anticholinesterase pesticides (metabolism, neurotoxicity, and epidemiology) || central mechanisms of...

12CENTRAL MECHANISMS OF SEIZURES ANDLETHALITY FOLLOWING ANTICHOLINESTERASEPESTICIDE EXPOSURE

ANDRZEJ DEKUNDY

Department of Toxicology, Institute of Agricultural Medicine, Jaczewskiego 2, 20-950 Lublin, Poland, and In Vivo Pharmacology,RþD CNS, Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt am Main, Germany

RAFAL M. KAMINSKI

UCB Pharma S.A., CNS Research, Epilepsy Pharmacology, Chemin du Foriest, R9, B-1420 Braine-l’Alleud, Belgium

12.1 Introduction 14912.1.1 Use of Anticholinesterase Pesticides 14912.1.2 Risks Associated with Anticholinesterase

Pesticides 15012.1.3 Symptoms of Acute Intoxication with

Anticholinesterase Pesticides 15012.1.4 Current Therapy for Poisoning with

Anticholinesterase Pesticides 150

12.2 Neurotransmitter Systems Implicated in Seizures andLethality of Anticholinesterases 15112.2.1 Muscarinic Mechanisms 15112.2.2 Nicotinic Mechanisms 15212.2.3 N-Methyl-D-Aspartate (NMDA)-Related

Mechanisms 15212.2.4 AMPA/Kainate-Related

Mechanisms 15412.2.5 Adenosinergic Mechanisms 154

12.2.6 GABA-ergic Mechanisms 15412.2.7 Monoaminergic Mechanisms 155

12.3 Role of Neuroinflammation inAnticholinesterase-Induced Seizures 155

12.4 Mechanistic Considerations 15612.4.1 Proposed Mechanisms of Neurochemical

Events Following Acute AnticholinesteraseIntoxication 156

12.4.2 Putative Mechanisms of Interactionsbetween Neurotransmitter SystemsImplicated in the Acute Toxicity ofAnticholinesterases 156

12.4.3 Targeting Neuroinflammation: An EmergingApproach to the Treatment ofAnticholinesterase-Induced Seizures 157

12.5 Conclusions 158

References 158

12.1 INTRODUCTION

12.1.1 Use of Anticholinesterase Pesticides

Organophosphates (OPs) are esters of phosphoric acid, whichwere first synthesized in the nineteenth century. The historyof agricultural use of OPs goes back to the 1930s when a

series of stable yet very toxic organophosphorus esters wasfirst synthesized. Ever since, the number of insecticides ofthis group has increased rapidly (Satoh, 2006) and organo-phosphorus esters have been widely utilized for crop protec-tion (Gupta, 2006). OPs have also been applied in medicine(Gelinas et al., 2000; Hiatt, 1983; Holmstedt et al., 1978).Diisopropyl fluorophosphate (DFP), a drug formerly used

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

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in ophthalmology (Hiatt, 1983), has been widely used as aresearch tool for studying mechanisms of toxicity of OPs(Galli and Mori, 1996; Gupta and Dekundy, 2005, 2007;Kadriu et al., 2009; Tuovinen, 2004). OP nerve gases (e.g.,tabun, sarin, and soman) were synthesized in the course ofwork on insecticides (Satoh, 2006). As the threat of use ofOP nerve gases against humans remains a serious concern(Watson et al., 2006), these compounds have been thoroughlytested in experimental animals. Results of the numerousstudies with DFP and nerve gases can be largely extrapolatedto the whole group of OP pesticides.

Carbamates are esters of unstable carbamic acid. Physo-stigmine, a natural compound derived from calabar beans,was the first thoroughly investigated carbamate (Holmstedt,1972; Somani and Dube, 1989). Synthetic carbamates arepotent blockers of acetylcholinesterase (AChE) and have beenbroadly used in medicine (Woltjer and Milatovic, 2006). Thefirst carbamates with strong insecticidal properties were syn-thesized in the 1950s. In the 1980s carbamates along withOPs were the most commonly used insecticides (Gupta, 2006).

12.1.2 Risks Associated with AnticholinesterasePesticides

OPs and carbamates may enter the human organism throughmultiple routes: gastrointestinal tract, respiratory tract, skin,and mucosa. By virtue of their high lipophilicity the com-pounds are able to rapidly permeate through cell membranes,and to readily cross the blood-brain barrier (Marrs and Vale,2006; Tang et al., 2006).

Poisonings induced by AChE inhibitors remain an impor-tant clinical problem (Bardin et al., 1994). Each year largenumbers of humans worldwide undergo poisonings withthese compounds. Acute poisonings with OPs or carbamatesmost often result from suicidal attempts or erroneousingestion; however, occupational exposure may also occur(Cable and Doherty, 1999).

12.1.3 Symptoms of Acute Intoxication withAnticholinesterase Pesticides

Inhibition of AChE leads to an abrupt accumulation of acetyl-choline (ACh) in the autonomic ganglia, the neuromuscularjunction, and the central nervous system. Symptoms ofacute poisoning with OPs and carbamates are similar andresult from overstimulation of both muscarinic and nicotinicACh receptors. The muscarinic symptoms include miosis,salivation, dacryorrhea, nausea, excessive sweating, urinaryincontinence, abdominal cramps, diarrhea, bradycardia,broncho- and laryngospasm, and excessive secretion of tra-cheobronchial glands. Major nicotinic symptoms compriseblurred vision, vomiting, increased muscle tone and muscularfasciculation, and paralysis of respiratory muscles. A highexposure may lead to the occurrence of symptoms of central

origin: partial or generalized seizures, mental disturbance,impaired coordination and respiratory failure due to aninhibition of the respiratory center in the brain stem. Thesymptoms usually appear within minutes following exposure.The respiratory failure of both peripheral and central originoften leads to death (Marrs and Vale, 2006).

12.1.4 Current Therapy for Poisoning withAnticholinesterase Pesticides

Medical treatment of poisonings with AChE inhibitors hasnot changed for years. The mainstay of the therapy is themuscarinic ACh receptor antagonist atropine. Atropine coun-teracts most of the life-threatening peripheral effects of AChEinhibitors. In acute poisonings with OP AChE inhibitors,AChE reactivators (oximes) are used in addition to atropine(Marrs and Vale, 2006). Oximes permeate weakly to thecentral nervous system, but they are very efficient at theneuromuscular junction, where atropine is considered to beless effective. It is generally accepted that combination ofatropine and an AChE reactivator affords virtually completeperipheral protection. If seizures occur, benzodiazepineanticonvulsants are used (Holstege et al., 1997; Marrs andVale, 2006).

The current treatment of poisonings with OPs and car-bamates, however, has clear limitations. In AChE inhibitor-related poisonings atropine is usually administered atextremely high doses, which may lead to an occurrenceof adverse effects; moreover, a sufficient supply of thedrug may not always be readily available (Bowden andKrenzelok, 1997). Furthermore, atropine counteracts onlythe muscarinic symptoms of poisonings with AChE inhibi-tors (Bardin and Van Eeden, 1990; Beards et al., 1994).Utility of the oxime treatment has long been a subject ofdebate. The ability of a given oxime to reactivate the inhibitedenzyme is toxicant specific, while in an emergency it is notalways possible to establish what the exact cause of a poison-ing was (van Helden et al., 1996). The utility of oximes isfurther limited by aging of the enzyme-inhibitor complex.Generation of alkyl bonds prevents further reactivation ofthe inhibited enzyme (Worek et al., 1997). Moreover, the qua-ternary structure of oximes does not allow them to cross theblood–brain barrier and thus reactivate AChE in the centralnervous system. Finally, the toxicity of oximes themselvesmay be an issue (Dawson, 1994; Marrs, 1991; Munro et al.,1990; van Helden et al., 1996; Worek and Szinicz, 1993).Benzodiazepine treatment of seizures associated with poison-ings with AChE inhibitors may depress the respiratory andcirculatory centers of the brain stem. It is hypothesized thatbenzodiazepines further potentiate a widely describeddepression of these centers by OPs or carbamates (Johnsonand Wilcox, 1975; Munro et al., 1990).

Increasing danger of exposure, limitations of contempor-ary therapy for acute poisonings, and unclear mechanisms

150 CENTRAL MECHANISMS OF SEIZURES AND LETHALITY FOLLOWING ANTICHOLINESTERASE PESTICIDE EXPOSURE

of toxicity raised research interest in OPs and carbamates.New experimental findings led to several hypotheses on apossible involvement of noncholinergic systems in the toxicand convulsant effects of AChE inhibitors. Among the neuro-transmitter systems implicated in the neurotoxic actions ofAChE inhibitors, the excitatory amino acid system attractedparticular attention [see e.g., Dekundy (2006) and Solbergand Belkin (1997) for reviews]. However, the adenosinergic,the g-aminobutyrergic (GABA-ergic), and the monoaminer-gic systems may also participate in the seizures and lethalityassociated with OP- or carbamate-induced poisonings.Recent studies provide evidence that neuroinflammatory con-sequences of exposure to anticholinesterases may contributeto seizures and pathology associated with these agents.

12.2 NEUROTRANSMITTER SYSTEMSIMPLICATED IN SEIZURES AND LETHALITYOF ANTICHOLINESTERASES

12.2.1 Muscarinic Mechanisms

An obvious consequence of the inhibition of brain AChEactivity is increased brain level of ACh. Indeed, severalstudies unequivocally demonstrated a marked increase inconcentration of this neurotransmitter in experimental somanintoxication (Lallement 1992a; Shih and McDonough, 1997;Shih et al., 1993).

Overabundance of ACh and overstimulation of muscarinicACh receptors in the central nervous system may lead to anoccurrence of epileptic seizures (Honchar et al., 1983;Turski et al., 1989; Zimmerman et al., 2008). Indeed, anincrease in ACh could be observed 3 min after soman admin-istration. The increase was particularly pronounced at the timewhen seizure activity was observed on an electroencephalo-gram (Shih and McDonough, 1997). Increased ACh concen-trations were found in the brain stem (Shih and McDonough,1997), the striatum (Shih et al., 1993), and the amygdala(Lallement 1992a). In the medial septum and in the hippo-campus the ACh level was markedly elevated in the perioddirectly preceding the occurrence of seizures. ACh leveldecreased after 10 to 20 min, and secondarily increasedafter 50 min of continuing seizures (Lallement 1992a).Thus, antagonism at muscarinic ACh receptors by atropineshould efficiently prevent seizures induced by compoundsinhibiting AChE. However, atropine (1.8 mg/kg) did not pro-tect mice against seizures produced by chlorphenvinphos,dichlorvos, physostigmine, or methomyl (Dekundy et al.,2001, 2003, 2007). This observation remains in agreementwith clinical reports evidencing lack of efficacy of atropinein interrupting epileptic seizures related to poisonings withOPs or carbamates (Bardin and Van Eeden, 1990; Bardinet al., 1994; Karalliedde, 1999). Also in laboratory rodents,atropine failed to afford any protection against seizures

induced by AChE inhibitors even though it was administeredat a very high dose (McLean et al., 1992).

On the other hand, many authors demonstrated efficacyof atropine in preventing and interrupting seizures inducedby different AChE inhibitors (Capacio and Shih, 1991;Carpentier et al., 2000; Ligtenstein and Moes, 1991; Shihand McDonough, 2000; Shih et al., 1991). However, dosesof atropine used in these studies were relatively high, usuallyexceeding 10 mg/kg (Ligtenstein and Moes, 1991;McDonough and Shih, 1997; Shih and McDonough, 1999,2000). The ED50 of atropine, a statistically determinedhypothetical dose counteracting seizures produced by OPnerve gases soman or sarin in 50% of rats, was found to beas high as 60 mg/kg (Capacio and Shih, 1991; Shih andMcDonough, 1999). Moreover, in most of the studies otherantidotes were used in addition to atropine, like oximesand/or peripherally active reversible AChE inhibitors likepyridostygmine (Ligtenstein and Moes, 1991; Shih andMcDonough, 2000). Although atropine seems to act syner-gistically with some oximes, like HI-6 (Ligtenstein andMoes, 1991), ED50 of atropine in the presence of HI-6 insoman-treated rats was still as high as 21 mg/kg (Capacioand Shih, 1991).

Shih and McDonough investigated efficacy of atropine incounteracting seizures induced by soman administered atfivefold LD50 dose in guinea pigs. Atropine was administered5 min after the first appearance of electroencephalographicseizures. ED50 of atropine in this model was found to be12.2 mg/kg. It should be mentioned, however, that pyrido-stygmine was administered directly before, and a smalldose of atropine (2 mg/kg) and oxime 2-PAM directlyafter, soman administration (Shih and McDonough, 2000).Some studies showed that atropine (5 mg/kg) was able toalleviate behavioral seizures, electroencephalographic altera-tions, and associated neuropathological changes followingadministration of soman in guinea pigs. However, also inthis case atropine was co-administered with pyridostygmine(Carpentier et al., 2000).

Efficacy of atropine in counteracting lethality followingpoisonings with OP or carbamate AChE inhibitors is evi-denced by many clinical and experimental studies (Holstegeet al., 1997; Minton and Murray, 1988). Administration ofatropine (1.8 mg/kg) protected mice against death producedby OP pesticides, chlorphenvinphos and dichlorvos, andcarbamates, physostigmine or methomyl (Dekundy et al.,2001, 2003, 2007). On the other hand, some authors reportedlack of efficacy of even relatively high doses of atropinein counteracting lethal consequences of poisonings withsome other OP compounds (Clement, 1994; Karalliedde,1999). The conventional treatment with atropine may notalways be able to afford sufficient protection against centralneurotoxicity. Moreover, it may not even afford sufficientcontrol of some peripheral symptoms, in particular excessiveexcretion of tracheobronchial glands (Bardin and Van Eeden,

12.2 NEUROTRANSMITTER SYSTEMS IMPLICATED IN SEIZURES AND LETHALITY OF ANTICHOLINESTERASES 151

1990). Some clinical reports on poisonings with OP anticho-linesterases give evidence that even initially effective treat-ment with atropine may be followed by late reappearance ofsymptoms (Bardin et al., 1994). Similarly, administration ofatropine in mice did not prevent an occurrence of lethalityproduced by the OP pesticide chlorphenvinphos as assessed120 hours following its administration (Dekundy et al.,2001).

Several muscarinic ACh antagonists (e.g., scopolamine,biperiden, or benactizine) were found to be more potentthan atropine in counteracting seizures and lethality producedby anticholinesterases in rodents. However, this phenomenonhas been associated with some effects of the compoundson glutamatergic and/or dopaminergic neurotransmission(Capacio and Shih, 1991; Raveh et al., 1999; Shih andMcDonough, 1997, 2000; Shih et al., 1991). Evidence accu-mulated thus far suggests that cholinergic mechanismsmay play a crucial role in the patomechanism of seizuresassociated with poisonings with AChE inhibitors. However,epileptogenic and acute toxic effects of OPs or carbamatescannot be fully explained by their ability to increase brainACh level.

12.2.2 Nicotinic Mechanisms

The first neurochemical alteration caused by AChE inhibitorsin many regions of the central nervous system is a consi-derable elevation of ACh concentration (Shih andMcDonough, 1997). It is conceivable that surplus ACh result-ing from AChE inhibition overstimulates not only themuscarinic but also the nicotinic ACh receptors. Possiblerole of nicotinic mechanisms in AChE inhibitor-inducedseizures and lethality should not be neglected (Jafari-Sabet,2006; Klemm, 1983; Shih et al., 1991). Superabundance ofACh may be critically involved in the propagation of excit-atory postsynaptic potentials via neuronal nicotinic receptors(Chu et al., 2000). Continuous release of ACh and glutamatecould also be a result of activation of presynaptic nicotinicreceptors (McGehee et al., 1995). Interestingly, in our recentstudy a nicotinic antagonist mecamylamine attenuated theconvulsions produced by carbamate methomyl, but notthose resulting from administration of OP dichlorvos. Acombination of otherwise ineffective doses of mecamylamineand MK-801 diminished lethality and blocked behavioralseizures produced by both anticholinesterases (Dekundyet al., 2003). This finding correlated well with observationsof Chu et al. (2000), who demonstrated reduction of gluta-mate antagonist-resistant excitatory postsynaptic currentsin rat neocortex by mecamylamine. It seems conceivablethat discrepant effects of NMDA antagonists in OP- andcarbamate-induced seizures observed in some studies(Dekundy et al., 2001, 2003, 2007) may be at least to someextent due to different actions of OPs and carbamates onneuronal nicotinic receptors.

In fact, carbamates have been reported to interact directlywith the nicotinic ACh receptors (Sherby et al., 1985).Among several carbamates, physostigmine was found to bethe most potent agonist at the nicotinic receptors (Sherbyet al., 1985). Interestingly, nicotine-induced seizures, simi-larly to carbamate-induced seizures, are not universallyblocked by NMDA receptor antagonists (Kis et al., 2000)whereas the neuronal nicotinic receptor antagonist meca-mylamine inhibits seizures induced by both nicotine (Kiset al., 2000) and carbamates (Dekundy et al., 2003). Takentogether, these data seem to suggest the direct activationof nicotinic receptors by carbamates may contribute to theirconvulsant properties.

12.2.3 N-Methyl-D-Aspartate (NMDA)-RelatedMechanisms

Many authors have suggested that glutamatergic mechanismsmay be implicated in epileptogenic effects of OPs (reviewedin Solberg and Belkin, 1997). Indeed, a noncompetitiveNMDA antagonist, dizocilpine, afforded protection againstseizures produced by a systemic administration of OP pesti-cides chlorphenvinphos and dichlorvos in mice (Dekundyet al., 2001, 2003, 2007). This observation remains in agree-ment with other published experimental data. In the firstreport suggesting possible involvement of the excitatoryamino acid system in the neurotoxicity of AChE inhibitorsit has been demonstrated that prophylactic administrationof dizocilpine at 1 mg/kg alleviates, and at 5 mg/kg fullyblocks the behavioral and electroencephalographic seizureactivity following administration of the OP nerve gassoman in guinea pigs (Braitman and Sparenborg, 1989).Antiseizure effects of this NMDA antagonist were muchstronger than those of diazepam, a compound used in theclinical treatment of seizures associated with anticholinester-ase poisonings (Shih, 1990; Shih et al., 1991). Some authorsdemonstrated anticonvulsant efficacy of dizocilpine adminis-tered after the initiation of seizure activity (McDonough andShih, 1993). Other studies proved that dizocilpine is able toprevent seizure-related neuronal necrosis in brains of animalssurviving experimental soman intoxication (Sparenborget al., 1992). Dizocilpine is not the only NMDA antagonistexerting anticonvulsant effects in AChE inhibitor-inducedpoisonings. The antidementia drug and uncompetitiveNMDA antagonist memantine (Parsons et al., 2007) redu-ced seizures and neuropathology in soman-treated rats(Deshpande et al., 1995). The compound efficiently protectedagainst soman-induced convulsions both when given prophy-lactically and curatively (Gupta, 1994; Gupta and Kadel,1989; McLean et al., 1992). Also the phencyclidyne deriva-tive and NMDA antagonist trienylcyclohexylpiperidine(TCP) prevented occurrence of seizure activity in soman-treated guinea pigs (Carpentier et al., 1994). Unlike diaze-pam, TCP was able to interrupt epileptic seizures and


normalize electroencephalographic alterations produced bythis nerve gas (Carpentier et al., 1994; Lallement et al.,1993, 1994a, 1994b). Another NMDA antagonist, gacycli-dine, prevented the occurrence of convulsions associatedwith systemic administration of soman (Lallement et al.,1999). Antiseizure efficacy of anticholinergic drugs likebenactizine, biperiden, or caramiphen in experimentalsoman intoxications has been associated with their anti-NMDA properties (Capacio and Shih, 1991; McDonoughand Shih, 1995; Raveh et al., 1999; Shih et al., 1991).

Effects of NMDA antagonists on carbamate-inducedseizures were not thoroughly investigated. Some reportsdemonstrated that memantine was able to protect againstseizures induced by experimental intoxication with a com-monly used carbamate insecticide, carbofuran (Gupta,1994; Gupta and Kadel, 1989). However, other NMDAantagonists, dizocilpine and CPP, failed to modify seizuresinduced by systemic injections of a reference compoundphysostigmine or a carbamate insecticide methomyl(Dekundy et al., 2001, 2003, 2007).

Results of many studies with experimental animalsstrongly suggest that the excitatory amino acid system maybe critically involved in acute toxicity of AChE inhibitors.Dizocilpine (1 mg/kg or higher) attenuated seizures, butfailed to protect against lethality following experimentalsoman intoxication. Moreover, higher doses of dizocilpineincreased acute toxicity of soman (McDonough and Shih,1993; Shih, 1990; Sparenborg et al., 1992). However, whenco-administered with atropine and an AChE reactivator,dizocilpine markedly decreased mortality following somanadministration (Braitman and Sparenborg, 1989). It hasbeen suggested that dizocilpine alone, like diazepam, mayaugment soman-induced depression of the activity of therespiratory center of the brain stem (McDonough andShih, 1993). Concurrent administration of atropine withdizocilpine prevented the occurrence of this disadvantageousinteraction and afforded a very effective protectionagainst soman-induced death (McDonough and Shih, 1993;Shih, 1990).

Memantine increased survival of rats administered highdoses of soman (Deshpande et al., 1995). The same drug,co-administered with atropine, protected against lethalityfollowing acute experimental intoxications with both carba-mates and OPs (Gupta, 1994; Gupta and Kadel, 1989;McLean et al., 1992). Likewise, TCP protected guineapigs against soman-induced death in the presence of atropineand pyridostigmine (Carpentier et al., 1994). Memantine andTCP were effective both when given prophylactically andcuratively in acute soman intoxication (Carpentier et al.,1994; McLean et al., 1992). Another NMDA antagonistgacyclidine, co-administered with a reversible AChEinhibitor and a cholinolytic drug, prevented mortalityassociated with administration of soman in guinea pigs(Lallement et al., 1999). A competitive NMDA antagonist

CPP co-administered with atropine effectively counteractedacute toxicity of OP pesticides (chlorphenvinphos anddichlorvos) and carbamates (physostygmine or methomyl)in mice (Dekundy et al., 2001, 2003, 2007).

Some authors have demonstrated that several cholinolyticcompounds, for example, benactizine, biperiden, or carami-phen, possess NMDA-antagonistic properties. Interestingly,the substances were found to be superior to atropine inpreventing lethal consequences of soman administration inlaboratory rodents (Capacio and Shih, 1991; McDonoughand Shih, 1995; Raveh et al., 1999; Shih et al., 1991).

The results of experimental studies provided unquestion-able evidence of implication of NMDA receptors in acutetoxicity of AChE inhibitors. However, studies aimed atelucidating the mechanisms by which NMDA antagonistsmay influence processes underlying seizures and lethality byOPs and/or carbamates are sparse. There is some evidencesuggesting that some NMDA antagonists may exert non-specific effects, like interaction with brain AChE. Forexample, dizocilpine has been demonstrated to inhibitAChE in the rat brain and to prevent inactivation of thisenzyme by DFP in vitro (Galli and Mori, 1996). Co-adminis-tration of memantine with atropine prior to systemic injec-tions of either carbamate insecticide carbofuran or OPnerve gas soman reduced AChE inhibition by both com-pounds (Gupta and Kadel, 1989; McLean et al., 1992). Onthe other hand, memantine and/or related compoundneramexane have been shown not to affect AChE inhibitionby edrophonium (McLean et al., 1992) or by AChE inhibitorsused in the treatment of Alzheimer’s disease, like galanta-mine, rivastigmine, donepezil, or tetrahydroaminoacridine(Gupta and Dekundy, 2005, 2007; Wenk et al., 2000).Likewise, both dizocilpine and CPP, alone or in combinationwith atropine, failed to influence AChE inhibition by physos-tigmine, methomyl, chlorphenvinphos, or dichlorvos in themouse brain (Dekundy et al., 2001, 2007). It seems that thenonspecific effects of some NMDA antagonists on brainAChE may, to some extent, contribute to their beneficialactions observed in toxicological studies with OPs and carba-mates. However, these effects cannot fully explain thesuperior efficacy of NMDA antagonists in AChE inhibitor-induced poisonings.

There is some discrepancy in the reports on changes inconcentrations of excitatory amino acids in the course ofsoman poisoning. In the CA3 and CA1 regions of the hippo-campus, an early increase in extracellular glutamate levelwas observed (Lallement et al., 1991c). An even more rapidincrease in glutamate was demonstrated in the amygdala,which suggested a critical involvement of this brain structurein the seizures produced by soman (Lallement et al., 1991b).In the piriform cortex, a brain area particularly vulnerable toneuropathological changes related to seizure activity, gluta-mate concentration was found to be increased in the ratshaving electroencephalographic seizures following systemic

12.2 NEUROTRANSMITTER SYSTEMS IMPLICATED IN SEIZURES AND LETHALITY OF ANTICHOLINESTERASES 153

soman application (Wade et al., 1987). Interestingly, in thestriatum, a significant increase in aspartate but not glutamaterelease has been observed following an administration ofa convulsion-inducing dose of soman (Jacobsson et al.,1997a). No increases in excitatory amino acids were foundin the cerebral cortex. Furthermore, 80 min after the initiationof seizure activity, concentration of glutamate in the cerebralcortex was found to be below the basal value (Shih andMcDonough, 1997). It has been suggested, that in generationof seizures following soman administration, excitatory aminoacids may play a crucial role in the hippocampus, but not inthe striatum (Jacobsson et al., 1997a).

12.2.4 AMPA/Kainate-Related Mechanisms

AMPA/kainate receptors play an important role in epilepsyand are targets for antiepileptic drugs (Rogawski andDonevan, 1999). Nevertheless, there are only few reportsconcerning their protective effects in anticholinesterase-induced poisonings. NBQX, an AMPA/kainate receptorantagonist, given prophylactically or curatively, preventedor reduced, respectively, soman-induced seizures. Delayedtreatment with NBQX and atropine was also able to attenuatesoman-induced convulsions (Lallement et al., 1994b).Furthermore, co-administration of atropine, NBQX, andTCP prevented or interrupted occurrence of soman-inducedseizures and neuropathology (Lallement et al., 1994a,1994b). In line with this, AMPA/kainate antagonists blockedsoman-induced second population spikes and further spon-taneous discharges in the hippocampal CA1 or CA3 region(Wood and Tattersall, 2001). On the other hand, NBQXfailed to reduce acute toxic effects of chlorphenvinphos,dichlorvos, physostigmine, and methomyl (Dekundy et al.,unpublished observations). Sarin had no effect on the ampli-tude of AMPA receptor-mediated postsynaptic currentsevoked by field stimulation of hippocampal CA1 pyramidalneurons (Chebabo et al., 1999). Soman was demonstratedto produce a downregulation of [3H]AMPA binding to fore-brain membrane preparations (Raveh et al., 2002). Glutamatebinding to kainate receptors in the hippocampal CA3 andCA1 areas remained unchanged within the first 40 min ofseizures in rats exposed to a convulsive dose of soman(Lallement et al., 1991c). Very recent evidence suggeststhat selective antagonists of GluR5 kainate receptorsmay hold promising potential in treatment of OP-inducedseizures (Apland et al., 2008).

12.2.5 Adenosinergic Mechanisms

Adenosine is an endogenous modulator of brain functionhaving a profound influence on the integration of excitatoryand inhibitory neurotransmission (Boison, 2008). Acti-vation of adenosine receptors or increase of adenosine syn-thesis exerts powerful anticonvulsant action (Dragunow,

1988; Boison, 2008). Consequently, it has been suggestedthat the adenosine system may participate in the seizuresand lethality associated with exposure to OPs or carbamates(van Helden and Bueters, 1999; Zarrindast et al., 1995).Organophosphorus anticholinesterases sarin, tabun, andsoman have been shown to directly bind to A1 adenosinereceptors on synaptic membranes of guinea pig brain(Lau et al., 1988). Adenosine A1 receptor agonists 50-N-ethylcarboxamido-adenosine or N6-cyclopentyl-adenosine,administered immediately after a lethal dose of soman,prevented or delayed occurrence of cholinergic symptomsand increased survival in rats. These effects were associatedwith decreased brain ACh concentration (van Helden et al.,1998). N6-cyclopentyl-adenosine was also found to behighly effective against soman, tabun, or sarin poisoning(Bueters et al., 2002). A combination of the drug with atro-pine reduced the toxicity of DFP in rat (Tuovinen, 2004).However, N6-cyclopentyl-adenosine failed to protect againstVX or parathion (Bueters et al., 2002). Another A1 adenosinereceptor agonist, phenylisopropyl adenosine, was ineffectiveagainst the metamidophos-induced cholinergic symptomsand mortality (Kalkan et al., 2005). However, more recentdata suggests that the observed beneficial effects of adenosineagonists seem to be mediated not only by central mechan-isms. It has been demonstrated that N6-cyclopentyl-adenosine is able to protect AChE against its inhibitionby sarin (Bueters et al., 2003). Moreover, N6-cyclopentyl-adenosine treatment altered the distribution of sarin intothe brain, presumably through its peripheral adenosine A1receptor-mediated cardiovascular side effects and reductionin blood pressure (Joosen et al., 2004).

12.2.6 GABA-ergic Mechanisms

Aggressive treatment with GABAA receptor potentiatingbenzodiazepines, diazepam or clonazepam, is generally effec-tive against seizures induced by OPs. However, as alreadymentioned, it is associated with inhibition of respiratory cen-ters of the brain stem and marked sedation. Interestingly,novel therapies taking the advantage of nonsedative benzo-diazepines are beginning to emerge (Kadriu et al., 2009;Pibiri et al., 2008). Imidazenil, a positive and selective allo-steric modulator of a5-containing GABAA receptors, ismore potent, effective, and safer than diazepam in protectingrats from diisopropyl fluorophosphate-induced seizures andthe associated neuronal damage (Kadriu et al., 2009).Furthermore, the combination of huperzine A, a reversibleAChE inhibitor, with imidazenil provides a prophylactic,safe and very effective approach for protection against diiso-propyl fluorophosphate seizures (Pibiri et al., 2008).

Several neurosteroids act as positive allosteric modulatorsof GABAA receptors and produce robust anticonvulsantand neuroprotective effects after exogenous administration(Gasior et al., 1999). Recently, the neurosteroid pregnanolone


has been reported to show protective effects against mortalityand seizures induced by soman. Importantly, treatment withpregnanolone 30 min after seizure onset was more effectivethan diazepam administered at the same time (Lumleyet al., 2008).

Activation of interneurons and elevation of GABA levelsin the brain is often associated with seizure activity. Somanmarkedly increased GABA release in the cerebral cortex, thestriatum, and the hippocampus (Jacobsson et al., 1997a; Shihand McDonough, 1997). The change in cortical GABA wassignificant 20 min after the initiation of electroencephalo-graphic seizures, while in the striatum and the hippocampusonly 80 min thereafter (Jacobsson et al., 1997b; Shih andMcDonough, 1997). The increase in GABA may reflectnonspecific compensatory mechanisms aimed at regaining alost balance between stimulatory and inhibitory processesin the brain.

12.2.7 Monoaminergic Mechanisms

Monoamines and/or their receptors have repeatedly beenimplicated in epileptic seizures (Kobayashi and Mori, 1977;Starr, 1996). Indeed, dopaminergic (Bourne et al., 2001;Coudray-Lucas et al., 1987; Jacobsson et al., 1997a, 1997b;Shih et al., 1991), serotonergic (Bodjarian et al., 1995),and noradrenergic (Buccafusco and Aronstam, 1987;Coudray-Lucas et al., 1987) systems may participate in theseizures and lethality associated with exposure to OPs orcarbamates.

Dopamine is a potent source of free radicals and is knownto produce cytotoxic effects per se. The cytotoxic effects ofdopamine in cultured chick telencephali cells are additive(Jacobsson and Fowler, 1999). Soman was repeatedlydemonstrated to increase dopamine levels in experimentalrodents (Bourne et al., 2001; Jacobsson et al., 1997a;Reithmann et al., 1988; Shih and McDonough, 1997).Also, the pesticidal anticholinesterases monocrotophos orcarbofuran caused a marked increase in dopamine (Guptaet al., 1984). In fact, the increase in striatal dopamine concen-tration was the major neurochemical event following theinitial ACh surge in soman-treated rodents (Jacobssonet al., 1997a). Moreover, the level of this neurotransmitterwas found to be positively correlated with seizure activityon the electroencephalogram (Jacobsson et al., 1997b). Inline with the above findings, systemic administration of theD1 receptor antagonist SCH 23390 inhibited seizure activity(Bourne et al., 2001). It should be mentioned that someauthors indicated that administration of OPs or carbamatesmay be associated with increased dopamine turnover ratherthan the elevation of the brain level of this neurotransmitter(el-Etri et al., 1992; Soininen et al., 1990).

Some data suggest that decreased striatal noradrenalinelevel may play a role in soman-induced neurotoxicity andseizures (Buccafusco and Aronstam, 1987; Coudray-Lucas

et al., 1987; el-Etri et al., 1992; Fosbraey et al., 1990; Shihand McDonough, 1997). Levels of noradrenaline werereduced by soman in guinea pig cortex, hippocampus, andstriatum (Fosbraey et al., 1990). Noradrenaline levels specifi-cally declined by up to 70% in forebrain of soman-treated convulsive rats. In nonconvulsive rats noradrenalinelevels remained unchanged (el-Etri et al., 1992). In line withthis finding, a-adrenergic agonists (methyldopa, xylazine,and clonidine) effectively protected against lethal effectsof soman intoxication. Potency for protection was relatedto affinity for a-adrenergic binding sites labeled with[3H]clonidine. Atropine acted synergistically with adrenergicagonists to potentiate protection (Buccafusco and Aronstam,1987). Moreover, pharmacological elevation of brain cate-cholamines also resulted in significant protection againstsoman toxicity, additive with that of clonidine (Buccafuscoet al., 1988). However, ability to decrease brain noradrenalinelevel may not be characteristic for all AChE inhibitors. Forexample, OPs paraoxon, metrifonate, dichlorvos, or naledproduced no changes in brain noradrenaline (Coudray-Lucas et al., 1987; Soininen et al., 1990), whereas anticholin-esterase pesticides monocrotophos or carbofuran caused anincrease in noradrenaline (Gupta et al., 1984).

Anticholinesterases induced inconsistent changes in brainserotonin levels. For example, the pesticides monocrotophosor carbofuran caused an increase in this neurotransmitterin the brain (Gupta et al., 1984). On the other hand, someauthors suggested that the turnover rather than the level ofserotonin may be increased in AChE-treated rodents (el-Etriet al., 1992).

12.3 ROLE OF NEUROINFLAMMATION INANTICHOLINESTERASE-INDUCED SEIZURES

Mounting evidence indicates that seizure activity triggersinflammatory response in the brain, significantly impact-ing neuronal excitation and processes leading to epilepsy(Vezzani and Granata, 2005). Various proinflammatory cyto-kines are produced during seizures, while leukocyte infiltra-tion, and microglial and astrocytic activation contribute toneuronal injury that follows convulsions (Ravizza et al.,2008a; Vezzani et al., 2008; Wetherington et al., 2008).Furthermore, seizure-induced blood–brain barrier leakageenables entry of peripheral inflammatory molecules andproteins that may further augment the damage (Tomkinset al., 2007). Although OP toxicity has been almost exclu-sively linked with their cholinergic activity other mecha-nisms may also play an important role. In fact, OPs havebeen demonstrated to produce robust neuroinflammatoryresponse following exposure to seizure-inducing dosesof these agents (Chapman et al., 2006; Collombet et al.,2005; Cowan et al., 1996; Svensson et al., 2001; Williamset al., 2003; Zimmer et al., 1997). Furthermore, chronic

12.3 ROLE OF NEUROINFLAMMATION IN ANTICHOLINESTERASE-INDUCED SEIZURES 155

exposure to OPs elicits strong immunotoxic response(Galloway and Handy, 2003; Sharma, 2006).

One of the first reports linking OP-induced seizures withinflammatory response was a landmark study by Zimmeret al. (1997). This group demonstrated strong astrocyticactivation as early as 1 h after injection of a convulsantdose of soman. Activation of microglia followed 4 to 8 h later(Zimmer et al., 1997). The highest intensity of these morpho-logical changes was detected in limbic areas that are heavilyengaged during OP-induced seizures (Zimmer et al., 1997).Collombet et al. (2005) extended these observations demon-strating that microgliosis peaked 3 days after soman-inducedseizures, while delayed astrogliosis was still present even after30 days.

It appears tempting to speculate whether anti-inflamma-tory treatments could provide any protection against seizuresor neuropathology associated with toxic doses of AChEinhibitors.

12.4 MECHANISTIC CONSIDERATIONS

12.4.1 Proposed Mechanisms of NeurochemicalEvents Following Acute AnticholinesteraseIntoxication

Experiments aimed at demonstrating neurochemical events inanticholinesterase-treated animals shed light on mechanismsunderlying seizures and lethality associated with OPs orcarbamates. In vivo microdialysis techniques allowedsimultaneous local administration of a toxicant, monitoringneurochemical parameters and electroencephalogram, andobservation of animal behavior (Jacobsson et al., 1997a,1997b). Changes in brain neurotransmitter levels were alsoevaluated after systemic administration of a neurotoxin(Lallement et al., 1991a; Shih et al., 1993; Wade et al.,1987). These studies provided strong evidence that bothlocal and peripheral administration of soman induce rapidand pronounced inhibition of AChE activity in the striatum(Jacobsson et al., 1997a; Shih and McDonough, 1997; Shihet al., 1993), hippocampus (Lallement 1992b; Shih andMcDonough, 1997), and brain stem (Shih et al., 1993). Ithas been determined that inhibition of activity of brainAChE by more than 65% leads to an occurrence of epilepticdischarges on electroencephalogram (Tonduli et al., 1999). Ahypothesis on the mechanism of toxicity of AChE inhibitorshas been created based on results of several neurochemicalstudies. According to this hypothesis, the blockade ofAChE leads to an early increase of ACh concentration,which initiates seizures. The seizure activity leadsto secondary changes in release and turnover of monoamines(in particularof dopamine). This in turn is followed by changesin levels of excitatory amino acids, which leads to neuro-pathology and lethality, and in inhibitory neurotransmitters,

which may reflect compensatory mechanisms (Shih andMcDonough, 1997; Solberg and Belkin, 1997). However,the hypothesis does not convincingly explain the lack offull efficacy against lethal and convulsant effects of AChEinhibitors after NMDA antagonists administration as singletreatment (Dekundy et al., 2001, 2003, 2007). Furthermore,the theory is not fully consistent with the widely documentedinefficacy of atropine in preventing and treating seizuresinduced by AChE inhibitors.

The causes of the apparent discrepancies may be sought inreciprocal relationships between cholinergic and other neuro-transmitter systems in the brain. According to the Bouillontheory, the synaptic and extracellular spaces in the centralnervous system are filled with a mixture of modulatoryneurotransmitters (including ACh, glutamate, dopamine,and inorganic ions), which control the excitability of the post-synaptic membrane through their presynaptic effects. Loss ofa balance between particular components of the neurotrans-mitter milieu may lead to the occurrence of neuropathologicalchanges (Sivilotti and Colquhoun, 1995).

12.4.2 Putative Mechanisms of Interactions betweenNeurotransmitter Systems Implicated in the AcuteToxicity of Anticholinesterases

Neurochemical studies have clearly demonstrated that aneurotransmitter composition in the synaptic cleft and inthe extracellular space changes dramatically in the course ofpoisoning with an AChE inhibitor. It seems to be certainthat increased ACh is the very first neurochemical changein many areas of the central nervous system (Lallement1992a; Shih and McDonough, 1997; Shih et al., 1993).Excessive concentration of this neurotransmitter may initiatea cascade of events. ACh acting at postsynaptic M1 muscar-inic ACh receptors depolarizes neurons (Cheramy et al.,1996). Spreading depolarization increases massive ACh andglutamate release from synaptic vesicles (McGehee et al.,1995; Zapata et al., 1998). Stimulation of a7-containingneuronal nicotinic receptors by excess ACh may produce acontinuous increase in ACh and glutamate release in a posi-tive feedback mechanism (Gray et al., 1996; McGeheeet al., 1995; Tani et al., 1998).

The published data suggests that cholinergic system isclosely interrelated with the glutamatergic one. In vitrostudies markedly increased the understanding of these inter-actions. It has been demonstrated that ACh augments gluta-mate release both through membrane depolarization and astimulation of presynaptic nicotinic receptors (McGeheeet al., 1995). Glutamate, through its effect on NMDA recep-tors, increases in turn the ACh release, which leads to ageneration of a positive feedback loop between the twoneurotransmitters (Anderson et al., 1994). Moreover,stimulation of NMDA receptors activates phospholipases,which may markedly increase choline release from


phosphatidylcholine in neuronal membrane. This mayadditionally lead to increased permeability of the cellularmembrane and thus facilitate cell damage. The released cho-line may then be taken up with a high-affinity choline uptakemechanism and used for ACh resynthesis (Zapata et al., 1998,2000). It has been shown that muscarinic M1 and NMDAreceptors are colocalized at the same neurons (Ikarashiet al., 1998; Marino et al., 1998). Docherty et al. (1987)have observed that glutamate and ACh are simultaneouslyreleased from the same neurons suggesting that the twoendogenous substances may function as cotransmitters.Indeed, a stimulation of M1 muscarinic ACh receptors poten-tiated NMDA-induced currents in hippocampal pyramidalcells in vitro (Marino et al., 1998). The mechanism of theeffect is not clear, but several subcellular mechanisms maybe considered, for example, removal of physiological mag-nesium block of NMDA receptor channel with M1 receptorstimulation (Egorov et al., 1999), and activation of proteinkinase C leading to calcium release from intracellular storesand to direct or indirect phosphorylation of NMDA receptorproteins (Girod et al., 2000; Lu et al., 1999; Markram andSegal, 1992a, 1992b). In line with these findings, the poten-tiation of NMDA receptor-mediated responses by ACh invitro could be reversed by a selective M1 muscarinic receptorantagonist pirenzepine, but not an M2 muscarinic receptorantagonist methocramine. Targeting pathways downstreamthe M1 muscarinic receptors also effectively decreased theACh potentiation of NMDA signalling (Aramakis et al.,1999; Calabresi et al., 1998; Markram and Segal, 1992a,1992b; Wang and Salter, 1994). The above evidence suggeststhat neurons of the central nervous system may be particularlyvulnerable to the sequelae of overstimulation of both NMDAand M1 muscarinic ACh receptors. This may explain theobserved relative inefficacy of NMDA antagonists givenalone, and a superior efficacy of a concurrent blockade ofmuscarinic and NMDA receptors in experimental AChEinhibitor-induced intoxications.

Some authors have implied that a robust increase in striataldopamine concentration may play an important role in acutetoxicity and seizures induced by OP compounds (Jacobssonet al., 1997a, 1997b; Shih and McDonough, 1997). Thisbrain structure has been implicated in the pathogenesis ofepileptic seizures (Deransart et al., 1998; Jacobsson et al.,1997b). Synapses of afferent glutamatergic and dopaminergicneurons can be found on the striatal cholinergic neurons(Smith and Bolam, 1990). Stimulation of NMDA receptorsmay increase dopamine in the striatum, an effect potentiatedby ACh (Cheramy et al., 1996; Jin and Fredholm, 1997).Moreover, stimulation of presynaptic muscarinic and nico-tinic ACh receptors on dopaminergic neurones may resultin a massive release of dopamine (Bourne et al., 2001;Puttfarcken et al., 2000; Reid et al., 1999), which in turnincreases ACh release from cholinergic neurons via D1 dopa-mine receptors (Cheramy et al., 1996). The above data may at

least partly explain the consistently demonstrated increases inboth ACh and dopamine (Shih and McDonough, 1997) andbeneficial effects of nicotinic receptor antagonists (Chiouand Li, 1994; Dekundy et al., 2003; Klemm, 1983; Shihet al., 1999), and a selective D1 dopamine receptor antagonistSCH 23390 (Bourne et al., 2001) in AChE inhibitor-inducedexperimental poisonings. As NMDA receptors seem to play acritical role in dopamine release, the blockade of NMDA-mediated neurotransmission may alleviate the toxicity ofAChE-inhibiting compounds partly through decrease ofdopamine in some brain regions.

Several authors suggested that neurotoxicity of someAChE inhibitors may be induced or modified by direct,AChE-independent effects of these compounds on targetsin the central nervous system (Bakry et al., 1988). It hasbeen suggested that various AChE inhibitors may act directlyat nicotinic (Bakry et al., 1988; Rao et al., 1987; Rocha et al.,1999), muscarinic (Bakry et al., 1988), and NMDA (Johnsonand Michaelis, 1992) receptors. Some of the anticholin-esterases were also shown to prolong the open time of calciumchannels (Rocha et al., 1999). Some OPs may produce directeffects on monoamine metabolism (Coudray-Lucas et al.,1987). Indeed, OP pesticides monocrotophos, dichlorvos,and phosphamidon inhibited monoaminooxidase (MAO)-Aand MAO-B activities in rat brain mitochondria (Nag andNandy, 2001).

A detailed review of all studies conducted extends beyondthe scope of this chapter. It seems that the direct effects ofanticholinesterases may account for the observed differencesin anticonvulsant effects of NMDA antagonists in OP- orcarbamate-induced seizures (Dekundy et al., 2007).

12.4.3 Targeting Neuroinflammation: An EmergingApproach to the Treatment of Anticholinesterase-Induced Seizures

The above-mentioned inefficacy of inhibition of key neuro-transmitter pathways suggests that additional mechanismsare involved in mediation of AChE-induced seizures.Indeed, following years of disregard astrocytes and microgliaare beginning to be viewed as active players during seizureactivity (Rogawski, 2005). Glutamate released from glialcells can generate excitatory currents by activation ofNMDA receptors and could contribute to the overall excito-toxicity and form a basis for neuronal synchronization,which is a hallmark of epileptic seizures (Rogawski, 2005;Wetherington et al., 2008). In fact, a recent report by Tianet al. (2005) suggests that astrocytic release of glutamateacting on AMPA and NMDA receptors can trigger paro-xysmal depolarization shifts underlying seizure activity.More importantly, paroxysmal depolarization shifts can betriggered by astrocytic glutamate even in the absence ofsynaptic interactions among the neurons (Tian et al., 2005).The glial response to OP-induced seizures is well established

12.4 MECHANISTIC CONSIDERATIONS 157

and reported in several studies (Baille et al., 2005; Baille-LeCrom et al., 1995; Collombet et al., 2005; Zimmer et al.,1997). Interestingly, early glial reaction, within 24 h, wasdetected in uninjured brain regions, while late (7 days) gliosisis observed in damaged regions and linked with neuronaldeath (Baille et al., 2005). The early astroglial reaction maytherefore be at least partially responsible for compromisedhomeostasis of glutamate and excitotoxicity after convulsantdoses of OPs (Baille-Le Crom et al., 1995).

More recent data indicates that activated glia acts as asource of inflammatory cytokines that may drive the excit-atory processes triggered by seizures (Vezzani et al., 2008).For example, proinflammatory cytokines such as interleukin(IL)-1b, IL-6, and tumor necrosis factor (TNF)-a are pro-duced during seizure activity and their levels together withmRNA message remain elevated for several hours after sei-zures induced by pilocarpine or kainic acid (Vezzani andGranata, 2005). Furthermore, IL-1b exacerbates kainicacid-induced seizures (Vezzani and Granata, 2005), whileits inhibition yields an anticonvulsant response (Ravizzaet al., 2008b; Vezzani and Granata, 2005). Upregulated mess-age levels for IL-1b, IL6, and TNF-a were consistentlyreported following exposure to convulsant doses of OPnerve agents such as soman (Dhote et al., 2007; Svenssonet al., 2001; Williams et al., 2003) and sarin (Chapmanet al., 2006). To date no data is available that could linkseizures induced by acute exposure to OP pesticides withenhanced inflammatory cytokine production. However,chronic exposure to compounds like cyfluthrin, chlorpyrifos,or diazinon causes alterations in the levels of variousproinflammatory cytokines (Alluwaimi and Hussein, 2007;Mense et al., 2006). It is very likely that inflammatoryresponses to convulsions induced by OP pesticides could besimilar to those described above for OP nerve agents. Inter-estingly, exogenous administration of an anti-inflammatorycytokine (IL-10) significantly decreased peripheral organdamage associated with OP pesticide poisoning (Yurumezet al., 2007).

Another important fact that may contribute to OP-inducedpathology is the blood–brain barrier damage observedfollowing exposure to the convulsant doses of these com-pounds (Carpentier et al., 1990; Sinha and Shukla, 2003).A marked seizure-related and reversible blood–brain barrieropening following OP-induced seizures peaks at the time ofthe most pronounced paroxysmal electroencephalographicactivity, with signs of cerebral hyperactivity and hypoxia(Carpentier et al., 1990). Plasma protein leakage is alsomost pronounced in the areas associated with strong epilepticactivity, with subsequent parenchymal edema and neuronaldamage (Carpentier et al., 1990). Plasma albumin leakagethrough a compromised blood–brain barrier is one of thekey factors responsible for delayed neurodegeneration anddevelopment of the epileptic focus following seizures(Tomkins et al., 2007). Systemic inflammation triggered

by a cholinergic agent, pilocarpine, increases blood–brainbarrier permeability and promotes entry of cytokinesinto the brain, leading to status epilepticus (Marchi et al.,2009). Consequently, inhibition of systemic inflammationand tightening of the blood–brain barrier permeabilityreduces the severity of pilocarpine-induced seizures(Marchi et al., 2009).

It remains to be determined whether similar therapeuticinterventions, that is, suppression of central and/or peripheralinflammation, would be also beneficial in treatment ofseizures induced by OPs.

12.5 CONCLUSIONS

AChE inhibitors, widely used as drugs or pesticides andbeing a potential threat as agents of chemical warfare, stillendanger human lives. There is still a great need for moreeffective treatment and/or prevention of poisonings inducedby OPs or carbamates.

It has been demonstrated that NMDA antagonists specifi-cally potentiate preventive effects of atropine against OP orcarbamate AChE inhibitor-induced lethality and are able toblock or attenuate OP-induced seizures.

It seems that combined treatment with atropine andNMDA antagonists might be clinically used for the treatmentof anticholinesterase-induced poisonings. Finally, inhibitionof neuroinflammatory actions of AChE inhibitors mayprovide novel therapeutic avenues for treatment of seizureand pathology associated with the exposure to toxic dosesof these agents.

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