APPENDIX I-P:  Abou-Donia, Organophosphorus Ester-Induced Chronic Neurotoxicity, Archives of Environmental Health, v.58, n.8, 1 Aug. 03


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Organophosphorus Ester-Induced Chronic Neurotoxicity 

MOHAMED B. ABOU-DONIA / Archives of Environmental Health v.58, n.8, 1aug03


MOHAMED B. ABOU-DONIA Department of Pharmacology and Cancer Biology Duke University Medical Center Durham, North Carolina

ABSTRACT. Organophosphorus compounds are potent neurotoxic chemicals that are widely used in medicine, industry, and agriculture. The neurotoxicity of these chemicals has been documented in accidental human poisoning, epidemiological studies, and animal models. Organophosphorus compounds have 3 distinct neurotoxic actions. The primary action is the irreversible inhibition of acetylcholinesterase, resulting in the accumulation of acetylcholine and subsequent overstimulation of the nicotinic and muscarinic acetylcholine receptors, resulting in cholinergic effects. Another action of some of these compounds, arising from single or repeated exposure, is a delayed onset of ataxia, accompanied by a Wallerian-type degeneration of the axon and myelin in the most distal portion of the longest tracts in both the central and peripheral nervous systems, and is known as organophosphorus ester-induced delayed neurotoxicity (OPIDN). In addition, since the introduction and extensive use of synthetic organophosphorus compounds in agriculture and industry half a century ago, many studies have reported long-term, persistent, chronic neurotoxicity symptoms in individuals as a result of acute exposure to high doses that cause acute cholinergic toxicity, or from long-term, low-level, subclinical doses of these chemicals. The author attempts to define the neuronal disorder that results from organophosphorus ester-induced chronic neurotoxicity (OPICN), which leads to long-term neurological and neurobehavioral deficits. Although the mechanisms of this neurodegenerative disorder have yet to be established, the sparse available data suggest that large toxic doses of organophosphorus compounds cause acute necrotic neuronal cell death in the brain, whereas sublethal or subclinical doses produce apoptotic neuronal cell death and involve oxidative stress.

<Key words: acetylcholinesterase, apoptosis, brain, necrosis, nervous system, neurobehavioral, neuropathological, neuropsychological, OPICN, OPIDN, organophosphorus compounds>


ORGANOPHOSPHORUS COMPOUNDS are chemicals that contain both carbon and phosphorus atoms.1 They are derivatives of phosphoric (H3PO4), phosphorus or phosphonic (H3PO3), and phosphinic (H3PO2) acids (Fig. 1). The biological action of organophosphorus compounds is related to their phosphorylating abilities. This is dependent on the electrophilicity (positive character) of the phosphorus atom, which is determined by its substituent groups. Steric factors of substituents also play a major role in determining the biological activity of these chemicals. Lipid solubility is important because it enhances the ability of these compounds to cross biological membranes and the blood-brain barrier, leading to increased biological activity. Organophosphorus compounds are an economically important class of chemical compounds with numerous uses, such as in pesticides, industrial fluids, flame retardants, therapeutics, and nerve gas agents. Most modern synthetic organophosphorus compounds are tailor-made to inhibit acetylcholinesterase (AChE), an enzyme essential for life in humans and other animal species. Tetraethylpyrophosphate was the 1st organophosphate synthesized as an AChE inhibitor in 1854.2 Later, dimethyl and diethyl phosphorofluoridates were synthesized.2 During World War II, organophosphorus compounds were developed primarily as agricultural insecticides, and later as chemical warfare agents. The majority of organophosphorus insecticides are organophosphorothioates; nerve agents are organophosphonates or organophosphonothioates; industrial chemicals are typically organophosphates3 (Table 1). Biologically, organophosphorus compounds are neurotoxic to humans and other animals via 3 distinct actions: (1) cholinergic neurotoxicity, (2) organophosphorus ester-induced delayed neurotoxicity (OPIDN), and (3) organophosphorus ester-induced chronic neurotoxicity (OPICN).

Fig. 1.    Chemical structures of phosphoric acid, phosphinic acid, phosphorus acid, and phosphonic acid.


Table 1.   Compounds Cited in the Text and Their IUPAC Designations

Compound               IUPAC designation                                      .
Chlorpyrifos           O,O-diethyl O-3,5,6-trichloro-2-pyridylphosphorothioate
Cyclosarin (GF)        O-cyclohexyl methylphosphonofluoridate
DFP                    O,O-diisopropyl phosphorofluoridate
DEET                   N,N-diethyl-m-toluamide
Diazinon               O,O-diethyl O-2-isopropyl-6-methylpyrimidin-4-yl phosphorothioate
Fenthion               O,O-dimethyl O-4 methylthio-m-tolylphosphorothioate
Malathion              S-1,2-bis(ethoxycarbonyl)ethyl O,Odimethylphosphorodithioate
Methamidophos          O,S-dimethyl phosphoramidothioate
Permethrin             3-phenoxybenzl (1RS)-cis-trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate
Quinalphos             O,O-diethyl O-quinoxalin-2-yl phosphorothioate
Tabun (GA)             O-ethyl N,N-dimethylphosphoamidocyanidate
Sarin (GB)             O-isopropyl methylphosphonofluoridate
Soman (GD)             O-2,2-trimethypropyl methylphosphonofluoridate
TOCP                   Tri-ortho-cresylphosphate
VX                     O-ethyl S-2-diisopropylaminoethylmethylphosphonothioate
VR                     O-isobutyl S-2-diethylaminoethylmethylphosphonothioate


Fig. 2.    Active center gorge of mammalian acetylcholinesterase (AChE).


Fig. 3. Sarin phosphonyl enzyme: isopropyl methylphosphonyl acetylcholinesterase (AChE).


Fig. 4. Sarin aged phosphonyl enzyme: methylphosphonyl acetylcholinesterase (AChE).


Cholinergic Neurotoxicity of Organophosphorus Compounds

Organophosphorus compounds cause cholinergic neurotoxicity by disrupting the cholinergic system that includes AChE and its natural substrate, the neurotransmitter acetylcholine.3 Acetylcholine is released in response to nerve stimulation and binds to post-synaptic acetylcholine receptors, resulting in muscle contraction or gland secretions. Its action is rapidly terminated by hydrolysis with AChE via the serine hydroxyl in the catalytic triad of AChE.2 The 3-dimensional structure of AChE reveals an active center located at the base of a narrow gorge about 20 Ĺ in depth.4 The active center includes the following sites (Fig. 2): (a) the catalytic triad: Glu 334, His 447, and Ser 203; (b) an aceyl pocket: Phe 295 and Phe 297; (c) a choline subunit: Trp 86, Glu 202, and Tyr 337; and (d) a peripheral site: Trp 286, Tyr 72, Tyr 124, and Asp 74.

Organophosphorus ester inhibition of AChE. Organophosphorus esters inhibit AChE by phosphorylating the serine hydroxyl group at the catalytic triad site (Fig. 3). The phosphoric or phosphonic acid ester formed with the enzyme is extremely stable and is hydrolyzed very slowly.1 If the phosphorylated enzyme contains methyl or ethyl groups, the enzyme is regenerated in several hours by hydrolysis. On the other hand, virtually no hydrolysis occurs with an isopropyl group (e.g., sarin) and the return of AChE is dependent upon synthesis of a new enzyme. Phosphorylated AChE undergoes aging—a process that involves the loss of an alkyl group, resulting in a negatively charged monoalkyl enzyme (Fig. 4).3 Organophosphorus compounds undergo detoxification by binding to other enzymes that contain the amino acid serine. These enzymes include plasma butyrylcholinesterase (BChE)5,6 and paraoxonase.7,8

Inhibition of AChE results in the accumulation of acetylcholine at both the muscarinic and nicotinic receptors in the central nervous system (CNS) and the peripheral nervous system (PNS). Excess Acetylcholine initially causes excitation, and then paralysis, of cholinergic transmission, resulting in some or all of the cholinergic symptoms, depending on the dose size, frequency of exposure, duration of exposure, and route of exposure, as well as other factors such as combined exposure to other chemicals and individual sensitivity and susceptibility.

Human exposure. Human exposure—mostly via inhalation— to the organophosphorus nerve agent sarin was recently documented in 2 terrorist incidents in Japan. At midnight on June 27, 1994, sarin was released in Matsumoto City.9 Of the 600 persons who were exposed, 58 were admitted to hospitals, where 7 died. Although miosis was the most common symptom, severely poisoned patients developed CNS symptoms and cardiomyopathy. A few victims complained of arrhythmia and showed cardiac contraction. The 2nd terrorist attack by sarin was in the Tokyo subway trains, at 8:05 A.M. on March 20, 1995, when a total of 5,000 persons were hospitalized and 11 died.10 Patients with high exposure to sarin in the Tokyo subway incident exhibited marked muscle fasciculation, tachycardia, high blood pressure (nicotinic responses), sneezing, rhinorrhea, miosis, reduced consciousness, respiratory compromise, seizures, and flaccid paralysis.11 Patients with mild exposure complained of headaches, dizziness, nausea, chest discomfort, abdominal cramps, and miosis. Interestingly, patients had pupillary constriction, even when their cholinesterase activity was normal. Furthermore, inhibition of red blood cell AChE activity was a more sensitive indicator of exposure than serum BChE activity. 12 The absence of bradycardia and excessive secretions— which are common in dermal or ingestion exposures— suggested that the major route of exposure to the sarin gas in these instances was via inhalation. The patients were treated with atropine eye drops for marked miosis, and with pralidoxime iodide (2-PAM).

Organophosphorus Ester-Induced Delayed Neurotoxicity (OPIDN)

Characteristics of OPIDN. OPIDN is a neurodegenerative disorder characterized by a delayed onset of prolonged ataxia and upper motor neuron spasticity as a result of a single or repeated exposure to organophosphorus esters.13–16 The neuropathological lesion is a central-peripheral distal axonopathy caused by a chemical transection of the axon (known as Wallerian-type degeneration), followed by myelin degeneration of distal portions of the long and large-diameter tracts of the CNS and PNS.17 Incidents of OPIDN have been documented for over a century. The earliest recorded cases were attributed to the use of tri-o-cresyl phosphate (TOCP)-containing creosote oil for treatment of pulmonary tuberculosis in France in 1899.13,15,16 In 1930, TOCP was identified as the chemical responsible for an estimated 50,000 cases of OPIDN in the Southern and Midwestern regions of the United States.13,15,16 More recently, Himuro et al.18 reported that a 51-yr–old man who was exposed to sarin during the Tokyo subway incident and survived its acute toxicity, then died 15 mo later. Neuropathological alterations and neurological deficits observed in this individual were consistent with the dying-back degeneration of the nervous system characteristic of OPIDN. This incident indicated that humans are more sensitive than experimental animals to sarin-induced OPIDN, inasmuch as it required 26–28 daily doses of LD50 (25 µg/kg, i.m.) sarin to produce OPIDN in the hen.19 OPIDN has been divided into 3 classes: Type I is caused by the pentavalent phosphates and phosphonates, as well as their sulfur analogs; Type II is produced by the trivalent phosphites; 20 and Type III is induced by phosphines.21,22 All 3 OPIDN types are produced by organophosphorus compounds and characterized by central-peripheral distal axonopathy. Type II differs from Type I in terms of the susceptibility of rodents and the presence of neuropathological lesions in neuronal cell bodies.20 Type III OPIDN is not accompanied by inhibition of the neurotoxicity target esterase (NTE), thus casting further doubt on this enzyme as the target for OPIDN.21,22

Mechanisms of OPIDN. Early studies on the mechanisms of OPIDN centered on the inhibition of the esterases AChE23 and BChE24 by organophosphorus esters. Subsequent studies eliminated both enzymes as targets for OPIDN.25 Johnson26 proposed an NTE—an enzymatic activity preferentially inhibited by organophosphorus compounds capable of producing OPIDN as its target. Despite numerous studies since the introduction of this concept 35 yr ago, the NTE hypothesis has not advanced our understanding of the mechanism of OPIDN because: (a) evidence for the involvement of NTE in the development of OPIDN is only correlative; (b) it has not been shown how inhibition and aging of NTE leads to axonal degeneration; (c) NTE, which is present in neuronal and non-neuronal tissues and in sensitive and insensitive species, has no known biochemical or physiological function; (d) some organophosphorus pesticides that produce OPIDN in humans do not inhibit or age NTE;27–30 and (e) phosphines that produce Type III OPIDN do not inhibit NTE.21,22 However, the most convincing evidence against this hypothesis is the recent finding that NTE-knockout mice are sensitive to the development of OPIDN,31–33 indicating that this enzyme is not involved in the mechanisms of OPIDN.

Protein kinases as targets for OPIDN. Because research on esterases did not increase our understanding of the mechanisms of OPIDN, we have been studying the involvement of protein kinase-mediated phosphorylation of cytoskeletal proteins in the development of OPIDN. These studies were prompted by the following observations: (a) Since organophosphorus compounds are effective phosphorylating agents, it is reasonable to expect that they would interfere with normal kinasemediated phosphorylation of a serine or threonine group at the target protein. (b) The earliest ultrastructural alterations in OPIDN are seen mostly as aggregation and accumulation of cytoskeletal proteins, microtubules, and neurofilaments, followed by their dissolution and disappearance. (c) The structural and functional status of cytoskeletal proteins are affected significantly by protein kinase-mediated phosphorylation.

Anomalous hyperphosphorylation of cytoskeletal elements is associated with OPIDN, a neurodegenerative disorder characterized by distally located swellings in large axons of the CNS and PNS, with subsequent axonal degeneration. Central to our hypothesis is the observation that increased aberrant protein kinase-mediated phosphorylation of cytoskeletal proteins could result in the destabilization of microtubules and neurofilaments, leading to their aggregation and deregulation in the axon.34 Protein kinases are able to amplify and distribute signals because a single protein kinase can phosphorylate many different target proteins. Several protein kinases are turned on by 2nd messengers. For example, calcium/calmodulin-dependent protein kinase II (CaM kinase II) is inactive until it is bound by the calcium-calmodulin complex that induces conformational changes and causes the enzyme to unfold an inhibitory domain from its active site.35 We have demonstrated substantial increases in the autophosphorylation, 36,37 enzymatic activity,38 protein levels, and mRNAs of CaM kinase II in hens treated with diisopropylphosphorofluoridate (DFP).39 These aberrant alterations have resulted in increased phosphorylation of the following cytoskeletal proteins: tubulin, neurofilaments, microtubule associated proteins-2 (MAP-2), and tau proteins.39–42 Increased activity of CaM kinase II can affect the stability of cytoskeletal proteins through posttranslational modification. Phosphorylation of these proteins interrupts their interaction, polymerization, and stabilization, leading to their degeneration.39,43,44

On the other hand, early studies identified transcription factors as critical phosphoproteins in signaling cascades. Immediate early genes control gene expression and therefore affect long-term cellular responses. We have demonstrated that the transcription of c-fos is elevated in OPIDN, perhaps through the activation of cAMP (adenosine monophosphate) response element binding (CREB), which is phosphorylated by CaM kinase II. Subsequent to c-fos activation,45 we observed altered gene expression of CaM kinase II,43 neurofilaments,46 glial fibrillary acidic protein (GFAP), and vimentin.47 Our results also showed an increase in medium (NF-M) and a decrease in low (NF-L) and high (NF-H) molecular weight neurofilaments in the spinal cords of hens treated with DFP. 46 This imbalance in the stoichiometry of neurofilament proteins interferes with their interaction with microtubules and promotes neurofilament dissociation from microtubules, leading to the aggregation of both cytoskeletal proteins.48 Immunohistochemical studies in nervous system tissues from TOCP- and DFPtreated hens demonstrated aberrant aggregation of phosphorylated neurofilament, tubulin, and CaM kinase II.49

Organophosphorus Ester-Induced Chronic Neurotoxicity (OPICN)

Various epidemiological studies have demonstrated that individuals exposed to a single large toxic dose, or to small subclinical doses, of organophosphorus compounds have developed a chronic neurotoxicity that persists for years after exposure and is distinct from both cholinergic and OPIDN effects.50 This disorder has been variously referred to in the literature as: “chronic neurobehavioral effects,”11 “chronic organophosphateinduced neuropsychiatric disorder (COPIND),50 “psychiatric sequelae of chronic exposure,”51 “central nervous system effects of chronic exposure,”52 “psychological and neurological alterations,”53 “long-term effects,”54 “neuropsychological abnormalities,”55 “central cholinergic involvement in behavioral hyperreactivity,” 56 “chorea and psychiatric changes,”57 “chronic central nervous effects of acute organophosphate pesticide intoxication,”58 “chronic neurological sequelae,”59,60 “neuropsychological effects of long-term exposure,”61 “neurobehavioral effects,”62 and “delayed neurologic behavioral effects of subtoxic doses.”63 Our review of the literature indicated that these studies describe a nervous system disorder induced by organophosphorus compounds which involves neuronal degeneration and subsequent neurological, neurobehavioral, and neuropsychological consequences. We will next define and describe this disorder, and refer to it as “organophosphorus ester-induced chronic neurotoxicity” or OPICN.

Characteristics of OPICN. OPICN is produced by exposure to large, acutely toxic—or small subclinical— doses of organophosphorus compounds. Clinical signs, which continue for a prolonged time ranging from weeks to years after exposure, consist of neurological and neurobehavioral abnormalities. Damage is present in both the PNS and CNS, with greater involvement of the latter. Within the brain, neuropathological lesions are seen in various regions, including the cortex, hippocampal formation, and cerebellum. The lesions are characterized by neuronal cell death resulting from early necrosis or delayed apoptosis. Neurological and neurobehavioral alterations are exacerbated by concurrent exposure to stress or to other chemicals that cause neuronal cell death or oxidative stress. Because CNS injury predominates, improvement is slow and complete recovery is unlikely.

OPICN following large toxic exposure to organophosphorus compounds. Several studies have reported that some individuals who were exposed to large toxic doses of organophosphorus compounds, and who experienced severe acute poisoning and subsequent recovery, eventually developed the long-term and persistent symptoms of OPICN. Many of the adverse effects produced by organophosphorus compounds are not related to AChE inhibition.64 Individuals with a history of acute organophosphate exposure reported an increased incidence of depression, irritability, confusion, and social isolation.65 Such exposures resulted in decreased verbal attention, visual memory, motoricity, and affectivity. 66 Rosenstock et al.58 reported that even a single exposure to organophosphates requiring medical treatment was associated with a persistent deficit in neuropsychological functions. A study of long-term effects in individuals who experienced acute toxicity with organophosphorus insecticides indicated dose-dependent decreases in sustained visual attention and vibrotactile sensitivity.59 In another study, one-fourth of the patients who were hospitalized following exposure to methamidophos exhibited an abnormal vibrotactile threshold between 10 and 34 mo after hospitalization.67

Callender et al.60 have described a woman with chronic neurological sequelae following acute exposure to a combination of an organophosphorothioate insecticide, pyrethrin, piperonyl butoxide, and petroleum distillates. Initially, she developed symptoms of acute cholinergic toxicity. One month after exposure, she experienced severe frequent headaches, muscle cramps, and diarrhea. After 3.5 mo, she developed numbness in her legs, tremors, memory problems, anxiety- depression, and insomnia. One year following exposure, she developed weakness, imbalance, and dizziness, and was confined to a wheelchair. Her symptoms were all characteristic of OPIDN. Twenty-eight months after exposure, she developed “delayed sequelae of gross neurologic symptoms,” consisting of coarse tremors, intermittent hemiballistic movements of the right arm and leg, flaccid fasciculations of muscle groups, muscle cramps, and sensory disturbances.

Some victims of the Tokyo subway sarin incident, who developed acute cholinergic neurotoxicity, also developed long-term, chronic neurotoxicity characterized by CNS neurological deficits and neurobehavioral impairments.11 Six to 8 mo after the Tokyo poisoning, some victims showed delayed effects on psychomotor performance, the visual nervous system, and the vestibule-cerebellar system.68 It is noteworthy that females were more likely than males to exhibit delayed effects on the vestibular-cerebellar system. Three years after the Matsumoto attack in Japan, some patients complained of fatigue, shoulder stiffness, weakness, and blurred vision.9 Others complained of insomnia, bad dreams, husky voice, slight fever, and palpations. Colosio et al.62 reviewed the literature on the neurobehavioral toxicity of pesticides, and reported that some individuals who were acutely poisoned with organophosphorus compounds developed long-term impairment of neurobehavioral performance. They also concluded that these effects were only “an aspecific expression of damage and not of direct neurotoxicity.”

OPICN following subclinical exposures to organophosphorus compounds. Reports on OPICN in individuals following long-term, subclinical exposures, without previous acute poisoning, have been inconsistent, mostly because of difficulty in the quantitative determination of exposure levels, but also because of problems with selection of controls. Several studies of workers exposed to low subclinical doses of organophosphorus insecticides failed to show neurobehavioral alterations between pre- and post-exposure measurements.69–75 It has been suggested that the levels of exposure of subjects in these reports might have been below the threshold level needed to cause neurobehavioral deficits, and that studies of prolonged low-level exposures may eventually reveal neurobehavioral deficits.62 Consistent with this opinion are the reports of impairment in neurobehavioral performance in individuals exposed to low-levels of organophosphorus insecticides. Professional pesticide applicators and farmers who had been exposed to organophosphorus pesticides showed elevated levels of anxiety, impaired vigilance and reduced concentration.76 Kaplan et al.77 reported persistent long-term cognitive dysfunction and defects in concentration, word finding, and short-term memory in individuals exposed to low subclinical levels of the organophosphorus insecticide chlorpyrifos. A significant increase in hand vibration threshold was reported in a group of pesticide applicators,78 and significant cognitive and neuropsychological deficits have been found in sheep dippers who had been exposed to organophosphorus insecticides.72,73 Male fruit farmers who were chronically exposed to organophosphorus insecticides showed significant slowing of their reaction time.79 Female pesticide applicators exhibited longer reaction times, reduced motor steadiness, and increased tension, depression, and fatigue compared with controls.74 Workers exposed to the organophosphorus insecticide quinalphos during its manufacture exhibited alterations in CNS function that were manifested as memory, learning, vigilance, and motor deficits, despite having normal AChE activity.80 Rescue workers and some victims who did not develop any acute neurotoxicity symptoms nevertheless complained of a chronic decline in memory 3 yr and 9 mo after the Tokyo attack.81 Pilkington et al.55 reported a strong association between chronic low-level exposure to organophosphate concentrates in sheep dips and neurological symptoms in sheep dippers— suggesting that long-term health effects may occur in at least some sheep dippers exposed to these insecticides over their working lives.

Neurological and neurobehavioral alterations. Although the symptoms of OPICN are a consequence of damage to both the PNS and CNS, they are related primarily to CNS injury and resultant neurological and neurobehavioral abnormalities. Studies on the effects of exposure to organophosphorus compounds over the past half century have shown that chronic neurological and neurobehavioral symptoms include headache, drowsiness, dizziness, anxiety, apathy, mental confusion, restlessness, labile emotions, anorexia, insomnia, lethargy, fatigue, inability to concentrate, memory deficits, depression, irritability, confusion, generalized weakness and tremors.51,52,82,83 Respiratory, circulatory, and skin problems may be present as well in cases of chronic toxicity.1 It should be noted that not every patient exhibits all of these symptoms. Gershon and Shaw51 reported that most of the symptoms that develop after organophosphate exposure resolve within 1 yr. Jamal50 conducted an extensive review of the health effects of organophosphorus compounds and concluded that either acute or long-term, low-level exposure to these chemicals produces a number of chronic neurological and psychiatric abnormalities that he called “chronic organophosphate-induced neuropsychiatric disorder,” or COPIND. Jamal recommended a multifaceted approach to the evaluation of the toxic effects of chronic, subclinical, repeated, low-level exposures to organophosphorus compounds; included were structural and quantitative analyses of symptoms and clinical neurological signs. In the present article, our concept of OPICN encompasses structural, functional, physiological, neurological, and neurobehavioral abnormalities, including neuropsychiatric alterations or COPIND. OPICN may be caused by an acute exposure that results in cholinergic toxicity, or by exposure to subclinical doses that do not produce acute poisoning.

Neuropathological alterations. Petras84 investigated the neuropathological alterations in rat brains 15–28 days following intramuscular injections of large, acutely toxic doses (79.4–114.8 µg/kg) of the nerve agent soman. He reported that the brain damage in all 4 animals that developed seizures was comparable to damage present in 3 of the 4 animals that exhibited only limb tremor. Neuropathological lesions were characterized by axonal degeneration, seen in the cerebral cortex, basal ganglia, thalamus, subthalamic region, hypothalamus, hippocampus, fornix, septum, preoptic area, superior colliculus, pretectal area, basilar pontine nuclei, medullary tegmentum, and corticospinal tracts. Although the mechanism of soman-induced brain injury was not known, Petras noted that the lesions did not resemble those present in experimental fetal hypoxia85 or OPIDN.17 These results are consistent with later findings obtained after acute soman exposure,86,87 exposure to the nerve agent sarin,88 and neuronal necrosis induced by the organophosphorus insecticide fenthion.89 Petras also indicated that soman-treated rats did not need to experience a seizure to develop lesions. Abdel-Rahman et al.90 demonstrated neuropathological alterations in rat brain 24 hr after administration of an intramuscular LD50 dose (100 µg/kg) of sarin. Neuronal degeneration was present in the cerebral cortex, dentate gyrus, CA1 and CA3 subfields of the hippocampal formation, and the in Purkinje cells of the cerebellum. Neuronal degeneration of hippocampal cells is consistent with organophosphorus compound-induced alterations in behavior, and cognitive deficits such as impaired learning and memory. 91–94 Furthermore, chronic exposure to organophosphorus compounds resulted in long-term cognitive deficits, even in the absence of clinical signs of acute cholinergic toxicity.95,96 Shih et al.97 demonstrated that lethal doses (2 × LD50) of all tested nerve agents (i.e., tabun, sarin, soman, cyclosarin, VR, and VX) induced seizures accompanied by neuropathological lesions in the brains of guinea pigs, similar to those lesions reported for other organophosphorus compounds in other species.98–103 Recent reports have indicated that anticonvulsants protected guinea pigs against soman- and sarin-induced seizures and the development of neuropathological lesions.104,105 Time-course studies also have reported that sarin-induced brain lesions exacerbated over time and extended into brain areas that were not initially affected.88,106 Kim et al.107 found that that an intraperitoneal injection of 9 mg/kg (1.8 × LD50) DFP in rats protected with pyridostigmine bromide and atropine nitrate caused tonic-clonic seizures, followed by prolonged mild clonic epilepsy accompanied by early necrotic and delayed apoptotic neuronal degeneration. Early necrotic brain injury in the hippocampus and piriform/ entorhinal cortices was seen between 1 and 12 hr after dosing. On the other hand, typical apoptotic terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL)-positive cell death began to appear at 12 hr in the thalamus. Daily dermal administration of 0.01 × LD50 of malathion for 28 days caused neuronal degeneration in the rat brain that was exacerbated by combined exposure to the insect repellent DEET and/or the insecticide permethrin.108

Correlation between neuropathological lesions and neurological and neurobehavioral alterations. Neuropathological changes—the hallmark of OPICN— could explain the neurological, neurobehavioral, and neuropsychological abnormalities reported in humans and animals exposed to organophosphorus compounds. A subcutaneous dose of 104 µg/kg soman induced status epilepticus in rats, followed by degeneration of neuronal cells in the piriform cortex and CA3 of the hippocampus.103 Similar results have been reported in a variety of species.98,109,110 In another study, only those mice treated with a subcutaneous dose of 90 µg/kg of soman which developed long-lasting convulsive seizures exhibited the neuropathological alterations. 111 Twenty-four hours after dosing, numerous eosinophilic cells and deoxyribonucleic acid (DNA) fragmentation (TUNEL-positive) cells were observed in the lateral septum, the endopiriform and entorhinal cortices, the dorsal thalamus, the hippocampus, and the amygdala. Animals that had only slight tremors and no convulsions did not show any lesions.111 Guinea pigs given a subcutaneous dose of 200 µg/kg soman (2 × LD50) developed seizures and exhibited neuropathological lesions in the amygdala; the substantia nigra; the thalamus; the piriform, entorhinal, and perirhinal cortices; and the hippocampus between 24–48 hr following injection.104 Male guinea pigs developed epileptiform seizures after receiving 2 × LD50 subcutaneous doses of the following nerve agents: tabun (240 µg/kg), sarin (84 µg/kg), soman (56 µg/kg), cyclosarin (114 µg/kg), VX (16 µg/kg), or VR (22 µg/kg). The seizures were accompanied by necrotic death of neuronal cells, with the amygdala having the most severe injury, followed by the cortex and the caudate nucleus.97

An intraperitoneal injection of 9 mg/kg (1.8 × LD50) DFP caused severe early (15–90 min) tonic-clonic limbic seizures, followed by prolonged mild clonic epilepsy. 107 Necrotic cell death was seen 1 hr after DFP administration, primarily in the CA1 and CA3 subfields of the hippocampus and piriform/entorhinal cortices, and manifest as degeneration of neuronal cells and spongiform of neuropils. Whereas the severity of hippocampal injury remained the same for up to 12 hr, damage to the piriform/entorhinal cortices, thalamus, and amygdala continued to increase up to 12 hr. Furthermore, apoptotic death of neuronal cells (TUNEL-positive) was seen in the thalamus at 12 hr, and peaked at 24 hr. Rats that survived 1 × LD50 sarin (95 µg/kg) exhibited persistent lesions, mainly in the hippocampus, piriform cortex, and thalamus.88 Furthermore, brain injury was exacerbated over time; at 3 mo after exposure, other areas that were not initially affected became damaged. A recent study has described the early neuropathological changes in the adult male rat brain 24 hr after exposure to a single intramuscular dose of 1.0, 0.5, 0.1, or 0.01 × LD50 (100 µg/kg) sarin.90 Sarin at 1.0 × LD50 caused extensive severe tremors, seizures, and convulsions accompanied by damage involving mainly the cerebral cortex, the hippocampal formation (dentate gyrus, and CA1 and CA3 subfields) and the cerebellum. Damage was evidenced by (a) a significant inhibition of plasma BChE, brain region AChE, and M2 M-acetylcholine receptor ligand binding; (b) an increase in permeability of the blood-brain barrier; and (c) diffuse neuronal cell death coupled with decreased MAP-2 expression within the dendrites of surviving neurons. The 0.5 × LD50 sarin dose did not cause motor convulsions, and only moderate Purkinje neuron loss. The 0.1 and 0.01 × LD50 doses of sarin caused no alterations at 24 hr after dosing. These results indicate that sarin-induced acute brain injury is dose-dependent.

In animals treated with 1 × LD50 sarin, both superficial layers (I–III) and deeper layers (IV–V) of the motor cortex and somatosensory cortex showed degeneration of neurons. In the deeper layers of the cortex, neuron degeneration was seen in layer V. Pyramidal neurons in layers III and V of the cortex are the source of the axons of the corticospinal tract, which is the largest descending fiber tract (or motor pathway) from the brain controlling movements of various contralateral muscle groups. Thus, sarin-induced death of layers III and V neurons of the motor cortex could lead to considerable motor and sensory abnormalities, ataxia, weakness, and loss of strength. Furthermore, disruption of the hippocampal circuitry because of the degeneration of neurons in different subfields can lead to learning and memory deficits. Lesions in the cerebellum could result in gait and coordination abnormalities. Because the severely affected areas (e.g., the limbic system, corticofugal system, and central motor system) are associated with mood, judgment, emotion, posture, locomotion, and skilled movements, humans exhibiting acute toxicity symptoms following exposure to large doses of organophosphates may also develop psychiatric and motor deficits. Inasmuch as the damaged areas of the brain do not regenerate, these symptoms are expected to persist long-term.112–114 These findings are in agreement with a recent study by Kilburn115 which evaluated the neurobehavioral effects of chronic low-level exposure to the organophosphorus insecticide chlorpyrifos in 22 patients. Kilburn demonstrated, for the 1st time, an association between chlorpyrifos sprayed inside homes and offices and neurophysiological impairments in balance, visual fields, color discrimination, hearing reaction time, and grip strength. These patients also had psychological impairment of verbal recall and cognitive function, and two-thirds of them had been prescribed antidepressant drugs. In addition, the patients exhibited severe respiratory symptoms, accompanied by airway obstruction. Other chlorpyrifos-induced neurotoxicity incidents in humans have been reported.116 These results are consistent with the report that daily dermal application of 0.1 mg/kg chlorpyrifos to adult rats resulted in sensorimotor deficits.117 Also, maternal exposure to a daily dermal dose of 0.1 mg/kg chlorpyrifos during gestational days 4–20 caused an increased expression of GFAP in the cerebellum and hippocampus of offspring on postnatal day 30.118 A major component of astrocytic intermediate neurofilaments, GFAP is up-regulated in response to reactive gliosis resulting from insults such as trauma, neurodegenerative disease, and exposure to neurotoxicants.119

Mechanisms of OPICN. Recent studies have shown that large toxic doses of organophosphorus compounds cause early convulsive seizures and subsequent encephalopathy, leading to the necrotic death of brain neuronal cells, whereas small doses produce delayed apoptotic death. Pazdernik et al.103 have proposed the following 5 phases that result in organophosphorus compound-induced cholinergic seizures: (1) initiation, (2) limbic status epilepticus, (3) motor convulsions, (4) early excitotoxic damage, and (5) delayed oxidative stress. The mechanisms of neuronal cell death in OPICN that appear to be mediated through necrosis or apoptosis, which may involve increased AChE gene expression, are discussed below.

Necrosis. The large toxic doses of organophosphorus compounds which induce early seizures activate the glutamatergic system and involve the Ca2+-related excitotoxic process,120,121 possibly mediated by the Nmethyl- D-aspartate (NMDA) subtype of glutamate receptors. 122,123 De Groot104 hypothesized that accumulated acetylcholine, resulting from acute inhibition of AChE by organophosphorus compounds, leads to activation of glutamatergic neurons and the release of the excitatory L-glutamate amino acid neurotransmitter. This in turn produces increased depolarization and subsequent activation of the NMDA subtype of glutamate receptors—and the opening of NMDA ion channels— resulting in massive Ca2+ fluxes into the post-synaptic cell and causing neuronal degeneration. Thus, glutamate- induced neuronal degeneration during seizures may occur as a result of lowering of the threshold for glutamate excitation at NMDA receptor sites.

Activation of nitric oxide synthase, following stimulation of NMDA receptor sites, increases the level of nitric oxide, which functions as a signaling or cytotoxic molecule responsible for neuronal cell death.124 As a retrograde messenger, nitric oxide induces the release of several neurotransmitters, including excitatory amino acid L-glutamate125 which alters neurotransmitter balance and affects neuronal excitability. The production of nitric oxide is enhanced in AChE-inhibitor–induced seizures.126,127 Kim et al.107 demonstrated the involvement of nitric oxide in organophosphate-induced seizures and the effectiveness of nitric oxide synthesis inhibitors in preventing such seizures.

Apoptosis. Neuronal degeneration caused by apoptosis or programmed cell death may have physiologic or pathologic consequences. Elimination of precancerous, old, or excess cells is carried out by apoptosis without injury to surrounding cells as seen in necrosis.128 Small doses of organophosphorus compounds cause delayed neuronal cell death that involves free radical generation (i.e., reactive oxygen species [ROS]). Organophosphates that cause mitochondrial damage/dysfunction also cause depletion of ATP and increased generation of ROS, which results in oxidative stress.129,130 ROS can cause fatal depletion of mitochondrial energy (ATP), induction of proteolytic enzymes, and DNA fragmentation, leading to apoptotic death.129,131,132 These results are consistent with the DNA damage detected in the lymphocytes in peripheral blood in 8 individuals, following residential exposure to the organophosphorus insecticides chlorpyrifos and diazinon.133

The brain is highly susceptible to oxidative stressinduced injury for several reasons: (a) its oxygen requirements are high; (b) it has a high rate of glucose consumption; (c) it contains large amounts of peroxidizable fatty acids; and (d) it has relatively low antioxidant capacity.131,132 A single sublethal dose of 0.5 × LD50 sarin, which did not induce seizures, nevertheless caused delayed apoptotic death of rat brain neurons in the cerebral cortex, hippocampus, and Purkinje cells of the cerebellum 24 hr after dosing.90,134 Furthermore, rats treated with a single 0.1 × LD50 dose of sarin, and which did not exhibit brain histopathological alterations 1, 7, or 30 days after dosing, nevertheless showed apoptotic death of brain neurons in the same areas mentioned above, 1 yr after dosing.90,135 These results are consistent with the sensorimotor deficits exhibited by sarin-treated animals 3 mo after exposure; the animals showed continued deterioration when tested 6 mo after dosing.

Increased AChE gene expression. Recent studies have suggested that AChE may play a role in the pathogenesis of OPICN similar to that reported for Alzheimer’s Disease.136,137 We have demonstrated that sarin induced the AChE gene in the same regions of the brain that underwent neuronal degeneration.138 AChE has been shown to be neurotoxic in vivo and in vitro; it accelerates assembly of amyloid peptide in Alzheimer’s fibrils, leading to cell death via apoptosis.139 Some studies have demonstrated increased AChE expression in apoptotic neuroblastoma SK-N-SH cells after long-term culturing.139 Brain AChE has been shown to be toxic to neuronal (Neuro 2a) and glial-like (B12) cells.137 There are also reports that transgenic mice overexpressing human AChE in brain neurons undergo progressive cognitive deterioration.140 These results suggest that sarin may provoke an endogenous cell suicide pathway cascade in susceptible neurons (e.g., in the caspase-3 pathway), resulting in the release of AChE into adjacent brain tissues. The aggregation of AChE initiates more apoptotic neuronal death. Amplification of this cascade thus may result in the progressive neuronal loss that is the hallmark of sarin-induced chronic neurotoxicity. It is noteworthy that a common symptom of both OPICN and Alzheimer’s Disease is memory deficit, suggesting that the aging process may be accelerated following exposure to organophosphorus compounds in OPICN.

Other factors. The occurrence and severity of OPICN is influenced by factors such as environmental exposure to other chemicals, stress, or individual genetic differences. For example, cholinotoxicants such as organophosphates or carbamates—which do not have a positive charge and are capable of crossing the blood-brain barrier—act at the same receptors and thus exacerbate OPICN. Individuals with low levels of the plasma enzymes BChE5,6,141 or paraoxonase7,8 that act as the 1st line of defense against neurotoxicity (by removing organophosphates from circulation through scavenging or hydrolysis) are vulnerable to the development of persistent OPICN. All of these factors may be involved in development of the phenomenon known as “chemical sensitivity” or “multiple chemical sensitivity.”142 Thus, prior chemical exposure, stress, or genetic factors might make individuals predisposed or susceptible to CNS injury upon subsequent exposure to other chemicals.

Combined exposure to other chemicals that cause oxidative stress can intensify OPICN which results from exposure to organophosphorus compounds.108 Furthermore, stress that also causes oxidative stress decreases the threshold level required to produce neuronal damage and results in increased OPICN following combined exposure to stress and organophosphates. Thus, OPICN may explain the reports that Persian Gulf War veterans showed a higher than normal propensity toward persistent neurological complaints such as memory and attention deficits, irritability, chronic fatigue, muscle and joint pain, and poor performance on cognition tests.143–147 A large number of these personnel were exposed to low levels of sarin during the demolition of Iraqi munitions at Khamisiya,148,149 as well as to other chemicals and to stress.150,151 Also, OPICN may explain the recent report that Persian Gulf War veterans are at an almost 2-fold greater risk of developing amyotrophic lateral sclerosis (ALS) than other veterans,152 which is consistent with Haley’s153 suggestion that the increase in ALS is “a warrelated environmental trigger.” Furthermore, OPICN induced by low-level inhalation of organophosphates present in jet engine lubricating oils and the hydraulic fluids of aircraft154 could explain the long-term neurologic deficits consistently reported by crewmembers and passengers, although organophosphate levels may have been too low to produce OPIDN.155


Previous reports have indicated that, subsequent to exposure to organophosphorus compounds, an individual could develop acute cholinergic neurotoxicity, followed by OPICN. In a few cases, OPIDN may occur with or without the development of cholinergic neurotoxicity, with OPICN developing later. Furthermore, OPICN may occur following long-term, low-level exposure to organophosphorus compounds, and without the development of acute neurotoxicity. Because the long-term, persistent effects of OPICN result from neuronal degeneration of the PNS and CNS, induced by organophosphates, it is unlikely that improvement is the consequence of the regeneration of brain neurons, inasmuch as such repair is not typical of the CNS. Clinical improvement may occur, however, through repair of the PNS. Also, reversible changes in the CNS that might be present initially (e.g., edema) could later subside, giving the appearance of repair. Furthermore, if damage is not too extensive, other neurons having the same function could meet the added demands and maintain normal activity. When the CNS is severely damaged, however, neither of these repair mechanisms is possible and some loss of function will likely occur.


Herein we have described the long-term, persistent neurodegenerative disorder induced by exposure to organophosphorus compounds. We define this effect as organophosphorus ester-induced chronic neurotoxicity, or OPICN. Numerous cases documenting this disorder have been reported since the extensive use of these chemicals in industry and agriculture began more than 50 yr ago. Although largely characterized by chronic neurobehavioral alterations, OPICN involves other molecular, neurochemical, neurophysiological, neuropathological, neuropsychological, and neurological changes. The term “neurotoxicity” encompasses all of these, and adequately describes this neurodegenerative disorder.

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The author would like to express his appreciation for the artwork of Anjelika Dechkovskaia. Submitted for publication May 14, 2004; revised; accepted for publication May 30, 2004. Requests for reprints should be sent to Mohamed B. Abou- Donia, Ph.D., Department of Pharmacology and Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC 27710.

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1. Abou-Donia MB. Organophosphorus pesticides. In: Chang LW, Dyer RS (Eds). Handbook of Neurotoxicology. New York: Marcel Dekker, 1994; pp 419–47.

2. Koelle GB. Protection of cholinesterase against inevitable inactivation by diisopropyl fluorophosphate in vitro. J Pharmacol Exp Ther 1946; 88:232–37.

3. Abou-Donia MB. Neurotoxicology. Boca Raton, FL: CRC Press, 1992; pp 3–24.

4. Sussman JL, Harel M, Frolow F. Atomic structure of acetylcholinesterase from Torpedo californica. A prototypic acetylcholine-binding protein. Science 1992; 253:872–78.

5. Whittaker M. The pseudocholinesterase variants: esterase levels and increased resistance to fluoride. Acta Genet Basel 1994; 14:281–85.

6. Lockridge O. Genetic variants of human serum cholinesterase influence metabolism of the muscle relaxant succinylcholine. Pharmacol Ther 1990; 47:35–60.

7. Mackness B, Mackness MI, Arrol S, et al. Effect of the molecular polymorphisms of human paraoxonase (PONI) on the rate of hydrolysis of paraoxon. Br J Pharmacol 1997; 122:265–68.

8. Davies HG, Richter RJ, Keifer M, et al. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman, and sarin. Nat Genet 1996; 14: 334–36.

9. Morita H, Yanagisawa N, Nakajima T, et al. Sarin poisoning in Matsumoto, Japan. Lancet 1995; 346:290–93.

10. Okumura T, Takasu N, Ishimatsu S, et al. Report on 640 victims of the Tokyo subway sarin attack. Ann Emerg Med 1995; 28:129–35.

11. Yokoyama K, Araki S, Murata K, et al. Chronic neurobehavioral effects of Tokyo subway sarin poisoning in relation to posttraumatic stress disorder. Arch Environ Health 1998; 53:249–56.

12. Masuda N, Takatsu M, Morinari H. Sarin poisoning in Tokyo subway. Lancet 1995; 345:1446–47.

13. Smith MI, Elvove E, Frazier WH. The pharmacological action of certain phenol esters with special reference to the etiology of so-called ginger paralysis. Public Health Rep 1930; 45:2509–24.

14. Johnson MK. The delayed neuropathology caused by some organophosphorous esters: mechanism and challenge. Crit Rev Toxicol 1975; 2:289–316.

15. Abou-Donia MB. Organophosphorous ester-induced delayed neurotoxicity. Annu Rev Pharmacol Toxicol 1981; 21:511–48.

16. Abou-Donia MB, Lapadula LM. Mechanisms of organophosphorus ester-induced delayed neurotoxicity: type I and type II. Annu Rev Pharmacol Toxicol 1990; 30:405–40.

17. Cavanagh JB, Patangia GN. Changes in the central nervous system in the cat as the result of tri-o-cresylphosphate poisoning. Brain 1995; 88:165–80.

18. Himuro K, Murayama S, Nishiyama K, et al. Distal sensory axonopathy after sarin intoxification. Neurology 1998; 51:1195–97.

19. Davies OR, Holland PR. Effect of oximes and atropine upon the development of delayed neurotoxic signs in chickens following poisoning by DFP and sarin. Biochem Pharmacol 1972; 21:3145–51.

20. Abou-Donia MB. Triphenyl phosphite: a type II organophosphorus compound-induced delayed neurotoxic agent. In: Chambers JE, Levi PE (Eds). Organophosphates: Chemistry, Fates, and Effects. Part IV: Toxic Effects – Organismal. San Diego, CA: Academic Press, 1992; pp 327–51.

21. Abou-Donia MB, Wilmarth KR, Jansen KF, et al. Triphenylphosphine (TPP): a type III organophosphorous compound-induced delayed neurotoxic agent (OPIDN). Toxicologist 1996; 30:311.

22. Abdel-Rahman AA, Jensen KF, Farr CH, et al. Daily treatment of triphenylphosphine (TPP) produces organophosphorous-induced delayed neurotoxicity (OPIDN). Toxicologist 1997; 36:19.

23. Bloch H, Hottinger A. Uber die spezifitat der cholinesterase- hemmung durch tri-o-kresyl phosphat. Int Z Vitaminforsch 1943; 13:90.

24. Earl CJ, Thompson RHS. The inhibitory action of triortho- cresyl phosphate and cholinesterases. Br J Pharmacol 1952; 7:261–69.

25. Aldridge WN, Barnes JM. Further observations on the neurotoxicity of organophosphorus compounds. Biochem Pharmacol 1966; 15:541–47.

26. Johnson MK. The delayed neurotoxic effect of some organophosphorus compounds. Br Med Bull 1969; 114:711–17.

27. Lotti M. The pathogenesis of organophosphate delayed neuropathy. Crit Rev Toxicol 1992; 21:465–87.

28. Curtes JP, Develay P, Hubert JP. Late peripheral neuropathy due to acute voluntary intoxication by organophosphorous compounds. Clin Toxicol 1981; 18:1453.

29. de Jager AE, van Weerden TW, Houthoff HJ, et al. Polyneuropathy after massive exposure to parathion. Neurology 1981; 31:603–05.

30. Stamboulis E, Psimaras A, Vassilopoulos D. Neuropathy following acute intoxication, with mercarbam (Opester). Acta Neurol Scand 1991; 83:198.

31. Winrow CJ, Hemming ML, Allen DA, et al. Loss of neuropathy target esterase in mice links organophosphate exposure to hyperactivity. Nat Genet 2003; 33:477–85.

32. O’Callahan JP. Neurotoxic esterase: not so toxic? Nat Genet 2003; 33:1–2.

33. Bus J, Maurissen J, Marable B, et al. Association between organophosphate exposure and hyperactivity? Nat Genet 2003; 34(3):235.

34. Abou-Donia MB. Involvement of cytoskeletal proteins in the mechanisms of organophosphorous ester delayed neurotoxicity. Clin Exp Pharmacol Physiol 1995; 22:358–59.

35. Schulman H. Advances in Second Messenger and Phosphorylation Research. New York: Raven Press, 1988; pp 39–111.

36. Patton SE, Lapadula DM, Abou-Donia MB. Relationship of tri-o-cresyl phosphate-induced delayed neurotoxicity to enhancement of in vitro phosphorylation of hen brain and spinal cord. J Pharmacol Exp Ther 1986; 239: 597–605.

37. Suwita E, Lapadula DM, Abou-Donia MB. Calcium and calmodulin-enhanced in vitro phosphorylation of hen brain cold-stable microtubules and spinal cord neurofilament triplet proteins after a single oral dose of tri-ocresyl phosphate. Proc Natl Acad Sci USA 1986; 83: 6174–78.

38. Abou-Donia MB, Viana ME, Gupta RP, et al. Enhanced calmodulin binding concurrent with increased kinasedependent phosphorylation of cytoskeletal proteins following a single subcutaneous injection of diisopropyl phosphorofluoridate in hens. Neurochem Res 1993; 22:165–73.

39. Gupta RP, Abou-Donia MB. Tau proteins-enhanced Ca2+/calmodulin (CaM)-dependent phosphorylation by the brain supernatant of diisopropyl phosphorofluoridate (DFP)-treated hen: tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II. Brain Res 1998; 813:32–43.

40. Gupta RP, Abou-Donia MB. In vivo and in vitro effects of diisopropyl phosphorofluoridate (DFP) on the rate of hen brain tubulin polymerization. Neurochem Res 1994; 19:435–44.

41. Gupta RP, Abou-Donia MB. Neurofilament phosphorylation and [125I] calmodulin binding by Ca2+ /calmodulin- dependent protein kinase in the brain subcellular fractions of diisopropyl phosphorofluoridate (DFP)- treated hen. Neurochem Res 1995; 20(9):1095–105.

42. Gupta RP, Abou-Donia, MB. Diisopropyl phosphorofluoridate (DFP) treatment alters calcium-activated proteinase activity and cytoskeletal proteins of the hen sciatic nerve. Brain Res 1995; 677:162–66.

43. Gupta RP, Bing G, Hong JS, et al. cDNA cloning and sequencing of Ca2+ /calmodulin-dependent protein kinase _ subunit and its mRNA expression in diisopropyl phosphorofluoridate (DFP)-treated hen central nervous system. Mol Cell Biochem 1998; 181:29–39.

44. Gupta RP, Abou-Donia MB. Tau phosphorylation by diisopropyl phosphorofluoridate (DFP)-treated hen brain supernatant inhibits its binding with microtubules: role of Ca2+/calmodulin-dependent protein kinase II in tau phosphorylation. Biochem Pharmacol 1999; 53: 1799–1806.

45. Gupta RP, Damodaran TV, Abou-Donia MB. C-fos mRNA induction in the central and peripheral nervous systems of diisopropyl phosphorofluoridate (DFP)-treated hens. Neurochem Res 2000; 25(3):327–34.

46. Gupta RP, Abdel-Rahman AA, Jensen KF, et al. Altered expression of neurofilament subunits in diisopropyl phosphorofluoridate (DFP)-treated hen spinal cord and their presence in axonal aggregates. Forthcoming.

47. Damodaran TV, Abou-Donia MB. Alterations in levels of mRNAs coding for glial fibrillary acidic protein (GFAP) and vimentin genes in the central nervous system of hens treats with diisopropyl phosphorofluoridate (DFP). Neurochem Res 2000; 25:809–16.

48. Jensen KF, Lapadula DM, Anderson JK, et al. Anomalous phosphorylated neurofilament aggregations in central and peripheral axons of hens treated with triortho- cresyl phosphate (TOCP). J Neurosci Res 1992; 33:455–60.

49. Abou-Donia MB, et al. Unpublished results, 2004.

50. Jamal G. Neurological syndromes of organophosphorus compounds. Adverse Drug React Toxicol Rev 1997; 16:133–70.

51. Gershon S, Shaw FB. Psychiatric sequelae of chronic exposure to organophosphorous insecticides. Lancet 1961; 1:1371–74.

52. Dille JR, Smith PW. Central nervous system effects of chronic exposure to organophosphate insecticides. Aerosp Med 1964; 35:475–78.

53. Metcalf DR, Holmes JH. EEG, psychological and neurological alterations in humans with organophosphorous exposure. Ann NY Acad Sci 1969; 160:357–65.

54. Duffy FH, Burchfield JL, Bartels PH, et al. Long-term effects of an organophosphate upon the human electroencephalogram. Toxicol Appl Pharmacol 1979; 47: 161–76.

55. Pilkington A, Buchanan D, Jamal GA, et al. An epidemological study of the relations between exposure to organophosphate pesticides and indices of chronic peripheral neuropathy and neuropsychological abnormalities in sheep farmers and dippers. Occup Environ Med 2001; 58(11):702–10.

56. Russell R, Macri J. Central cholinergic involvement hyper-reactivity. Pharmocol Biochem Behav 1979; 10: 43–48.

57. Joubert J, Joubert PH. Chorea and psychiatric changes in organophosphate poisoning. A report of 2 further studies. S Afr Med J 1988; 74:32–34.

58. Rosenstock L, Keifer M, Daniell WE, et al. Chronic central nervous system effects of acute organophosphate pesticide intoxication. Lancet 1991; 338:223–27.

59. Steenland K, Jenkins B, Ames RG, et al. Chronic neurological sequelae to organophosphate pesticide poisoning. Am J Public Health 1994; 84:731–36.

60. Callender TJ, Morrow L, Subramanian K. Evaluation of chronic neurological sequelae after acute pesticide exposure using SPECT brain scans. J Toxicol Environ Health 1994; 41:275–84.

61. Stephens R, Spurgeon A, Calvert IA, et al. Neuropsychological effects of long-term exposure to organophosphates in sheep dip. Lancet 1995; 345:1135–39.

62. Colosio C, Tiramani M, Maroni M. Neurobehavioral effects of pesticides: state of the art. Neurotoxicology 2003; 24:577–91.

63. Scremin O, Shih TM, Huynh L, et al. Delayed neurologic and behavioral effects of subtoxic doses of cholinesterase inhibitors. J Pharmacol Exp Ther 2003; 304:1111–19.

64. Echbichon DJ, Joy RM. Pesticides and Neurological Diseases. 2nd ed. Boston and London: CRC Press, 1995.

65. Savage EP, Keefe TF, Mounce LM, et al. Chronic neurological sequelae of acute organophosphates pesticide poisoning. Arch Environ Health 1988; 43:38–45.

66. Maroni M, Jarvisalo J, La Ferla L. The WHO-UNDP edidemiological study on the health effects of exposure to organophosphorous pesticides. Toxicol Lett 1986; 33:115–23.

67. McConell R, Keifer M, Rosenstock L. Elevated quantitative vibrotactile threshold among workers previously poisoned with methamidophos and other organophosphate pesticides. Am J Ind Med 1994; 25:325–34.

68. Yokoyama K, Araki K, Murata K, et al. A preliminary study on delayed vestibulo-cerebellar effects of Tokyo Subway sarin poisoning in relation to gender difference: frequency analysis of postural sway. J Occup Environ Med 1998; 40(1):17–21.

69. Rodnitzky RL, Levin HS, Mick DL. Occupational exposure to organophosphate pesticides. Arch Environ Health 1975; 30:98–103.

70. Maizlish N, Schenker M, Weisskopf C, et al. A behavioral evaluation of pest control workers with shortterm, low-level exposure to the organophosphate diazinon. Am J Indust Med 1987; 12:153–72.

71. Daniell W, Barnhart S, Demers P, et al. Neuropsychological performance among agricultural pesticide applicators. Environ Res 1992; 59(1):217–28.

72. Beach JR, Spurgeon A, Stephens R, et al. Abnormalities on neurological examination among sheep farmers exposed to organophosphate pesticides. Occup Environ Med 1996; 53(8):520–25.

73. London L, Myers JE, Neil V, et al. An investigation into neurological and neurobehavioral effects of long-term agrochemical use among deciduous fruit farm workers in the Western Cape, South Africa. Environ Res 1997;


74. Bazylewicz-Walckzak B, Majzakova W, Szymczak M. Behavioral effects of occupational exposure to organophosphorous pesticides in female greenhouse plantin workers. Neurotoxicology 1999; 20(5):819–26.

75. Steenland M. Chronic neurological effects of organophosphate pesticides. Br Med J 1996; 312: 1311–12.

76. Levin HS, Rodnitzsky RL, Mick DL, et al. Anxiety associated with exposure to organophosphorous compounds. Arch Gen Psychiatry 1976; 33:225–28.

77. Kaplan JG, Kessler J, Rosenberg N, et al. Sensory neuropathy associated with Dursban (chlorpyrifos) exposure. Neurology 1993; 43:2193–96.

78. Stokes L, Stark A, Marshall E, et al. Neurotoxicity among pesticide applicators exposed to organophosphates. Occup Environ Health 1995; 52:648–53.

79. Fielder N, Feldman RG, Jacobson J, et al. The assessment of neurobehavioral toxicity: SOGOMSEC joint report. Environ Health Perspect 1996; 104(Suppl 2): 179–91.

80. Srivastava AK, Gupta BN, Bihari V, et al. Clinical, biochemical and neurobehavioral studies on workers engaged in the manufacture of quinalphos. Food Chem Toxicol 2000; 38(1):65–69.

81. Nishiwaki Y, Maekawa K, Ogawa Y, et al. Effects of sarin on the nervous system in rescue team staff members and police officers 3 years after the Tokyo subway sarin attack. Environ Health Perspect 2001;109: 1169–73.

82. Durham WF, Wolfe HR, Quinby GE. Organophosphorus insecticides and mental alertness. Arch Environ Health 1965; 10:55–66.

83. Tabershaw IR, Cooper WC. Sequelae of acute organic phosphate poisoning. J Occup Med 1966; 8:5–20.

84. Petras JM. Soman neurotoxicity. Fundam Appl Toxicol 1981; 1:242–49.

85. Faro MD, Windle WF. Transneuronal degeneration in brains of monkeys asphyxiated at birth. Exp Neurol 1969; 24:38–53.

86. McDonough JH, Dochterman LW, Smith CD, et al. Protection against nerve agent-induced neuropathology, but not cardiac pathology, is associated with the anticonvulsant action of drug treatment. Neurotoxicology 1995; 15:123–32.

87. Kadar T, Cohen G, Sarah R, et al. Long-term study of brain lesions following soman, in comparison to DFP and metrazol poisoning. Hum Exp Toxicol 1992; 11: 517–23.

88. Kadar T, Shapira S, Cohen G, et al. Sarin induced neuropathology in rats. Hum Exp Toxicol 1995; 14: 252–59.

89. Veronesi B, Jones K, Pope C. The neurotoxicity of subchronic acetylcholinesterase (AChE) inhibition in rat hippocampus. Toxicol Appl Pharmacol 1990; 104: 440–56.

90. Abdel-Rahman A, Shetty AK, Abou-Donia MB. Acute exposure to sarin increases blood brain barrier permeability and induces neuropathological changes in the rat brain: dose-response relationships. Neuroscience 2002; 113(3):721–41.

91. McDonald BE, Costa LG, Murphy SD. Spatial memory impairment and central muscarinic receptor loss following prolonged treatment with organophosphates. Toxicol Lett 1988; 40:47–56.

92. Rafaelle K, Olton D, Annau Z. Repeated exposure to diisopropylfluorophosphate (DFP) produces increased sensitivity to cholinergic antagonists in discrimination retention and reversal. Psychopharmacology (Berl) 1990; 100:267–74.

93. Bushnell PJ, Padilla SS, Ward T, et al. Behavioral and neurochemical changes in rats dosed repeatedly with diisopropyl fluorophosphate. J Pharmacol Exp Ther 1991; 256:741–50.

94. Kassa J, Koupilova M, Vachek J. The influence of lowlevel sarin inhalation exposure on spatial memory in rats. Pharmacol Biochem Behav 2001; 70:175–79.

95. Prendergast MA, Terry AV Jr, Buccafusco JJ. Chronic, low-level exposure to diisopropyl fluorophosphate causes protracted impairment of spatial navigation learning. Psychopharmacology (Berl) 1997; 130: 276–84.

96. Prendergast MA, Terry AV Jr, Buccafusco JJ. Effects of chronic low-level organophosphate exposure on delayed recall, discrimination and spatial learning in monkeys and rats. Neurotoxicol Teratol 1998; 20: 115–22.

97. Shih TM, Duniho SM, McDonough JH. Control of nerve agent-induced seizures is critical for neuroprotection and survival. Toxicol Appl Pharmacol 2003; 188:69–80.

98. Lemercier G, Carpentier P, Setenac-Roumanou H, et al. Histological and histochemical changes in the central nervous system of the rat poisoned by an irreversible anticholinesterase organophosphorous compound. Acta Neuropathol 1983; 61:123–29.

99. Petras JM. Neurology and neuropathology of soman-induced brain injury: an overview. J Exp Anal Behav 1994; 61:319–29.

100. Carpentier P, Delamanche IS, Lebert M, et al. Seizurerelated opening of the blood brain barrier induced by soman: possible correlation with the acute neuropathology observed in poisoned rats. Neurotoxicology 1990; 11:493–508.

101. Clement JG, Broxup B. Efficacy of diazepam and avizafone against soman-induced neuropathology in brain of rats. Neurotoxicology 1993; 14:485–504.

102. Shih TM, Koviak TA, Capacio BR. Anticonvulsants for poisoning by the organophosphorous compound Soman: pharmacological mechanisms. Neurosci Biobehav Rev 1991; 15:349.

103. Pazdernik TL, Emerson MR, Cross R, et al. Soman-induced seizures: limbic activity, oxidative stress, and neuroprotective proteins. J Appl Toxicol 2001; 21: S87–S94.

104. deGroot DMG, Bierman EPB, Bruijnzeel PLB, et al. Beneficial effects of TCP on soman intoxication in guinea pigs: seizures, brain damage, and learning behavior. J Appl Toxicol 2001; 21:S57–S65.

105. Taysee L, Calvet JH, Buee J, et al. Comparative efficacy of diazepam and avizafone against sarin-induced neuropathology and respiratory failure in guinea pigs: influence of atropine dose. Toxicology 2003; 188: 197–209.

106. Abou-Donia MB, et al. Unpublished results, 2004.

107. Kim YB, Hur GH, Shin S, et al. Organophosphate-induced brain injuries: delayed apoptosis mediated by nitric oxide. Environ Toxicol Pharmacol 1999; 7:147–52.

108. Abdel-Rahman AA, Dechkovskaia AM, Goldstein LB, et al. Neurological deficits induced by malathion, DEET and permethrin, alone or in combination in adult rats. J Toxicol Environ Health A 2004; 67:331–56.

109. McLeod CG, Singer AW, Harrington DG. Acute neuropathology in soman-poisoned rats. Neurotoxicology 1984; 5:53–58.

110. Churchill L, Pazdernik TL, Jackson JL. Soman-induced brain lesions demonstrated by muscarinic receptor autoradiography. Neurotoxicology 1985; 6:81–90.

111. Baille V, Dorandeu F, Carpentier P, et al. Acute exposure to a low or mild dose of soman: biochemical, behavioral and histopathological effects. Pharmacol Biochem Behav 2001; 69:561–69.

112. Sidell FR. Soman and sarin: clinical manifestations and treatment of accidental poisoning by organophosphates. Clin Toxicol 1974; 7(1):1–17.

113. West I. Sequelae of poisoning from phosphate ester pesticides. Ind Med Surg 1968; 37(11):832.

114. Namba T, Nolte CT, Jackrel J et al. Poisoning due to organophosphate insecticides. Am J Med 1971; 50:475.

115. Kilburn KH. Evidence for chronic neurobehavioral impairment from chlorpyrifosa, and organophosphate insecticide (Dursban) used indoors. Environ Epidemiol Toxicol 1999; 1:153–62.

116. Blondell J, Dobozy VA. Review of Chlorpyrifos Poisoning Data. Washington, DC: U.S. Environmental Protection Agency, 14 Jan 1997.

117. Abou-Donia MB, Abdel-Rahman AA, Goldstein LB, et al. Sensorimotor deficits and increased brain nicotinic acetylcholine receptors following exposure to chlorpyrifos and/or nicotine in rats. Arch Toxicol 2003; 77: 452–58.

118. Abdel-Rahman AA, Dechkovskaia AM, Mehta-Simmons H, et al. Increased expression of glial fibrillary acidic protein in cerebellum and hippocampus: differential effects on neonatal brain regional acetylcholinesterase following maternal exposure to combined chlorpyrifos and nicotine. J Toxicol Environ Health A, 2003; 66:2047–66.

119. Eng LF, Ghirnikar RS. GFAP and astrogliosis. Brain Pathol 1994; 4:229–37.

120. Olney JW, de Gubareff T, Labruyere J. Seizure-related brain damage induced by cholinergic agents. Nature 1983; 301:520–22.

121. Dawson VL, Dawson TM, Lonedon ED, et al. Nitric oxide mediates glutamate neurotoxicity in primary cortical cultures. Proc Natl Acad Sci USA 1991; 88: 6368–71.

122. Solberg Y, Belkin M. The role of excitotoxicity in organophosphorus nerve agents central poisoning. Trends Pharmacol Sci 1997; 8:183–85.

123. Raveh L, Chapman S, Cohen G, et al. The involvement of the NMDA receptor complex in the protective effect of anticholinergic drugs against soman poisoning. Neurotoxicology 1999; 20:551–60.

124. DawsonTM, Dawson VL, Snyder SH. A novel neuronal messenger molecule in brain: the free radical, nitric oxide. Ann Neurol 1992; 32:297–31.

125. Montague PR, Gancayco CD, Winn MJ., et al. Role of NO production in NMDA receptor-mediated neurotransmitter release in cerebral cortex. Science 1994; 263(5149):973–77.

126. Bagetta G, Massoud R, Rodino P, et al. Systematic administration of lithium chloride and tacrine increases nitric oxide synthase activity in the hippocampus of rats. Eur J Pharmacol 1993; 237:61–64.

127. Kim YB, Hui GH, Lee YS, et al. A role of nitric acid oxide in organophosphate-induced convulsions. Environ Toxicol Pharmacol 1997; 1(3):53–56.

128. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267:1456–62.

129. Tsujimoto Y. Apoptosis and necrosis: intracellular ATP level as a determinant for cell death modes. Cell Death Differ 1997; 4:429–34.

130. Murphy AN, Fiskum G, Beal MF. Mitochondria in neurodegeneration: cell life and death. J Cereb Blood Flow Metab 1999: 19:231–45.

131. Floyd RA. Antioxidants, oxidative stress, and degenerative neurological disorders. Proc Soc Exp Biol Med 1999; 222:236–45.

132. Gupta RP, Milatovic D, Dettbarn WD. Depletion of energy metabolites following acetylcholinesterase inhibitor- induced status epilepticus: protection by antioxidants. Neurotoxicology 2001; 22:271–82.

133. Lieberman AD, Craven MR, Lewis HA, et al. Genotoxicity from domestic use of organophosphate pesticides. J Occup Environ Med 1998; 40(11):954–57.

134. Abou-Donia MB, et al. Unpublished data, 2004.

135. Abou-Donia MB, et al. Unpublished results, 2004.

136. Sberna G, Saez-Valero J, Li QX, et al. Acetylcholinesterase is increased in the brains of transgenic mice expressing the C-terminal fragment (CT100) of the B-amyloid protein precursor of Alzheimer’s Disease. J Neurochem 1998; 71:723–31.

137. Calderon FH, von Bernhardi R, De Ferrari G, et al. Toxic effects of acetylcholinesterase on neuronal and glial-like cells in vitro. Mol Psychiatry 1998; 3:247–55.

138. Damodaran TV, Jones KH, Patel AG, et al. Sarin (nerve agent GB)-induced differential expression of mRNA coding for the acetylcholinesterase gene in the rat central nervous system. Biochem Pharmacol 2003; 65: 2041–47.

139. Yang L, Heng-Yi H, Zhang XJ. Increased expression of intranuclear AChE involved in apoptosis of SK-N-SH cells. Neurosci Res 2002; 42:261–68.

140. Andres C, Seidman S, Beeri R, et al. Transgenic acetylcholinesterase induces enlargement of murine neuromuscular junctions but leaves spinal cord synapses intact. Neurochem Int 1998; 32:449–56.

141. Abou-Donia MB, Wilmarth KR, Jensen KF. Neurotoxicity resulting from coexposure to pyridostigmine bromide, DEET, and permethrin: implications of Gulf War chemical exposures. J Toxicol Environ Health 1996; 48: 35–56.

142. Rae WJ. Chemical Sensitivity. Vol 1. Boca Raton, FL: Lewis Publishers, 1992; pp 47–154.

143. Kurt TL. Epidemiological association in U.S. veterans between Gulf War illiness and exposure to anticholinesterases. Toxicol Lett 1998; 11:1–5.

144. Anger WK, Storzbach D, Binder LM. Neurobehavioral deficits in Persian Gulf veterans: evidence from a population- based study. J Int Neuropsychol Soc 1999; 5:203–12

145. McCauley LA, Rischitelli G, Lambert WE. Symptoms of Gulf War vertrans possibly exposed to organophosphate chemical warfare agents at Khamisiyah, Iraq. Int J Occup Environ Health 2001; 7:3170–75.

146. Storzbach D, Campbell KA, Binder LM. Psychological differences between veterans with and without Gulf War unexplained symptoms. Psychosom Med 2000; 726–35.

147. White RF, Proctor SP, Heeren T. Neuropshchological functions in Gulf War veterans: relationship to self-reported toxicant exposures. Amer J Ind Med 2001; 40: 42–44.

148. Institute of Medicine of the National Academies. Health Consequences of Service during the Persian Gulf War: Initial Findings and Recommendation for Immediate Action. Washington, DC: National Academies Press, 1995.

149. Augerson WS. A Review of The Scientific Literature as it Pertains to Gulf War Illnesses. Vol 5. Chemical and Biological Warfare Agents. Santa Monica, CA: Rand Corporation, 2000.

150. Hyams KC, Wignall FS, Roswell R. War syndromes and their evaluation: from the U.S. Civil War to the Persian Gulf War. Ann Intern Med 1995; 125:398–405.

151. Baker DG, Mendenhall CL, Simbart LA. Relationship between posttraumatic stress disorder and self-reported physical symptoms in Persian Gulf War veterans. Arch Intern Med 1997; 157:2076–78.

152. Horner RD, Kamins KG, Feussner JR, et al. Occurrence of amyotrophic lateral sclerosis among Gulf War veterans. Neurology 2003; 61:742–49.

153. Haley RW. Excess incidence of ALS in young Gulf War veterans. Neurology 2003; 61:750–56.

154. Freudenthal RL, Rausch L, Gerhart JK, et al. Subchronic neurotoxicity of oil formulations containing either tricresyl phosphate or tri-orthocresyl phosphate. J Am Coll Toxicol 1993; 12:409–16.

155. Daughtrey W, Biles R, Jortner B. Subchronic delayed neurotoxicity evaluation of jet engine lubricants containing phosphorus additives. Fundam Appl Toxicol 1996; 32:244–49.

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