fact that this treatment significantly decreased the brain glutamate and lactate levels following PO intoxication suggests that it could become a new effective neuroprotective agent.

Blood glutamate scavenging as a novel neuroprotective treatment for paraoxon intoxication.

Organophosphate-induced brain damage is an irreversible neuronal injury, likely because there is no pharmacological treatment to prevent or block secondary damage processes. The presence of free glutamate (Glu) in the brain has a substantial role in the propagation and maintenance of organophosphate-induced seizures, thus contributing to the secondary brain damage. This report describes for the first time the ability of blood glutamate scavengers (BGS) oxaloacetic acid in combination with glutamate oxaloacetate transaminase to reduce the neuronal damage in an animal model of paraoxon (PO) intoxication. Our method causes a rapid decrease of blood Glu levels and creates a gradient that leads to the efflux of the excess brain Glu into the blood, thus reducing neurotoxicity. We demonstrated that BGS treatment significantly prevented the peripheral benzodiazepine receptor (PBR) density elevation, after PO exposure. Furthermore, we showed that BGS was able to rescue neurons in the piriform cortex of the treated rats. In conclusion, these results suggest that treatment with BGS has a neuroprotective effect in the PO intoxication. This is the first time that this approach is used in PO intoxication and it may be of high clinical significance for the future treatment of the secondary neurologic damage post organophosphates exposure.

[Echinocytosis and changes of medium weight molecules content in endo- and exogenous intoxications].

The purpose of this study was to detect the interrelationship between the increased serum concentration of the medium weight molecules (MWM) and echinocytosis, to establish the rate of echinocyte appearance in blood in endogenous and exogenous intoxications and the dependence of echinocytosis on phosphacol and carbophos doses or the degree of endogenous intoxication. Two series of studies were conducted. In the 1st series, the experiments were conducted on outbred albino rats. Rats of the 1st group received phosphacol in doses equal to 0.5, 5.0 and 50 LD50, while the rats of the 2nd group were given carbophos in doses equal to 0.1 and 1.0 LD50. In the 2nd series, the blood of cats with chronic renal failure at uremic stage was studied (as an example of endogenous intoxication). In all the animals, serum MWM concentrations were measured together with the relative echinocyte content and echinocytes were examined with electrone microscope. The ability of MWM to influence erythrocyte deformation was evaluated. Increased MWM concentrations and echinocyte content was found in association with the increase of intoxication severity, while echinocyte percentage in cats' blood was augmented under the action of MWM.

Use of atropine-treated Daphnia magna survival for detection of environmental contamination by acetylcholinesterase inhibitors.

The toxicity of cholinesterase-inhibiting compounds (e.g., carbamates and organophosphates) is due to a decrease in acetylcholine metabolism, which results in a continuous stimulation of cholinergic receptors (muscarinic and nicotinic) that can be fatal. The goal of this study was to evaluate the protective effect of atropine (muscarinic receptor antagonist) against paraoxon-induced toxicity to Daphnia magna using its survival rate for the detection of environmental contamination by cholinesterase-inhibiting compounds. As expected, paraoxon was lethal to D. magna in a concentration-dependent manner. Noteworthy, the pretreatment of these organisms with atropine dramatically increased their survival against paraoxon. These results indicate that muscarinic stimulation plays an important role in paraoxon-induced lethality in D. magna. Therefore, simply by using the survival of atropine-treated and nontreated D. magna, water contamination by cholinesterase-inhibiting compounds may be rapidly and specifically detected.

Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides.

The in vitro rates of spontaneous reactivation and aging in human erythrocyte acetylcholinesterase were studied after inhibition by a dimethoxy (R1R2) and diethoxy substituted (R1R2) organophosphate pesticide (OP) of general structure R1R2P(O)X. These have been compared with data for human plasma cholinesterase previously reported using a similar methodology. A significantly slower rate of aging for erythrocyte acetylcholinesterase was found compared to plasma cholinesterase, whether inhibited by dimethoxy or diethoxy substituted OPs. For diethoxy OPs the rate of spontaneous reactivation of the inhibited plasma enzyme was significantly slower than for the inhibited red cell enzyme. This acetylcholinesterase, and previously published plasma cholinesterase, data suggest that in practise a blood sample taken 30-40 h after significant acute OP exposure will still show inhibition in either plasma or erythrocyte cholinesterase when analysed, but that any inhibited plasma enzyme is more likely to be in the aged form. In contrast a substantial proportion of the erythrocyte acetylcholinesterase is found unaged and therefore sensitive to reactivation by oximes. Samples from an occupational exposure where depressions in plasma or erythrocyte cholinesterase activity from baseline measurements were reactivated ex vivo using the oxime 2-PAM support this hypothesis. These data also confirm that the plasma enzyme is a more sensitive than erythrocyte acetylcholinesterase as an indicator of OP exposure and thus the potential value of ex vivo oxime reactivation of erythrocyte acetylcholinesterase in a blood sample to indicate subclinical OP exposure may be limited. However, this study is too small to draw conclusions on the sensitivity of ex vivo oxime reactivation of acetylcholinesterase as a novel biomarker of excessive OP absorption. Given that there is a better relationship between anticholinergic symptoms and red cell acetylcholinesterase inhibition, and that the slower resynthesis rate of any aged or inhibited red cell enzyme may be interpretatively useful when venepuncture is delayed, it is suggested that red cell acetylcholinesterase activity does have a place in monitoring potential OP exposure.

Differential expression of muscarinic subtype mRNAs after exposure to neurotoxic pesticides.

We have recently reported an increase in the density of muscarinic cholinergic receptors in mice neonatally exposed to a persistent environmental agent, dichlorodiphenyltrichloroethane (DDT), and a subsequent exposure as adults to nonpersistent toxicants, such as bioallethrin or paraoxon. Here we have examined the effects of an exposure like this on muscarinic receptor mRNA expression. Ten-day-old Naval Medical Research Institute mice received a single oral dose of DDT (0.5 mg/kg body weight). When aged 5 months, they received bioallethrin (0.7 mg/kg body weight per day for 7 days) or paraoxon (1.4 mg/kg body weight every second day for 7 days). mRNA expression of subtypes m1, m3, and m4 was studied in 7-month-old animals. Changes could only be discovered in the DDT-bioallethrin treated mice, where expression of subtype m4 was elevated in cortex and caudate putamen. Moreover, the expression pattern of the subtypes m1, m3, and m4 in mouse brains was found to be very similar to that seen in rats, except for slight differences in the pyramidal cell layer of the hippocampus, where the outermost part of the CA3 region did not show any m4 hybridization. The present study indicates that the earlier observed increase in muscarinic receptor density in mice exposed as neonates to DDT and as adults to bioallethrin can be attributed to changes in the expression of m4.

Regional blood flow during paraoxon infusion in rabbits.

In anaesthetized and artificially ventilated rabbits an intravenous infusion of paraoxon (0.8 mg/kg) was given over 30 min. The effects on cardiac output, blood flow to various vascular beds, and on the mass discharge of the postganglionic sympathetic efferents to the spleen and kidney were monitored. Immediately following paraoxon infusion atropine (0.5 mg/kg) was injected intravenously. Within 20 min of commencing the infusion signs of increased cholinergic stimulation were observed. Between the 20th and 25th min mean arterial blood pressure, heart rate, and cardiac output fell markedly. Even before arterial blood pressure fell total peripheral resistance and regional resistance to flow through the subclavian and coeliac arteries increased significantly, whereas resistance was below control, increased regional resistance being found only in the vascular beds of the subclavian and splenic arteries. The activity in the splenic sympathetic efferents increased, while the activity in the renal efferents was sharply reduced. While an effective antidote, atropine elicited transient intestinal vasodilation and a further transient decrease in total peripheral resistance. These and other results suggest that muscarinic mechanisms are mainly responsible for the paraoxon-induced changes in regional blood flow and regional sympathetic activity. The vasodilatory effect of atropine in the intestine was probably due to a local autoregulatory mechanism.