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

18NEUROTOXICITY OF ORGANOPHOSPHATES ANDCARBAMATES

KIRAN DIP GILL

Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India

GOVINDER FLORA

41779 Bristow Manor Dr, Ashburn, VA 20148, USA

VIDHU PACHAURI AND SWARAN J. S. FLORA

Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Gwalior-474002, India

18.1 Introduction 237

18.2 Acute Cholinergic Toxicity 238

18.3 Clinical Signs and Symptoms 239

18.4 Organophosphorus Ester-Induced DelayedNeurotoxicity/Neuropathy (OPIDN) 239

18.5 Delayed Onset of Encephalopathy and Coma in AcuteOP Poisoning in Humans 240

18.6 Chronic Neurotoxicity 241

18.7 OPs and the Gulf War Syndrome 242

18.8 Pesticides Exposure and Parkinson’s Disease 243

18.9 Effects on Receptors 243

18.10 Oxidative Stress 245

18.11 Seizure Activity and Oxidative Stress 247

18.12 Signaling Path Way 248

18.13 Effects on Gene Expression 250

18.14 Treatment of Organophosphate Poisoning 25218.14.1 Counteracting the Muscarinic Effects of

Excess Acetylcholine 25218.14.2 Counteracting the Nicotinic Effects of

Excess Acetylcholine 253

18.15 Additional Therapies 25418.15.1 Benzodiazepines 25418.15.2 Sodium Bicarbonate 25418.15.3 Glutamate-Receptor Antagonists 25418.15.4 Clonidine 25418.15.5 Annealed Erythrocytes 254

18.16 Anticonvulsants 25418.16.1 Diazepam 255

18.17 Other Methods of Antidotal Treatment 25518.17.1 Enzymes 25518.17.2 Phosphotriesterase 25518.17.3 Calcium Channel Blockers 25518.17.4 Adenosine Receptor Agonists 25518.17.5 N-Methyl-D-Aspartate Receptor

Antagonists 25518.17.6 Blockade of Acetylcholine Synthesis or

Uptake 255

18.18 Prevention and Treatment of Organophosphate-InducedDelayed Polyneuropathy 256

18.19 Conclusion 256

References 257

18.1 INTRODUCTION

The widespread use of cholinesterase inhibitors, especially aspesticides, produces a significant number of human poison-ing events annually. The World Health Organization(WHO) has estimated that one million serious accidental

poisonings and two million suicide attempts due to pesticidepoisoning occur every year worldwide (WHO, 1973). Thisnumber also accounts for a substantial fraction of almost900,000 people worldwide who die by suicide every year.Use of organophosphate (OP) and carbamate (CM) com-pounds in developing countries is particularly widespread

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

237

because of hot climatic conditions; the number of deaths maybe high. Pesticide poisonings are relatively common incountries such as Sri Lanka, Venezuela, Indonesia, SouthAfrica, and Brazil (Choi et al., 1995). Among the numerouspesticides that can result in death, OP and CM insecticidesare the most common because of their high toxicity.Sodium methyldithiocarbamate (SMD) is the third mostabundantly used conventional pesticide in the United States(U.S. Environmental Protection Agency, 2001).

Patterns of pesticide consumption have been shifting in thelast three decades, and the developing countries’ share ofglobal pesticide use increased from 20% to 40% (PAHO,2002). The Pan American Health Organization (PAHO) esti-mates about 3% of exposed agricultural workers suffer fromacute pesticide poisoning annually out of a total population ofabout 1.3 billion agricultural workers worldwide (PAHO,2002). A study involving 228 Indonesian farmers andprofessional pesticide applicators found that 21% of the totalsuffered from three or more symptoms per spray operation(Kishi et al., 1995). The first controlled study assessing work-ers who suffered with acute poisoning from cholinesteraseinhibitor compounds were reported by Savage et al. (1988).They reported that OPs inhibit the acetylcholine (ACh)hydrolyzing enzyme acetylcholinesterase (AChE), leadingto symptoms of hypercholinergic activity such as abdom-inal cramps, nausea, diarrhea, salivation, miosis, dizziness,tremor, anxiety, and confusion. Symptoms usually occurwithin minutes to hours of exposure and typically disappearwithin days or weeks, depending on the OP or CM compoundinvolved. A recent study conducted in Costa Rica comparedneurobehavioral performance between two groups of farmerswith previous acute intoxications by OP or CM (Wesselinget al., 2002). Plasma cholinesterase activity was assessedfor each group of subjects. Two years later, the subjects(farmers) showed long-term sequelae deficits in visuo- andpsychomotor tasks. In addition, vibrotactile sensitivity offingers and toes of the OP-poisoned subjects was worsethan that of the subjects who had been poisoned by CMs.

OPs are chemically organic esters of phosphorus-containing acids. These substances are anticholinesteraseinsecticides, widely used in agriculture, horticulture, veterin-ary medicine, public hygiene, and also used as nerve agents inchemical warfare (Gupta, 2006; Waddell et al., 2001).

CM compounds are esters of carbamic acid with a func-tional group –NH(CO)O–. Carbamates were originallyextracted from the Calabar bean Physostima venenosum, aperennial plant found in tropical West Africa. Currently, inaddition to their major use as pesticides, CMs are also indi-cated in the treatment of neurodegenerative disease likeAlzheimer’s, myasthenia gravis, glaucoma, and urine voidingdysfunction, and as a prophylactic in OP nerve agentexposure (Gupta, 2006). OPs and CMs are not generally per-sistent in the environment. The lack of bio-persistence of OPsin comparison with the organochlorines has meant that most

countries have tended to replace the organochlorines withOPs. Consequently their scale of use has increased in thelast few decades.

CMs are present naturally in hemoglobin. Carbamategroups are formed when carbon dioxide molecules bondwith the amino terminus of the globin chains. Ribulose 1,5-biphosphate carboxylase also requires the formation of a car-bamate to function. The most common routes of OPs andCMs exposure are oral and dermal. Humans likely to beaffected are occupationally exposed workers like insecticideformulators, applicators, and farm workers. Figure 18.1shows crops that are treated with OPs and CMs. Exposureto these pesticides can be easily monitored by measuringblood cholinesterase (ChE) activity. However, in the case ofCM poisoning the reduction in ChE activity remains onlyup to 48 hours, thus it is essential to collect blood samplesfor measuring enzyme activity as soon as possible afterexposure.

Exposure to OPs and CMs may lead to several distinctneurotoxic effects depending on the dose, frequency ofexposure, chemical constituent of the OP or CM, and host fac-tors that influence susceptibility and sensitivity. These effectsinclude acute cholinergic toxicity, a delayed ataxia knownas organophosphorus ester-induced delayed neurotoxicity(OPIDN), chronic neurotoxicity, and developmental neuro-toxicity (Salvi et al., 2003; Yang and Deng, 2007).

18.2 ACUTE CHOLINERGIC TOXICITY

The main known neurotoxic effect of OPs and CMs is chol-inesterase inhibition, which causes cholinergic overstimula-tion (Russel and Overstreet, 1987). Acute cholinergicabnormality develops within a few minutes to several hoursafter exposure, and affects peripheral muscarinic and nic-otinic receptors, as well as the central nervous system,through the inhibition of serine-containing esterases, of

Figure 18.1 Name of crops typically receiving organophosphateand carbamates application.

238 NEUROTOXICITY OF ORGANOPHOSPHATES AND CARBAMATES

which AChE is clinically the most important (Lotti et al.,1986). Despite the prominence of the anticholinesteraseeffects of most OPs and CMs, it is clear that some of themhave acute effects that may contribute qualitatively or quanti-tatively to the overall syndrome. There are few that interactdirectly with muscarinic receptors (Silveira et al., 1990),and pathways other than cholinergic ones can be affected(Fosbraey et al., 1990; Lau et al., 1988). Diisopropyl phos-phorofluoridate (DFP), for example, affects dopaminergicas well as somatostatinergic pathways in rats (Naseem,1990), while leptophos affects gamma-aminobutyric acid(GABA)-regulated chloride channels, unlike certain nerveagents (Gant et al., 1987). In addition, parathion, methyl para-thion, and malathion affect calmodulin-dependent phospho-diesterase activity (Pala et al., 1991). Inhibition of AChEenzyme is reversible in the case of CMs and irreversible inthe case of OPs. This inhibition leads to an accumulation ofACh at synapses and neuromuscular junctions (NMJ) causingover stimulation and subsequent disruption of transmission ofimpulses in the central, peripheral, and autonomic nervoussystems (Gupta et al., 1986, Martin-Rubi et al., 1995;Misulis et al. 1987; Storm et al., 2000).

The acute neurobehavioral effects of CM insecticides areprimarily due to overstimulation of the cholinergic systemas a result of central and peripheral AChE inhibition. Theseeffects include lowered activity levels, fasciculation ofmuscles, salivation, lachrymation, with body tremors anddyspnea at high doses (Orzel and Weiss, 1966). CM pesti-cides are relatively short acting in terms of AChE inhibitionand the resultant acute neurotoxicity as compared to OPpesticides, because they inhibit AChE by carbamylationand OPs by phosphorylation, that is, reversible vs. irrevers-ible inhibition (O’Brien et al., 1966).

18.3 CLINICAL SIGNS AND SYMPTOMS

Although these pesticides have been widely used for decades,much of the available toxicity literature has focused on rela-tively few of them. Among these, even fewer have beenexamined for functional outcomes as a direct consequenceof exposure. The clinical signs and symptoms of OP andCM insecticide and nerve agent poisoning are generallyattributed to ACh accumulation and are commonly dividedinto three groups, muscarinic, nicotinic, and central(Table 18.1).

The effects on the respiratory system are complex: bronch-oconstriction and increased bronchial secretions are charac-teristic signs of OP poisoning, while pulmonary edema israrely seen (Lainee et al., 1991). Death in fatal poisoningsis normally caused by respiratory paralysis, which may beof central or peripheral origin (Tsao et al., 1990), dependingon the individual OP, provided the patient survives the acutecholinergic crisis.

Most studies in which ChE inhibition is measured reportthe presence (or lack) of overt signs of toxicity (tremors, sali-vation, lachrymation, diarrhea, miosis, etc.). In some studies,toxic signs were reported at doses producing greater thanabout 50% inhibition of brain and/or blood ChE, althoughthis level varied with the different chemicals assessed. Suchobservations are available for oxamyl, methomyl, aldicarb(Fayez and Kilgore, 1992; Gupta and Kadel, 1991; Gupta,1994), carbofuran (Ferguson et al., 1984; Gupta and Kadel,1989), and carbaryl (Orzel and Weiss, 1966).

Patients who experience CM poisoning show specificsymptoms like dry mouth, fasciculation, tremor, agitation,ataxia, weakness, tension, anxiety, irritability, restlessness,and headaches (Steenland, 1996; Stephens et al., 1995).However, many of these symptoms usually disappear whencholinergic imbalance has reversed.

18.4 ORGANOPHOSPHORUS ESTER-INDUCEDDELAYED NEUROTOXICITY/NEUROPATHY(OPIDN)

Organophosphate-induced delayed neurotoxic/neuropathiceffect, which is commonly referred to as OPIDN, occurs2 to 3 weeks after acute exposure to certain organophosphateinsecticides (Abou-Donia and Lapadula, 1990; Johnson,1969). OPIDN is characterized by a delayed onset of ataxia

TABLE 18.1 Major actions of OP and CM anticholinesterasesat various organs in the body

Receptor Target Organ Symptoms and signsCentral Central nervous system Giddiness, anxiety,

restlessness, headache,tremor, confusion,failure to concentrate,convulsions,respiratory depression

Muscarinic GlandsNasal mucosa RhinorrheaBronchial mucosa BronchorrheaSweat SweatingLachrymal LachrymationSalivary Salivation

Smooth muscleIris MiosisCiliary muscle Failure of accomodationGut Abdominal cramps,

diarroheaBladder Frequency

Heart BradycardiaNicotinic Autonomic ganglia Sympathetic effects:

Pallor, tachycardia,hypertension

Skeletal muscle Weakness, fasciculation

Source: Modified from Fuortes et al. (1993).

18.4 ORGANOPHOSPHORUS ESTER-INDUCED DELAYED NEUROTOXICITY/NEUROPATHY (OPIDN) 239

accompanied by a Wallerian-type degeneration of the axonand myelin in the most distal portion of the longest axontracts in both the central nervous system (CNS) and the per-ipheral nervous system (PNS) (Cavanagh and Patangia,1965). The clinical features are predominantly motor neuro-pathy and primarily manifested as numbness and weaknessof the lower extremities, followed by progressive ascend-ing weakness of limb muscles (Yang and Deng, 2007).Early studies were conducted to delineate the mechanismsof OPIDN as inhibition of AChE or butyrylcholinesterase(BuChE) by OPs; however, subsequent studies eliminatedboth esterases as targets for OPIDN (Aldridge and Barnes,1966). Neurotoxic esterase (NTE) has since been proposedto be a critical molecular target in OPIDN because OP com-pounds that cause OPIDN preferentially inhibit its enzymaticactivity (Johnson, 1969). We also reported delayed neurotoxicpotential of an OP whose role in the development of OPIDNhad previously been questionable (Choudhary et al., 2002b).Dichlorvos treatment in vitro caused a concentration andtime-dependent decrease in the activity of NTE (Ehrichet al., 1997). In an in vivo study, dichlorvos (200 mg/kgbody wt) caused inhibition of NTE in rat brain (Sarin andGill, 1997). However, inhibition of NTE is not the onlyfactor for axonal degeneration (Abou-Donia, 2003), andreports further suggest that OPs exert neurotoxic effectsin NTE knockout mice targets other than NTE mediatingOPIDN (Glynn, 2003). Potential alternate molecular targetsinclude calcium/calmodulin-dependent protein kinase II(CaM kinase II; Choudhary et al., 2006). The evidencesupporting this hypothesis, which has been reviewed byAbou-Donia (2003), includes observations that aberrant phos-phorylation of cytoskeletal proteins is present in OPIDN andmay be related to the OP-induced axonal degeneration anddemyelination, and that CaM kinase II, which phosphorylatescytoskeletal proteins, is activated by OPs that cause OPIDN.

In our studies, single subcutaneous doses of dichlorvos(200 mg/kg body weight) led to a consistent increase in theactivity of both microtubule associated protein kinases,namely Ca2þ/calmodulin-dependent and cAMP-dependentprotein kinases, at all postexposure intervals (day 7, 15, and21) as compared to that of controls (Choudhary et al.,2002a). Autoradiography followed by microdensitometricstudies demonstrated enhanced phosphorylation of 55 kDaand 280 kDa proteins in dichlorvos-exposed animals. Thesetwo proteins were confirmed to be tubulin and microtubuleassociated protein-2 (MAP-2). Further studies have shownthat the hyperphosphorylation of these two proteins interfereswith the assembly of neuronal microtubules, eventuallyleading to possible disruption of neuronal cytoarchitecture,resulting in axonal degeneration (Choudhary et al., 2006).Compounds reported to cause OPIDN in humans includechlorpyrifos (Lotti et al., 1986), mipafox (Bidstrup et al.,1953), isofenfos (Moretto and Lotti, 1998), trichlorfon(Hierons and Johnson, 1978; Vasilescu and Florescu, 1980;

Vasilescu et al., 1984), methamidophos (Aygun et al.,2003; McConnell et al., 1999; Senanayake and Johnson,1982), trichlornate (De Kort et al., 1986; Jedrzejowskaet al., 1980), and phosphamidon/mevinphos (Chuang et al.,2002). For a number of other OPs, claims of OPIDN wereless convincing, for example, parathion (De Jager et al.,1981), fenthion (Aygun et al., 2003; Martınez Chuecos,1992), and malathion (Dive et al., 1994).

Dickoff (1987) studied a patient who ingested 27 gm (500mg/kg) of carbaryl (1-naphthyl N-methylcarbamate), a pop-ular carbamate pesticide. After he recovered from acutecholinergic toxicity, acute weakness of arms and legs wasaccompanied by electrophysiologic findings consistent withaxonal peripheral neuropathy. Recovery began at 1 weekand continued for 9 months. Dithiocarbamates are currentlysuspected not only for neurotoxicity, but also as endocrine-disrupting chemicals.

18.5 DELAYED ONSET OF ENCEPHALOPATHYAND COMA IN ACUTE OP POISONING INHUMANS

Recently, A. Peter et al. (2008b) described the clinical charac-teristics and course of delayed onset of OP poisoning. Theyhave noticed patients with onset of deep coma 4 to 7 daysafter hospital admission. Thirty-five patients admitted to theintensive care unit (ICU) with severe OP poisoning and trea-ted with atropine and supportive therapy were followed up.Oximes were not administered to any of the patients. Threepatients developed delayed-onset coma after presentingwith normal or near normal Glasgow coma score (GCS).They developed altered conscious state rapidly progressingto deep coma, days after OP ingestion. During this period,the patients had miosis, nonreacting pupils, and no clinicallydetectable cortical or brainstem activity. Computed tomogra-phy of the brain and cerebrospinal fluid analysis were normal.Electroencephalogram showed bihemispheric slow wave dis-turbances. Two patients required atropine during this periodto maintain heart rate and reduce secretions. In all threepatients, no metabolic, infective or noninfective cause ofaltered conscious state was identified. All patients survivedto hospital discharge. Three other patients who developed areduction in GCS but did not progress to coma and recovered(GCS 10T) in 3 days may have manifested delayed onset ofencephalopathy. Delayed onset of coma appears to have a dis-tinct clinical profile and course, with complete resolution ofsymptoms with supportive therapy. Although persistent chol-inesterase inhibition is likely to have contributed to the mani-festations, the mechanism of coma and encephalopathy needto be explored in further trials. The good outcomes in theseresults suggest that therapy should not be limited in OP-and CM-poisoned patients developing profound coma orencephalopathy during hospitalization.


18.6 CHRONIC NEUROTOXICITY

There has been continuous interest over the last few years instudies concerning the effects of sublethal acute (Brown andBrix, 1998) or chronic (Salvi et al., 2003) exposure to OPs.There is increasing evidence that OPs may also cause along-term, persistent chronic neurotoxicity following eithera single acute high-dose exposure or repeated exposures tolow-level, subclinical doses of OPs. The clinical and epide-miological data in support of chronic OP neurotoxicity pre-sents with pathological lesions in both the PNS and CNS,but it is the latter that is primarily responsible for presentingneurologic symptoms and changes in neurobehavioral per-formance, reflecting cognitive and psychomotor dysfunction.The most sensitive manifestation of chronic OP neurotoxicityis a general malaise lacking in specificity and related to mildcognitive dysfunction, similar to that described for Gulf Warsyndrome (Kamel and Hoppin, 2004; McCauley, 2006,2009). The mechanisms underlying these effects are notknown, and the role of AChE inhibition is controversial(Abou-Donia, 2003; Kamel and Hoppin, 2004) and mayvary depending on the conditions of exposure (Lotti, 1995).Chronic neurotoxicity subsequent to a single acute exposureto OPs may be triggered by AChE inhibition. In fact, acutesublethal doses of OPs were shown to have long-term effectsin humans (Ohbu et al., 1997; Proctor et al., 2006). It shouldbe noted that although there were some fatalities (less than 15)in the Tokyo subway incident, most victims were intoxicatedby low and undetermined sarin levels and suffered from acuteand chronic symptoms. Kassa (2001) reported that a singleinhalation episode of clinically asymptomatic concentrationsof sarin (1.25 mg/L) in rats that induced a 30% inhibition oferythrocyte AChE activity yielded a significant change instereotype, an effect that was present 3 months after the endof the experiment. Such repetitive exposure also resulted inalterations in CNS excitability. Chronic exposure of rats toone-tenth of the LC50 of sarin for 30 days induced a decreasein M1 receptors in the olfactory tubercle, changes in bloodand brain ChE activities, and the expression of cytokinemRNA levels (Henderson et al., 2002). Guinea pigs receiving0.3, 0.4, or 0.5 � LD50 of repeated sarin injections exhibiteddisrupted sleep pattern in the EEG (Shih et al., 2006) and adecrease in red blood cell AChE activity to a low level ofbaseline. Symptoms of cholinergic toxicity were observedonly in animals receiving 0.5 � LD50 sarin. An experimentinvolving the application of multiple low doses of soman-induced alterations in long-term potentiation was determinedin rats (Armstrong et al., 1997).

Oral administration of dichlorvos to rats (70 mg/kg)inhibited not only AChE activity but also hexokinase, phos-phofructokinase, lactate dehydrogenase, and glutamatedehydrogenase activity (Sarin and Gill, 1998). Dichlorvosadministration also caused significant depletion in thebrain glycogen content along with increased glycogen

phosphorylase activity (Sarin and Gill, 1998). We alsoreported that dichlorvos administration caused a markeddecrease in both the ambulatory and stereotypic componentsof spontaneous locomotor activity of rats. The musclestrength and coordination of the dichlorvos-treated animalswas also significantly impaired. Besides, a marked deterio-ration in the memory function assessed in terms of the con-ditioned avoidance response was discernible at the end ofthe treatment schedule in the experimental animals (Sarinand Gill, 1997).

Neurobehavioral sequelae of acute and chronic OPexposure have been described in the literature for decades(Gershon and Shaw, 1961; Tabershaw and Cooper, 1966).Alvin et al. (2007) have demonstrated that rats, when injectedwith chlorpyrifos (CPF) subcutaneously (dose range, 2.5 to18.0 mg/kg) every other day over a period of 30 days, andfollowed by a 2 week CPF-free washout period, dose-depen-dent decrements in a water maze hidden platform task and aprepulse inhibition procedure were observed during the wash-out period, without significant effects on open field activity,rotarod performance, grip strength, or a spontaneous novelobject recognition task. After washout, levels of CPF andits metabolite 3,5,6-trichloro-2-pyridinol (TCP) were mini-mal in plasma and brain; however, ChE inhibition was stilldetectable. Further, the 18.0 mg/kg dose of CPF was associ-ated with (brain region-dependent) decreases in nerve growthfactor receptors and cholinergic proteins, including the ves-icular ACh transporter, the high affinity choline transporter,and the nicotinic acetylcholine receptor. These deficits wereaccompanied by decrease in anterograde and retrogradeaxonal transport measured in sciatic nerves ex vivo. Thus,low-level (intermittent) exposure to CPF has persistent effectson neurotrophin receptors and cholinergic proteins, possiblythrough inhibition of fast axonal transport. Such neurochemi-cal changes may lead to deficits in information processingand cognitive function. Stephens et al. (1995) studied therelationship between chronic (nonreversing) neuropsycholo-gical effects and acute exposure effects and investigated 77OP-exposed male sheep dippers. Acute exposure effectswere assessed prospectively using a purpose-constructedsymptoms questionnaire administered pre-, and 24 hourspost-exposure. Urine was analyzed for dialkylphosphatelevels to confirm acute exposure. Chronic effects wereassessed in a cross-sectional neuropsychological study inthe absence of recent exposure using computerized neuropsy-chological tests, the General Health Questionnaire, and thesubjective Memory Questionnaire. Simple correlation andmultiple linear regression analyses (adjusting for confoun-ders) were used to assess relationships between the changein total symptom reporting from baseline to 24 hours afterexposure and chronic effect outcomes. There was no evidenceof any association between reported symptom levels andchronic neuropsychological effects. The result of this studysuggests that chronic effects of OP exposure appear to

18.6 CHRONIC NEUROTOXICITY 241

occur independently of symptoms that might immediatelyfollow acute OP exposure. This has implications for exposurecontrol: individuals may experience chronic effects withoutthe benefit of earlier warning signs of toxic effects duringacute exposures.

Being an apical measure of nervous system function,motor activity is sensitive to perturbations of the motor, sen-sory, and/or integrative systems. This behavior may thereforereflect subtle effects of CMs and serve as an early indicator oftoxicity. There are numerous studies to correlate motoractivity with ChE inhibition. Carbaryl (1.5 to 75 mg/kg po)produced a linear correlation between RBC ChE inhibitionand motor activity decreases, and at the higher doses, activitywas maximally depressed while RBC ChE was only about60% inhibited (Padilla et al., 1996). In studying active avoid-ance behavior, it was concluded that the behavioral disruptionfrom carbaryl (5 and 10 mg/kg ip) was greater than could beaccounted for by the degree of ChE inhibition (25% to 42%;Goldberg et al., 1965). A few other reports also suggest be-havioral alterations including decreased motor activity (8 to28 mg/kg ip) with 16 mg/kg carbaryl producing 58% brainChE inhibition (Ruppert et al., 1983), hypothermia, anddecreased activity within 24 hours after dosing at 25 and 75mg/kg (Gordon and Mack, 2001), and changes in measuresof a functional observational battery (10 and 30 mg/kg ip;Moser et al., 1988). Behavioral studies have reporteddecreased open-field activity and rearing at 6.2 mg/kg po(Agarwal et al., 1988). The importance of the conditions ofthe assay used to measure carbamate-induced ChE inhibitionis well known. Motor activity decreases were highly predic-tive of ChE inhibition for N-methyl carbamates and viceversa. Furthermore, with the possible exception of oxamyl,the data support the use of brain ChE activity over RBCwhen evaluating neurotoxicity for these chemicals.

Slotkin et al. (2006) have reported three major findingsregarding developmental neurotoxicity: (a) A single agentmay target multiple events in neural cell replication anddifferentiation, thus spanning a wide range of developmentalstages; (b) unrelated chemicals that likely possess differentoriginating mechanisms of action can nevertheless convergeon a common set of final events in cell development, produ-cing similar outcomes; and (c) the potential utility of anapproach using neuronotypic cells in culture to screen sus-pected developmental neurotoxicants, enabling characteriz-ation of vulnerable stages, likely outcomes, and rankcomparisons of related and unrelated chemicals.

18.7 OPs AND THE GULF WAR SYNDROME

Although it is more than 15 years after the Gulf War(GW), the etiology behind the large number of veteranswho have unexplained illnesses still remains a mystery.More than a decade after the end of operation “Dessert

Storm” in 1991, a large number of veterans who servedduring the war continued to experience an array of chronicsymptoms including memory loss, fatigue, cognitive pro-blems, somatic pain, skin abnormalities, and gastrointestinaldifficulty. The possibility of long-term health effects associ-ated with low dose exposure to chemical warfare agents hasbeen a controversial issue. The National Academy ofScience Report (1982) could not rule out the possibility oflong-term effects due to a combination of chemicals.Japanese terrorist attacks with sarin in the mid-1990s pro-vided evidence of the long-term effects of toxic exposure tosarin, but individuals who did not reveal the symptoms ofacute toxicity at the time of the attacks have not been followedin longitudinal studies (Murata et al., 1997; Okumura et al.,2009). The Department of Veteran Affairs ResearchAdvisory Committee (2004) reviewed the animal studies onchronic effects of low-level sarin exposure and concludedthat low dose sarin exposure is associated with chronic indi-cators of both neurological and immunological impairments.These animal studies report a number of effects, includingdecreased immune function, down regulation of muscarinicreceptors in the hippocampus, chronic depression of AChEactivity, memory loss and cognitive changes, and persistentchanges on electroencephalograph reading in differentanimals. The animals studies indicate the potential forresidual effects from low-dose exposure to these agents andprevalence of multisymptom complexes among GW veter-ans. All points indicate the need for more studies of thisphenomenon.

The most sobering indication of neurological diseaseamong GW veterans comes from two studies indicating anincreased risk of amyotrophic lateral sclerosis (ALS) atapproximately twice the rate of comparison in the yearssince the GW (Haley, 2003; Horner et al., 2003). Haleyet al. (1999) found physiological measure of functionalbrain mass to differ between ill GW veterans and matchedveteran controls. They also assessed the level of central dopa-mine activity in the basal ganglia of ill GW veterans andcontrols and found evidence suggesting an injury of dopa-minergic neurons in the basal ganglia. A separate team ofinvestigators reported that GW veterans have evidence ofneuronal damage in the hippocampus (Menon et al., 2004).Three investigative teams have explored the possibility ofautonomic nervous system disorders in GW veterans(James et al., 2004). GW veterans have been found to haveabnormal responses to tilt-table testing when compared tohealthy controls. Davis et al. (2004) found that other indi-cations of autonomic dysfunction, including heart rate blunt-ing during sleep. Peckerman et al. (2000) also reportedblunted cardiovascular response among ill GW veterans.

Multiple investigators have examined the potential role ofpolymorphisms in veterans with unexplained illness, but theresults have been mixed. Haley et al. (1999) reported thatthe most severely symptomatic GW veterans exhibited


particularly low activity of paraoxonase (PON1) type Q, thetype that would be most active in neutralizing nerve gases.The decreased capacity of these veterans to metabolize OPchemicals might have contributed to their likelihood of devel-oping GW illness. Hotopf et al. (2003) found that PON1activity, which is a major determinant of OP toxicity inhumans, was significantly decreased in British veteransdeployed to the GW compared to nondeployed veterans.The PON1 gene presents several polymorphisms in thecoding and promoter regions that affect the catalytic effi-ciency of the enzyme toward different substrates (theQ192R polymorphism) and its level of expression (e.g., theC-108T polymorphism). Extensive research in transgenicanimal models clearly indicates that PON1 “status”, encom-passing both the Q192R polymorphism and the level ofPON activity, plays a most relevant role in modulating theacute toxicity of some but not all OPs (Costa and Furlong,2009; Costa et al., 2006). The important determinant is thecatalytic efficiency of each PON1 allozyme toward a specificsubstrate; thus, in case of chlorpyrifos oxon, PON1 providesprotection in vivo, and PON1R192 provides better protectionthan PON1Q192; in case of diazoxon, both alloforms providethe same degree of protection, while in case of paraoxon, thesubstrate after which the enzyme was named, PON1 does notprovide any protection due to an overall low catalytic effi-ciency of PON1 toward this substrate. These studies in trans-genic mice provide a convincing case of extrapolating theresults obtained in animals to humans; however, direct andconclusive confirmation of the relevance of PON1 status indetermining relative susceptibility to OP toxicity is still lack-ing. This too is expected to be a fruitful area of futureresearch.

18.8 PESTICIDES EXPOSURE ANDPARKINSON’S DISEASE

In terms of environmental toxins, those related to agriculturalwork have been closely studied in relation to neurodegenera-tive diseases. Multiple studies have evaluated potential riskfactors including environmental toxin exposure as a contri-buting factor for Parkinson’s disease (PD). Of particularinterest to researchers have been herbicides, pesticides, fungi-cides, to a lesser extent rural living, and well water consump-tion. Unfortunately, the literature in these areas is fraught withcontradictory findings, probably because of the methodologi-cal differences that exist between studies. Parkinson’s diseaseis a neurodegenerative disorder resulting, in part, from theprogressive loss of dopamine (DA) neurons in the substantianigra pars compacta (SNpc) (Tanner, 1989). Although theexact mechanisms by which low chronic exposures to pesti-cides induce PD phenotype in experimental models are notknown, several have been shown, even at relatively lowlevels, to produce excessive generation of reactive oxygen

species (ROS). Baldereschi et al. (2003) showed that occu-pational pesticide exposure is significantly associated withPD. Furthermore, their results suggest that by virtue ofobtaining a pesticide use license, regardless of the actualamount of time spent in contact with pesticides, it is relatedto an increased risk of PD. Additional evidence comes froma cohort study of French elderly that describes a significantassociation, in men only, between PD and occupationalexposure to pesticides (Baldi, 2003). The neurotoxic effectof rotenone, a plant-derived pesticide, is to increase the for-mation of cytoplasmic inclusions in the substantia nigra neur-ons and a-synuclein aggregation. Data from rat studies (Garyet al., 2003) demonstrate that rotenone promotes degenerationof the dopaminergic neurons and induces Parkinsonian symp-toms. Dithiocarbamate fungicides, including maneb (MB),have been implicated in selective dopaminergic neurotoxicityand mitochondrial dysfunction in rodents and humans, result-ing in motor deficits, and ultimately, Parkinsonism (Mecoet al., 1994; Soleo et al., 1996).

Future studies will need to improve assessment of pesti-cide exposure in individuals and consider the role of geneticsusceptibility. More studies are needed with different classesof OPs and CMs.

18.9 EFFECTS ON RECEPTORS

Due to the ubiquitous distribution of both nicotinic andmuscarinic cholinergic receptors throughout the body,exposure to OP and CM compounds has widespread toxicconsequences in several target organs. Virtually all cholin-ergic synapses can be affected by exposure to anticholinester-ase compounds such as OPs and CMs (Ovsepian, 2008;Slotkin et al., 2008). These include autonomic postganglionicparasympathetic nerve endings, sympathetic and parasympa-thetic ganglia, motor end plates of skeletal muscle, and, ofcourse, various regions of the CNS (Gupta, 2004; Guptaet al., 1985, 1986; Jett and Lein, 2006; Kobayashi et al.,2010). Hyperactivity at these synapses due to accumulationof ACh causes a variety of symptoms mediated by overstimu-lation of muscarinic and nicotinic receptors.

The density of receptors with a stereo specific binding sitefor nicotine (Romano and Goldstein, 1980) in the mammalianbrain is only 1% to that of muscarinic receptors. In the brain,the highest concentrations of nicotinic receptors are foundin the thalamus, cortex, superior colliculus, and striatum,whereas the lowest concentrations occur in the piriformcortex and hippocampus. Thus, the distribution of nicotinicreceptors in the CNS clearly differs from that of muscarinicreceptors. For further details on brain regional distributionof muscarinic and nicotinic ACh receptors, readers arereferred to a recent review published elsewhere (Gupta,2004). It is also quite evident that most of the cholinergiceffects of OPs in the CNS are mediated via muscarinic

18.9 EFFECTS ON RECEPTORS 243

rather than nicotinic receptors (Patial and Kapoor, 1998). Thisis important because the most dramatic toxic actions of OPsare mediated via their effects on cholinergic receptors in theCNS and subsequent stimulation of other neurotransmittersystems in the brain, as well as via cholinergic receptorstimulation in other target organs, subsequent to the initialeffects of OPs on AChE and other key cholinergic elements(Pope, 2006).

Several CMs have also been shown to interact with cholin-ergic receptors. The CM physostigmine and related ChEIsinteract with muscarinic ACh receptors (mAChRs)(Lockhart et al., 2001), as well as nicotinic ACh receptors(nAChRs). Low concentrations of physostigmine and ana-logues agonize or potentiate neuronal nAChRs, whereashigh concentrations of these drugs block neuronal mAChRs(Zwart et al., 2000). Several CM insecticides like ChEIs, ami-nocarb, aldicarb, and carbaryl at 100 mM, displace 3H–AChfrom muscle type nAChRs in Torpedo electric organ mem-branes (Eldefrawi and Eldefrawi, 1983). Additionally, car-baryl concentration-dependently potentiates and inhibitsneuronal nAChR channels in rat pheochromocytoma PC12cells (Nagata et al., 1997). Smulders et al. (2004) demonstratethat the CM pesticides fenoxycarb and EPTC inhibit rat a4b2type neuronal nAChRs in a way that depends on the concen-tration of the agonist ACh, which is used to activate theligand-gated ion channels. Channel opening is not requiredfor a block and the CM inhibits the ACh induced ion currentindependent of the state of the ion channel. A Recent study ofthe kinetics of blocking of human muscle type nAChR gatedion channels expressed in HEK 293 cells by tacrine also con-cludes that single-site channel blocking cannot account forthe effects observed, whereas multiple-site sequential blockmodels do (Prince et al., 2002). Like fenoxycarb, tacrinewas shown to interact with the agonist recognition site ofthe nAChR, but only at much higher concentrations thanthose required to inhibit ion channels (Prince et al., 2002).CMs are also known to affect some transient receptors.TRPA1 is a member of the transient receptor potential(TRP) family and is restrictively expressed in sensory neuronsof dorsal root ganglia, trigeminal ganglia, and hair cells of theinner ear. TRPA1 channels are known to be activated by 30-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).URB597 was described previously as an inhibitor of fattyacid amide hydrolase (FAAH), which degrades the endogen-ous cannabinoid anandamide. Using Caþ2 influx assays andpatch-clamp electrophysiology, it has been demonstratedthat URB597 activated recombinant human and rat TRPA1channels transiently expressed in HEK293-F cells, as wellas rat TRPA1 expressed in cultured DRG neurons(Niforatos et al., 2007).

In addition to their indirect effects on muscarinic receptorsthrough AChE-mediated changes in ACh levels, many OPand CM pesticides can affect their expression and functiondirectly (Marinovich et al., 2002). Paraoxon, dichlorvos,

and tetraethyl pyrophosphate (TEPP) were found to be non-competitive antagonists of muscarinic receptors in bovinecaudate nuclei labeled with [3H] quinuclidinyl benzilate([3H]-QNB) at concentrations that had no effect on AChEactivity (Volpe et al., 1985). It was found that the activemetabolite of the pesticide chlorpyrifos, chlorpyrifos- oxon,bound to muscarinic receptors in rat striatum identified with[3H]-CD noncompetitively with an IC50 value of approxi-mately 22 nM and resulted in a covalent modification of thereceptor (Huff et al., 1994). It was suggested that because[3H]-CD binds to M2 receptors (Huff and Abou-Donia,1995) with high affinity, direct actions on a subset of muscar-inic receptors, in addition to their actions on AChE, couldaccount for some of the toxicity of OP compounds.Inhibition of [3H]-CD binding by OPs was observed at nano-molar to micromolar concentrations (Bakry et al., 1988).Other studies confirmed that binding of ligands to muscarinicreceptors is inhibited by OPs at concentrations far belowthose that inhibit AChE, as low as the picomolar range(Katz and Marquis, 1989; Silveira et al., 1990).

Direct effects of OP compounds on muscarinic receptorswere studied by using rat brain membranes or cultures ofhuman neuroblastoma N1E-115 cells (Bakry et al., 1988).Op nerve agents of G series (sarin, soman, or tabun) hadno effect on the receptors, but a nerve agent of V series(VX) and echothiophate inhibited, in a competitive man-ner, the binding of 1-quinuclidinyl(phenyl-4[3H])-benzilate([3H]QNB) and of [3H]pirenzepine ([3H]pZ) to muscarinicreceptors. Ward and Mundy (1996) explored the interactionof eight OP compounds with muscarinic receptors withregard to their ability to inhibit AChE activity in vitro intissue homogenates from rat hippocampus and frontalcortex. Of the compounds tested, only ecothiopate competedfor [3H] QNB binding and only at concentrations exceeding100 mM. The OP anticholinesterases did compete, however,with a muscarinic receptor agonist, [3H]CD ([3H]cis-methyldioxolane) that binds with a high affinity to 10% and3% of muscarinic receptors in the frontal cortex and hippo-campus, respectively. Ecothiopate and DFP were potentinhibitors of [3H] CD binding as were the active oxonforms of parathion, Malathion, and disulfoton. A similar pat-tern of potency was observed for the inhibition of brain AChEactivity, indicating that there was a strong correlation betweenthe abilities of OP compounds to inhibit [3H] CD binding andto inhibit AChE activity. Yagle and Costa (1996) exposedSprague Dawley rats to doses of 2 mg/kg/day of disulfoton[S-(2-(ethylthio)ethyl) phosphorothionate] for 14 consecu-tive days, and measured messenger ribonucleic acid(mRNA) levels of muscarinic receptor M1, M2, and M3 sub-types, immediately after the cessation of the exposure, as wellas after a 28-day recovery period. There was a markedreduction in the levels of muscarinic receptor subtypes in sev-eral brain regions immediately after the exposure, but after therecovery period only the M2 subtype mRNA levels remained


decreased, indicating that this receptor subtype may be moresensitive than the others toward OP-induced alterations.Recently, it has been reported that dichlorvos exposurecause significant reduction in the expression of M1, M2,and M3 muscarinic receptor subtypes in high dose group ani-mals whereas, in low dose group animals the expression ofonly M2 receptor were found to be reduced significantly(Verma et al., 2008b). Also, marked reductions in [3H]QNB binding were seen immediately after the cessation ofthe exposure, indicating a marked reduction in muscarinicreceptor numbers. The findings of Yagle and Costa (1996)are consistent with earlier observations by Doebler et al.(1983a), who showed that repetitive s.c. injections ofsoman at a 0.5 LD50 dose level caused a marked and progress-ive RNA reduction in caudate and cortex. Soman inducedreductions of overall brain RNA levels were mediated viamuscarinic receptor stimulation because the same could becompletely blocked by pretreatment with atropine whengiven together with pralidoxime (Doebler et al., 1983b).Feeding mice with parathion (0.4-500 mg/kg/day) in theirdiet for 14 days inhibited mouse brain AChE activity andtransiently reduced the maximal binding of [3H]QNB,[3H]NMS, and [3H]4-DAMP ([3H]-4-diphenylacetoxy-N-methylpiperidine methiodide) binding without affectingreceptor affinities for these ligands (Jett et al., 1993).Inhibition of whole brain AChE varied between 10% and80% in a dose-dependent fashion. These results suggestthat dietary doses of parathion induced a transient down regu-lation of different muscarinic receptor subtypes in the mousebrain (Churchill et al., 1985; Jett et al., 1994).

OP anticholinesterases may have direct actions on nic-otinic receptors. There are data to suggest that OP anti-cholinesterases bind to allosteric sites of the cholinergicnicotinic receptors as identified by inhibition of[3H]phencyclidine binding, but some can also bind to thereceptor’s recognition site because they inhibit [125I] a-bungarotoxin binding (Bakry et al., 1988). Soman andecothiopate at micromolar concentrations acted like partialagonists of the nicotinic receptors and induced receptordesensitization. On the other hand, VX acted like an openchannel blocker of the activated receptor (i.e., a compoundthat can only gain access to the ion channel when it is inthe open configuration) and also enhanced receptor desensiti-zation. Bakry et al. (1988) suggested that the toxicity of OPcompounds may include some direct actions on the nicotinicreceptor if their concentration in the circulation exceeds themicromolar level. The mechanism of this nicotinic recep-tor-OP compound interaction remains to be elucidated. Chiand Sun (1995) found that soman, sarin, tabun, and phency-clidine did not modify the binding of [125I]a-cobratoxin tothe nicotinic receptor. It seems that OP compounds haveboth direct and indirect effects, mediated mainly via AChaccumulation subsequent to AChE inhibition. Even if OPshave some direct effects on a wide variety of receptors and

their subtypes, it seems that they only slightly modify theoverall effects of these agents. The vast majority of theiractions are attributable to ACh accumulation on cholinergicreceptors and subsequent glutamatergic activation (Ravehet al., 2003).

The inhibition of brain AChE by CMs affects differentsubtypes of neuronal nicotinic receptors, independently ofAChE inhibition. This implies that neuronal nicotinic recep-tors are additional targets for some CM pesticides and thatthese receptors may contribute to carbamate pesticide toxi-cology, especially after long-term exposure (Smulderset al., 2004).

18.10 OXIDATIVE STRESS

Pesticide exposure in experimental rodent and cell culturemodels have been linked to reactive oxygen species (ROS)and reactive nitrogen species (RNS) generation, and/or aninflammatory response that potentiates ROS and RNS pro-duction. The brain regions accountable for initiation andpropagation of AChEIs convulsions have been foundvulnerable to oxidative/nitrative stress (Gupta et al., 2001,2007; Milatovic et al., 2006, 2009). The substantia nigra(SN) region of the brain is also found vulnerable to oxidativestress. The auto-oxidation of dopamine (Hastings, 1995), theenzymatic deamination of DA by monoamine oxidases(Halliwell, 1992), and the high iron content which catalyzesFenton reactions make SN vulnerable to oxidative stressand cellular injury. There is growing evidence supportingthe involment of ROS and RNS in excitotoxicity injury.Excessive activation of cholinergic and glutamatergicreceptor is thought to be responsible for excitotoxicity(Olney et al., 1986). Mancozeb (MZ) is neurotoxic tomesencephalic DA and GABA neuronal cell populationsfollowing acute exposure (Domico et al., 2006; Soleo et al.,1996). In addition, MZ and other similar Ethylenebisdithio-carbamates (EBDCs), like maneb (MB), have been reportedto be inhibitors and/or uncouplers of the mitochondrialelectron transport chain (Domico et al., 2006; Zhang et al.,2003). It has been reported that 15 mM MZ or MB uncouplethe mitochondrial electron transport chain (ETC), whilehigher doses (30 mM) inhibit respiration (Domico et al.,2006). Moreover, Zhang et al. (2003) found that MBselectively inhibits complex III of the ETC. Duringnormal respiration, small amounts of ROS are produced asbyproducts of the ETC process. However, perturbations inmitochondrial respiration can lead to excessive ROS andRNS generation and inundate cellular antioxidant capacity,leading to DNA damage, lipid peroxidation, proteinmodification, and eventually cell death (Rao andBalachandran, 2002).

The common initiating mechanism of excitotoxicity isthought to be frequent stimulation of nicotinic acetylcholine

18.10 OXIDATIVE STRESS 245

receptor at the mammalian neuromuscular junction and mus-curanic, nicotic, and glutamatergic receptor in the brain(Dettbarn et al., 2001; Gupta et al., 1985; Kobayashi et al.,2010). It has been hypothesized that increased ACh levelsfollowing AChE inhibition activate glutamatergic neuronscausing the release of glutamate, which ultimately results inexcitotoxicity via increased intracellular calcium and acti-vation of nitric oxide synthase following NMDA receptoractivation (De Groot et al., 2001; Gupta and Milatovic, 2009).

It has been shown that an over-expression of AChE intransgenic mice produces progressive neurochemical,neuro-morphological, and neurocognitive alteration, at leastin spatial memory in adult mice (Andres et al., 1997).Butyryl cholinesterase (BuChE), also called pseudo cholin-esterase or plasma cholinesterase is an enzyme geneticallydifferent from AChE, although it shares some important func-tions, such as ACh hydrolysis (Darvesh et al., 2003).Individual susceptibility to cholinesterase inhibitor com-pounds is due, in part, to individual genetic variations ofthis enzyme (Fontoura-da-Silva et al., 1996). A cholinergiccrisis, together with reduced levels of plasma BuChE activity,leads to the diagnosis of over cholinergic syndrome (Martin-Rubi et al., 1995). Figure 18.2 depicts major cellular events inOP and CM induced reactive oxygen species.

A proposed mechanism for chronic OP neurotoxicity issupported by the fact that nitric oxide synthesis inhibi-tors block OP-induced seizures (Aschner et al., 1999).However, the role of AChE inhibition in this sequence of

events has yet to be established. In contrast, chronic OP neu-rotoxicity induced by repeated exposure to subclinical OPdoses has been reported to occur in the absence of AChE inhi-bition (Abou-Donia, 2003; Kamel and Hoppin, 2004),suggesting that mechanisms other than anticholinesteraseactivity mediate the neurotoxic effects elicited by thisexposure scenario. We recently reported that chronic OPexposure (dichlorvos) may lead to significant increase inmitochondrial Ca2þ uptake (Kaur et al., 2007). Our resultsalso indicated decreased mitochondrial electron transferactivities of cytochrome oxidase (complex IV) along withaltered mitochondrial complex I, and complex II activity,which might have resulted from elevated mitochondrial cal-cium uptake (Kaur et al., 2007). The alterations in the mito-chondrial calcium uptake and mitochondrial electrontransfer enzyme activities in turn might have caused anincrease in malondialdehyde, protein carbonyl, and 8-hydoxydeoxyguanosine formation as a result of enhancedlipid peroxidation, as well as protein and mtDNA oxidation.All this could have been because of enhanced oxidative stress,decreased GSH levels, and decreased Mn-SOD activity in themitochondria isolated from dichlorvos treated rat brain. Thus,chronic OP exposure has the potential to disrupt cellular anti-oxidant defense system which in turn triggers the release ofcytochrome c from mitochondria to cytosol as well as cas-pase-3 activation in dichlorvos treated rat brain as revealedby immunoblotting experiments. Low-level long-term orga-nophosphate exposure finally resulted in oligonucleosomal

Figure 18.2 Major events in organophosphate and carbamate induced reactive oxygen species.


DNA fragmentation, a hallmark of apoptosis. These studiesprovide evidence of impaired mitochondrial bioenergeticsand apoptotic neuronal degeneration after chronic low-levelexposure to OPs (dichlorvos) (Kaur et al., 2007). Theseresults also agree with the other reports (Abou-Donia,2003) which suggest an intriguing possibility that repeatedexposures to sublethal or subclinical doses of OPs increasesapoptotic neuronal death via oxidative stress.

The DAergic neuronal cell population has been hypoth-esized to be vulnerable to oxidative stress because of theauto-oxidation of DA itself, that is, DA is metabolized to3,4-dihydroxyphenyl acetic acid via monoamine oxidase,producing H2O2 (Halliwell, 1992; Hastings, 1995).Mesencephalic GABAergic neurons are not at risk of suchintrinsic oxidative stress, but are equally as vulnerable asDA. Intracellular oxidases, including xanthine oxidase,monoamine oxidase, and cyclooxygenase-2, are availableto transfer electrons to exogenous ligands like MZ, resultingin ROS generation. In addition, the oxidation of dopamineproduces dopamine quinones, reactive species that can alsocause damage to lipids, proteins, and DNA (Hastings,1995). Mn-EBDCs, like MB and MZ, can catalyze the oxi-dation of catechols (Fitsanakis et al., 2002). If DA becomesavailable to MZ or MB in the cytosol or extracellularly, theEBDC-catalyzed oxidation of catecholamines denotesanother potential source of highly reactive free radicals andROS. Mechanism for CMs induced ROS generation is sum-marized in Figure 18.3 below.

18.11 SEIZURE ACTIVITY AND OXIDATIVESTRESS

Pirimicarb, a CM insecticide, is known to produce clinicalsymptoms ranging from the classic cholinergic syndrome toflaccid paralysis and intractable seizures. The similaritybetween EEG patterns, repetitive clonic convulsions and neu-ropathology in following status epilipticus (SE), and seizuresinduced by AChEIs suggests a common mechanism ofinitiation and propagation of the lesions. The AChEI-inducednormal cell death appears to be a consequence of a series ofextra- and intracellular events leading to the accumulationof Ca2þ ions in the cell and the generation of oxygen derivedfree radicals causing irreversible destruction of cellular com-pounds such as plasma lemma, mitochondria, and other intra-cellular membranes of the cytoskeleton. Hirokazu et al.(2000) demonstrated disulfoton exposure caused synapticgenes encoding AChE in muscle and sciatic nerves were sig-nificantly decreased at 12 hours after the administration; thisdown regulation lasted for up to 30 days after administration.These results indicated that administration of OP can decreaseAChE and nAChR expression in the neuromuscular junctionand is suggestive of multiple mechanisms of down regulationof both AChE and nAChR, some of which might involvealterations at the transcriptional level.

On the other hand, if laboratory animals are pretreated withsublethal doses of OPs and then treated with a higher, moretoxic dose of the same compound, they develop

Figure 18.3 Mechanism of carbamate induced oxidative stress (1) Carbamate ligand attack on NADPH oxidase; (2) cytosolic and trans-membrane subunits of enzyme couples with one another; (3) in the presence of NADPH oxidase electron transfers from NADPH to oxygenmolecule; (4) generation of superoxide anion and other free radical species and ultimately oxidative stress.

18.11 SEIZURE ACTIVITY AND OXIDATIVE STRESS 247

“Tolerance”. Tolerance to anti-AChE insecticides was firstdescribed by Barnes and Denz (1951) when they noticedthat rats survived a long-term feeding trial with the highlytoxic parathion. Down regulation of muscarinic receptorshas been demonstrated repeatedly in a variety of experimentalparadigms with several OPs (Abdallah et al., 1992; Costaet al., 1982; Gupta et al., 1985; Russell and Overstreet,1987). It has been shown that the degree of AChE inhibitioncorrelates well with decreases in muscarinic receptors (Jettand Lein, 2006; Jett et al., 1993). The rats exposed to para-thion or methyl parathion during development did not showany signs of overt toxicity, or the signs subsided followingsubsequent exposure due to tolerance development as indi-cated by decreases in AChE inhibition and muscarinic recep-tors binding sites (Gupta et al., 1985; Stamper et al., 1988).However, these rats exhibited spatial memory deficits in theradial arm and T-mazes. This was one of the first studies tosuggest that “compensatory” changes in receptors mayresult in tolerance to some effects (e.g., lethal) but may infact be an underlying mechanism for other more subtle effectssuch as memory impairment. In light of the prominent rolethese receptors play in cognitive function, it is not surprisingthat changes in the availability of muscarinic binding sitesdue to OP-induced down regulation have been associatedwith altered behavior. Jett et al. (1993, 1994) also observedthat protein and mRNA of certain subtypes of muscarinicreceptor may be more affected by OP exposure than others.Other laboratories have corroborated these findings (Yagleand Costa, 1996) and pointed to the M2 subtype as beingespecially vulnerable to OPs (Bakry et al., 1988; Katz andMarquis, 1989; Silveira et al., 1990). Paraquat (PQ) isknown as a potent redox cycler, which in the presence of mol-ecular oxygen can generate superoxide anions leading to oxi-dative stress and consequently neuronal cell damage or death(Jones and Vale, 2000). Intrahippocampal injection of PQcauses neuronal cell death in rats, mostly by apoptosis(Melchiorri et al., 1998). Direct delivery of manganese-ethy-lenebis-dithiocarbamate (Mn-EBDC) to the lateral ventriclesproduces selective degeneration of dopaminergic neurons inrats (Zhang et al., 2003). Most importantly, the ability ofMn-EBDC to inhibit preferentially mitochondrial complexIII has been demonstrated. A recent study (Zhou et al.,2004) provided evidence that in vitro exposure to Mn-EBDC is able to inhibit proteasomal function and inducea-synuclein aggregation. It was suggested that the neurotoxiceffects as well as proteasomal inhibition were associated withoxidative stress because they were prevented by pretreatmentwith antioxidants. Mn-EBDC is metabolized to the EBDCanion and manganese, both of which are neurotoxic.

18.12 SIGNALING PATH WAY

The experimental evidence supports the hypothesis that OPsmodulate intracellular signaling pathways downstream of

receptors and suggests that the diverse neurotoxic effects ofmany OPs may reflect their influence on multiple intracellularsignaling pathways (Izrael et al., 2004). Functional studiesexamining the effects of OPs on signaling events downstreamof muscarinic receptor activation further support the hypoth-esis that OPs can interact directly with M2 receptors (Vermaet al., 2008a). Activation of M2 and M4 receptors generallyreduces the activity of adenylyl cyclase, which decreasescAMP production; whereas activation of M1, M3, or M5

receptors increases phosphoinositide-specific phospholipaseC activity, which increases release of inositol triphosphate(Schuh et al., 2002). A comparative study of paraoxon,malaoxon, and chlorpyrifos oxon in slice cultures of rat fron-tal cortex indicated that all three OPs inhibited cAMP for-mation in a concentration dependent manner (Ward andMundy, 1996). In contrast, none of these OPs affectedeither basal or carbachol-stimulated phosphoinositide turn-over. These data suggest that OPs activate M2/M4 receptorsthrough direct interactions and not as the result of increasedlevels of endogenous ACh consequent to AChE inhibition.Other in vitro studies of rat striatum predicted and confirmedthat, generally, OPs act to stimulate M2/M4 receptor function(Axelrad et al., 2002; Huff and Abou-Donia, 1994; Jett et al.,1991). Chlorpyrifos-oxon was also found to inhibit c-AMPsynthesis in striatal dissociated cells (Huff et al., 1994) butin an atropine-insensitive manner. Chlorpyrifos-oxon wasalso shown to inhibit c-AMP synthesis in NG108-15 cellsand in Chinese Hamster Ovary (CHO) cells transfectedwith muscarinic receptor subtypes (Huff and Abou-Donia,1995), but only at relatively high concentrations.

Among the many potential developmental neurotoxicants,the greatest attention has been paid to pesticides, in light oftheir widespread use in home and in agriculture. The develop-ing nervous system is more sensitive than the mature nervoussystem to the neurotoxic effects of OPs and CMs. PC12 cells,is a standard in vitro model for neuronal development that hasalready been used to characterize essential features of thedevelopmental neurotoxicity of OPs and CMs (Jamesonet al., 2006). PC12 model enables the detection of toxicantactions that target cell replication as well as the eventsinvolved in differentiation and the phenotypic emergence ofspecific neuronal features.

Song et al. (1997) suggested that cAMP signaling mayalso be a target for the developmental neurotoxicity of chlor-pyrifos based on evidence that postnatal exposure in neonatalrats decreases adenylyl cyclase expression and function andalters cAMP levels under a variety of experimental manipula-tions. The actions of the OPs and CMs in vivo are clearlymodified by their binding to serum and tissue proteins.Also, the concentration of these proteins is lower in thefetus than in the adult (Gupta et al., 1984; Thom et al.,1967; Yaffe and Stern, 1976), so that at comparable concen-trations of each neurotoxicant, the fetus will bear a dispropor-tionate burden of adverse effects. It is important to note that


the OPs show strong binding to serum proteins both in vivoand in vitro (Qiao et al., 2001), which reduces their bio-effective concentrations. The effect is highest for chlorpyri-fos, less important for parathion, even lower for diazinon,and lowest for physostigmine (Whelpton and Hurst, 1990;Wu et al., 1996). Diazinon and physostigmine, with theirlower binding, exert greater net effects than would otherwisebe expected. Physostigmine is effective as a cholinesteraseinhibitor and shares some OP like effects on cell differen-tiation, but it is much less capable of eliciting immediate anti-mitotic actions (Jameson et al., 2006; Qiao et al., 2001); it isconsiderably less effective than OPs as a developmental neu-rotoxicant in lower organisms (Buznikov et al., 2003).

Further studies indicated that the effects of both pre- andpostnatal chlorpyrifos exposure on adenylyl cyclase signalingpersist in the adult brain (Dresbach et al., 2004). We have alsoreported that chronic dichlorvos exposure (6 mg/kg b.w./day) for a period of eight weeks caused significant reductionin both high affinity (HA) and low affinity (LA) cholineuptake (CU), with maximal effect being observed in thebrain stem followed by cerebellum and cerebrum (Rahejaand Gill, 2007). Muscarinic receptor binding was signifi-cantly decreased in brainstem and cerebellum as reflected inthe decreased receptor number (Bmax), without any changein the binding affinity (KD) of the receptors. Dichlorvos treat-ment caused marked inhibition in cAMP synthesis as indi-cated by decreased adenylate cyclase activity as well ascAMP levels in cerebrum, cerebellum and brain stem. Ourstudy shows that OPs may interact with a muscarinic recep-tor-linked second messenger system and this could be apotential mechanism for the neurotoxic effects observedafter repeated exposure to low levels of OPs, which are unex-plainable on the basis of cholinergic hyperactivity (Rahejaand Gill, 2007).

Phosphatidyl inositol (PIs) also play a key role in muscar-inic cell signaling as precursors of second messengers whichare responsible for transducing the signal from the cell surfacemuscarinic receptors into the cell (Berridge, 1989). PIs aredifferent from all other membrane phospholipids becausekinases are able to further phosphorylate their inositol headgroups. Although PIs account for about 10% of the total phos-pholipid composition of the cell membrane in most cells,phosphatidylinositol-4,5-bisphosphate (PIP2) is a minormembrane component that makes up between 1% and 10%of the total PI pool. Its concentration is higher in the brainthan in any other tissue, which suggests that it plays an impor-tant role in the specialized functions of the nervous system.Stimulation of calcium-mobilizing receptors, to whichmuscarinic receptor subtypes belong, initiates a bifurcatinghydrolysis pathway of PIP2, an acidic membrane-boundphospholipid. Hydrolysis of this membrane phospholipidresults in the formation of two second messengers, InsP3and DAG (Downes and Michell, 1981). DAG stimulatesPKC, an enzyme vital for several important cellular

functions, including receptor-mediated activation, whereasInsP3 diffuses into the cytosol to release calcium from non-mitochondrial internal stores and, perhaps indirectly, tostimulate the entry of extracellular calcium into the cell(Fig. 18.4) (Berridge, 1987). Ultimately, the PI pathwayleads to the reformation of PIP2, and the cycle is againprimed. Lithium inhibits the metabolism of inositol phos-phates in the final dephosphorylation step. Thus, lithium islikely to reduce the supply of free inositol required to main-tain the formation of lipid precursors used for cell signaling.These pathways regulate several cellular processes, includingmetabolism, contraction, neural activity, and cell proliferation(Berridge, 1989; Berridge and Irvine, 1989). Katz andMarquis (1992) exposed human SK–N–SH neuroblastomacells to low concentrations of paraoxon or carbachol, adirect muscarinic agonist. They reported that Paraoxon inhib-ited the N-[3H]methylscopolamine ([3H]NMS) muscarinicreceptor binding. However, Paraoxon at low concentrations(0.1 nM), caused a time-dependent increase in the PI turn-over, whereas high concentrations of carbachol were requiredfor the same effect. Pertussis toxin, a G-protein inhibitor, andneomycin, a PLC inhibitor, inhibited cholinergic-inducedfacilitation of PI hydrolysis. It seems that paraoxon maymodulate signal transduction in neuronal cells by indirectactivation of muscarinic receptors; that is, by elevatinglevels of ACh, as well as by acting at a site distal to the recep-tor (Katz and Marquis, 1992). Bodjarian et al. (1992) demon-strated that soman also facilitates PI hydrolysis inhippocampal slices from rats. The effect was mediatedthrough muscarinic receptor subtypes M1 and M3 sub-sequent to AChE inhibition and ACh accumulation. Eventhough the M2 muscarinic receptor subtype is preferentiallycoupled with inhibition of adenylate cyclase, leading toreduction of levels of cAMP, it was also shown to be associ-ated with PLC-mediated hydrolysis of PIs. Thus, findingsfrom in vitro studies are consistent with the assumption thatOP compounds affect neuronal PI signaling and that this ismediated via cholinergic muscarinic receptor activation.

Interestingly, the mechanism of action for the OP steroido-genesis inhibitor, diethylumbelliferyl phosphate, is alsobelieved to be mediated through an interaction with thecAMP/PKA pathway (Choi et al., 1995). OPs can activateCaM kinase II (Abou-Donia, 2003). We have reported thatchronic dichlorvos administration caused significant rise inthe intrasynaptosomal calcium levels (Raheja and Gill,2002). The activity of major calcium expelling enzymeCa2þATPase was found to be declined. Also, the depolariz-ation induced calcium uptake via voltage operated calciumchannels increased significantly. Concomitant to the increasein intrasynaptosomal calcium, calpain activity was found tobe increased. Dichlorvos could be mediated through modifi-cations in the intracellular calcium homeostasis which maylead to impaired neuronal function. Studies of CHOK1cells indicate that OPs may also activate extracellular

18.12 SIGNALING PATH WAY 249

signal-regulated kinase (ERK) signaling pathways (Bomserand Casida, 2000), possibly via increased levels of diacylgly-cerol (DAG) subsequent to OP inhibition of DAG lipase(Bomser et al., 2002). It has been reported that chlorpyrifosalso interferes with muscarinic receptor mediated transloca-tion of protein kinase C (PKC)-�/and decreases the basallevels of both PKC-�/and PKC-[3II, the two isoformsknown to be relevant to behavioral performance (Izraelet al., 2004).

Dithiocarbamates is an important class of carbamate com-pounds known to produce neurotoxic effects. Two classes ofcommonly used dithiocarbamates include mono and dialkyldithiocarbamates. N-methyldithiocarbamate is a mono alkylsubstituent used principally as soil fumigant, whereas N,N-dimethyldithiocarbamate (DMDC) and N,N-diethyldithiocar-bamate (DEDC) are used in agricultural, medical, andindustrial fields. Metabolic pathways observed for decompo-sition of dithiocarbamtes are presented in Figure 18.5.

18.13 EFFECTS ON GENE EXPRESSION

The signaling pathways identified as potential targets in OPneurotoxicity can modulate gene expression via alterationsin the expression levels or activational status of transcriptionfactors. One transcription factor of considerable interest in OP

neurotoxicity is Ca2þ/cAMP response element bindingprotein (CREB), which is activated via phosphorylation bya variety of signaling pathways, including cAMP/PKA,MAP kinase/ERK, p38, and CaM kinase II (Lonze andGinty, 2002). Numerous studies have indicated that CREBis critical to several forms of use-dependent synaptic plas-ticity and transcription-dependent forms of memory, and evi-dence supports a major role for CREB in cell survival anddifferentiation during brain development (Lonze and Ginty,2002; Shaywitz and Greenberg, 1999). Since impairmentsof brain development and memory function are two primaryneurological effects observed in laboratory studies with OPs,Schuh et al. (2002) hypothesized that the mechanisms under-lying these effects may include alteration of the expression oractivational status of CREB. OPs caused similar effects in pri-mary cultures of hippocampal neurons. The mechanism(s) bywhich OPs activate CREB is not known but is probably notmediated by OP effects on adenylyl cyclase activity, whichare predominantly inhibitory. Chlorpyrifos and its oxonmetabolite phosphorylate CREB directly (Bomser et al.,2002). However, direct phosphorylation of CREB cannotbe the mechanism by which TCP induces increased pCREBbecause TCP does not contain a phosphorus atom. Possiblemechanisms that have yet to be addressed experimentallyinclude activation of CaM kinase II (Abou-Donia, 2003) orenhanced DAG signaling (Bomser et al., 2002). There is

Figure 18.4 Receptor-mediated phosphoinositide turnover: An agonist (A) such as ACh binds to receptor (R), causing the activation of a Gprotein (Gp) that, in turn, stimulates PLC. The PLC hydrolyses PIP2, generating InsP3 and DAG. InsP3 binds to a specific receptor on the mem-brane of a nonmitochondrial cell organelle that contains Ca2þ, causing the release of calcium from the intracellular stores. The binding of InsP3to its receptors is inhibited by heparin and increased intracellular Hþ and Ca2þ. InsP3 can further be phosphorylated to InsP4, which may facili-tate the entry of calcium into the cell through the plasma membrane or may trigger the movement of calcium within the cell. DAG activates PKC,which phosphorylates a large number of substrates. Activation of cAMP-dependent protein kinase A (PKA) leads to phosphorylation of InsP3receptor protein.


documentation of OP effects on other transcription factorsimportant in neurodevelopment and synaptic plasticity.Thus, OPs elevate levels and activation of c-fos (Adamkoet al., 1999; Gupta et al., 2007), cause developmental stage-specific changes in AP-1 and Sp-1 expression and DNAbinding activity (Crumpton et al., 2000), and stimulate phos-phorylation of c-Jun (Caughlan et al., 2004). RecentlyVerma et al. (2008a) reported dichlorvos low dose exposureleads to reduction in the signal transduction cascade linkedto receptor subtypes and adenylyl cyclase-linked signalingpathway was impaired. Finally, the phosphorylation ofCREB was significantly reduced in both low dose and highdose group animals. These reveal the significance of M2

muscarinic receptor linked adenylyl cyclase signaling path-way and phosphorylation of CREB in the development ofneurobehavioral impairments after chronic low-levelexposure to dichlorvos.

Newhouse et al. (2004) used human dopaminergic SH-SY5Y cells to study mechanisms of rotenone-inducedneuronal cell death. Their results suggest that rotenone, atnanomolar concentrations, induces apoptosis in SH-SY5Ycells that are caspase-dependent. Furthermore, rotenone treat-ment induces phosphorylation of c-Jun, the c-Jun N-terminal

protein kinase (JNK), and the p38 mitogen activated protein(MAP) kinase, indicative of activation of the p38 and JNKpathways. Importantly, expression of dominant interferingconstructs of the JNK or p38 pathways attenuated rotenone-induced apoptosis. These data suggest that OPs (rotenone)induce apoptosis in the dopaminergic SH-SY5Y cells thatrequire activation of the JNK and p38 MAP kinases and cas-pases. These studies provide insights concerning the molecu-lar mechanisms of OPs-induced apoptosis in neuronal cells.

Convulsions may be associated with rapid and majorincreases in gene expression. This may be a direct conse-quence of convulsions, or at least be causally associatedwith them (Ceccatelli et al., 1989; Zimmer et al., 1997a, b).A high dose of soman (77.7 mg/kg, bw) caused tonic-clonic convulsions in the exposed rats and induced a robustprogressive expression of an immediate early gene, c-fos, areliable indicator of neuronal activation (Greenberg et al.,1986), in the piriform cortex and the noradrenergic locuscoereleus. Later, c-fos expression also occurred in the entorh-inal cortex, the endopiriform nucleus, the olfactory tubercle,the anterior olfactory nucleus, and the main olfactory bulb.At 2 hours the c-fos expression achieved its maximumand was then present in the cerebral cortex, thalamus,

Figure 18.5 Decomposition products and metabolic pathways for dithiocarbamates. 1, Combined mechanism of decomposition of both monoand dialkyl substituents; 2, Specific pathway for monoalkyldithiocarbamates; 3, Specific pathway for dialkyldithiocarbamates.

18.13 EFFECTS ON GENE EXPRESSION 251

caudate-putamen, and the hippocampus, brain regions, typi-cally metabolically activated subsequent to soman exposure.At 8 hours and beyond, c-fos expression returned to the con-trol level (Zimmer et al., 1997a, b). In general, c-fos promotesthe transcription of additional genes, including those thatencode proteins that are required for metabolic and physio-logic activities of the cell. Thus, c-fos expression indicatesthat the cell is adapting to external stimuli by producing theproteins necessary for continued cellular function. Underextreme stress, c-fos also promotes the transcription ofgenes that encode proteins that are critical to cell survival(Sheng and Greenberg, 1990). Several other investigators(Arenander et al., 1989; Seuwen et al., 1990) also haveshown that muscarinic receptor-mediated activation of PKCinduces the immediate early genes c-fos and c-jun. Theseare genes that encode nuclear proteins and act in tandem asa dimeric complex that binds to a specific DNA consensussequence in target genes to stimulate their transcription.Muscarinic receptor activation has induced c-fos expressionin PC12 pheochromocytoma cells (Arenander et al., 1989),and both c-fos and c-jun expression in fibroblasts that expressM1 muscarinic receptors (Seuwen et al., 1990) and glial celllines (Ashkenazi et al., 1989). There is an immediatetranscriptional regulation of gene coding for AChE, cholineacetyltransferase (ChAT), and vesicle ACh transporter(VAChT), which reduces the expression of ChAT andVAChT mRNA, increasing the AChE mRNA (Grisaruet al., 1999).

Dithiocarbamates exert a variety of striking molecularchanges in cell systems. These include: (a) the ability to oxi-dize protein thiols and inhibit hydroxyradical formation; (b)inhibit nuclear factor-kappa B (NF-kB) activation via mech-anism(s) that notably do not involve either oxidation-reduction dependent modification of the NF-kB protein orinterference with NF-kB-DNA binding; (c) induce AP-1-dependent cell differentiation and gene expression via denovo transcription of c-fos and c-jun (Aragones et al.,1996) and result in profound changes in some cells via amechanism that involve alterations in the intracellular trans-port of copper ions (Cereser et al., 2001; Yu et al., 2003;Zenzen et al., 2001).

Transcription of hIL-10 is controlled by the constitutivelyexpressed transcription factors, Sp1 and Sp3 (Naora et al.,1994), the induction of which has been demonstrated to besensitive to oxidation of critical protein thiols. Alternatively,the half-life of hIL-10 mRNA appears to be predominantlydetermined by post-transcriptional signals. Homeostaticregulation of the subcellular distribution of copper is highlyorchestrated, and alterations in intracellular copper concen-tration have been demonstrated to alter post-translationalgene expression via multiple mechanisms (Shiraishi et al.,2006). Taken together it can be hypothesized that DTC influ-ences cell-specific gene expression of vIL-10 via copper-dependent direct oxidation of protein thiols.

It has also been reported that dithiocarbamates directlysuppresses cell-mediated immune response and T lympho-cyte activation (Burkitt et al., 1998; Irons et al., 2001).Dithiocarbamates are historically considered not to be muta-genic, although Soloneski et al. (2001, 2002) recentlyreported increases in chromosome aberrations in cells treatedwith relatively high concentrations of ethylene bisdithiocarbamate.

The changes induced by OPs may produce permanentchanges in the gene levels in these cells. It is clear thatcholinergic-induced convulsions are associated withincreased expression of immediate early genes. The exactrole of these genes, whether they are consequences of neur-onal excitation or causally linked with it, remains to beelucidated.

18.14 TREATMENT OF ORGANOPHOSPHATEPOISONING

There has been a significant progress in the research work onthe development of specific therapies effective in poisoningby OP and CM pesticides. The usefulness of anticholinergicdrugs such as atropine was the first known antidote, whichwas soon followed in the 1950s by the demonstration ofoxime-induced cholinesterase reactivation (Wilson andGinsburg, 1955). A later development was the addition ofanticonvulsants to the atropine/oxime combination. N-Acetylcysteine (NAC) and aurintricarboxylic acid (ATA)ameliorated diethyldithiocarbamate (DDTC) induced cyto-toxicity (Kanno et al., 2003). The cytotoxicity and DNA frag-mentation were completely prevented by ATA. However,NAC blocked DDTC-induced DNA fragmentation on treat-ment for 12 hours, but not 24 hours. These data indicatedthat the inhibition effect of DDTC-induced cytotoxicity andapoptosis by only NAC is not complete, and is followed bythe activation of endonuclease, a critical pathway forDDTC-induced cytotoxicity via apoptosis.

18.14.1 Counteracting the Muscarinic Effects ofExcess Acetylcholine

18.14.1.1 Atropine There is a wide variation and lack ofevidence in current recommendations for atropine dosingschedules leading to subsequent variation in clinical practice.Atropine competes with ACh and other muscarinic agonistsfor a common binding site on the muscarinic receptor, thuseffectively antagonizing the actions of ACh at muscarinicreceptor sites, which leads to increased tracheobronchialand salivary secretions, bronchoconstriction, and bradycar-dia. The usefulness of atropine is virtually undisputed.Atropine may also be of value in treating acute dystonicreactions occasionally observed in acute OP poisoning(Wedin, 1988).


18.14.1.2 Glycopyrrolate Glycopyrrolate, with its highselectivity for peripheral cholinergic sites, has been usefulfor controlling secretions with less side effects such as flush-ing, tachycardia and a decreased level of consciousness. Theadvantages of better secretion control may be offset against alack of effect on central neurological symptoms caused byOPs and CMs. A combination of glycopyrrolate and atropinemay adequately control bronchorrhoea and bradycardia with-out causing tachycardia. A double-blind, randomized trial of39 patients comparing glycopyrrolate and atropine showed nodifferences in clinical outcomes and complications (Bardinand Eeden, 1990). A case of OP poisoning refractory to atro-pine and in which glycopyrrolate was successfully used toreduce cholinergic symptoms was recently reported (J.V.Peter et al., 2008). In South Africa, atropine is routinely com-bined with glycopyrrolate to limit the central stimulatoryeffects of atropine. Other antimuscarinic compounds (e.g.,lipophilic compounds like scopolamine and benactyzine,and less lipophilic agents like atropine methyl nitrate)which act mainly in the periphery have been used in animalexperiments and in human OP and CM poisoning to counter-act the effects of excess ACh at muscarinic synapses. MostOP compounds are lipophilic, especially the nerve gases.As a result, central effects often predominate, causing respir-atory depression. In such instances, an antimuscarinic com-pound that reaches the brain will be needed. In contrast, inpoisoning with more peripherally acting anticholinesterases,like neostigmine or pyridostigmine, peripheral respiratoryimpairment (striated muscles), bronchorrhoea and broncho-constriction will predominate and a peripherally acting anti-dote may be preferred. Atropine sulphate, acting bothperipherally and centrally, remains the antimuscarinic ofchoice for the treatment of OP compound poisoning. Inyoung children, Sofer et al. (1989) observed that atropine(0.05 mg/kg repeated at 5 to 10 min intervals when necess-ary) had an obvious beneficial effect on the predominant pre-senting signs that were related to the CNS. The centralcholinergic synapses were considered to be more sensitiveto atropine in the very young. Further, atropine may passthrough the blood–brain barrier more easily in children.

18.14.2 Counteracting the Nicotinic Effects of ExcessAcetylcholine

18.14.2.1 Oximes Oximes reactivate inhibited AChE bycleavage of phosphorylated active sites of the enzyme.Reactivation by oximes is most marked at the NMJ. Theydo not reverse the muscarinic manifestations of OP and CMpoisoning and they have a short half-life (1.2 h) when admi-nistered intravenously. Other effects attributed to oximes (invery high doses), which are charged molecules and actmainly peripherally are: anticholinergic effect; sympathomi-metic effect; depolarizing effect at the neuromuscular junc-tion; an ability to inhibit cholinesterase; direct influence on

synaptic transmission by mechanisms which are not knownprecisely at present (Petroianu and Lorke, 2008; Shrotet al., 2008).

A direct reaction of oximes with sarin, soman, and tabunhas been reported, although Eyer (2003) calculated that adirect reaction is likely to be of negligible therapeuticvalue. Oximes, being ionized compounds, do not easilycross the blood–brain barrier. Approximately 10% reactiva-tion of brain AChE claimed following oxime therapy is con-sidered to be an over-estimation. However, some workersbelieve that the limited passage of the oxime to the brainmay have a significant, albeit small, effect and promptimprovement has been reported in the level of consciousnessand in the EEG of an intoxicated child following an intra-venous infusion of pralidoxime chloride. They are ineffec-tive by themselves in counteracting the central effects ofOPs. The therapeutic effect of atropine together with anoxime, where the latter is effective, is more than merely addi-tive. In English-speaking countries, the oxime pralidoximeis preferred. In central Europe, obidoxime is the oxime ofchoice. Studies have shown that only after OP concentrationin the body falls below a critical value, where reactivation rateexceeds inhibition rate, the significant reactivation occurs.The experimental work by Worek et al. (2007) demonstratedunequivocally that oximes are not equally effective and thattheir rank order of effectiveness changes with the OP com-pound involved. These workers found obidoxime to be themost potent and efficacious oxime in reactivating AChEinhibited by OP insecticides, but inferior to HI6 against thenerve agents with the exception of tabun (i.e., soman, sarinand cyclosarin). One possible reason for this specificity isthe inevitable formation of phosphonyloximes duringreactivation. These metabolites are highly potent anticholi-nesterases by themselves and are possibly more reactivethan the parent OP compounds. Possibly, oximes delay theageing process of AChE during nerve agents poisoning(Luo et al., 2008). In recent studies, oximes of K serieshave been found more effective (Kuca et al., 2009).

18.14.2.2 Pralidoxime Pralidoxime chloride (2-PAM),methyl sulphate, or mesylate (P2S) is usually used, as theiodide increases the risk of adverse cardiac events and ofiodism; 2-PAM is the most effective when administered intra-venously. The pharmacokinetics of pralidoxime in poisonedchildren following continuous infusion vary widely anddiffer from those reported in both healthy and poisonedadults (Hilmas and Hilmas, 2009). The volume of distribution(Vd) and the plasma clearance of pralidoxime were found tobe greater in children and, because of this, a dose regimenbased on symptom severity has been recommended. Inpatients with mild to moderate symptoms, a loading dose of20 mg/kg should achieve plasma pralidoxime concentrationsof approximately 7 mg/l. Larger loading doses will berequired in patients with seizures, coma, bradycardia, or

18.14 TREATMENT OF ORGANOPHOSPHATE POISONING 253

respiratory depression. Concerns have been expressed overthe safety and efficacy of the use of pralidoxime in patientswith carbamate poisoning in general, and more so with car-baryl poisoning specifically. In appropriate doses pralidox-ime alone protects against carbaryl poisoning in mice.However, it fails to show beneficial effects which may bethe result of oxime overdose (Mercurio-Zappala et al.,2007). To improve the central effects of pralidoxime, thedihydropyridine derivative was synthesized. This derivativeknown as pro-2PAM acts as a “pro-drug” which is able topass through membranes, such as the blood–brain barrierand, once across the membranes, in vivo oxidation convertsthe pro-2-PAM to PAM to give a 13-fold higher level of2-PAM in the brain. The use of sugar oximes (the molecularcombination of glucose with 2-PAM derivatives) to promoteCNS penetration has also been considered. It should be notedthat until now, no controlled clinical study has been carriedout to assess the clinical efficacy of the oximes properly.

18.15 ADDITIONAL THERAPIES

18.15.1 Benzodiazepines

Benzodiazepines are useful adjuncts to atropine (and oximes)in the treatment of OP poisoning. They increase survival anddecrease the incidence of associated neuropathies (Raszewskiand Filip, 2004). The combination of atropine and diazepamwas more effective than atropine alone in reducing mortalitydue to soman (Svensson et al., 2005).

18.15.2 Sodium Bicarbonate

Alkalinization of the serum to pH 7.5 with sodium bicarbon-ate may be useful as hydrolysis of the esteratic portion of theOP inhibited AChE molecule increases as the pH increases.Clinicians in Iran report the successful management of OP-intoxicated patients using infusions of sodium bicarbonate(J.V. Peter et al., 2007).

18.15.3 Glutamate-Receptor Antagonists

There is interest in the role of glutamate in sustained OP-induced seizure activity and the possible therapeutic role ofnon-selective glutamate receptor antagonists, such as felba-mate, and of selective NMDA-receptor-channel blockers,such as dizocilpone and procyclidine, in the managementof OP poisoning.

18.15.4 Clonidine

Clonidine inhibits the release of acetylcholine from centraland peripheral cholinergic neurons, in addition to being a cen-trally active alpha-2-adrenergic agonist. In rodents, clonidinepre-treatment (0.3 mg/kg) increased the onset of latency of

tremor from 5 to 20 minutes, delayed death from 12 to 24minutes and increased the percentage of survivors to 50% fol-lowing poisoning with physostigmine (Pycock et al., 1977). Itwas suggested that central cholinergic neurons involved in theregulation of respiration and fine motor control, but not per-ipheral motor neurons, are inhibited by clonidine acting onalpha-receptors. The protective effects of clonidine arelikely to involve multiple sites of action, including block-ade of ACh release, of postsynaptic muscarinic receptorsand transient inhibition of AChE that could prove to beuseful in the treatment of OP and CM poisoning (J.V. Peteret al., 2008).

18.15.5 Annealed Erythrocytes

By placing phosphotriesterase within resealed annealederythrocytes in the circulation by a single injection can theor-etically persist for the life of an erythrocyte (120 days) andcan constantly remove OP that is being slowly released into the blood stream from fatty tissues. Presently, the annealederythrocytes are a convenient carrier system that have poten-tial utility as a prophylactic agent by autologous transfusionto individuals who are at risk to OP agents, for example,aerial crop dusters, agricultural workers, and soldiers exposedto nerve gases. Alternative carrier systems, for example, steri-cally stable liposome, may be practical for the actual treat-ment of poisoning (J.V. Peter et al., 2007).

18.16 ANTICONVULSANTS

A large number of anticonvulsants have been studied in ani-mals or used in OP and CM poisoning. Many anticonvulsantshave been investigated in attempts to improve the treatment ofOP nerve agent poisoning, such as: (a) the water-soluble dia-zepam pro-drug avizafone (Lallement et al., 2000); (b) otherbenzodiazepines, such as clonazepam and midazolam (Pieriet al., 1981); and (c) anticonvulsants of other types, such asbarbiturates and phenytoin. Other drugs that have beenstudied include tiagabine (GABA uptake inhibitor) and gluta-mate receptor antagonists (Shih et al., 1999). Only diazepamand midazolam have achieved widespread use in the treat-ment of OP pesticide poisoning. Anticonvulsants includingbenzodiazepines, especially diazepam, were originallystudied in OP poisoning for the symptomatic relief of OP-induced convulsions. Benzodiazepines are CNS depressants,anxiolytics, and muscle relaxants (Diamantis and Kletzkin,1966). The main site of action of benzodiazepines is the7-aminobutyric acid A (GABAA) receptor. The GABAAreceptor is a ligand-gated chloride ion channel (Ortells andLunt, 1995), the GABAergic system being the major inhibi-tory neurotransmission system in the mammalian CNS.Benzodiazepines including diazepam alter GABA bindingat the GABAA receptor in an allosteric manner, but these


drugs do not activate the GABAA receptor by direct action.Nevertheless, the overall effect is to increase the inhibitoryaction of the GABAergic system. Data from experimentalnerve agent poisoning (Hayward et al., 1990) suggest thatbenzodiazepines, such as diazepam and midazolam, amelio-rate or prevent the development of pathological changes inthe CNS.

18.16.1 Diazepam

Diazepam is the anticonvulsant that has been most studied foruse in OP pesticide poisoning (Dickson et al., 2003). In thiscontext, the most likely mode of administration is by intrave-nous injection, but other modes of administration have beenconsidered for self-administration or when administrationby those not trained in intravenous injection is required.

18.17 OTHER METHODS OF ANTIDOTALTREATMENT

A number of novel approaches to the antidotal treatment ofOPs and CMs have been studied, often using prophylacticprotocols and mostly in relation to nerve agent poisoning,but some may be applicable, at least in principle, to thosepesticide poisoning.

18.17.1 Enzymes

18.17.1.1 Cholinesterase AChE (Maxwell et al., 1999;Wolfe et al., 1992), BuChE (Masson and Rochu, 2009;Broomfield et al., 1999), and carboxylesterases (CarbEs)(Sterri and Fonnum, 2009) have been studied as bioscaven-gers for nerve agents.

18.17.2 Phosphotriesterase

McGuinn et al. (1993) described a study in which squid DFP-hydrolyzing enzyme (DFPase) was entrapped within mouseerythrocytes. These red blood cells were shown to be capableof hydrolyzing DFP. This approach would presumably beeffective against other OP esters that are hydrolyzed byDFPase. In a similar approach, Pei et al. (1995) reportedthat resealed murine erythrocyte cells containing recombinantphosphotriesterase protected against the lethal effect of para-oxon in mice. Also, when these carrier cells were adminis-tered in combination with 2-PAM and/or atropine, synergywas reported.

18.17.3 Calcium Channel Blockers

Calcium channel blockers have also been studied in OP andCM poisoning (e.g., nimodipine) (Choudhary et al., 2002a;Dretchen et al., 1992; Karlsson et al., 1994).

18.17.4 Adenosine Receptor Agonists

Adenosine receptor agonists were reported to prevent clinicalsigns and increased survival in soman, sarin, and DFP poi-soning (Harrison et al., 2003; Tuovinen, 2004).

18.17.5 N-Methyl-D-Aspartate Receptor Antagonists

Although the initial stimulus for seizures appears to becholinergic overactivity, as the seizures develop, other excit-atory neurotransmission systems become involved, includ-ing the glutamatergic system. The N-methyl-D-aspartate(NMDA) receptor is a subtype of glutamatergic receptor:antagonists at this receptor, dizocilpine and 3-[(R, S)-2-car-boxypiperazin-4-yl]-propyl-1-phosphonic avid, were foundin mice experimentally poisoned with chlorfenvinphos toblock seizures (Dekundy et al., 2001). It has been suggestedthat the beneficial activity of caramiphen, an anticholinergicdrug, in soman poisoning may be modulated throughactivity at the NMDA receptor (Raveh et al., 1999, 2003).Anticonvulsant and neuroprotective properties of NMDAantagonists encourage the investigation of their effects inAChE inhibitor-induced poisonings. It has been shown thatboth muscarinic ACh and NMDA receptor-mediated mech-anisms contribute to the acute toxicity of AChE inhibitors,and NMDA receptors seem critical to OP-induced seizures(Dekundy et al., 2007). In a series of in vivo studies, anNMDA receptor antagonist memantine (MEM, 18 mg/kg,sc), in combination with atropine sulfate (ATS, 16 mg/kg,sc), was reported to be very effective against OP nerveagents (soman, sarin, tabun, and VX), OP insecticidemethyl parathion and DFP, and CM pesticides (Gupta andDettbarn, 1992; Gupta and Kadel, 1989, 1990, 1991;McLean et al., 1992). In a recent study, MEM and ATS atte-nuated carbofuran-induced changes in AChE activity (markerof exposure and effect), levels of F2-isoprostanes and F4-neu-roprostanes (ROS markers), citrulline (RNS marker), declinesin high energy phosphates, as well as the alterations in mor-phology of hippocampal neurons in CA1 sector. MEM andATS pretreatment also protected rats from carbofuran-induced hyper cholinergic behavioral activity, including sei-zures (Gupta et al., 2007).

18.17.6 Blockade of Acetylcholine Synthesis or Uptake

An obvious therapeutic measure in OP poisoning would be todecrease the synthesis of ACh. Sterling et al. (1988) found thatadministration of acetylsecocholinium 30 minutes prior tosoman enhanced the protective effects of atropine and 2-PAMC1 in the rat, whereas administration of N-hydroxyethyl-naphthylvinylpyridine a few minutes before soman reducedmortality due to soman. Acetylsecocholinium is an inhibitorof high-affinity choline uptake and a choline acetyltransferase(ChAT) inhibitor, and N-hydroxyethylnaphthylvinylpyridine

18.17 OTHER METHODS OF ANTIDOTAL TREATMENT 255

is a ChAT inhibitor. N-allylquinuclinidol, another inhibitor ofhigh-affinity choline uptake, reduced mortality. Gray et al.(1998) studied naphthylvinylpyridine derivatives as antidotesfor nerve agent poisoning and concluded that their beneficialexperimental action in mice and guinea pigs were not relatedto ChAT inhibition.

18.18 PREVENTION AND TREATMENT OFORGANOPHOSPHATE-INDUCED DELAYEDPOLYNEUROPATHY

There is no recognized antidotal treatment for organo-phosphate-induced delayed polyneuropathy (OPIDP).Numerous substances, including certain carbamates, phenylmethane sulfonyl fluoride, n-butane-sulfonyl fluoride, andsome phosphinates, have been shown to prevent the develop-ment of OPIDP when given before neuropathic OPs to sensi-tive species such as hens (Johnson et al., 1992). Furthermore,it has been found that elements of the conventional treatmentused in humans (atropine, trimedoxime, and midazolam),if injected into hens prophylactically, ameliorated the sub-sequent development of OPIDP induced by DFE (Petrovicet al., 2000). Jokanovic et al. (2001) found that a combina-tion of trimedoxime, atropine, and methylprednisolonegiven 20 minutes before experimental poisoning of henswith DFP reduced the severity of the OPIDP that developedsubsequently. The effect was less if the treatment was givenafter the DFE. None of the previously mentioned studiesprovide any support for the efficacy of any post exposure anti-dotal therapy, so the treatment of OPIDP is essentiallysymptomatic.

Currently fielded treatments for nerve agent intoxicationpromote survival, but do not afford complete protectionagainst either nerve agent-induced motor and cognitivedeficits or neuronal pathology. Lenz et al. (2007) reportedthat the use of human plasma-derived butyrylcholinesterase(HuBuChE) to neutralize the toxic effects of nerve agentsin vivo has been shown to both aid survival and protectionagainst decreased cognitive function after nerve agentexposure. Recently, a commercially produced recombinantform of human butyrylcholinesterase (r-HuBuChE;PharmAthene Inc.) expressed in the milk of transgenicgoats has become available which is biochemically similarto plasma-derived HuBuChE in in vitro assays. The pharma-cokinetic characteristics of a polyethylene glycol coated(pegylated) form of r-HuBuChE were determined in guineapigs; the enzyme was rapidly bioavailable with a half-life(t1/2) and pharmacokinetic profile that resembled that ofplasma-derived HuBuChE. Guinea pigs were injected with140 mg/kg (i.m.) of pegylated r-HuBuChE 18 hours priorto exposure (sc) to 5.5 � LD50 VX or soman. VX andsoman were administered in a series of three injections of1.5 � LD50, 2.0 � LD50, and 2.0 � LD50, respectively,

with injections separated by 2 hours. Pretreatment withpegylated r-HuBuChE provided 100% survival againstmultiple lethal doses of VX and soman. Guinea pigs dis-played no signs of nerve agent toxicity following exposure.Assessments of motor activity, coordination, and acquisitionof spatial memory were performed for two weeks followingnerve agent exposure. There were no measurable decreasesin motor or cognitive function during this period. In contrast,animals receiving 1.5 � LD50 challenges of soman or VXand treated with standard atropine, 2-PAM, and diazepamtherapy showed 50% and 100% survival, respectively, butexhibited marked decrements in motor function and, in thecase of GD, impaired spatial memory acquisition. Theadvances in this field have resulted in the decision to selectboth the plasma-derived and the recombinant form ofBuChE for advanced development and transition toclinical trials.

Efforts have now been expanded to identify a catalyticprotein capable of not only binding, but also rapidly hydro-lyzing the standard threat nerve agents. Recent work hasfocused on paraoxonase-1 (PON1), a naturally occurringhuman serum enzyme with the capacity to catalyze thehydrolysis of nerve agents, albeit too slowly to afford dra-matic protection (Ali and Chia, 2008). Using rationaldesign, several amino acids involved in substrate bindinghave been identified, and site-directed mutations haverevealed that residue H115 plays an important role in binding.In addition, the stereo specificity of PON1 for the catalytichydrolysis of soman has been examined. The enzyme exhibitsa slight stereo specificity for the C þ Pþ isomer of soman.

18.19 CONCLUSION

The use of OPs and CMs as insecticides in the agriculturaland urban settings is still high and is expected to remain so,at least in the near future. While other classes of insecticidesare gaining market share (e.g., pyrethroids) and new classeshave been developed (e.g., neonicotinoids), the efficacy ofOPs and CMs, with their relatively low cost and their lackof bioaccumulation in the ecosystems, would support this pre-diction. Yet these pesticides display relatively limited selec-tivity (one exception may be malathion) between insectsand nontarget species, including humans. As such, concernson their potential adverse effects in human populations willcontinue. The issues discussed in this chapter still representreal-life problems, with clinical, societal, and legal ramifica-tions. Continuing research in all these areas and others notmentioned is welcome and warranted. The extreme toxicityof many OP and CM compounds highlights the need for amore complete understanding of their mechanisms of toxicactions. This information also provides new insights into neu-rotoxicity and opens new vistas for research to explore mech-anisms of OP and CM toxicity. Even though the overall


toxicity and the general mechanisms of their toxic actionshave been rather well clarified over the years and seem tobe quite similar within the group, a more thorough under-standing of the cascades of cellular and subcellular toxicevents of these compounds in the nervous systems isneeded for effective prevention and treatment of OP andCM-induced poisonings.

REFERENCES

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