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Organophosphorus Compounds
Organophosphorus Compounds RJ Richardson, University of Michigan, Ann Arbor, MI, USA GF Makhaeva, Institute of Physiologically Active Compounds, Russian Academy of Sciences, Chernogolova, Moscow Region, Russia Ó 2014 Elsevier Inc. All rights reserved.
Name Organophosphorus (OP) compounds are organic compounds containing trivalent or pentavalent phosphorus. This chapter focuses on the more common pentavalent phosphorus compounds.
Synonyms OP compounds are often referred to generically as organophosphates; however, this is a specific subset comprising derivatives of phosphoric acid. Many other derivatives are possible, such as phosphorothioates, phosphonates, phosphoramidates, and phosphinates. OP insecticides are typically organophosphates or organophosphorothioates. OP nerve agents (nerve gases) are usually organophosphonates; these are true organophosphorus compounds, as they contain a carbon– phosphorus covalent bond.
Molecular Formula Figure 1 shows a generic structure of an OP compound containing pentacovalent phosphorus. In such compounds, the phosphorus atom has tetrahedral geometry; thus, depending on the identity of the substituents attached to phosphorus, optical isomerism is possible.
When organochlorine insecticides were phased out owing to ecological concerns voiced in the 1960s and 1970s, the use of OP insecticides increased as replacements. More recently, OP insecticide use has been curtailed in response to health concerns and wider adoption of integrated pest management. Currently, new uses are being found for non-insecticidal OP compounds. For example, as certain flame retardants, such as the polybrominated diphenyl ethers (PBDEs), are being phased out, OP compounds are entering the market as replacements.
Uses OP compounds are incredibly diverse with respect to chemical structure and attendant physical–chemical properties; accordingly, the uses of these compounds cover a wide range. Thus, depending on their molecular structures, OP compounds have found uses as insecticides, nerve agents, hydraulic fluids, fuel additives, lubricants, plasticizers, and flame retardants. They are also used as drugs for a variety of indications, including cancer (cyclophosphamide), glaucoma (isoflurophate), and osteoporosis (certain bisphosphonates).
Environmental Fate and Behavior Because of the diversity of chemical structures and associated physical–chemical properties of OP compounds, no generalizations can be made concerning solubility, partitioning, environmental persistence, long-range transport, or bioaccumulation. Entries for individual compounds should be consulted.
Exposure and Exposure Monitoring
Figure 1 Generic structure of an organophosphorus (OP) compound containing pentacovalent phosphorus. The atom connected to phosphorus by a double bond is either oxygen or sulfur. Y and Z are linker atoms or groups that can include O, S, NH, or CH2. R1 and R2 can include substituted or unsubstituted alkyl or aryl groups. X can be a linker atom or group as for Y or Z, and R3 can be a substituted or unsubstituted alkyl or aryl group; in addition, X can be a halogen (typically F) or cyano group (–CN), in which case R3 is absent. The phosphorus atom has tetrahedral bonding geometry; thus, depending upon the groups attached to P, optical isomerism is possible.
Considering the many uses of OP compounds, exposures can occur through most of the common media (air, food, surfaces, and water) and routes of exposure (dermal, ingestion, and inhalation). Insecticidal OP compounds and nerve agents are designed to inhibit neuronal acetylcholinesterase (AChE; see the following). Because humans and other mammals have AChE in their erythrocytes, the activity of this enzyme can be assayed in blood samples as a biomarker of exposure. These compounds often inhibit plasma butyrylcholinesterase (BChE) as well, providing an additional biomarker of exposure. Neuropathic OP compounds inhibit neuropathy target esterase (NTE) in neural tissue; this enzyme is also found in blood lymphocytes, thereby furnishing a means of assessing exposure.
Background Toxicokinetics OP chemistry can be traced back to the nineteenth century, but it came to toxicological prominence in the 1930s in Germany with the development of OP insecticides and nerve agents.
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The toxicokinetics of OP compounds is as varied as the structures and uses associated with the members of this broad class
Encyclopedia of Toxicology, Volume 3
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Organophosphorus Compounds
of chemicals. Because the subset of OP insecticides receives special scrutiny by the US EPA, toxicokinetic data may be found for individual chemicals in this category. The insecticidal OP compounds and nerve agents will usually be readily absorbed and easily pass into both the peripheral nervous system (PNS) and central nervous system (CNS) by virtue of their generally high lipophilicity. At the same time, they are relatively reactive, and will be hydrolyzed catalytically by A-esterases such as paraoxonase-1 (PON-1) or stoichiometrically by B-esterases such as BChE or carboxylesterase (CaE). The hydrolysis products tend to be relatively water soluble and can be excreted in the urine.
Mechanisms of Toxicity Toxicologically, the most prominent subclasses of OP compounds are OP insecticides and nerve agents, which are designed to be toxic by virtue of inhibiting AChE in the nervous system. The mechanism of action of OP nerve agents is the same as that for OP insecticides; the insecticides are safer, because they are far less potent than the nerve agents. It so happens that another subset, with some overlap with the OP insecticides, can produce OP compound–induced delayed neurotoxicity (OPIDN), presumably by inhibiting and aging neuronal NTE (see the following). Because neurotoxicity is the most conspicuous and wellstudied untoward effect of OP compounds, the remainder of this chapter discusses the three forms of neurotoxicity associated with these compounds: acute cholinergic toxicity, intermediate syndrome, and OPIDN. For specific information on acute, short-term, and chronic toxicity in animals or humans, as well as special toxic responses (immunotoxicity, reproductive toxicity, genotoxicity, carcinogenicity, ecotoxicology, or other hazards), exposure standards, and additional details on clinical management of toxicities, entries for individual compounds should be consulted. In the sections on neurotoxicity that follow, some general aspects of clinical management are briefly discussed.
Acute Cholinergic Neurotoxicity OP insecticides and nerve agents exert their toxicity by inhibiting AChE in cholinergic synapses throughout the CNS and PNS. Each cholinergic synapse is a miniature transducer that converts a presynaptic electrical signal into a chemical signal (acetylcholine), which diffuses across the synaptic cleft, where it triggers another electrical signal on the postsynaptic side by interacting with acetylcholine receptors. The supply side of this transduction economics consists of production, storage, and release of acetylcholine; these processes are akin to flipping an electrical switch to the ‘on’ position. The demand side entails destroying the chemical signal by hydrolyzing acetylcholine, thus flipping the switch to the ‘off’ position – this is the job of AChE, which catalyzes the hydrolysis of acetylcholine. When AChE is inhibited, acetylcholine is not hydrolyzed, and the switch cannot be turned off. Having many of the cholinergic synapses in the body constantly ‘on’ produces a drastic overstimulation (and ultimately fatigue) of the many
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organs that receive cholinergic innervation. The toxicity is aptly named cholinergic, because the proximate toxic agent is the body’s own cholinergic neurotransmitter, acetylcholine, rapidly building up to toxic levels in cholinergic synapses. The signs and symptoms arising from AChE inhibition are a reflection of the cholinergic synapses affected (e.g., CNS versus PNS), organ system innervated, and type of cholinergic receptor involved (muscarinic versus nicotinic). In cases of severe poisoning, the overriding signs can consist of excess salivation/sweating, lacrimation, urination, and defecation; collectively, this set of signs is known by the acronym SLUD. The combination of central nervous system depression, bradycardia, bronchorrhea, and paralysis of respiratory muscles can lead rapidly to coma and death. Fortunately, effective treatments and prophylactic measures are available for poisoning by OP inhibitors of AChE. First, atropine is a competitive antagonist of acetylcholine at muscarinic acetylcholine receptors that is especially useful in acute life-threatening intoxications. Patients are ‘titrated’ with frequent small doses (1.0 mg s.c. or i.v.) to control initial muscarinic signs. Relatively large cumulative doses (up to 50 mg day–1) may be necessary to control severe muscarinic signs. Because of the toxicity of atropine, patients should be monitored by examining dilation of pupils (mydriasis), absence of secretions (dry mouth), facial flushing, and/or disappearance of sweating. To counteract certain central and peripheral effects of AChE inhibition not treated with atropine, diazepam should be used in doses of 10 mg s.c. or i.v., repeated as needed. Other CNSacting drugs should not be used, because of potential respiratory depression. Provided that the inhibited AChE has not undergone the ‘aging’ reaction (see Figure 2 and the discussion that follows), the full range of cholinergic effects can be controlled by giving oximes to reactivate inhibited AChE. Typically, pralidoxime (2-PAM) is given in doses of 1.0 g by slow i.v. infusion over 20 min. Pralidoxime treatment can be repeated; however, it can bind calcium, thereby inducing muscle cramps and mimicking one of the signs of OP poisoning, but this effect can be addressed by giving oral or i.v. calcium solutions. Figure 2 schematizes the interactions of two types of OP compounds, a phosphonate and a phosphinate, with AChE, and the toxicological consequences of each. Inhibition by either type of compound produces cholinergic toxicity, which can be treated to some extent with atropine. However, the phosphonate contains an RO-substituent that is susceptible to the aging reaction, involving net loss of the R-group and leaving a negatively charged organophosphoryl group covalently attached to the enzyme. The aged OP moiety resists reactivation because it is stabilized by the enzyme and it repels nucleophiles, such as oximes. In contrast, phosphinates are incapable of aging, owing to the strength of the carbon– phosphorus bonds linking the R-groups to phosphorus. Because phosphinylated AChE does not age, its OP group can be displaced by oximes. Thus, inhibition of AChE alone produces cholinergic toxicity irrespective of aging, but as aging transpires, the therapeutic efficacy of oximes diminishes. Aging is so named because it is a time-dependent reaction. Early investigations of AChE inhibition by OP compounds noted that the inhibited enzyme became increasingly
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Organophosphorus Compounds
Figure 2 Inhibition of AChE is sufficient for cholinergic neurotoxicity. AChE is represented by a wavy line containing the active site serine hydroxyl group. Pathway (1) shows inhibition by a phosphonate leading directly to cholinergic toxicity, treatable by both atropine (acetylcholine antagonist) and oximes (AChE reactivators). If aging occurs, the type of toxicity does not change, but oxime reactivators are no longer effective. Pathway (2) shows inhibition by a phosphinate, which cannot undergo aging. Cholinergic toxicity still occurs and is treatable by both atropine and oximes. R1 and R2 may be substituted or unsubstituted alkyl or aryl groups. X is the primary leaving group that is displaced by the serine hydroxyl of AChE and may be, for example, substituted or unsubstituted alkoxy, aryloxy, or fluorine. Neither inhibition nor aging of inhibited AChE can produce OPIDN – this requires inhibition and aging of NTE (see Figure 3).
intractable to reactivation with time; the resistant OP-AChE conjugate was said to have ‘aged,’ but the mechanism of this change was not known at the time. It is now known that halflives of aging depend on chemical structure and vary from minutes for some nerve agents to w40 h for diethyl phosphoryl insecticides. Accordingly, oximes can often be employed effectively in cases of poisoning by OP insecticides, but not so in cases of intoxication with some nerve agents. Because of the rapid rate of aging of AChE inhibited by some nerve agents, prophylactic measures have been instituted so that individuals at risk of exposure can be protected before inhibition and aging have occurred. Pretreatment with pyridostigmine is used to partially inhibit AChE with a spontaneously reactivatable carbamate, thereby protecting the enzyme from longer-term inhibition with a non-reactivatable OP compound. Prophylactic measures under development include intravenous injections of human BChE to serve as a stoichiometric bioscavenger or with PON-1, engineered to enhance its activity as a catalytic bioscavenger of OP compounds. Inhibition of AChE by OP compounds is also a timedependent reaction, and it can be characterized by a bimolecular rate constant of inhibition (ki) or an IC50 concentration at a given time of incubation of enzyme and inhibitor. Thus, the potency of an inhibitor increases with increasing ki or decreasing fixed-time IC50. These numbers can be used in a relative sense to predict the tendency of a compound to produce acute cholinergic toxicity versus delayed neurotoxicity (see the section on OPIDN).
Intermediate Syndrome The intermediate syndrome is usually related to a high-level exposure to an OP insecticide. Onset occurs w24–96 h after poisoning. When it occurs, it often affects conscious patients
who exhibit no cholinergic signs. Because the onset occurs between that of acute cholinergic toxicity and OPIDN (see the following), this effect has been dubbed the intermediate syndrome. The condition is characterized by marked weakness of muscles innervated by cranial nerves II–VII and X, sudden weakness of neck flexor muscles, weakness of proximal limb muscles, absence of fasciculations but occasional spasticity, hyperreflexia and dystonia, and decreased or absent tendon reflexes. Respiratory insufficiency may develop rapidly, causing patients to use accessory muscles for ventilation along with an increase in ventilatory rate, cyanosis, coma, and death if these signs and symptoms are not recognized and treated early enough. It is surmised that the intermediate syndrome arises from a persistent depolarizing blockade and resultant desensitization (with possible attendant downregulation) of nicotinic receptors in neuromuscular junctions caused by excess synaptic acetylcholine. The result is akin to myasthenia gravis, which partially inactivates neuromuscular junctions through a different mechanism, i.e., autoimmunity. In the intermediate syndrome, the pattern of muscle weakness is similar to that of the myopathy produced in experimental animal studies following high levels of localized AChE inhibition, but the myopathy and the intermediate syndrome are not thought to be causally linked.
OPIDN (Delayed Neurotoxicity) The term delayed neurotoxicity may be used to describe any type of toxicity to the nervous system involving a delay between the precipitating chemical exposure and the appearance of neurological signs or symptoms. However, this designation usually refers to OPIDN, also known as OP compoundinduced polyneuropathy (OPIDP).
Organophosphorus Compounds
The particular syndrome of OPIDN is produced by certain organic compounds of pentacovalent phosphorus. The less common and relatively unstable organic compounds of trivalent phosphorus, such as triphenyl phosphite, can produce a different spatial–temporal pattern of neurodegeneration, which is distinct from OPIDN. The underlying pathology in OPIDN involves bilaterally symmetrical degeneration of sensory and motor axons in distal regions of peripheral nerves and spinal cord tracts. Generally, the longest, largest diameter fibers tend to be preferentially affected. The most prominent lesions are often found in the dorsal columns of the cervical spinal cord, especially in the fasciculus gracilis. Injury to this tract results in specific sensory deficits, including loss of recognition of limb position (proprioception) and vibration sensitivity. Pathogenesis studies indicate that the primary lesion in OPIDN is in the axon rather than the myelin sheath or the cell body of the neuron, and that demyelination occurs secondarily to axonal degeneration. The process has been likened to a ‘chemical transection’ of the axon, with subsequent Wallerian-type degeneration, as opposed to a ‘dying back’ of the axon following an insult to the cell body as once hypothesized. Signs and symptoms of axonopathy appear after a delay of w8 days following absorption of an effective dose of an OPIDN-producing (neuropathic) OP compound and consist of abnormal sensations (paresthesias) in the extremities, including numbness and tingling. There may also be pain, particularly in the calves of the legs. Distal reflexes may be absent or attenuated. The feet and lower legs are usually affected predominantly and before involvement of the hands and arms, but severe cases involve the upper and lower limbs in a ‘glove and stocking’ distribution. Incoordination of movement (ataxia) develops at about the same time as the sensory disturbances and may progress to partial flaccid paralysis (paresis) after w10–21 days. Recovery from severe disease is usually poor, and there is no specific treatment. Over a period of months to years, flaccidity may be replaced by spasticity, reflecting regeneration of peripheral nerve injury with residual damage to descending upper motor neuron pathways in the spinal cord. Spasticity can be alleviated by treating with antispasticity drugs, such as baclofen. Because of the ubiquity of OP compounds and the serious and often irreversible nature of OPIDN, much effort has been expended to develop ways to identify the OP compounds that pose a genuine risk of causing this condition. Consequently, although the pathogenic mechanism remains unknown, human OPIDN is now an extremely rare disease, with a worldwide incidence of only about two cases per year, usually arising from intentional ingestion of massive doses of OP compounds in attempted suicides. Sporadic episodes of OPIDN affecting domestic animals and livestock also occur, largely from misapplication of OP compounds used directly on the animals for control of insect or arachnid pests. Most of the estimated 40 000 human cases that occurred between 1930 and 1960 arose from contamination of cooking oil or beverages with tri-o-cresyl phosphate (TOCP; also known as tri-o-tolyl phosphate, TOTP). More than half of the cases of OPIDN have been attributed to consumption of an alcoholic extract of Jamaica Ginger (‘Ginger Jake’) that had been adulterated with solvents containing TOCP. Ginger Jake was used as a source of
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alcohol during Prohibition in the United States. The resulting paralysis became known as ‘Jake Leg’ or ‘Jake Walk.’ Awareness of OPIDN coupled with the advent of improved methods for assessing the relative potential of OP compounds to produce the disease has led to the virtual elimination of human cases. Nevertheless, neuropathic OP compounds and OPIDN continue to be active fields of study. This apparent paradox arises from the importance of OP chemistry in diverse applications, the potential threat of neuropathic OP compounds as agents of terrorism or warfare, the promise of neuropathic OP compounds as tools in neurological research, and the recent discovery that certain mutations in the gene encoding NTE produce a form of motor neuron disease. Experimental studies have identified the adult chicken as the species of choice for testing OP compounds for their potential to cause OPIDN. Hens greater than w8 months of age are now used in routine testing. Other species in addition to humans and chickens that are known to be susceptible to single doses of neuropathic OP compounds include certain nonhuman primates, water buffalo, cattle, swine, sheep, dogs, and cats. Rats and mice have been considered resistant to the clinical manifestations of OPIDN. However, recent studies have shown that histopathological lesions, particularly in the spinal cord, can be produced in these species by compounds known to cause OPIDN in the adult hen. The apparent resistance of rodents to OPIDN may result, at least in part, from the fact that relatively young (less than w3 months of age) animals have been used in most studies. Generally, the young of a given species are much more resistant to OPIDN than adults are. For example, chicks younger than about 50 days of age will not develop OPIDN after a single dose of a neuropathic OP compound. Moreover, chicks are resistant to repeated doses if they are younger than w14 days of age. Species and age differences in susceptibility to OPIDN have been attributed to long axons in large animals and robust repair of neural injury in young animals. The complete mechanism of OPIDN has not been elucidated. However, there is good evidence that the disease is initiated by a concerted two-step reaction involving inhibition and aging of a critical amount of NTE in target neural tissues. The net result of the aging step is the rapid formation of a negatively charged species in the active site of the enzyme (Figure 3). Such a reaction can take place with OP inhibitors of NTE such as phosphates, phosphonates, or phosphoramidates, which have an ester or amide group in addition to the leaving group (Figure 4). Phosphates and phosphonates undergo aging by net loss of an R-group. Phosphoramidates having only a single R-group attached to the phosphoramidate nitrogen appear to age by loss of the phosphoramidate proton rather than by loss of an R-group. Compounds that do not inhibit NTE do not cause OPIDN, even if they belong to a structural class capable of undergoing the aging reaction. For example, although paraoxon belongs to the phosphate class of OP compounds, it does not produce OPIDN because it is a poor inhibitor of NTE. NTE inhibition and aging transpire within minutes to hours following absorption of an effective dose of a neuropathic OP compound. Thus, events that remain to be elucidated contribute to the delay of 8–21 days between the exposure and the initial signs of ataxia and paresis. However, if inhibition but
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Organophosphorus Compounds
Figure 3 Inhibition and aging of NTE are required for initiation of OPIDN. NTE is represented by a wavy line containing the active site serine hydroxyl group. Pathway (1) shows inhibition by a phosphonate, which undergoes rapid aging to yield a negatively charged phosphonyl adduct. OPIDN follows within 8–21 days and is not treatable. Pathway (2) shows inhibition by a phosphinate, which cannot undergo aging. The neutral phosphinylated adduct does not trigger OPIDN; however, it confers protection against subsequently administered neuropathic (ageable) NTE inhibitors. For each type of inhibitor, R1 and R2 may be substituted or unsubstituted alkyl or aryl groups. X is the primary leaving group that is displaced by the serine hydroxyl of NTE and may be, for example, substituted or unsubstituted alkoxy, aryloxy, or fluorine.
no aging occurs by dosing with an NTE inhibitor that is incapable of generating a negative charge at the active site, no OPIDN ensues. Furthermore, an animal whose NTE is inhibited with a nonaging compound is protected against a subsequent dose of an OP compound that would be neuropathic in a naïve animal (Figure 3). Nonaging inhibitors of NTE include representatives from the phosphinate class of OP compounds, certain carbamates, and sulfonyl fluorides, such as phenylmethanesulfonyl fluoride (PMSF) (Figure 4). The threshold of NTE inhibition in target neural tissue that correlates with the development of OPIDN after a single dose of a neuropathic OP compound is w70%. For many compounds, inhibition measured in brain is paralleled in spinal cord and peripheral nerve, and brain values are often used in screening tests in hens to assess relative neuropathic potency. Repeated dosing also appears to require that a high point of inhibition be reached before OPIDN will develop. The threshold appears to be the same as for acute dosing for some compounds, but for some others, the critical level of inhibition may be as low as 50%. With repeated dosing, there still appears to be a delay of w8–21 days between the time inhibition exceeds the threshold value and the appearance of signs of OPIDN. NTE has also been found in circulating lymphocytes and platelets, where its inhibition has found use as a biomarker of exposure to neuropathic OP compounds. There is a good correlation between inhibition of NTE in leukocytes and brain when the measurements are carried out within 24 h of an acute exposure. However, a good correlation might not be found later (even by 48 h) or under conditions of repeated exposures. Nevertheless, leukocytes provide an accessible source of NTE for detection of inhibition by neuropathic OP compounds. Currently, there is considerable interest in using protein mass spectrometry to detect OP adducts on NTE or other proteins as sensitive and specific biomarkers of exposure to neuropathic OP compounds.
It is important to realize that OPIDN depends on a particular type of chemical modification of NTE rather than mere inhibition of its enzymatic activity. Inhibition of NTE is a necessary, but not sufficient, condition for OPIDN. Aging of the inhibited enzyme results in a complete change in the toxicological outcome. Whereas inhibition without aging results in no clinically apparent injury, suprathreshold inhibition with aging triggers an inexorable neurodegenerative process leading to evident disease. The situation with NTE is completely different from that with acetylcholinesterase (AChE). Inhibition of a sufficient amount of AChE will produce cholinergic toxicity, regardless of whether or not aging of inhibited AChE occurs (Figure 2). Aging of inhibited AChE does not alter the type of toxic response, but it does change the options available for therapy against cholinergic toxicity. For example, oximes such as pralidoxime methiodide (2-PAM) are used to reactivate inhibited AChE, but these agents are ineffective if aging of the enzyme has occurred. Moreover, oximes do not appear to affect the clinical course of OPIDN following administration of a neuropathic OP compound, except to allow survival of an otherwise lethal dose of a compound that also has cholinergic toxicity. In a homologous series of OP compounds, increasing potency for AChE inhibition and cholinergic toxicity correlates with decreasing potency for NTE inhibition and OPIDN. The relative inhibitory potency (RIP) of an OP compound or its active metabolite for NTE versus AChE in vitro can be used as a convenient index of the probable neuropathic potential of the compound. A commonly used measure of inhibitory potency is the IC50, the concentration required to inhibit 50% of the enzyme activity under a standardized set of reaction conditions and time of incubation of the inhibitor with the enzyme preparation. A better measure of inhibitory potency is the bimolecular rate constant of inhibition, ki. When pseudo– first-order kinetics are observed, it is valid to use the
Organophosphorus Compounds
Figure 4 NTE inhibitors. For each type of inhibitor, R–R4 may be substituted or unsubstituted alkyl or aryl groups. For carbamates, R1 can be a hydrogen atom, and for phosphoramidates, R1 and/or R3 can be a hydrogen atom. X is the primary leaving group that is displaced by the serine hydroxyl of NTE and may be, for example, substituted or unsubstituted alkoxy or aryloxy. Fluorine can be a leaving group for the OP NTE inhibitors and is the most common leaving group for sulfonate NTE inhibitors. Type A inhibitors are neuropathic and include certain phosphates, phosphonates, and phosphoramidates. Mixtures of subtypes are possible. Phosphonates are intrinsically asymmetric and enantiomers may have different inhibitory and/or aging properties. Type B inhibitors are not neuropathic, but pretreatment protects against OPIDN from subsequent exposure to type A inhibitors. Type B inhibitors include certain phosphinates, sulfonates, and carbamates.
relationship, IC50 ¼ 0.693/kit, where t is the time of preincubation of the inhibitor with the enzyme. Comparisons of AChE/NTE ki ratios or NTE/AChE IC50 ratios in vitro (RIPs) with toxicity data in vivo have shown that RIP values <1 indicate that the dose required to produce OPIDN is less than the median lethal dose (LD50). In contrast, RIP values >1 correspond to doses greater than the LD50 required to produce OPIDN. The higher the RIP, the safer is the compound with respect to its capacity to produce OPIDN. Thus, insecticidal OP compounds generally are much more potent inhibitors of AChE than NTE and do not produce OPIDN except at doses that would require aggressive treatment for cholinergic toxicity. On the other hand, compounds can be made that are better inhibitors of NTE than AChE. If such compounds can also undergo aging, not only will they produce OPIDN; they will do so at doses that elicit little or no cholinergic toxicity. Marginal or subclinical OPIDN can be potentiated to fullblown disease by subsequent treatment with nonaging inhibitors of NTE. The phenomenon is called promotion by some authors, which is an appropriate term if the initial insult is undetectable. Potentiation was initially a surprising finding,
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especially in view of the fact that reversing the order of dosing of the nonaging and aging NTE inhibitors affords protection against OPIDN. However, it now appears that the outcome of many types of neural injuries, including physical crushing of nerves, can be exacerbated by dosing with nonaging NTE inhibitors as well as with inhibitors of other serine esterases or proteases. The apparent indifference to the method of producing the initial lesion suggests a general mode of action for potentiation, such as interference with regeneration and repair. Although the precise physiological function of NTE is currently unknown, the protein is known to be anchored in the endoplasmic reticulum and to catalyze the hydrolysis of membrane phospholipids and lysophospholipids. Moreover, it has recently been established that NTE belongs to a ninemember family of patatin-like phospholipase domain containing proteins (PNPLAs), of which NTE is PNPLA6. Conventional knock-out of the gene is embryonic-lethal in mice, indicating an essential role in development, and conditional knock-out in the brain results in neurodegeneration. In addition to its serine hydrolase domain, NTE contains tandem domains with homology to cyclic nucleotide-binding regions in other proteins, implying a regulatory or signaling function. Mutation of a homologous protein called SWS in Drosophila results in a spongiform neurodegenerative disease, suggesting that NTE might be associated with neurological or neurodevelopmental disorders. More recently, NTE mutations have been linked to a form of motor neuron disease in humans. Certainly, much work remains to be done to elucidate the normal and pathogenic roles of NTE, but the accomplishments thus far have proved to be useful in a wide range of fields, including toxicological risk assessment, developmental neurobiology, and clinical neurogenetics.
See also: Acetylcholine; A-esterase; Atropine; Azinphos-Methyl; Carboxylesterases; Chemical Warfare; Chlorpyrifos; Cholinesterase Inhibition; Cyclophosphamide; Diazinon; Dichlorvos; Fenthion; Ginger Jake; Malathion; Methamidophos; Methyl Parathion; Naled; Nerve Agents; Neurotoxicity; Parathion; Pyridostigmine; Pyridoxine; Soman; Tabun; Trichlorfon; V-Series Nerve Agents: Other than VX; VX.
Further Reading Moser, V.C., Aschner, M., Richardson, R.J., Philbert, M.A., 2008. Toxic responses of the nervous system. In: Klaassen, C. (Ed.), Casarett and Doull’s Toxicology: The Basic Science of Poisons, seventh ed. McGraw-Hill Professional, New York, pp. 631–665. Rainier, S., Albers, J.W., Dyck, P.J., Eldevik, O.P., Wilcock, S., Richardson, R.J., Fink, J.K., 2011. Motor neuron disease due to neuropathy target esterase gene mutation: clinical features of the index families. Muscle Nerve 43, 19–25. Richardson, R.J., 2010. Anticholinesterase insecticides. In: McQueen, C. (Ed.) Comprehensive Toxicology, second ed., vol. 13. Academic Press, Oxford, pp. 433–444. Richardson, R.J., Worden, R.M., Makhaeva, G.F., 2009. Biomarkers and biosensors of delayed neuropathic agents. In: Gupta, R.C. (Ed.), Handbook of Toxicology of Chemical Warfare Agents. Academic Press/Elsevier, Amsterdam, pp. 859–876. Thompson, C.M., Richardson, R.J., 2004. Anticholinesterase Insecticides. In: Marrs, T.C., Ballantyne, B., (Eds.), Pesticide Toxicology and International Regulation (Current Toxicology Series). John Wiley & Sons Ltd., Chichester, pp. 89–127. Wijeyesakere, S.J., Richardson, R.J., 2010. Neuropathy target esterase. In: Krieger, R. (Ed.), Hayes’ Handbook of Pesticide Toxicology, third ed. Elsevier/Academic Press, San Diego, pp. 1435–1478.
Name Organophosphorus (OP) compounds are organic compounds containing trivalent or pentavalent phosphorus. This chapter focuses on the more common pentavalent phosphorus compounds.
Synonyms OP compounds are often referred to generically as organophosphates; however, this is a specific subset comprising derivatives of phosphoric acid. Many other derivatives are possible, such as phosphorothioates, phosphonates, phosphoramidates, and phosphinates. OP insecticides are typically organophosphates or organophosphorothioates. OP nerve agents (nerve gases) are usually organophosphonates; these are true organophosphorus compounds, as they contain a carbon– phosphorus covalent bond.
Molecular Formula Figure 1 shows a generic structure of an OP compound containing pentacovalent phosphorus. In such compounds, the phosphorus atom has tetrahedral geometry; thus, depending on the identity of the substituents attached to phosphorus, optical isomerism is possible.
When organochlorine insecticides were phased out owing to ecological concerns voiced in the 1960s and 1970s, the use of OP insecticides increased as replacements. More recently, OP insecticide use has been curtailed in response to health concerns and wider adoption of integrated pest management. Currently, new uses are being found for non-insecticidal OP compounds. For example, as certain flame retardants, such as the polybrominated diphenyl ethers (PBDEs), are being phased out, OP compounds are entering the market as replacements.
Uses OP compounds are incredibly diverse with respect to chemical structure and attendant physical–chemical properties; accordingly, the uses of these compounds cover a wide range. Thus, depending on their molecular structures, OP compounds have found uses as insecticides, nerve agents, hydraulic fluids, fuel additives, lubricants, plasticizers, and flame retardants. They are also used as drugs for a variety of indications, including cancer (cyclophosphamide), glaucoma (isoflurophate), and osteoporosis (certain bisphosphonates).
Environmental Fate and Behavior Because of the diversity of chemical structures and associated physical–chemical properties of OP compounds, no generalizations can be made concerning solubility, partitioning, environmental persistence, long-range transport, or bioaccumulation. Entries for individual compounds should be consulted.
Exposure and Exposure Monitoring
Figure 1 Generic structure of an organophosphorus (OP) compound containing pentacovalent phosphorus. The atom connected to phosphorus by a double bond is either oxygen or sulfur. Y and Z are linker atoms or groups that can include O, S, NH, or CH2. R1 and R2 can include substituted or unsubstituted alkyl or aryl groups. X can be a linker atom or group as for Y or Z, and R3 can be a substituted or unsubstituted alkyl or aryl group; in addition, X can be a halogen (typically F) or cyano group (–CN), in which case R3 is absent. The phosphorus atom has tetrahedral bonding geometry; thus, depending upon the groups attached to P, optical isomerism is possible.
Considering the many uses of OP compounds, exposures can occur through most of the common media (air, food, surfaces, and water) and routes of exposure (dermal, ingestion, and inhalation). Insecticidal OP compounds and nerve agents are designed to inhibit neuronal acetylcholinesterase (AChE; see the following). Because humans and other mammals have AChE in their erythrocytes, the activity of this enzyme can be assayed in blood samples as a biomarker of exposure. These compounds often inhibit plasma butyrylcholinesterase (BChE) as well, providing an additional biomarker of exposure. Neuropathic OP compounds inhibit neuropathy target esterase (NTE) in neural tissue; this enzyme is also found in blood lymphocytes, thereby furnishing a means of assessing exposure.
Background Toxicokinetics OP chemistry can be traced back to the nineteenth century, but it came to toxicological prominence in the 1930s in Germany with the development of OP insecticides and nerve agents.
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The toxicokinetics of OP compounds is as varied as the structures and uses associated with the members of this broad class
Encyclopedia of Toxicology, Volume 3
http://dx.doi.org/10.1016/B978-0-12-386454-3.00173-1
Organophosphorus Compounds
of chemicals. Because the subset of OP insecticides receives special scrutiny by the US EPA, toxicokinetic data may be found for individual chemicals in this category. The insecticidal OP compounds and nerve agents will usually be readily absorbed and easily pass into both the peripheral nervous system (PNS) and central nervous system (CNS) by virtue of their generally high lipophilicity. At the same time, they are relatively reactive, and will be hydrolyzed catalytically by A-esterases such as paraoxonase-1 (PON-1) or stoichiometrically by B-esterases such as BChE or carboxylesterase (CaE). The hydrolysis products tend to be relatively water soluble and can be excreted in the urine.
Mechanisms of Toxicity Toxicologically, the most prominent subclasses of OP compounds are OP insecticides and nerve agents, which are designed to be toxic by virtue of inhibiting AChE in the nervous system. The mechanism of action of OP nerve agents is the same as that for OP insecticides; the insecticides are safer, because they are far less potent than the nerve agents. It so happens that another subset, with some overlap with the OP insecticides, can produce OP compound–induced delayed neurotoxicity (OPIDN), presumably by inhibiting and aging neuronal NTE (see the following). Because neurotoxicity is the most conspicuous and wellstudied untoward effect of OP compounds, the remainder of this chapter discusses the three forms of neurotoxicity associated with these compounds: acute cholinergic toxicity, intermediate syndrome, and OPIDN. For specific information on acute, short-term, and chronic toxicity in animals or humans, as well as special toxic responses (immunotoxicity, reproductive toxicity, genotoxicity, carcinogenicity, ecotoxicology, or other hazards), exposure standards, and additional details on clinical management of toxicities, entries for individual compounds should be consulted. In the sections on neurotoxicity that follow, some general aspects of clinical management are briefly discussed.
Acute Cholinergic Neurotoxicity OP insecticides and nerve agents exert their toxicity by inhibiting AChE in cholinergic synapses throughout the CNS and PNS. Each cholinergic synapse is a miniature transducer that converts a presynaptic electrical signal into a chemical signal (acetylcholine), which diffuses across the synaptic cleft, where it triggers another electrical signal on the postsynaptic side by interacting with acetylcholine receptors. The supply side of this transduction economics consists of production, storage, and release of acetylcholine; these processes are akin to flipping an electrical switch to the ‘on’ position. The demand side entails destroying the chemical signal by hydrolyzing acetylcholine, thus flipping the switch to the ‘off’ position – this is the job of AChE, which catalyzes the hydrolysis of acetylcholine. When AChE is inhibited, acetylcholine is not hydrolyzed, and the switch cannot be turned off. Having many of the cholinergic synapses in the body constantly ‘on’ produces a drastic overstimulation (and ultimately fatigue) of the many
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organs that receive cholinergic innervation. The toxicity is aptly named cholinergic, because the proximate toxic agent is the body’s own cholinergic neurotransmitter, acetylcholine, rapidly building up to toxic levels in cholinergic synapses. The signs and symptoms arising from AChE inhibition are a reflection of the cholinergic synapses affected (e.g., CNS versus PNS), organ system innervated, and type of cholinergic receptor involved (muscarinic versus nicotinic). In cases of severe poisoning, the overriding signs can consist of excess salivation/sweating, lacrimation, urination, and defecation; collectively, this set of signs is known by the acronym SLUD. The combination of central nervous system depression, bradycardia, bronchorrhea, and paralysis of respiratory muscles can lead rapidly to coma and death. Fortunately, effective treatments and prophylactic measures are available for poisoning by OP inhibitors of AChE. First, atropine is a competitive antagonist of acetylcholine at muscarinic acetylcholine receptors that is especially useful in acute life-threatening intoxications. Patients are ‘titrated’ with frequent small doses (1.0 mg s.c. or i.v.) to control initial muscarinic signs. Relatively large cumulative doses (up to 50 mg day–1) may be necessary to control severe muscarinic signs. Because of the toxicity of atropine, patients should be monitored by examining dilation of pupils (mydriasis), absence of secretions (dry mouth), facial flushing, and/or disappearance of sweating. To counteract certain central and peripheral effects of AChE inhibition not treated with atropine, diazepam should be used in doses of 10 mg s.c. or i.v., repeated as needed. Other CNSacting drugs should not be used, because of potential respiratory depression. Provided that the inhibited AChE has not undergone the ‘aging’ reaction (see Figure 2 and the discussion that follows), the full range of cholinergic effects can be controlled by giving oximes to reactivate inhibited AChE. Typically, pralidoxime (2-PAM) is given in doses of 1.0 g by slow i.v. infusion over 20 min. Pralidoxime treatment can be repeated; however, it can bind calcium, thereby inducing muscle cramps and mimicking one of the signs of OP poisoning, but this effect can be addressed by giving oral or i.v. calcium solutions. Figure 2 schematizes the interactions of two types of OP compounds, a phosphonate and a phosphinate, with AChE, and the toxicological consequences of each. Inhibition by either type of compound produces cholinergic toxicity, which can be treated to some extent with atropine. However, the phosphonate contains an RO-substituent that is susceptible to the aging reaction, involving net loss of the R-group and leaving a negatively charged organophosphoryl group covalently attached to the enzyme. The aged OP moiety resists reactivation because it is stabilized by the enzyme and it repels nucleophiles, such as oximes. In contrast, phosphinates are incapable of aging, owing to the strength of the carbon– phosphorus bonds linking the R-groups to phosphorus. Because phosphinylated AChE does not age, its OP group can be displaced by oximes. Thus, inhibition of AChE alone produces cholinergic toxicity irrespective of aging, but as aging transpires, the therapeutic efficacy of oximes diminishes. Aging is so named because it is a time-dependent reaction. Early investigations of AChE inhibition by OP compounds noted that the inhibited enzyme became increasingly
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Organophosphorus Compounds
Figure 2 Inhibition of AChE is sufficient for cholinergic neurotoxicity. AChE is represented by a wavy line containing the active site serine hydroxyl group. Pathway (1) shows inhibition by a phosphonate leading directly to cholinergic toxicity, treatable by both atropine (acetylcholine antagonist) and oximes (AChE reactivators). If aging occurs, the type of toxicity does not change, but oxime reactivators are no longer effective. Pathway (2) shows inhibition by a phosphinate, which cannot undergo aging. Cholinergic toxicity still occurs and is treatable by both atropine and oximes. R1 and R2 may be substituted or unsubstituted alkyl or aryl groups. X is the primary leaving group that is displaced by the serine hydroxyl of AChE and may be, for example, substituted or unsubstituted alkoxy, aryloxy, or fluorine. Neither inhibition nor aging of inhibited AChE can produce OPIDN – this requires inhibition and aging of NTE (see Figure 3).
intractable to reactivation with time; the resistant OP-AChE conjugate was said to have ‘aged,’ but the mechanism of this change was not known at the time. It is now known that halflives of aging depend on chemical structure and vary from minutes for some nerve agents to w40 h for diethyl phosphoryl insecticides. Accordingly, oximes can often be employed effectively in cases of poisoning by OP insecticides, but not so in cases of intoxication with some nerve agents. Because of the rapid rate of aging of AChE inhibited by some nerve agents, prophylactic measures have been instituted so that individuals at risk of exposure can be protected before inhibition and aging have occurred. Pretreatment with pyridostigmine is used to partially inhibit AChE with a spontaneously reactivatable carbamate, thereby protecting the enzyme from longer-term inhibition with a non-reactivatable OP compound. Prophylactic measures under development include intravenous injections of human BChE to serve as a stoichiometric bioscavenger or with PON-1, engineered to enhance its activity as a catalytic bioscavenger of OP compounds. Inhibition of AChE by OP compounds is also a timedependent reaction, and it can be characterized by a bimolecular rate constant of inhibition (ki) or an IC50 concentration at a given time of incubation of enzyme and inhibitor. Thus, the potency of an inhibitor increases with increasing ki or decreasing fixed-time IC50. These numbers can be used in a relative sense to predict the tendency of a compound to produce acute cholinergic toxicity versus delayed neurotoxicity (see the section on OPIDN).
Intermediate Syndrome The intermediate syndrome is usually related to a high-level exposure to an OP insecticide. Onset occurs w24–96 h after poisoning. When it occurs, it often affects conscious patients
who exhibit no cholinergic signs. Because the onset occurs between that of acute cholinergic toxicity and OPIDN (see the following), this effect has been dubbed the intermediate syndrome. The condition is characterized by marked weakness of muscles innervated by cranial nerves II–VII and X, sudden weakness of neck flexor muscles, weakness of proximal limb muscles, absence of fasciculations but occasional spasticity, hyperreflexia and dystonia, and decreased or absent tendon reflexes. Respiratory insufficiency may develop rapidly, causing patients to use accessory muscles for ventilation along with an increase in ventilatory rate, cyanosis, coma, and death if these signs and symptoms are not recognized and treated early enough. It is surmised that the intermediate syndrome arises from a persistent depolarizing blockade and resultant desensitization (with possible attendant downregulation) of nicotinic receptors in neuromuscular junctions caused by excess synaptic acetylcholine. The result is akin to myasthenia gravis, which partially inactivates neuromuscular junctions through a different mechanism, i.e., autoimmunity. In the intermediate syndrome, the pattern of muscle weakness is similar to that of the myopathy produced in experimental animal studies following high levels of localized AChE inhibition, but the myopathy and the intermediate syndrome are not thought to be causally linked.
OPIDN (Delayed Neurotoxicity) The term delayed neurotoxicity may be used to describe any type of toxicity to the nervous system involving a delay between the precipitating chemical exposure and the appearance of neurological signs or symptoms. However, this designation usually refers to OPIDN, also known as OP compoundinduced polyneuropathy (OPIDP).
Organophosphorus Compounds
The particular syndrome of OPIDN is produced by certain organic compounds of pentacovalent phosphorus. The less common and relatively unstable organic compounds of trivalent phosphorus, such as triphenyl phosphite, can produce a different spatial–temporal pattern of neurodegeneration, which is distinct from OPIDN. The underlying pathology in OPIDN involves bilaterally symmetrical degeneration of sensory and motor axons in distal regions of peripheral nerves and spinal cord tracts. Generally, the longest, largest diameter fibers tend to be preferentially affected. The most prominent lesions are often found in the dorsal columns of the cervical spinal cord, especially in the fasciculus gracilis. Injury to this tract results in specific sensory deficits, including loss of recognition of limb position (proprioception) and vibration sensitivity. Pathogenesis studies indicate that the primary lesion in OPIDN is in the axon rather than the myelin sheath or the cell body of the neuron, and that demyelination occurs secondarily to axonal degeneration. The process has been likened to a ‘chemical transection’ of the axon, with subsequent Wallerian-type degeneration, as opposed to a ‘dying back’ of the axon following an insult to the cell body as once hypothesized. Signs and symptoms of axonopathy appear after a delay of w8 days following absorption of an effective dose of an OPIDN-producing (neuropathic) OP compound and consist of abnormal sensations (paresthesias) in the extremities, including numbness and tingling. There may also be pain, particularly in the calves of the legs. Distal reflexes may be absent or attenuated. The feet and lower legs are usually affected predominantly and before involvement of the hands and arms, but severe cases involve the upper and lower limbs in a ‘glove and stocking’ distribution. Incoordination of movement (ataxia) develops at about the same time as the sensory disturbances and may progress to partial flaccid paralysis (paresis) after w10–21 days. Recovery from severe disease is usually poor, and there is no specific treatment. Over a period of months to years, flaccidity may be replaced by spasticity, reflecting regeneration of peripheral nerve injury with residual damage to descending upper motor neuron pathways in the spinal cord. Spasticity can be alleviated by treating with antispasticity drugs, such as baclofen. Because of the ubiquity of OP compounds and the serious and often irreversible nature of OPIDN, much effort has been expended to develop ways to identify the OP compounds that pose a genuine risk of causing this condition. Consequently, although the pathogenic mechanism remains unknown, human OPIDN is now an extremely rare disease, with a worldwide incidence of only about two cases per year, usually arising from intentional ingestion of massive doses of OP compounds in attempted suicides. Sporadic episodes of OPIDN affecting domestic animals and livestock also occur, largely from misapplication of OP compounds used directly on the animals for control of insect or arachnid pests. Most of the estimated 40 000 human cases that occurred between 1930 and 1960 arose from contamination of cooking oil or beverages with tri-o-cresyl phosphate (TOCP; also known as tri-o-tolyl phosphate, TOTP). More than half of the cases of OPIDN have been attributed to consumption of an alcoholic extract of Jamaica Ginger (‘Ginger Jake’) that had been adulterated with solvents containing TOCP. Ginger Jake was used as a source of
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alcohol during Prohibition in the United States. The resulting paralysis became known as ‘Jake Leg’ or ‘Jake Walk.’ Awareness of OPIDN coupled with the advent of improved methods for assessing the relative potential of OP compounds to produce the disease has led to the virtual elimination of human cases. Nevertheless, neuropathic OP compounds and OPIDN continue to be active fields of study. This apparent paradox arises from the importance of OP chemistry in diverse applications, the potential threat of neuropathic OP compounds as agents of terrorism or warfare, the promise of neuropathic OP compounds as tools in neurological research, and the recent discovery that certain mutations in the gene encoding NTE produce a form of motor neuron disease. Experimental studies have identified the adult chicken as the species of choice for testing OP compounds for their potential to cause OPIDN. Hens greater than w8 months of age are now used in routine testing. Other species in addition to humans and chickens that are known to be susceptible to single doses of neuropathic OP compounds include certain nonhuman primates, water buffalo, cattle, swine, sheep, dogs, and cats. Rats and mice have been considered resistant to the clinical manifestations of OPIDN. However, recent studies have shown that histopathological lesions, particularly in the spinal cord, can be produced in these species by compounds known to cause OPIDN in the adult hen. The apparent resistance of rodents to OPIDN may result, at least in part, from the fact that relatively young (less than w3 months of age) animals have been used in most studies. Generally, the young of a given species are much more resistant to OPIDN than adults are. For example, chicks younger than about 50 days of age will not develop OPIDN after a single dose of a neuropathic OP compound. Moreover, chicks are resistant to repeated doses if they are younger than w14 days of age. Species and age differences in susceptibility to OPIDN have been attributed to long axons in large animals and robust repair of neural injury in young animals. The complete mechanism of OPIDN has not been elucidated. However, there is good evidence that the disease is initiated by a concerted two-step reaction involving inhibition and aging of a critical amount of NTE in target neural tissues. The net result of the aging step is the rapid formation of a negatively charged species in the active site of the enzyme (Figure 3). Such a reaction can take place with OP inhibitors of NTE such as phosphates, phosphonates, or phosphoramidates, which have an ester or amide group in addition to the leaving group (Figure 4). Phosphates and phosphonates undergo aging by net loss of an R-group. Phosphoramidates having only a single R-group attached to the phosphoramidate nitrogen appear to age by loss of the phosphoramidate proton rather than by loss of an R-group. Compounds that do not inhibit NTE do not cause OPIDN, even if they belong to a structural class capable of undergoing the aging reaction. For example, although paraoxon belongs to the phosphate class of OP compounds, it does not produce OPIDN because it is a poor inhibitor of NTE. NTE inhibition and aging transpire within minutes to hours following absorption of an effective dose of a neuropathic OP compound. Thus, events that remain to be elucidated contribute to the delay of 8–21 days between the exposure and the initial signs of ataxia and paresis. However, if inhibition but
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Organophosphorus Compounds
Figure 3 Inhibition and aging of NTE are required for initiation of OPIDN. NTE is represented by a wavy line containing the active site serine hydroxyl group. Pathway (1) shows inhibition by a phosphonate, which undergoes rapid aging to yield a negatively charged phosphonyl adduct. OPIDN follows within 8–21 days and is not treatable. Pathway (2) shows inhibition by a phosphinate, which cannot undergo aging. The neutral phosphinylated adduct does not trigger OPIDN; however, it confers protection against subsequently administered neuropathic (ageable) NTE inhibitors. For each type of inhibitor, R1 and R2 may be substituted or unsubstituted alkyl or aryl groups. X is the primary leaving group that is displaced by the serine hydroxyl of NTE and may be, for example, substituted or unsubstituted alkoxy, aryloxy, or fluorine.
no aging occurs by dosing with an NTE inhibitor that is incapable of generating a negative charge at the active site, no OPIDN ensues. Furthermore, an animal whose NTE is inhibited with a nonaging compound is protected against a subsequent dose of an OP compound that would be neuropathic in a naïve animal (Figure 3). Nonaging inhibitors of NTE include representatives from the phosphinate class of OP compounds, certain carbamates, and sulfonyl fluorides, such as phenylmethanesulfonyl fluoride (PMSF) (Figure 4). The threshold of NTE inhibition in target neural tissue that correlates with the development of OPIDN after a single dose of a neuropathic OP compound is w70%. For many compounds, inhibition measured in brain is paralleled in spinal cord and peripheral nerve, and brain values are often used in screening tests in hens to assess relative neuropathic potency. Repeated dosing also appears to require that a high point of inhibition be reached before OPIDN will develop. The threshold appears to be the same as for acute dosing for some compounds, but for some others, the critical level of inhibition may be as low as 50%. With repeated dosing, there still appears to be a delay of w8–21 days between the time inhibition exceeds the threshold value and the appearance of signs of OPIDN. NTE has also been found in circulating lymphocytes and platelets, where its inhibition has found use as a biomarker of exposure to neuropathic OP compounds. There is a good correlation between inhibition of NTE in leukocytes and brain when the measurements are carried out within 24 h of an acute exposure. However, a good correlation might not be found later (even by 48 h) or under conditions of repeated exposures. Nevertheless, leukocytes provide an accessible source of NTE for detection of inhibition by neuropathic OP compounds. Currently, there is considerable interest in using protein mass spectrometry to detect OP adducts on NTE or other proteins as sensitive and specific biomarkers of exposure to neuropathic OP compounds.
It is important to realize that OPIDN depends on a particular type of chemical modification of NTE rather than mere inhibition of its enzymatic activity. Inhibition of NTE is a necessary, but not sufficient, condition for OPIDN. Aging of the inhibited enzyme results in a complete change in the toxicological outcome. Whereas inhibition without aging results in no clinically apparent injury, suprathreshold inhibition with aging triggers an inexorable neurodegenerative process leading to evident disease. The situation with NTE is completely different from that with acetylcholinesterase (AChE). Inhibition of a sufficient amount of AChE will produce cholinergic toxicity, regardless of whether or not aging of inhibited AChE occurs (Figure 2). Aging of inhibited AChE does not alter the type of toxic response, but it does change the options available for therapy against cholinergic toxicity. For example, oximes such as pralidoxime methiodide (2-PAM) are used to reactivate inhibited AChE, but these agents are ineffective if aging of the enzyme has occurred. Moreover, oximes do not appear to affect the clinical course of OPIDN following administration of a neuropathic OP compound, except to allow survival of an otherwise lethal dose of a compound that also has cholinergic toxicity. In a homologous series of OP compounds, increasing potency for AChE inhibition and cholinergic toxicity correlates with decreasing potency for NTE inhibition and OPIDN. The relative inhibitory potency (RIP) of an OP compound or its active metabolite for NTE versus AChE in vitro can be used as a convenient index of the probable neuropathic potential of the compound. A commonly used measure of inhibitory potency is the IC50, the concentration required to inhibit 50% of the enzyme activity under a standardized set of reaction conditions and time of incubation of the inhibitor with the enzyme preparation. A better measure of inhibitory potency is the bimolecular rate constant of inhibition, ki. When pseudo– first-order kinetics are observed, it is valid to use the
Organophosphorus Compounds
Figure 4 NTE inhibitors. For each type of inhibitor, R–R4 may be substituted or unsubstituted alkyl or aryl groups. For carbamates, R1 can be a hydrogen atom, and for phosphoramidates, R1 and/or R3 can be a hydrogen atom. X is the primary leaving group that is displaced by the serine hydroxyl of NTE and may be, for example, substituted or unsubstituted alkoxy or aryloxy. Fluorine can be a leaving group for the OP NTE inhibitors and is the most common leaving group for sulfonate NTE inhibitors. Type A inhibitors are neuropathic and include certain phosphates, phosphonates, and phosphoramidates. Mixtures of subtypes are possible. Phosphonates are intrinsically asymmetric and enantiomers may have different inhibitory and/or aging properties. Type B inhibitors are not neuropathic, but pretreatment protects against OPIDN from subsequent exposure to type A inhibitors. Type B inhibitors include certain phosphinates, sulfonates, and carbamates.
relationship, IC50 ¼ 0.693/kit, where t is the time of preincubation of the inhibitor with the enzyme. Comparisons of AChE/NTE ki ratios or NTE/AChE IC50 ratios in vitro (RIPs) with toxicity data in vivo have shown that RIP values <1 indicate that the dose required to produce OPIDN is less than the median lethal dose (LD50). In contrast, RIP values >1 correspond to doses greater than the LD50 required to produce OPIDN. The higher the RIP, the safer is the compound with respect to its capacity to produce OPIDN. Thus, insecticidal OP compounds generally are much more potent inhibitors of AChE than NTE and do not produce OPIDN except at doses that would require aggressive treatment for cholinergic toxicity. On the other hand, compounds can be made that are better inhibitors of NTE than AChE. If such compounds can also undergo aging, not only will they produce OPIDN; they will do so at doses that elicit little or no cholinergic toxicity. Marginal or subclinical OPIDN can be potentiated to fullblown disease by subsequent treatment with nonaging inhibitors of NTE. The phenomenon is called promotion by some authors, which is an appropriate term if the initial insult is undetectable. Potentiation was initially a surprising finding,
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especially in view of the fact that reversing the order of dosing of the nonaging and aging NTE inhibitors affords protection against OPIDN. However, it now appears that the outcome of many types of neural injuries, including physical crushing of nerves, can be exacerbated by dosing with nonaging NTE inhibitors as well as with inhibitors of other serine esterases or proteases. The apparent indifference to the method of producing the initial lesion suggests a general mode of action for potentiation, such as interference with regeneration and repair. Although the precise physiological function of NTE is currently unknown, the protein is known to be anchored in the endoplasmic reticulum and to catalyze the hydrolysis of membrane phospholipids and lysophospholipids. Moreover, it has recently been established that NTE belongs to a ninemember family of patatin-like phospholipase domain containing proteins (PNPLAs), of which NTE is PNPLA6. Conventional knock-out of the gene is embryonic-lethal in mice, indicating an essential role in development, and conditional knock-out in the brain results in neurodegeneration. In addition to its serine hydrolase domain, NTE contains tandem domains with homology to cyclic nucleotide-binding regions in other proteins, implying a regulatory or signaling function. Mutation of a homologous protein called SWS in Drosophila results in a spongiform neurodegenerative disease, suggesting that NTE might be associated with neurological or neurodevelopmental disorders. More recently, NTE mutations have been linked to a form of motor neuron disease in humans. Certainly, much work remains to be done to elucidate the normal and pathogenic roles of NTE, but the accomplishments thus far have proved to be useful in a wide range of fields, including toxicological risk assessment, developmental neurobiology, and clinical neurogenetics.
See also: Acetylcholine; A-esterase; Atropine; Azinphos-Methyl; Carboxylesterases; Chemical Warfare; Chlorpyrifos; Cholinesterase Inhibition; Cyclophosphamide; Diazinon; Dichlorvos; Fenthion; Ginger Jake; Malathion; Methamidophos; Methyl Parathion; Naled; Nerve Agents; Neurotoxicity; Parathion; Pyridostigmine; Pyridoxine; Soman; Tabun; Trichlorfon; V-Series Nerve Agents: Other than VX; VX.
Further Reading Moser, V.C., Aschner, M., Richardson, R.J., Philbert, M.A., 2008. Toxic responses of the nervous system. In: Klaassen, C. (Ed.), Casarett and Doull’s Toxicology: The Basic Science of Poisons, seventh ed. McGraw-Hill Professional, New York, pp. 631–665. Rainier, S., Albers, J.W., Dyck, P.J., Eldevik, O.P., Wilcock, S., Richardson, R.J., Fink, J.K., 2011. Motor neuron disease due to neuropathy target esterase gene mutation: clinical features of the index families. Muscle Nerve 43, 19–25. Richardson, R.J., 2010. Anticholinesterase insecticides. In: McQueen, C. (Ed.) Comprehensive Toxicology, second ed., vol. 13. Academic Press, Oxford, pp. 433–444. Richardson, R.J., Worden, R.M., Makhaeva, G.F., 2009. Biomarkers and biosensors of delayed neuropathic agents. In: Gupta, R.C. (Ed.), Handbook of Toxicology of Chemical Warfare Agents. Academic Press/Elsevier, Amsterdam, pp. 859–876. Thompson, C.M., Richardson, R.J., 2004. Anticholinesterase Insecticides. In: Marrs, T.C., Ballantyne, B., (Eds.), Pesticide Toxicology and International Regulation (Current Toxicology Series). John Wiley & Sons Ltd., Chichester, pp. 89–127. Wijeyesakere, S.J., Richardson, R.J., 2010. Neuropathy target esterase. In: Krieger, R. (Ed.), Hayes’ Handbook of Pesticide Toxicology, third ed. Elsevier/Academic Press, San Diego, pp. 1435–1478.