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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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Suggested Citation:"Anticholinesterases." National Research Council. 1982. Possible Long-Term Health Effects of Short-Term Exposure to Chemical Agents: Volume 1. Washington, DC: The National Academies Press. doi: 10.17226/740.
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2 ANTICHOLINEUE"SES Antichollnesterases (anti-ChEs) are toxic to h''mans principally because they interfere with molecular and cellular mechanisms required for the normal functioning of the central nervous system (CNS) and peripheral nervous system (PNS). Their adverse health effects are related Costly to inhibition of acetylcholinesterase (AChE), a critically important CNS and PNS enzyme that hydrolyzea the neurotransmitter acetylcholi.ne (ACh). Chemical-warfare (COO) agents exploit the acute, life-threatening properties of profound AChE inhibition; some of the antl-ChEs precipitate other clinically si gnif leant de let eriou s e f f ec t ~ on sensory and neuromuscular function. All the ma jor anei--ChEs are chemically reactive and potentially capable of allylating a variety of biologic macromolecules, but the long-term health implications of these reactions are not well known. This chapter therefore focuses on the acute toxic effects of anti-ChEs and the possible long-term effects on CNS and PNS function. The fundamental cellular component of the nervous system, the neuron, has long ~ branching ~ cylindric processes (dendrites and axon) that extend from the cell body. Dendrites are modified for signal reception end transduction end form extensive networks that permit interneuronal communication, coordination, and integration of ner~rous-system function. The axon typically is a long extension of the neuron specialized f or transmission of electric signals and, at its distal end, for chemical communication of informaelon to other neurons or to muscle at sites termed synapses and neuromuscular junctions, re spectively. ACh is the chemical transmitter of information from both somatic ant autonomic (PNS) neurons. On the somatic side, the lower motor neuron uses ACn to convey excitatory impulses to voluntary muscle. Cholinergic neurons of the autonomic division of the PNS are grouped in craniosacral (parasympathetic) and thoracolumbar (sympa thetic) outflows from the spinal cord . Parasympathetic pathways use ACh at both preganglionic and postganglionic neurons; in the sympathetic system, ACh is restricted to the preganglionic effecter. These cholinergic-dependent autonomic neurons play an important role in regulating the function of various Maritally import ant e f f ec tor organs. Cholinergic pathways are widely distributed in CNS tissue, but their functions are less well understood than those in the PNS. AChE terminates the transmitter action of ACh. Drugs that inhibit or inactivate AChE (anti-ChE agents) cause ACh to accumulate at cholinocepeive sites and thus produce effects equivalent to continuous stimulation cuff cholinergic nerve fibers. Before World War ~ I, only "reversible" antl-ChE agents were generally known, of which physostigmine (eserine) is the outstanding example. Shortly before and during World War ~ I, a class of highly toxic chemicals, the organophosphorus compounds ~ OPs ), were developed , chiefly by Schrader of I.G. Parbenindustrie,first as agricultural insecticides and later as CW agents. The high potency of these compounds was found to be due to "irreversible" inhibition of AChE; thus, they produced effects for considerably longer periods than the classical s

inhibitors. Since the pharmacologic actions of both classes of anti-ChE agents are qualitatively similar, they are discusses as a group, and important features of individual pleases or compounds are noted. - CHEMISTRY OF ANTICHOLINESTERASES A complete fiat of anti-ChE compounds used in the Edgewood program is contained in the master file (Appendix A). Structure-activity relationships have been reviewed extenalvely for the "reversible inblbitors tl,2) the OF agent a (3,4) and both classes of compounds (5-7). " REVERSIBLE " INHIBITORS After the structure of physostigmine wee eatabliahet, Stedman (8,9) undertook a systematic lovestigatlon of a number of related synthetic c-ompouads. The essential moiety of the physostigaine molecule we. found to be the methy~carba~ate of a basically substituted, simple ooaminophenol. The quaterna~y ammonium - derivative, Deo~tig~iD@9 i~ a compound of greater stability and equal or greater potency. Retention of the dimethy~carbamate side chain in the mete position, but with incorporation of the quaternary nitrogen atom Into the ring to fore a pyrityl nucleus, results in compounds with anti-ChE and other pharmacologic properties similar to those of neostigmine. Pyridostlgmine is a drug of this clasaO Although the carbamates are the most familiar and the most commonly encountered anei-ChEs, members of other chemical classes are also capable of inhibiting AChE. For example, edrophonium, an analogue of the phenolic residue of neostlgmine, is used extensively in clinical medicine. It was not administered to the volunteers at Edgewood, but two other noncarbamates were: 1,2,3, 4°tetrahydro-9-acridinamine (Tacrine) and hexafluorenium (Mylaxen). These compounds also are used in clinical medicine but are not as popular as neostigmine, pyridostigmine, or edrophonium. O RGANOP HO SP HORUS INHIBITORS The general formula for this class of cholinesterase inhibitors is shown in Figure 1. A great variety of substituents is possible: R1 and R2 may be alkyl, alkoxy, aryloxy, amido, mercapto, or other groups; and X (also called the "leaving group-) may be a halide, cyanide, thiocyanate, phenoxy, thiophenoxy' phosphate, alkyIthioethylmercaptide, dialkylaminoethyl~ercaptide, or carboxylate group. It is obviously impossible to discuss here more than a few representative compounds of the more than 50,000 that Fig. 1: Organophosphorus Compounds General Formula: R2 \ X Rl TO

have been prepared; a useful chemical classification · f the compounds in this class that are of particular pharmacologic or toxicologic interest has been developed by Holmstedt (3, 4) . 1'ilsopropyl phosphorofluoridate (DEP3 is perhaps the best known and the mos ~ extensively studied compound . Generally, the most acutely toxic compounds are those with one carbon-phosphorus bond (phosphonates). The compounds studied by the Army (Appendix A), with the exception of OPP, con~aln this feature. In GA ~ rabun), the leaving group is cyanide . In GB ~ sarin), GO (soman) ~ snot OF, the leaving group is fluoride. In the V agents, OX and EA 3148 ~ the most potent agent atmintstered to the volunteers), the leaving group is a dialkylaminoalk~rImercaptide. ABSORPTION, FATE, ANI) EXCRETION OF ANTI-CHES . Physostigmine is readily absorbed from the gastrointestinal tract, subcutaneous tissues, and mucous membranes. It undergoes hydrolytic cleavage at the ester linkage by cholinesterases, renal excretion plays only a minor role in its disposal. In man, a Beg dose of physostigmi-ne injected subcutaneously is largely destroyed in 2 h. Neostig~ine and related qua ternary ammonium drugs are absorbed poorly after oral administration, and much larger doses are needed for effect than when they are administered by injection; both are metabolized by hepatic microsomal enzymes (IO) and excreted in the urine . The commonly encountered OP anti-ChE agents are, with some exceptions ~ e.g., echothiophate), highly soluble in lipids . Consequently ~ e hey are rapidly and effectively absorbed when administered by almost any route, including the gastrointestinal tract, the skin and mucous membranes after contact with the liquid form, and the lungs after inhalation of vapors, finely dispersed dust, or aerosols. Most OP compounds are excreted almost entirely as metabolites in urine. Between the time of-absorption and excre Lion, there are varied periods during which -the original compound or its metabolizes remain bound to proteins in the blood and tissues. Both hydrolytic and oxidative enzymes are involved in metabolism of the OP compounds. The OP anti-ChE agents are hydrolyzed in the body by a group of enzymes, the phosphory~phosphatases. These are widely distributed and hydrolyze a large number of OP compounds (e.g., DFP, tabun, satin, paraoxon, and tetraethyl pyrophosphate, or TEPP) by splitting the anhydride-like P-E (or P-CN) bond. They also hydrolyze several aliphatic esters (e.g., ethyl acetate) and aromatic esters (e.g., phenyl acetate). The enzymes are not irreversibly inhibited by OP compounds, presumably because the phosphory3-ated active site reacts rapidly with water to regenerate the free form, in contrast with its high stability in the case of the cholinesterases. Because of the above reactions, the effects of exposure to two OP insecticides may be synergistic. For example, when malathion is administered to animals in combination with O-ethyl O~-nitrophenyl pheny~phosphonothionate (EPN), the resulting toxicity is as much as 50 times that expected from the sum of their individual toxicities, this results primarily from the inhibition by EPN of enzyme 8y8te~s that normally metabolize malathion to inactive products. Other combinations of OP insecticides also have shown supra-addition of toxic effects. 7

· Arc #ma - I - ~~ - Cx ACHED '0~ '0; I' · 1 ~ it_ Cow Cal ~~ ~ f cat I~Y_ S - ~~ ~ h - ~~ Iel ll. [~ ~C3^C~. I DAYS Be_ C ~e til. ~~ - '~ ~0 _ _ ~ ~,0 ~~ —V'~,— Be_ 3~ - oe~.~ ~ ~ ~~ ~ ~ Con - C — 't`~ #0 C - Ad ~. f`` '0 · ~~ I =^ ~ j "^ ,~,0 Of - ~_~e Ho ,0 ~ _ +~50 ESCROW COCCI. Fig. 2: Steps involved in the hydrolysis of acetylcholine (ACh) by acetylcholinesterase (ACHE) All, and in the inhibition of AChE by reversible (I), carb~myl eager (all), and organophosphorus clot) agents. Heavy, I1ght, and dashed arrows represent extremely rapid, intermediate, and extremely slow or insignificant reactions, respectively. Reproduced from Koelle, G.B., In: The Pharmacologic Basis of Therapeutics (Goodman, L.S. and Gils'ian, A., eds.), 5th ado Macmillan Publ. Co., 1975, pg. 448. 8

MECHANISMS OF ACTION INHIBITION OF ACET~CHOLI~STE~SE BY ~ICHOLI~STE"SES The mechanisms of action of compounds that typify the three c lasses of anti-ChE agent s are show in Figure 2. They differ primarily in quantitative respects from the reaction between AChE and i ts noneal substrate, ACh. simple quaternary compounds, such as edrophonium, form electrostatic bonds with the anionic site of the enzyme and hydrogen bonds with the imidazole nitrogen atom of the esteratic site. In all such case., inhibition is rapidly reversible, and such drugs have a Very short duration of inhibitory action. It was at one -time generally assumed that physostig~ine, neostigmine, and related inhibitors that possess a carbamyl ester linkage or urethane structure, in addition to a tertiary amino or quaternary ammonium group, inhibit the enzyme in the same reversible fashion. However, careful kinetic studies showed that physostigaine and neostigmine are hydrolyzed by cholinesterase ~ Il-14 ~ O Initially, inhibitors of this class form complexes in which the inhibitor is attached to the enzyme at both anionic and esteratic sites; subsequently, hydrolysis proceeds in a manner analogous to that of ACh, and the alcoholic moiety is split off, leaving a carbamylated ant inhibited enzyme. I`ater, this enzyme reacts with water to release a substituted carbamic acid and the regenerated enzyme. The main difference between the reaction of the natural substrate, ACh, and that of the carbamate inhibitors is the velocity of the final step; the half-life of dimethy~carbamy! ACh35:, formed by the reaction with neostigmine, is more than 40 million times that of the acetylated enzyme: 30 min and 42,us, respectively (15~. The reaction between AChE and most OP inhibitors, such as OFF, occurs only at the esteratic site. It proceeds in a comparable fashion, except that, as a result of the initial hydrolysis, the enzyme becomes phosphorylated. The reaulting phosphorylated enzyme is extremely stable: if the attached allays groups are methyl or ethyl, substantial regeneration of the enzyme by hydrolytic cleavage requires several hours; with isopropyl groups, as in DFP, virtually no hydrolysis occurs, and the return of AChE activity depends on synthesis of new enzyme, which requires days to months. Some quaternary OP compounds (e.g., echothiophate) combine at both the e~teratic and anionic sites, and that probably contributes to their extreme potency and specificity (4,16~. From the foregoing account, it 1. apparent that the terms "reversible" and "irreversible, a as applied to the carbamy] ester and OP anti-ChE agents, respectively, reflect only quantitative differences and that both classes of drugs react with the enzyme in essentially the same manner as does ACh. REACTIVATION OF ACHE Although the phosphorylated esteratic site of AChE undergoes hydrolytic regeneration at a low or negligible rate, Wilson (17) f ound that the nucleophilic agent hydroxyla~ine ~ H2NOH) can reactivate the enzyme much more rapidly. In the subsequent search for more effective Deactivators, a large number of hydroxam~c acids 9

(RCONHOH) and oximes (RCH-NOB) was shown to have this property, e.g., tiacetyl monoxide, or DAM. From the data that accrued, it was predicted that highly effective reactivation should be produced by a molecule containing both a quaternary nitrogen atom ant an oxime group, Spaced at an appropriate distance. Thla goal was achieved (18) with pryidlne~2-aldoxime meehy! chloride ( 2-fonsyI-~-~ethylpyridinium chloride oxime, pralidoxime), reactivation with this compound occurs in one millionth the time of that with hydroxylamine (19). Some bisquaternary oxides were later shown to be even more potent as reactivatore; an example is obitoxime chloride (Figure 3)- 1~1~-(oxydimethylene)o Lis(4-fonmy~pyridiniu~), dichloride dioxide (20~. 0 ~,c - ~~ D.oc~ "canoed 1~, 1 Cal CHe ~d~b8~. c~P~(~-J Ho~cH x" 0~.~ Chase Fig. 3: Cholinesterase Deactivators. Reproduced from Roelle, G.B. In:The Pharmacologic Basis of Therapeutics (Goodman, L.S. and Gilman, A., eds.), 5th ed. Macmillan Publo CO.. 1975, pg. 458. The mechanism of reactivation is sketched in Figure 4. When the quaternary ammonium group of pralitoxime is attracted electrostatically to the anionic site of the enzyme, the oxime group of the former is oriented optimally to exert nucleophillc attack on the electrophilic phosphorus atom of the phosphorylatet esteratic site; the oxime-phospho~ate is then split off, leaving the enzyme (21,22~. Although the oximes reactivate phoaphorylatet cholinestereae in vitro and increase recovery from intoxication produced in vivo by some organophosphates, they are not panaceas for the treatment of pig by anti-ChEs. Pralidoxime and obidoxime are Quarternary ammonium compounds, and their capacity to reactivate brain enzymes is inhibited by the blood-brain barrier. In is also debatable whether pralitoxime and related agents can effectively antagonize the manifestations of intoxication by neostig~ine and other carbamyl ester inhibitors. Moreover, coat phosphorylated AChEs undergo a fairly rapid process termed aging and within the course of Pollutes or hours, thereby become completely resistant to reactivatora.

Hox so H.C~O =.1~? :~J IC - ~I - - o ~tC3O j =3~' Kl -of Pock +~0. QC=N—OH CL7I .N—O—F' ~=9_ Come Ontme phospl~ot~o - ~CH~bJ—O—ted CH, HACK Amp. _~_ InI~ Fig. 4. Reactivation of alkylphoaphorylated acetylcholinesterase (AChE). After alkylphosphorylation of AChE by DFP (left), spontaneous hydrolytic reactivation occurs at an insignificant rate (upper reaction), as indicated by the dashes arrow. -4 1ng. is the loss of one of the isopropoxy residues that occurs more rapidly than spontaneous hydrolysis; the product is very realatant to regeneration by pralldoxlme. Pralidoxlme (lower reaction) combines with the anionic site by electrostatic attraction of ita qua ternary nitrogen atom, Which orients the nucleophlllc oxide group to react with the electrophilic phosphorus atom; the oxime-phoaphonate is split off, leaving the regenerated enzyme. Reproduced from Koelle, G.B.: In The Pharmacologic Basis of Therapeutles (Goodman, L.S. and Gilman, A., ads.), 5th ed. Macmillan Publ. Co., 1975, pg. 457. AGING OF I~IBI~ ACHE Aging causes Inhibited enzymes to become refractory to reactivation. The phenomenon was first reported In 1955 by Bobblger (23) and later observed with OE-inblbited AChEs (26,25) ant with an OP-lahibited atropinesterase (26). An extensive discussion of aging Way be found in Berend's thesis (27). The process determines the time during which reactivatore can be expected to be beneficial for those exposed to OF anti-ChEa. In the case of OF anti-ChEs, an Inhibited (e.g., phoaphorylated) 11

enzyme, which initially can be reactivated by oxides, is changed to a form that cannot be reactivated by these compounds (19). The term aging has been applied because the amount of inhibited enzyme refractory to reactivation increases with time. For example, Berry et al. (28) observed that, although pretreatment of animals with a combination of pralltoxime (2-PAM) and atroplne increased the LD,o values of several compounds (cog., TEPP and DFP), it hat little effect on the lethality of satin and none on the lethality of soman; they concluded that this was the result of the rapid aging of the ChEs inhibited by satin ant aortas. More direct evidence of in viva aging was obtained by Harris et al. (29), who injected 32P-labeled saris and aoman into rats and observed that the rate of aging of the inhibited rat-brain AChE was the same in viva as in parallel in vitro experiments. Aging is probably due to the aplitting-off of one alkyl or alkoxy group from the inhibited enzyme, leaving a more stable monoalkyl- or monoalkoxy-phosphoryl AChE (30,31). Wilson (32) pointed out that the difference in reactivatability may be associated with the relative reactivity of secondary and tertiary phosphate esters. Whereas the original inhibited enzyme is a tertiary phosphate ester, the dealkoxylated derivative is a secondary phosphate ester. Thus-, the rate of aging depends on the phosphoryl group (33) and not on the group hydrolyzed from the OP by cholinesterase (ChE). Berry and Davies (28) observed, as have many others, that soman yields the most rapidly aged-inhibited ChE obtained from any available anti-ChE; the half-life of the pinacolyl phosphonylatet enzyme was determined to be less than 1.5 min. The next most rapidly aged-inhibited enzyme also contained a branched-chain secondary group; (CH3)2-CH-CH(CX3)-0~-) Some straight-chain secondary groups, such as CH3-CH2-CH(CH3)-OH-, were also associated with relatively rapidly aged-inhibited enzymes, the phosphonylated enzyme half-life being about 0.5 h. Berry and Davies (28) noted that aging 'is slow when the alkyl group is a primary alcohol, whether or not the carbon chain is branched, but is much more rapid if the alkyl group is a secondary or cyclic alcohols CHOLINERGIC RECEPTOR AND ACH CHANNEL . In addition to reacting with ChEa, OPs ant other anti-ChEa also can react with other critical molecules in nerves or in effector organs. OP drugs may exert direct effects on the cholinergic receptor or on its phospholipid environment, at both CNS and ENS synapses (34-38~. OPa also react with other neural ant metabolic enzymes, ant some of them are capable of alkylating DNA. The biologic consequences of these reactions are not as well understood as are those inhibiting ChE. White and Stedman (39) Suggester that, in addition to inhibiting AChE, OP compounds have an effect on the aite where the ACh molecule reacts at the neuromuscular Junction. Riker and Wescoe (40) showed a direct agonist action of neostlgmlne at the neuromuscular junction, ant many others have found that neoatigmine and some other anti-ChE agents have anticurare effects not apparently related to inhibition of ChE (41). Additional observations indicated that preparations that had been denervated for over 20 d responded with a contracture when exposes to satin, 12

and findings described below indicate that anti-ChE agents like neostigmine cause marked destruction of denervated muscle (42,43~; this phenomenon is most likely correlated with the partial agonist effect of neostigmine. Similarly' Mique] (44) suggested that OP compounds react with other sites on the muscle, in addition to the enzyme itself. Studies by Xavier ant Valle (45) disclosed that Phos~rinR, an OP insecticide, was able to affect both the ACh receptor and the ion channel associated with it, but without affecting AChE itself. They also found, using two different methods, that physostig~ine and neostigmine, in addition to producing blockade of AChE, potentiated the muscle response to ACh when applied in the presence of complete AChE blockade. Albuquerque's studies with OP compound. (46) have suggested an additional effect on the ionic channel that is unrelated to the sites of reaction of ACh on AChE or on the ACh receptor. The simple presence of OP compounds makes the reaction of many agonists, particularly ACh and anatoxin (AnTX),, more intensive and speed ~ the ef feet s of some compounds that block the ion channel. Several agents that react both with the ACh receptor and the channel appear to antagonize the action of the OP compounds in this manner. Conversely, the binding rate of OPs is increased when increasing concentrations of the agonist are present (46), and the rate of binding induced by the presence of large quantities of agonist can occur whether ACh is the agonist or other agents--such as AnTX, subaryldicholine, and succiny~choline--are used in place of ACh. PRESY.NAPTIC ACTION Anti-a~E agents have presynaptic effects that are related mostly to a reaction with the presynaptic nerve seminal (47-55~. A number of workers reported that compounds such as neostigmine and physostig~ine augment and prolong - spontaneous release of ACh (miniature endplate potentials or MEPPs) ant increase the size of endplate potentials (EPPs) (48,56-59~. Boy d and H - rtin (48) reported a biphasic effect of ChE inhibitors: an increase at low concentrations and a decrease at high concentrations. In a detailed study of the action of neostigmine, ambenonium, edrophonium, and methoxyambenonium on cat tenuissimus muscle, Blaber and Christ (60) reported that MEPP frequency was increased by several ChE inhibitors, and concluded that the effect is probably not related to ChE inhibition but might be related to excitation-secretion coupling, the process by which an action potential releases ACh. The only published study on presynaptic effects of the more potent ChE inhibitors is that of Abraham and Edery (49), who examined the effect of soman on synaptic transmission in rat diaphragm. In vitro soman increased the frequency of MEPPs (an effect blocked by Mgl'), caused muscle depolarization that reversed spontaneously, ant increased quantal content. It was suggested that the observed changes in transmitter release resulted from an effect on the action potential invadlag the nerve terminal, although no direct evidence was offered. 13

In addition, several ChE inhibitors generate an.tidromic action potentials in motor nerves; these may occur spontaneously (57,619 62) or after an orthodromic nerve volley (47,50,62-64~. The potentials are apparently not caused by action potentials originating in muscle, inasmuch as the antidromic repetitive firing has been observed in nerves from muscle incapable of twitching (62)0 Riker en al. (50) and Werner (5l,52) concluded that the antidromic discharges were produced by direct actions on the motor nerve terminal. Although ChE inhibitions smith later accumulation of ACh and an increase in extracellular potassium concentration, may be the cause of the observed effects, these posalbilittes are unlikely. NERVE MEMBRANE _ ~ .. Another example of an effect apparently unrelated to ChE inhibition is found in studies of the actions of ChE inhibitors on ionic conductances of electrically excitable membranes. When single frog nerve fibers were used, physostigmine (~-10 - ) attenuated action potential and current, markedly prolonged the duration of the current, and slowed conduction. This mechanism might be involved in functional sensory deficit aad--$f selective for inhibitory fibers, as is the case with local anesthetics (65)o~might play a role in generation of facilitation before depression. CENTRAL NERVOUS SYSTEM The situation is still more complex in the CNS. Even if the action of anti-ChEs were limited to the inhibition of postsynaptic AChE, the complex circuitry of the brain provides ample opportunity for effects at other sites. Because brain cholinergic pathways are diffuse ant connect with many other systems, overactivity or blockade of cholinergic synapses can lead to aboonmal activity in many other neurons. Apparently, there is no end to the lint of transmitters and bioactive substances that can be affected indirectly or directly by cholinergic agonists (66~. Among the effects in question are those on the Y -aminobutyric acid (GABA) system which are important in brain excitability and epileptogenesis (67), as well as those involving peptide transmitters and bioactive peptides (68)0 It it unknown whether these effects are brief or long-lasting. For example, it is unlikely that a perturbation in GABA content would be long-lived after the initial effect of the ant i-ChE on the GABA system. However, the circuit ~ are complex, and even a temporary perturbation: might lead to reverberations that persist for a long time. The CIIS has only a limited capacity to regenerate, and recovery after tissue damage might be slow or incomplete. It is also known that a brief presence of excess transmitter in a synapse can lead to compensatory changes in the number of postsynaptic receptors. "NECtROTOXIC" ESTERASE (NTE) Some OPs react with a poorly cnaracterizet enzyme--"neurotoxic esterase" (NTE)--of unknown function resident in CNS, ENS, and some o ther tissues and precipitate a delayed CHS-PtIS distal axonal degeneration, which is expressed clinically as a sensorimotor

The aubJectIve manifeatatio4e of brain dysfunction usually disappear shortly after cessatlo4 of exposure; in most 14atances, BEG abnontallties reportedly dleappear vlthin 2 Ok of acute exposure or termination of chronic exposure (90~92). ACUTE TOXIC EFFECTS Toxicity of particular OF compounds does not vary greatly ~IBODg mammals. Slgna and ·y'rptoole differ eal41y to sequence and 14 indivltual prominence (93-96). Rapidity of appearance, 14te4~1ty, and timecouree depend 04 close and route of adel4istratlo4. The effects of acute latoslcatlo4 with antl-ChZ agents are manifest by o~uscarinic and 4icotl41c ·ig4e and ·"ptoes and, except for compounds of extremely low colubllity 14 lipids, ·1gns referable to the CNS. Effects may be local or general. Local effects are due to the action of vapors or aerosols at their elte of contact filth the eye a or respiratory tract or to the local absorptlo4 after liquid contamluatIon of the skin or oucoua membranes, 14clutlDg those of the geatrointestl~l tract. Genere1 effects rapidly follow systemic absorption by any route; they appear moat Tepidly after inhalation of vapors or aerosols, In which case severe effects any appear within a few minutes. In contrast, the onset of Captor after gastrointestinal and percutaneous abeorptlo4 1e delayed. The duration of effects is determined largely by the nature of the compound; it may vary from minutes, as after an overtone of edrophonlum, to several days or even veeka after lrreverelble alkylphoaphorylation of AChE, as by DFP or sable. After exposure to vapors or aerosols or after lchalatlo4, ocular and respiratory effects generally appear first. Ocular effects include marked miosia, con junctival congestion, clliary spasm, and brovache, along with watery nasal dlecharge; respiratory effects consist of tightnesa" to the cheat and-'rheezing due to the combination of bronchoconatriction and increased bronchial secretion. After ingestion, geatrointestinal effects appear first, including anorexia, nausea and vomiting, abdominal cramps, and diarrhea. After percutaneous absorption of liquid, localized sweating and muscular faaciculatlon in the immediate vicinity are generally the earliest olanifeatations. Severe intoxication la manifest by extreme salivation, Involuntary defecatlo4 and urination, sweating, lacri~atlon, bradycardia, and hypote4eIo4. .Nlcotinic actions at the neuromuscular junctlo4e of ·Iceleeal mnacle usually consist of fatigability and generalized weakness, involuntary trltchi~, scattered faaciculatlo4, and eventually severe weakness and paralysis. The most ·erloua co4eequence of the neuromuacular actions la paralyala of the respiratory muscles. The effects 04 the CNS include co~uaio4, atasla, slurred speech, lose of reflexes, coca, and central respiratory paralysis. Actions on the vasomotor and other cardiovascular centers add to the peripheral actions to complicate the hemodynamic patted. Afeer large doses or lchalation of high coocentratlo4s, the Dime course may be telescoped into a few minutes and "my of the above ·1gns overshadowed by dyspnea, apnea, and collapse. ~ case of severe accidental poisoning in man is illustrative (97). Development of signs after smaller doses has been described by Grob (98,99) and others (100-104). 21

neuropathy (69). Although a causal association between NTE inhibition and delayed neurotoxicity has not been demonatrated, they are strongly correlated. NTE is operationally defined as the eateratic activity agaicat phenyl phenylvalerate (the preferred substrate), phenyl valerate, or closely related esters that is resiatant" to paraoxon and sensitive to DFP and mipefox (N,N'-diisopropylphoaphorotiamidlc fluoride). Two groups of compounda inhibit NTE tFlgure 5): one group conaiata of various phosphates, phosphoramidates, and phoaphonates, which induce neuropathy; and the Secant contaloa sulfonatea, phoaphinates, and carbamatea, which do not. The latter can react covalently at the phosphorylation site involved in delayed neurotoxicity and thereby protect against later dosea of 0P compounta that would otherwise induce neuropathy. En contrast, neuropathic 0P compounta induce irreversibly raged) inhibited NTE. Aging involves transformation of the phosphorylated enzyme to a further modified fore in which one R group has been cleaves from the phosphorus and a negatively charges residue remains attached to the enzyme. Once this process occurs, regeneration of the active Site of the enzyme is no longer possible, ant neuropathy will ensue if the NTE-inhlbition threshold has been reached or exceeded withla a required period. The relationahip between this phenomenon ant the onset of atonal degeneration is unknown, but It acema likely from recent experimental Studies with DFP (70,71) that the target site in nervous tissue in in the nerve fiber itself. Ro ~ R-~ '0 P ~ RO x R- - X p~ospt`~e phosphoromi~ole R0 0 R x pro - ~~e o R—1' - X RHO R HO 11 UP N—C · O B' OX R' BOX "Itono'. - ~~e C - bate Figure 5. Two groups of NTE inhibitors; Group A (upper) induce delayed neuropathy, Group B (lower) protect against the neuropathic potency of the upper group. R and R' may be alkyl, aryl or heterocyclic aubatituenta; X is the leaving group which is ejected when the inhibitor reacts covalently with the enzyme. Redrawn from Johnson, M.K., 1974: A comparison of the Structures of inhibitors of neuro toxic estereae . J. Neurochemistry 23:786, Fig. 1, Raven Press, N.Y. BIOCHEMICAL AND METABOLIC CHANGES 0P ant relates drugs have effects on dehydrogeneaea and on gluconeogenesis and oxygen uptake (6,72). Failure to gain weight 15

normally accompanies chronic intoxication with some OP agents, ant weight loss accompanies OP-induced neuropathy. Some of these biochemical changes may not be specifically related to the anti-ChEs; e.g., changes in blood lactate and acid-base relationships could be related to severe hypoxia, and the hyperglycemia could result from discharge of catecholamines via cholinergic control of the adrenal medulla. OP ALKYLATION e Some organophosphates are strong alkyla~cing agents in vitro (73~74). Examples are tetramethyl pyrophosphate, dlchlor~ros, methyl paraoxon, tetrachlorfenvinphos, me~rinphos, crotoxyphos, ant methyl parathion. Review of several studies on the alkylation of DNA with dichlorvos indicated that dicl~lorvos induced methylation in isolated ONA or in DNA of bacterial and animal cells treated in vitro. The extent to which OP esters alkylate DtlA in vivo is not clear. On the one hand, them existence In Canals of considerable extrahepatic esterase activity provides an important detoxifying capability that diminishes the chances of deleterious alkylation (74, 75) . On the other hand, it was reported (76) that [14C]parathion administered in the diet or by intraperitoneal injection resulted in binding of metabolites to liver TUNA. It was Also assumed that the esterase detoxification pathways may operate only slowly, or not at all, for simple alkyl phosphates like trimethy.1 phosphate ~ 74, 75) . BIOLOGIC AND CLINICAL EFFECTS ACUTE EFFECTS: PH~COL~Y The characteristic acute pharmacologic effects of the anti-ChE agents classically are ascribed to the inhibition or inactivation of AChE at sites of cholinergic transmission, with the consequent accumulation and action of endogenous ACh liberated both by stimulated cholinergic nerve and (in much smaller amounts) by continual leakage during the resting stage. AChE is present in most tissues in a quantity in excess of that required for normal function; to exert a marked effect in viva, an anti-ChE agent must generally inhibit 50-9OX of the functional AChE at a given site. Thin can be achieved readily, because most of the anti-ChEs produce 50X inhibition of the enzyme at concentrations of 10-7 M or lower. In principle, it should be possible to predict the ~ pharmacologic properties of anti-ChE agents merely by knowing the loci at which ACh is physiologically released by nerve impulses, and the responses of the corresponding effector organs to the chemical mediator. Potentially, the anti-ChE agents can produce all the following effects: stimulation at autonomic parasympathetic effecter organs; stimulation, followed by depression or paralysis, of skeletal muscle and of all autonomic ganglia (nicotinic actions); and stimulation, followed by depression, of cholinoceptive sites in the CHS. Because cholinergic stimulation of ganglia increases activity in the sympathetic, AS well as the parasympathetic, postganglionic nerveS, all the autonomic effectors are activated. These assumptions are only broadly correct, inasmuch as, with smaller doses of anti-ChEs, particularly those uset therapeutically, several modifying factors are present. Host importantly, an

enormous number of compounds can inhibit cholineatersse, and 30 two are identical in either biochemical propertioa or pharmacokinetlca. Thus, although they all share some general characteriatica, the Retailer effects vary considerably from compound to compound. For example, compounds containing a quaternary ammonium group do not penetrate cell membranes readily; hence, some anti-ChE agents are excluded by the bloot-brain barrier from exerting substantial action on the CNS. But quaternary ammonium compounds act relatively strongly at the neuromuscular Junctions of skeletal muscle through both their anti-ChE ant their direct cholino~lmetic mechanisms and have comparatively lesa effect at autonomic effecter sites. Their ganglionic actions are generally intermediate. The more lipid-soluble agents, such as tertiary aminea and most OP co~pounda, have ubiquitous effects at both ENS and CNS cholinoceptive aitea. The main acute pharmacologic actions of anti-ChE agent a that are of concern here are those on the eye, the intestine and other organs innervated by the autonomic division of the ENS, the skeletal neuromuscular Junction, and the brain. Effects of cholinergic and adrenergic stimulation on effecter organs are summarized in Table 1. Eye When applied to the conjunctive, anti-ChE agents cause conjunctival hyperemia and constriction of the iris sphincter (miosis) and ciliary muscle (spasm of accommotatlon). Koala is apparent in a few minutes and becomes maximal in 0.5 h. The pupil may be pinpoint sized, but it generally contracts even further when exposed to light. It returns to its normal atze in a few hours to several days, depending on the drug and its concentration. The spasm of accommodation is more transient and generally vanes considerably before termination of the miosis. Intraocular pressure usually decreases concomitantly, but in some cases anti-ChE agents may cause an initial increase in intraocular pressure owing to dilatation of the finer blood vessels and increased permeability of the bloot-aqueous humor barrier; this is generally followed by a decrease to below initial pressure. Systemically administered anti-ChEs Similarly affect the cholinergic terminals Supplying the circular muscles of the eye to produce miosis, but they also affect Sympathetic ganglia that operate the apposing radial Oracles, and they act on the brain in ways that may reduce activity in choline An to nerves to the eye. Thus, the effect a of systemic administration are not as predictable as those of local administration, and either constriction or dilatation of the pupils may be seen. Gastrointestinal Tract Although the actions of various anti-ChE agents on the geacrointestinal tract are nearly identical, neoatigmine has been studied most extensively in this regard. In man, neoatigmine - increases gastric contractions and increases the secretion of acidic gastric Juice. The drug tends to counteract the inhibition of gastric tone and motility induced by atropine and increases the stipulatory effect of morphine. Neostigmine augments the motor activity of the small and large bowel; the colon is particularly Stimulated. Atony is overcome or prevented, propulsive waves are increased in amplitude and frequency, and transport is thus promoted. Atropine inhibits, but 17

does not abolish, the intestinal effects of neostig~lne. The total effect of anti-ChE agents on intestinal motility probably represents a combination of actions at the ganglion cells of Auerbach's plexus and at the muscle fibers, as a result of the preservation of ACh released by the cholinergic preganglionic and postganglionlc fibers, respectively. Ac Lions at Other Autonomic Sites Secretory glands thee are innervated by postgas~glionic cholinergic fibers include the bronchial, lacrimal, sweat, salivary, gastric, intestinal, and acinar pancreatic glands; low doses of anti-ChE agents cause, in general, an augmentation of their secretory responses to nerve stimulation, and higher doses increase the resting rate of secretion. Smooth muscle fibers of the bronchioles and ureters are contracted by these drugs, and the ureters may show increased pert staltic -activity. The cardiovascular actions of anti-ChE agents are extremely complex, in that they reflect at any given moment the sum of the excitatory and inhibitory actions of accumulated endogenous ACh at several levels. The predominant cardiac effect of the peripheral action of accumulated ACh is bradycardia, which results in a de creas e i n ca rdiac outpu t and in hypotension. The of f ec tive refractory period of cardiac muscle fibers is shortened, and the refractory period and conduction time of the conducting tissue are prolonged. The blood vessels are in general dilated, although the coronary and pulmonary circulation may show the opposite response. The sum of the foregoing effects should result in hypotension, but at the ganglionic level ACh has first an excitatory and, at higher concentrations, an inhibitory action. Hence, the excitatory action on parasympathetic ganglion cells tends to reinforce the above effects, whereas the opposite sequence results from the action of ACh on sympathetic ganglion cells. Excitation followed by inhibition is also produced by ACh at the medullary vasomotor and cardiac centers. All these effects are further complicated by the hypoxia resulting from bronchoconstriction sot other actions on the respiratory system. The hypoxia reinforces both sympathetic tone and ACh-induced discharge of epinephrine from the adrenal medulla. It is not surpriaing, therefore, that a wide variety of hemodyn~mic effects of anti-ChE agents has been reported, depending on drug, dose, route of administration, species, ant other factors. Ne uromuscular Junct ion The actions of anti-ChEs are thought to be due to inhibition of ChE at the motor endplate and retention of ACh at the Junctional region. This combination culminates in a marked reaction of the transmitter with the receptor and a great activation of the entire receptor-ion-channel complex. If this process continues, a number of undesirable reactions can occur, among them paralysis, the desensitization of the junctional receptor, and structural changes in the muscle and nerve ending. Normally, a single nerve impulse in a terminal motor axon li berate s enough ACh to produce a localized depolarization ~ the endplate potential) that initiates a propagated muscle action potential. The liberated ACh is rapidly hydrolyzed by AChE, and the muse le relaxes . Theref ore, each motor-nerve impulse ~ nit fates only one muscle contraction. Af ter Dartial inhibition of AChE. however,

the ACh liberated by a single nerve impulse may persist long enough to set up repetitive muscle action potentials, with a resulting increase in strength of contraction. Furthermore, sufficient ACh may diffuse to neighboring muscle fibers and excite them as well, causing asynchronous contractions (fibrillation). In addition, the action of anti-ChE agents on the axon terminal can initiate antidromic firing, which results in aceivatlon of the motoneuro3 and leads in turn to the synchronous contraction of an entire motor unlE (fasciculation). In the presence of a Sufficiently high tone of an anti-ChE agent, the local concentration of ACh may produce a depolarislug blockade of the neuromuscular Junction and paralysis. Thus, a small dose of anti-ChE may increase the skeletal muscle contraction produced by a Single maximal nerve stimulus, but larger doses, or repetitive nerve stimulation at a high physiologic rate, may result in depression or block of neuromuscular transmisaton. Brain The mechanisms by which anti-ChEs perturb brain function are more complex, harder to study, and consequently less well understood. The brain is an extraortinarlly complex network of neurocellular pathways which uses electrochemical mechanisms to conduct signals needed to perform and integrate cognition, awareness, memory, language, sleep and wakefulness, locomotion, sensation, and hormonal ant autonomic functions. Cholinergic neurons are probably involved in many Intraregional and interregional pathways of the brain, although their identity and specific functions are poorly understood. Several sets of presumptive central cholinergic pathways have been proposed: medial septal nucleus to dentate gyrua, and aubiculum of hippocampus habenula to interpeduncular nucleus; cortical internurons to cortical pyramidal neurons; and thalamus, putamen, and caudate to neurons in the caudate (77). The presence of cholinergic synapses in central motor pathways (pyramidal and extrapyramidal) and of afferent systems involving both the reticular formation and the thalamus, hippocampus, and limbic system, suggests the poasibility of their participation (with other types of chemical Synapses) in initiation and control of movement, in sleep, arousal, and wakefulness, in memory, and in emotional regulation. This, in turn, implies susceptibility of these functions to anti-ChEs and cholinomimetic drugs. The electroencephalogram (EEG), a record of changes in the voltage-field distribution over the head as a function of time, is a tool for studying Some aspects of brain function. Adults have characteristic EEG patterns under standard conditions, but these vary according to the state of consciousness (alert, startled, drowsy, dreaming, or deeply aleeping3. Awake Subjects display a high-voltage (50 TV), low-frequency (~-14 Hz) alpha rhythm. This is most prominent in the occipital region of the scalp and when the eyes are closed. The resting alpha rhythm is replaced during periods of attention and problem-solving by a low-voltage (5-lO uV), high-frequency (15-30 Hz) beta rhythm, most prominent in frontal and parietal regions. The changeover from alpha to beta rhythm, tensed "desynchronization', can be induces by mental concentration or external Stimuli (including anti-ChEs). Other frequencisa of electric activity commonly observed in the EEG are theta (4-7 Hz) and delta (e 3 Hz, waves. 19

Among brain functions, Sleep is particularly well studied with the EEG. When sleep occurs, the alpha pattern disappears and, over a period of 4-5 min. the EEG changes from a low-voltage to a higher-amplitute, 4- to 6-Hz pattern with intermittent 14- to 16-Hz "spindle" activity. Later, over the course of 1-2 h, the voltage increases, the frequency decreases (to 1-3 Hz), and spindle activity becomes less frequent. The EEG then becomes desynchronized, rapid eye movement (REM) occurs, and the person dreams. REM sleep lasts approximately 15-20 min and occurs three to five times during a normal sleep cycle of 7-8 h. These characterlatlcs are constant from night to night If the person Is healthy, Is well adapted to the environment, ant has an habitual, normal, 24-h sleep-wake cycle. The control of the sleep-wake cycle appears to be a complex phenomenon involving several groups of neurons (nuclei) In the brainstem that use different synaptic transmitter chemicals: the locus ceruleus (norepinephrine), the dorsal rapine nuclei (serotonin), and the gigantocellularis nuclei (ACh) of the pontlne reticular formation (78). Cholinergic neurons in the gigantocellularis nuclei concentrate their electric discharges during REM sleep: activity begins minutes before the onset of REM sleep, continues at a high rate during the REM period, and abruptly ceases as REM sleep terminates. An attractive postulate of the control of the sleep-wake cycle is that, during non-REM sleep (and waking), an inhibitory system composed of neurons in the locus ceruleus or the dorsal Raphe nuclei tonically prevents cholinergic neurons in the gigantocellularis nuclei from firing. At a critical point, the latter escape this inhibition and fire at very high rates, thereby initiating the REM period. The inhibitory neurons then increase their activity ant inhibit the cholinergic discharge, and REM sleep ceases. Dependence on ACh as the excitatory transmitter implies the sensitivity of this system to aDti-ChEa, which, a priori, tend to increase neuronal activity. This postulate is consistent with empirical evidence from humans and animals treated with cholinergic agonists, which tend to decrease the latency of REM sleep ant increase the number of REM-sleep episodes. Atropine exerts opposite effects. Table 2 lists some of the effects of anti-ChEs and cholinomimetic drugs on brain function (79-84~. Doses of OP compounds that are toxic, but too small to threaten life, produce a variety of clinical manifestations, including miosia, muscular fasciculation, and apprehension t85,86). The acute behavioral alterations are usually accompanied by marked desynchronization of the EEG (87). Larger doses of OP compounds--which may induce convulsions, muscular paralysis, ant teeth--cause slowing of the EEG pattern followed by the appearance of spike waves that herald the onset of seizures. Symptomatic recovery is normally complete within 2-9 wk. at which time the erythrocyte cholineaterase content usually has returned to normal (88,89). Repeated low-dose administration of OP compounds can produce symptoms and Signs that are not seen after single exposures to the same toses. For example, subjects given daily injections of FOP reported the additional symptoms of insomnia, excessive dreaming, emotional lability, increased libido, paresthesias, visual hallucinations, and tremor (90~; and prolonged administration in animals induces sensorimotor neuropathy. 20

After a ·logle exposure, death any come vlthin S win or not for acme 24 h, dependlag on dose, route of add nistratioc, drug, ant other factors. She cause of death la primarily reeplratory failure, usually accompanied by a cardloveacular Component. Muccarlaic, nlcotlnic, and central effects all conttlbuce to respiratory embaresament; they include lary~gospeam, bronchoconstriction, increased tracheobronchial and salivary secretion, and peripheral and central respiratory paralysis. Although the blood pa sour may fall alarmingly and cardiac Irregularities ma, interveDe, these effects probably result as much from ~yposle me f roe the specific actions ~entloned, inasmuch as they can often be redemand by the eatabliah~ent of adequate pulmonary ventilation. Differences In hazard aeon" aeebere of the OF group arise from differences in laherent potency (EA 3148 is the moat potent), vapor pressure (GB is hazardous by inhalation), and ability to penetrate the skin (VX ant GD) (102,105,106). GB as a liquid a~n~ntatered cutaneously has also been reported to cause severe poisoning (107, 108). Absorption of some of these compounds f roe the respiratory tract has been estimated to approach absorption after intravenous administration in completenesa (105,109,110). Incapacitate concentrations of GB ant others by inhalation have ~ en estimated in animal teats, (111) and extrapolations to can have been attempted (105,112,113) Two antidotes are currently used for acute potaont~g by antl-ChEs One is atroplne, which acts 88 a auscarinic receptor antagonist and reduces the excessive atimulation of parasy~pathetie functlona, thus reversing the effects on the eye, lung, gastroluteatinal muscles, ant, moat laportant, the bears. Aeroplne also relieves effects of poisoning at central euacarinic synspaea, but not at central nicotinic synapses. She other antidote is an oxide (such as 2-PAM or obitoxime) that relieves poisoning at skeletal muscle entplatea (20). It acts as an activator of phoaphorylated AChE; through nucleophllic attack, it removes the phosphate group, thus restoring enzyme function at motor entplatea. The oximea are inefficient if the phoaphorylated enzyme has aged O These optima contain quaternary ultrogen ant therefore do cot penetrate the bloot-brain barrier ant have no effect at cholinergic aynapeea in the brain or spinal cord. The acute effects of the anti-ChEa are ahort-lived and do not outlast the inhibition of the enzyme. Indeed, come systeea develop tolerance rapidly, co function returns to normal even before there is substantial regeneration of measurable enzyme activity. It has been amply doc'~entet that, even with over 99: inhibition of all ChEa, animals (and presumably humsna) can survive without oxide or atropine treatment, if they are supported for a couple of hours by pharmacologic or nonphar~scologic means, such as artificial respiration, (72). Shia recovery from the "irreveralble effects of inhibitors may depend on rapid regeneration of ChEs, particularly some AChE isoenzymes (114); desenaitization of the postaynaptic membrane, a phenomenon that limits the reaponce to accumulated ACh; or compensatory changes In presynaptic and poatsynaptic receptors (115). 22

DELAYED NEUROPATHY (CtIS-PNS DISTAL AXONOPA1.HY) C1 inical Features Degeneration of particular regions of the nervous system is a well-characterized adverse health effect of human and animal exposure to many OP esters (phosphates, phosphoroamidates, and phosphonates) that may or may not also display anti-AChE properties. Some neuropathic OP esters can precipitate prominent necrologic abnormalities after a single exposure (as well as after multiple exposures), the clinical disease usually beginning within 2-3 wk. At some time during this clinically quiescent periods a stereotyped sequence of neuropathologic changes takes place that leads to the appearance of sensorimotor neuropathy. The degree of clinical impairment and the prognosis for functional recovery depend directly on the extent of nervous system damage, which in turn depends on the neuropa~chic potency of the responsi ble OP compound, as well as the dose and duration of exposure. The first recorded cases of paralysis from OP intoxication occurred at the end of the nineteenth century, when patients with tuberculosis were treated with phosphocreosote, an uncharacterized mixture of esters derived fro's phosphoric acid and coal-ear phenols ( 116 ~ . Several thousand cases appeared in the southern states in 1930 when alcoholic extracts of Jamaica ginger, widely consumed during Prohibition, were adulterated (3~17) with 2X triorthocresy~phosphate (TOCP). Adulteration of cooking oil c ontaminated with lubricating of} containing cresy! phosphates has proved responsible for several outbreaks of OP neurotoxicity, including a major epidemic in Morocco in which more than 10,000 people reportedly were affected I. More recently, the OP -pesticide leptophos has been associated with an outbreak of occupational neurotoxicity among workers at a plant in Texas; the victims displayed pronounced clinical features of ~pinal-cord damage, and some had psychologic manifestations (119) . Approximately 2 wk after ingesting cresyl phosphates, during which gastrointestinal dis~curbances may be manifest, affected persons experience pain, aches, and tingling in the feet and calves, followed within days by progressive weakening of leg and foot muscles that leads to paralysis. The thighs and then the hands and arms may become weak during succeeding days. Weakness is always more severe in the legs than in the arms and in both limbs is greatest in distal muscles. During the progressive phase of the illness, which lasts I-2 wk (depending on dose), the weakness spreads steadily, but usually stops short of complete quadriplegic. leurologic examination reveals signs of damage to the spinal cord (hyperactive knee jerks) and peripheral nerves (hypoacti~re ankle jerks) . Foot drop is pronounced, and victims adopt a high-stepping gait. Muscle denervation is evident from electromyography, and atrophy in the lower legs and hands may become secrete. Recovery in mild cases takes months or years, but severely affected persons who recover some muscle strength may have ataxia and spasticity permanently ~120) . Many experimental species are vulnerable t o the delayed neurotoxic effects of OP compounds, such as TOCP, although it Is accepted that neuro toxic doses vary markedly from one species to another. Rodents are relatively resistant and fowl very susceptible; hens are widely used to assay AChE compounds for 23

ability to induce paralysis (121). In all sensitive speclea, there is a period of 1-2 wk before the onset of necrologic algna, during which body weight changes and nerve fibers degenerate. Hens given TOCP develop a steadily increasing flaccid paresis of the hindli~ba, with an ataxic, broad-based gait. Paresis spreads over the course of several days and, if respiratory muscles become involved, may lead to death. Neuropathologic Features The limited neuropathologic information available from studisa of affected persons demonstrates that OP poisoning induces degeneration of nerve fibers in spinal cord and peripheral nerves (122). Neuropathologic examination of experimentally poisoned animals reveals a characteristic pattern of distal, retrograde degeneration of peripheral nerves. Long nerve fibers of large diameter asem to be affected before shorter and smaller fibers, so sensory and motor manifestations of nerve damage generally affect the legs before the ares. A similar principle holds for involvement of the spinal cord: long ascending (gracile ant spinocerebellar) ant long deseendiDg (egg., corticospinal) tracts are-eyametrlcally involved in the degeneration process. Neuropathologic changes initially appear distally and multifocally in affected pathways, leading to degeneration of distal axons and structural and functional disconnection of sensory and motor terminals. Axonal degeneration progresses steadily toward, but stops short of ,, the nerve cell bodies ~ 71) . Loss of axons precipitates a secondary loss of the normal myelin sheath in affected regions of spinal cord and peripheral nerves; this phenomenon has been erroneously described as "d emyelina t ion" --a t era reserved t o descry be t he neurotoxlc properties of substances, such as hexachiorophene, that damage myelin without causing atonal degeneration. In sum, the clinical and pathologic features of delayed CP neuropathy are classifies as a central~peripheral distal axonopathy (123~. Neuropathic Potency Neuropathlc potency can be assayed by determining the response of a vulnerable species (the hen, Gallua gallua do~eaticus) to OF intoxication or predicted from the degree of inhibition of the ner~rous-system enzyme NTE. It is important to note that chemical reactivity of an OP compound with NTE is unrelated to its ability to inhibit AChE (69~. Some OF agents designed for chemical warfare can inhibit both NTE and AChE. However, doses needed to inhibit N1rE and induce neuropathy may be much higher than those which would prove fatal to animals or humans without protection against the acute anti-ChE effects. Alternatively, the degree of NTE inhibition may be insufficient to induce clinical neuropathy. Phosphorofluoridates induced delayed neuropathy in chickens after 9-15 d at Loses of 0.3-2.5 mg/kg; the dimethyl compound required doses of 30 mg/kg. Five alkyiphosphorofluoridates were active at 1-5 mg/kg given in divided daily doses. Diethyl phosphofluoridothionate induced neuropathy at 0.7S mg/kg, but not at 0.5 mg/kg. Various d ialkylphosohinic f luorides and dialkylpyrophosphonates were negative ~124). Neuropathologic studies designed to detect subclinical damage to spinal cord or peripheral nerves in animals treated with CW agents are not available.

LONG-TERM BRAIN DYSFUNCTION Several studies have suggested that some subjects experience long-term sequelae from a single OP exposure or a period of low-level exposure. Minor diverters of affect, emotion, and memory were reported by Tabershaw and Cooper (125) in 38% of 114 subjects after acute OP poisoning. Rowntree et al. (126) suggested that OP exposure might exacerbate psychiatric problems. Metcalfe and Holmes (82) claimed that OP exposure may lead to persistent EEG changes; they also reported that workers with histories of both OP and chlorinatet-hydrocarbon exposure, but with no recent exposures, had EEG patterns that showed excessive slowing during drowsiness and after hyperventilation. Moreover, all-night-sleep EEGs reportedly displayed patterns commonly associated with narcolepsy. Psychologic dysfunction in this group included disturbed memory and difficulty in maintaining alertness and appropriate focusing of attention. Duffy ant co-workers (127) examined the brain electrical activity of workers occupationally exposed to satin and with documented single or repeated accidental exposure to toxic concentrations of it at least a year before EEG recording. Standard clinical EE& measurement, computer-derived EEG spectral analysis, and standard overnight-sleep EEGs, were examined in 77 exposes workers, and the results were compared with those from a control group of 38 nonexposed industrial workers. Statistically significant group differences in sarin workers included increased beta activity, delta ant theta slowing, decreased alpha activity, and increased REM sleep. The results of Ouffy et al. were consistent with those from a previous study that examined EEG changes in rhesus monkeys exposed to sarin or dieldrin (87~. Two dose schedules were uses: a single "large dose" (sarln at S,ug/kg or dieldrin at 4 mg/kg, administered intravenously), which produced overt signs of toxicity, ant a series of 10 weekly "small doses" (satin at 1 ~g/kg or tieldrin at 1 mg/kg, administered intramuscularly). The effects of anoxia in the first group were precluded by pretreating animals with gallamine triethiodide and providing artificial respiration. Animals treated with single doses of either satin or tieldrin displayed significant increases in the relative amount of beta voltage (15-50 Hz) in the EEG that persisted for a year. For sarin. the predominant effect was in the EEG derivation from the temporal cortex, and for dieldrin, from the frontal cortex. For both drugs, the increase in beta activity was most prominent when subjects were awake in darkness or drowsy. In summery, research results have indicated that a single symptomatic exposure or a series of subclinical exposures to sarin can alter the frequency spectrum of the spontaneous EEG for up to a year (83). The effect of anti-ChEs on the structural integrity of brain tissue has rarely been investigated, although the destructive effects of some of these compounds on spinal cord and peripheral nerves are well known. There is a clear need to study, with current ultrastructural techniques, the possibility of selective brain damage underlying the reported long-term changes in EEG patterns of animals and humans exposes to anti-ChEs ~ especially in light of a recent provocative report of widespread atonal ant terminal degeneration in the brains of rats treated with soman. As an incidental finding in an unrelated study of the effects of this 25

agent on behavior of rats, Petras (128) described nerve-ter~inal degeneration in the limbic System, corticofugal system, and central motor system-areas associated with mood, affect' Judgment, emotion' posture, and locomotion. The author pointed out that tanager regions would be unlikely to regenerate and that long-term psychiatric and motor deficits might be anticipated. Only 16 animals were studier by Petras, ant only seven brains hat observable damage. The severs had significant acute toxicity at the time of exposure, including muscle fasciculations, tremors9 and seizures. Only an abstract of this work has been published9 and the research has not been pursued systematically. It cannot now be concluded that the tissue damage was a direct effect of the OP, rather than an indirect effect (egg., related to brain hypoxia). Nor can it be ascertained whether the response was specific for sodas. JUNCTIONAL NEUROMYOPATHY Pathologic changes develop aubacutely ant reversibly in some motor-nerve terminals, neuromuscular Junctions, and associated muscle fibers after a/ministration of anti-ChE drugs to laboratory animals. ChE inhibitors with long-lasting effecta-such as paraoxon, DFP, tabun, sarln, soman, ant parathion-and reveraible inhibleors, such as physostlgmine and neostlg~lne. induce these neuromyopathic changes (129-131). The effect may result not only from AChE inactivation, but also from an increase in the rate of spontaneously released ACh secondary to a prejunctional action of anti-ChE drugs (S4,132). Thus, guanitine, which increases ACh release, alao causes a subacute myopathy similar to that produced by anti-ChE agents ( 133) e The diaphragm is most severely affected in treated animals (134), but abnormalities are also prominent in the soleus9 gastrocnemius, and quadriceps muscles. Ultrastructural changes may appear within hours of drug adminiatration, progress otter a period of a few days, and resolve within a couple of weeks. Pathologic changes in motor-nerve terminates, neuromuscular junctions9 and muscle fibers are associated with an initial decrease tn contractile strength after a few days and then a return to nearly normal strength after several more days (135~. These pathophysiologic events may be clearly delineated from the degeneration of motor-nerve terminals and atrophy of muscles that follow administration of agents that induce delayed neuropathy (131), in thee subacute neuromyopathic changes not only resolve before ache onset of delayed retrograde atonal degeneration, but also fad] to develop with TOCP, an O-P compound that inhibits NTE but has Little AChE activity. They may also be prevented by protecting the neuromuscular junction with curare or a deactivator of phosphorylated ChE, such as 2-PAM. The myopathic process seems to depend on the degree and duration of ChE inhibition (136~; this suggests that skeleeal~uscle hyperactivity is causally associated with the phenomenon. The significance of these observations for humans exposed to OF agents is unknown, but it seems likely from animal experiments that myopathy does not develop in the absence of muscle hyperactivity induced by anti-ChEs. There is some evidence from human autopsy material of focal necrosis of diaphragmatic and intercostal muscles after accidental exposure co a single large dose of OF insecticide (137) .

Motor-nerve terminals show various degrees of subcellular changes within 30 min to 2 h after injection of soman or paraoxon ~138 ); soman induces the more severe changes. Nerve terminal alterations include the appearance of intra-axonal myelin figures, membrane enclosures, ant an increased number of large-coated vesicles. Three days after FOP injection, soleus motor-nerve terminals are reduced in number and naked endplates are common. Pathologic changes also appear in the subaeural apparatus and in the immediate subjacent muscle; the latter displays swollen reitochondria, myelin figures, enlarged nucleoli, dilatation of the sarcoplasmic reticulum, loss of myofibrillar striation, and, later, myofilament loss and fragmentation of Z bands (132~. Focal muscle necrosis then ensues. Prejunctional and post~unctional subacute changes are resolved within 2 wk after administration of DEP; however, because this compound also inhibits ME and induces delayed neuropathy, a week after recovery from the subacute neuromyopathic changer motor-nerve terminals undergo a second phase of degeneration and regeneration, with reinnervation of damages endplates 6-8 wk later ~131). OTHER ADVERSE HEALTH EFFECTS Mutagenic or carcinogenic action is- not a general feature of the OP compounds, although some may have- these properties. There appears to be a lack of information on the mutagenicity or. carcinogenicity of carbonate anti-ChEs. Some agents on the list of compounds under scrutiny belong to chemical classes other than OP compounds and carbamates. Edrophonium is a quarernary ammonium inhibitor of ChE. Hethacholine is a stable choline ester and, although urecholine is a carbamate, it is not hydrolyzed by ChE. These two compounds are direct-acting cholinomimetic agent s. They are approved f or use in clinical medicine, but have few indications. Hexafluorenium is a bisquaternary ammonium distantly related to tubocurarine, and it has both anti-ChE and curariform actions. None of these quaternary ammonium compounds is known to have genotoxic action. Another compound is tacrine, 9-amino-l,2,3,4-tetrahydroacridine; as a class, acritines are notorious for mutagenicity and carcinogenicity, but this particular chemical has been used in clinical practice for more than 20 yr without known sequelae. Mutagenicity The OP malathion has been investigated extensively in a number of mutagenesis test systems. Some studies were done with a metabolic activating system, some without (malathion requires metabolic activation for its AChE-inhibitlug effect). Malathion also has been tested in somatic cells; tests f or chromosomal aberrations in human hematopoletic cell lines, sister chromatic exchange (SCE) in human fetal fibroblasts, and the micronucleus test in mice have been performed. A mouse tominant-lethal mutation assay has also been carried out with malathion, although the study was compromised, in that the dose (300 mg/kg) was well below that which is maximally tolerated. Among these studies, only one (reported in an abstract) claimed positive findlags (139~. These occurred in the mouse micronucleus and SCE tests and in a host-mediated Salmonella. 27

assay. The findings concerning the mutagenicity of malathion are the re f ore e quivocal. A 1975 review (140) concluded that the OPs bidrin, dichlorvoa, dimethoate, methyl parathion, and oxydemeton methyl, were mutagens in a single microbial mutation assay. In addition, trio~ethyl phosphate ts a clear-cut mutagen in male mouse germ cells; six other OP compounds (bromophos, dlazinon, fenitrothion, malathion parathion trichlorphon) apparently are not. Results of assays in four microbial test systems were inconclusive for several OPs (141~. Further study of these compounds has not been undertaken. Care inogenicity Malathion given In the diet for 103 wk wee not carclnogenlc In male and female rats, although the dose used may have been less than that which is maximally tolerated. No increased tumor incidence could be found in mice given malathion for 80 wk. An Internaelonal Agency for Research in Cancer (IARC) monograph reported strong evidence of the carclaogeniclty of ttlmethyl phosphate ~142 ~ in one species and evidence of care inogenicy in another There in equivocal evidence of the carcinogenicity of parathion ant dichiorvo~ in animals. A single dose of dichlor~ros reportedly damages the germinal epitheliums of mouse testes (143~. With the exception of urethane Methyl carbamate), a potent mutagen and established carcinogen in laboratory animals (144,14S), carban~ate compounds have been largely unstudied. Fetal and Teratogenic Effects Parathion reportedly increases resorption and lowers the weight of rat fetuses (146~. Results for malathion are questionable (147~. No data appear to be available on the potential mammalian terarogenicity of military nerve agents. Ef f ec t s on Male Re Droductior A s ingle dose of dichlorvos reportedly damages the germinal epithelium of mouse testes severely (143~. Ca taractoRenic Ef facts The introduction of DFP and other logy-acting anti-ate agents in the late 1940s appeared to represent an important arc e in the treatment of open-angle glaucoma. These drugs maintained control of intraocular tension when administered frequently and were effective in cases that were no longer controllable with the shorter acting compounds. Then, in 1966, independent reports simultaneously implicated the long-acting anti-ChE agents in the causation of lenticular opacities (14B, 149~. The incidence of this effect appeared to be as high as 50X; its development varied directly the strength of the solution, frequency of instillation, duration of therapy, and age of the patient . With physostig~ine and other short-acetug anti-ChE agents, the occurrence of lenticular opacities appeared to be no greater than that in nontreated subjects in the same age groups.

The mechanism of the cataractogenic action of the long-acting anti-ChE agents has remained obscure (150~. It has been shown that verve t monkeys can serve as a model for the production of an identical picture with the daily, lon8-tem instillation of echothiophate (151~. In an extensive series in which children were treated for accommodative esotropia (strabismus) with the instillation of short- and long-acting anti-ChE agents, no lenticular opacities or other serious effects were detected (152~. Hemocoagulation Aberrations of the clotting mechanism attend exposure to OF anti-ChEs (-153~. Study of 31 persons exposed to saris or parathion revealed a biphasic reaction consisting of rapid coagulation l~mediately after exposure, followed by a prolongation of clotting time ~ 91 ,154) . Anti-ChEs also increase f ibrinolysis. Fibrinolysis and hypercoagulability were noted in two patients with mevinphos poisoning (155). Increased coagulation was associated with increased prothrombin activity (secondary to increased Factor VII activity) ant with increased prothrombin consumption. Coagulation abnormalities nomalize several weeks after exposure, and there is no overt liver damage. IMMEDIATE AND DELAYED EFFECTS IN VOLUNTEERS AFTER SINGLE OR REPEATE ~ _ ~ T ~ _ According to the available information, 1,406 subjects were tested with 16 agents of unstated purity (Table 3-3~. Some of the subjects also were treated with protective or reactivating agents (case file data of Edgewood subjects, Appendix E). For this review, approximately 15: (219) of the medical records were selected on the basis of high dosage, repetitive exposure, or the presence of additional physiologic stress. Case records were also selected randomly on the terminal digit of the case number (i.e., ending in 3~. Brief summaries were made available to the Panel. In addition, complete records of 32 persons given EA 3148 intravenously were examined. These were chosen because this agent is considered the most potent of the anti-ChEs tested (1563. In all cases, subjects were identified by pharmacologic class, by agent, and by letter code; names were withheld. The case summaries are brief and anecdotal. With the exception of one case, they deal only with the period immediately after the test dose. There are no reporte-of neurologic or psychologic examinations (with the exception of subject A53), and only four report ~ of EEGs--three made before treatment with the anti-ChE agent, and one after. The medical records of subjects tested with EA 3148 comprise doctors' orders, observations of the patient recorded largely by nurser, a ciluical master log recording only blood pressure and pulse before and after exposure, results of a test of numerical facility, and ChE concentrations in blood, plasma, and red cells, before and at intervals af ter exposure. Physician' workup, progress ~ and discharge notes are absent. Descriptions of the subjects' reactions are bather vague and are certainly not sufficient for careful analysis of long-term effects of these agents. The various subjective complaints listed in the ca~e-file data of Appendix E are not further documented by examination of f indings. 29

The mayor focus of this Panel's investigation Is the possibility of long-term or delayed effects. Easentially, anecdotal information is provided in the case a~m~arles and some of this information seems to suggest that immediate psychological effects can follow the administration of both reverotbie and irreversible cholinesterases. However, the dries do not provide hard data that would allow the panel to address, in a definitive manner, the question of whether or not there is a possibility of long-term or delayed effect. As noted above, the case summaries were concerned with the period immediately after administration of the agent and therefore give no indication of the possibility of long-term adverse effects. The paucity of data in the medical records prevents further Study in relation to the goal of this report. However, there are published papers which appear to demonstrate long lasting EEG changes from single or repeated exposure to OPa (83). In reviewing the detailed case report of the Single subject who experienced long-lasting paychologlc symptoms, the Panel notes that he had had :both physical ant psychologic evaluations before acceptance in the volunteer program. Evidently, this included examinations and paychologic teatlag (the MMPI wee mentionet)O It is possible that these data, if available, can be used as a besia for comparison with a long-term followup study' if such is undertaken. ~ MORTALITY DATA A preliminary review of the standardized mortality ratios (SMRs) suggests that mortality was not significantly increased by exposure to anti-ChE chemicals, as tested at Edgewood (Table 4). Whether one looks at subjects exposes to sarin only or saris plus another chemical (aarin total), VX only or VX plus another chemical (VX total), anti-ChE only or anti-ChE plus another chemical, the SMRs are roughly 80% (or less) of the rates expected for the U.S. population as a whole. This presumably reflects the fact that those who enter the military service do not have chronic diaessea. These findings provide a first approximation of mortality ant were intended to reveal trends. A more thorough evaluation of mortality findings is contained in Chapter 4. EVALUATION OF THE LIKE LINOOD OF LONG-TERM _ . . . . _ ADVERSE HEALTH EFFECTS . The following commentary is based on evaluation of the known adverse health effects of anei-ChEa on humans ant animals, the type, number, amount, and route of administration of agents, and medical records of subjects released by the Army. The subjects were experimentally given anti-ChE agents in the CW testing program during the 1950s and l960s. The mortality data, collected during 1981, led to the conclusion that, during the elapsed time since testing, subjects were no more likely to have died than comparable soldiers outside the testing program. Morbidity data are unavailable and should be collected; special focus should be placed on the posalble long-term adverse health effects highlighted below. It must be emphasized that the following opinions represent essentially extrapolations from known datee Although there are

published and nonpublished results indicating that EEG changes can occur for one year after exposure to organophosphorus drugs, there is no documentation available as to longer-lasting effects. The statements offered below therefore represent conjecture on the part of Panel members and their consultants, who collectively have broad experience with and expertise on anti-ChEs and their effects in humans and animals. BRAIN DYSFUNCTION There are no records to indicate that the soldiers might have experienced subtle changes in brain function that lasted for long periods after discharge from the test environment (except, perhaps, in the one case cited above). But examination of primates ant humans exposed to single or repeated doses of satin has revealed statistically significant changes in the EEG that are apparent for at least a year after exposure (83). These observations have yet to receive laboratory confirmation, and their exact importance is unknown. There are also a number of anecdotal reports of minor disorders of affect, sensation, memory, and sleep in humans accidentally exposed to OP war gases or insecticides. The underlying changes in brain structure and function are unknown. The EEG changes are compatible with abnormalities of sleep and behavior. It is also possible that pre-existing psychopathologic conditions were exacerbated. Some subjects were treated with aoman, which has been alleged (in a single, limited experimental study) to induce in laboratory rats profound and irreversible neuropathologic changes in vitally important regions of the brain (128). This laboratory study has not been replicated, and the duration ant lifetime significance, if any, of the above-noted effects in humans are not known; they should be looked for in both adult life ant old age. There is no evidence from the medical records that Subjects given potent anti-ChE chemicals (e.g., military nerve agents) experienced the more severe acute effects that were produced in animals with large doses of soman. In particular, there are no reports of respiratory insufficiency or convulsions that could precipitate periods of hypoxia and lead to permanent damage of brain tissue. JUNCTIONS NEURO=OPATHY AM DELAYED NEUROPAT~ Medical records of the vast majority of subjects tested do not refer to induction of the muscle hyperactivity that is associated with acute neuromyopathy in laboratory animals and humans poisoned by OP insecticides. Although there was no systematic attempt to inspect tested subjects for fibrillation or fasciculation of skeletal musculature during or after testing, the doses of anti-ChEs used in relation to the route of application are considered unlikely to have induced muscle damage in most of the subjects. Even if a subacute neuromyopathy had occurred in a few subjects, especially those who reportedly displayed weakness and muscle twitching after exposure, experience with laboratory animals suggests that these changes would have been resolved within weeks. 31

Although some of the OP agents tested can induce peripheral neuropathy in laboratory animals, the doses needed to induce clinical signs greatly exceed the LD'o for that species (1OS, 112, 113). Humans are generally understood to develop neuropathy after receivlag doses comparable with those which induce clinical algna in hens (69, 121); because the toses used on the teat subjects were far below those needed to induce experimental neuropathy, it is moat unlikely that any subject experienced delayed onset of distal limb paralysis. However, clinical algna of neuropathy in experimental animals occur some time after the onset of CHS-PNS distal axonopathy, after a particular number of nerve fibers have undergone degeneration. It is therefore not possible to rule out the chance that subclinical pathologic changes occurred in vulnerable nerveofiber pathways in subjects treated with agents known to be capable of inducing neuropathy. ENS nerve fibers probably undergo regeneration and re-eatabliahment of end-organ connection promptly after the degenerative phase has terminated; damaged CNS fibers are unlikely to regenerate functional connections, but minor damage of this type usually does not induce noticeable functional impairment. Development in the test subjects of anything more than minor and transient sensortmotor manifestations as an expressio.n of such putative damage is conaitered moat unlikely and no such adaptors were recorded. Neurologic examination would reveal 8igO8 of long-term CNS changes, such as the Babinaki sign (extensor planter response), hyperactive ankle or knee Jerk, and spastic or ataxic gait. Such signs would be permanent attributes of a person who suffered this type of CNS damage, but such changes in the test subjects are considered unlikely. OTHER ADVERSE HEALTH EFFECTS Mutagenicity, Carcinogenicity, and Male Reproductive Effects There is little information on mutagenicity, carcinogenicity, and male reproductive effects, in relation to anti-ChE chemicals. Experimental evidence suggests that malathion does not pose a mutagenic or carcinogenic risk to exposes hens, nevertheless, some scientists disagree with this conclusion (157). The safety of the other compounds could not be confidently determined, because of the absence of laboratory atudica. Neverthelesa, the Panel ts unaware of any reports linking these adverse health effects to single or repeated exposure to anti-ChE agent a. Furthermore, there is no suggestion of increased mortality from carcinogenicity, although final Judgment on this issue must be reserved until mortality and morbidity data are collected and analyzed. Information on whether the tested aubJecta have sired children and on the state of health of their offspring since testing would be helpful in evaluating the possibility of anti-ChE-induced mutagenicity or adverse effects on the male reproductive system.

Cataractogenicity On the basis of a review of the literature, it appears highly unlikely the t si ngle or occasional systemic exposure of young atul t subjects to anti-ChE agents would result in the development of initially undetectable, long-Berm damage to the eye. Blood Chances Abnormalities of blood coagulation reported in persons exposes to particular anti-ChEs are considered reversible. Therefore, no long-term adverse effects on hemocoagulatlon in the test subjects are f ore seen. CONCLUSIONS The panel concludes that although no evidence has been developed ~ to date) that any of the anticholinesterase test compounds surveyed carries long-range adverse human health effects in the doses used, the results of an ongoing NAS/NRC morbidity study may shed further light on this issue. The panel therefore is unable to rule out the possibility that some anti-ChE agents produced long-term adverse health effects in some individuals. Exposures to low doses of OP compounds have been reported (but not confirmed) to produce subtle changes in BEG, sleep pattern, and behavior that persist f or at least a year. Whether the subjects at Etgewood incurred these changes and to what extent they might now show these effects are not known. If such changes occurred and persisted, they would be difficult to detect now. They could be determined scientifically only by a new study in which BEG, sleep state, and psychologic-test scores were compared with those from nonexposed control subjects. This might be considered, if reasonable suspicion develops, based on responses obtained in the referenced morbidity study, that selected subjects experienced behavioral changes traceable in onset to experimental exposure to the anti-ChE agents. 33

TABLE 1 RF~IsOtSEI; OF }~'PE~lI QltGANS SO AUIU~MIC Nt:RW. Jl~lM)loC~; ~ I, , . _ , _ . . _ Fats— ~- Radlol muscle' iris Sphine~er much. iris Cil'ary muscle I~a" S ~ Sac Atria Sac ·- His Purlrinic system Ventncics ,`eriol" Coronary Sl`'r, and mucous SicJctal muscic Cerebral Pulmons~ Abdominal Piecers, rebel Seli`ery Abode ~~jR5 (S'li' - i,) taint Bronchial muselc Bronchial glands Stomper · Motility and tone Sphincle" Seeretios Ja'es`~e Motility and tone Sphincters S=retion Gollbl~r - ` Due Ki^,, U'ino~ 81~~ De~, Tn~one and sphincter U'"er hlotili~ and tone {Jt~ Srs O~o~s, Af~# S,in Filomolor mu~cles S.cc' gbn~ S'~ C - ~h ~o! ^~0o , . _ _ A~epl" J,pr P B. B~ ~t B. o~' @~2 .~2 .~92 .92 `? o3'B2 $! B a~ aA ar~aral a`~w I-PUl,~' _ 6-1~RJ - I;~;R J - ~lJI..~' o._ . . _~ . .. o. C-nstr;~ction (m,Jrisa,) ~ + Rcla's~ion (OJ fir ~nsion Incres" in hcar' nte ~ ~ Incres~e ir' contnctil.~ and 0nduaine "~ty ~ ~ Increasc ir~ automa~ici~ and 0nduaior' vel~ + ~ leaes" ir' sutomatici~ an~d conduaion ~doci~ Incresse ita cor~~racoli~ con° duciio~a veloa~, susoma° ~i~, u~d reae of idiowen~c ubr pacem~en ~ ~ ~ Con3triction +; dibtation, ~ + Constnenon ~ + Constaction ~ +; dib"~.on&. + Constaction (sli~ht) Constaction +, diblation, Constaction ~ + +; dila='6on~ Constne~or' ~ ~ + Constnetion + +; dilata~ion ~ + Relex~ior Inhibition (.~) Deere~ (usualq)' ~ Con~amon (usual~} + Inhibitton (?) ~eue' Costreaion (usually) Inhibiiion (~ Rel~iion + Rceir~ secretion + Rel"~uon (u - al~q) Contreciion ++ 1~ (Ul~) Pregn~t; e~treaKm (~); r~onpre~ast; rebxation (O ~18~ + + C - ~on ++ ~ ~e Con~ ~ ~ +; lel~ 34 #~.~.~.~. Contrectim (minci') C.ontraction fior neat visiar, +++ Deerc~e in hear' rat6; val~t a" - +++ DeercAse in cort~actili~O and (u.~ally) inctca. in con° duaion ~i~ ~ + Occrcne in condu6tior~ vcloc- i~o ~ ~ Uock ~ + + Littic c~em Sligh! decrcasc in contremilin elatmed by ~ome Dilatation ~ Dib"tion. Dilatation' Diletation' Dile - ~'on. Dilatetion ~ ~ Contr~ion ++ Stimulat~on ~ + Inc~e +~+ Relaxaiion (usus11~) Slimubtion ~ + l~se +~+ Rela'soor. (usualI, Slim~btion Co. - ction + . _ ' Co~elion ~ ~ + Rd~ion + Ine~se (?) v - ~e E~ ~ + _ G - c~lized ~eaction ~ + ~ S~ of 4inephnne sad no~hnee

TABLE ~ (coned) alum - to 1)' t2,t't:(~-~N (~(;~.N 1-0 AtOO~MIC NF.~. IM~' S1~ _) al l I ( 1~s l)~;~s Lit - '.ncreos Aeini l~lct' (A cells) For Ce'Js SoJvory Glands rimol Clot ~c'4o - Sol Good ''.~1~' Type . ~2 asp, O A08~' .~.1C 1-r'JI.~.~' It ~ben~enn~r~ise IlumnengmetiS10 n~re:~`cd secretion Dcerca~cd seerc''on Increa~cd-secrction Lipolysis'° ~ + Potassium and water accretion Amplest seerct~on Melatonin synthesis | ¢~tOLt-~.~;~t 'arvulis' C~cosen Synthesis Scere~ion ++ _ _ Potassium and weler secretion ~ ~ ~ ~ ~ . S~i'on ~ + Koelle, G. B. In: The Pharmacological Basis of Therspeutics9 Goodman, L. S . and Gilman, A, eds. ), 5th edition. Macmillan Publishing Co., New York, 1975. Pg. 408. 1 Responses are designated 1 + 103 + 10 provide an approximate indication of the importance of adrenergic and cholinerglc nerve activity in the control of the various organs and functions listed. 2 Dilatation predominates in situ due to metabolic autoregulatory phenomena . 3 Cholinergic vasodilatation at these sites is of questionable physiological signif icance . 4 Over the usual concentration range of physiologically released, circulating epinephrine, B-receptor response (vasodilation) predominates in blood vessels of skeletal muscle and liver; a-receptor (vasoconstriction), in blood vessels of other abdominal viscera. The renal and mesent.eric vessels also contain specific dopsminergic receptors, activation of which causes dilatation, but their physiological significance has not been established. ' Sympathetic cholinergic system causes ~ra~odilat:ation in skeletal muscle, but this is not involved in most phy siological responses. 6 It has been proposed that adrenergic f lbers terminate at inhibitory B receptors on smooth muscle fibers, and at inhibitory a receptors on parasympathetic cholinergic (excitatory) ganglion cells of Auerbach's plexus. 7 Depends on stage of menstrual cycle, amount of circulating estrogen and progesterone, and other factors. Palms of hands and some other sites ~ ' adrenergic sweating" ~ . 9 There is significant variation among species in the type of receptor that mediates certain metabolic responses. 35

TABLE 2 Effects, of Cholinergice tn Nor.~1 and Paychotic Persone* Drug ~ffect on Norea1 P rson Effect on PsYchotlc Person Phy8O8~tg~DC Depreselon; Paychoootor retertation beproveeent, particularly antichEa of thought disorder; No effect; antagonise of eathylphenid-te activat,lon Organophoaphorus Dysphorta; Dlghteares; ascessive So~e ~sproveeent tn antichEa dreaming; hallucinations a~d hebrephrenice; exacerbation deluston,~; ·chizold reactions; in most ceses (paranolda) ·uditory halluctnatione; paranoid and rellg,Ious talustone; agr.~sion Choline Depression Improvement Arecoline, Incressed interaction, oxotremorlne lucid interval *Derived from review papera by Singh and Lal (79) and by Rarezmar and Rlchardson (80) and papera of Gerahon and Shew (81), Hetcalf and Bol~es (82), Duffy and Burchftel (83)' and Karezmar and Ohts (84). l 36

TABLE 3 Su=.ary of Tests Conducted snd Recorde Selected for Anticholinesterase Chemicale Tox No. Compound Subjects Tested Records S;lected A_lb Sarin, GB, 1208 246 25 `_2b VX, 1701 740 75 A_3b GA, tabun 26 13 A_4b GF, 1212 21 10 A_5b GD, 1210, ·oman 83 10- A_6b DFP, tilaopropylfluorophoaphate 11 5 A-7 EA 3148 32 *_gc TRA 15 5 A_lodd Eserlne, physostlg~lne 138 23 A-ll Proatigmine, neoatig~ice 22 5 A-12e Bexafluorenlu. (Hylaxen) 11 11 A-13 Pyridostig~ice 27 A-14b Halathion 10 5 Ao20 Methacholine 9 A 21f Urechollne 1S 5 Sotal 1~406 aTvo 6eta of recorts, one based on high dose, another beaed on random selection; each set contains 219 recorde. bOrganophosphate ester. CAcridine~ dCarbamate. eQuaternary a~onium inhibitor. fCholinerg ic agonist . 37 219

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109. *Creathull, P., Coon, W.S., t2cGrath, F.P., and Oberat, F.W., ~957: Inhalation effects incapacitation and mortality) for monkeys exposed to GA, GB and GE Capote (U). CWLR 2179. 110. *Lubash, G.D., Grlbetz, I., and Johnson, R.P., 1960: Pulmonary and cardiovascular effect o of satin in treated dabs. CHAR 2389, Edgewood, MD. 111. *Cresthull, P., Christensen, M.K., and Oberot, FeWe ~ 1961: Estimated Speed of action of GB vapor for death and various degrees of incapacitation tn man. CRDI*R 3050, Edgewood, tID. 112. *Silver, S.D., 1953: The estimation of the toxicity of GB to man MLSR 23, Edgewood, MD. 113. *Punts, C.L. and Atkinson, 1960: Ichalatlon toxicity and speed of action of VX with suggested estimates for man based on animal data ~ U) . Edgewood, HD . 114. S~inivasan, R. ,~ Karczmar, AoGe ~ and Bershon, J., 1976: Rat brain acety~cholinesterase and its isoenzymes after intracere brat administration of DFP . Biochem. Pharmacol 25: 2739-2745. 115. Russel, R.W., Carson, V.G., Booth, R.A., and Jeuden, D.~., 1981: Neuropha ntaco l . 20 :1197 . 116. I`orot, C., 1899: I`es combinations de la creosote dans le traitmant de la tuberculose pulmonaire, Thesis, Paris. Cited by lIuntter, D. In: Industrial Toxicology, Clarenden Press, Oxford, 1944. 117. Burley, B.T., 1932: Polyneuritia from tricresylphosphate. J. Am. Hed. Assoc. 98 :298-304. 118. Smith, H. ant Spalting, J.~.K., 1959: Outbreak of paralysis in Morocco due to orthocresyl phosphate poisoning. Liancet 277 :1019-1021. ~ 119. Xintaras, C. and Burg, J.R., 1980: Screening and prevention of human neurotoxic outbreaks: Issues and Problems In: Experimental and Clinical Neurotoxicology ~ Spencer, P. S . and Schaumberg, B.~. ede. ~ pp. 663-674. Williams and Wilkins Co., Baltimore, MD. 120. Morgan' J.P. and Penovlch, P., 1978: Jamaica ginger paralysis. Forty-seven-year follow~up. Arch. Neurol. 35:530-532. *Found only at the Edgewood library. 47

121. Abou-Donla, M.D., Grahaot, D.G. and Komcil, A.A., 1979: Delayet neurotoxlci ty of 0~( 2, 4-dichlorophenyl) -O~e thy 1 phoaphonothiate. Effect of a aingle oral tose on hena. Tox. Appl. Pharmacol. 49:293. 122. Airing, C.D., 1942. The aystemic nervous affinity of trtorthocresylphoaphate (Jamaice Gi DgeF Palay). Brain 65:45-47 123. Spencer, P.S. and SchauD'burg, H.H., 1980: Claasification of neurotoxic disease. ~ morphological approach. In: Experio~ental and Clinical Neurotoxicology, (Spencer, P.S. ant Schaumburg, H.H., eda. ) pp. 92-99, Williao~e ant Wilkins Co., Baltimore . 124. Davies, D.R., Holland, P. and Rumens, H.J., 1960: The relationship between the chemical structure and aeurotoxicity of allyl organophosphorus compounde. Brit. J. phar~acol. 15 o2 71-2713 0 125. Tabershaw. I.R. and Cooper, W.C., 1966: Sequellae of ac~ate organic pho sphate poisoning ~ JO Occup ~ Med ~ 8 0 5-10 ~ 126. Rowntree, D.W., Nevin, S., ant Wilaon, A., 1950: The effects of disopropylfluorophosphonate in schi20phrenia ant manic tepreaslve paychosis. J. Neurology, Neurosurgery and Paychiatry 13:47~62. 127. Duffy, F.~., Burchfiel, J.I`., Bartela, P.~., Gaon, M. and Sim, V. 1979: 1.ong-tene effects of an organophoaphate upon the human electroencephalogram. Toxicol. Appl. Phar~acol. 47 :161-176. 128. Petrea, J.M., 1981: Soman Neurotoxicity. Funt. and Applo Toxicol. ~ :242 ~ 1290 Fenichel, G.M., Ribler, W.B., Olson, M.D. and Dettbarn, W.D., 1972: Chronic inhibition of cholinesterase as a cause of myopathy, Neurology 22:1026~33. 130. Engle, A.G., Iambert, F.~. and Santa' T., 1973: Study of long-ter~ anticholinesterase therapy: Effects on neuro~uscular transmission and on motor end-plate structure. Neurology 23:12 73-1281. 131. Glazer, E.J., Balcer, T. and Riker, Si.F., Jr., 1978: The neuropathology of DFP at cat soleus neuromuscular Junction. J. Neurocytol. 7: 741-758. 132. Laskowski, M.B., 0180n, W.Il., and Dettbarn, W.~., 1975: Ultrsatructural ehangea at the motor end-plate produced by an irreversible cholinesterase inhi bitor. E5cp. Neurol. 47: 290~3 06. 48

133. Fenlchel, G.M., ICibler, W.B. ant Dettbarn, lI.D., 1974: l~he effect of immobilization and exerclae on acetylcholine~etiated myopathic 8 ~ ~urology 24:1086-1090. 134. Wecker, Id. ant Dettharn, W.D., 1976: Paraoxon-intuced myopathy: ^acle specifict~cy and acety~choline tnvolvement. Exp. Neurol. 51:281-291. 135. Lowntes, H.E., Bal~er, T., and Riker, W.F., Jr., 1974: Motor nerve dy~function in telayet DFP neuropathy. European J. Phamacol. 29:66-73. 136. Wecker, L. and Dettbarn, W.D., 1976: Paraoxon-inducet myopathy: Mbscle specificity ant acetylcholine in~olvement. Exp. Neurol. 51:281-291. 137. Wecker, I.., Mrak' R.E., and Dettbarn' W.D., 1981: J. Environ Pathol. Toxicol. 13 8. Laskowski, M. B. , Olson, W.H. ant Dettbarn, W.D. , 1976: Motor end-plate degeneration coincident with cholinesterase (ChE) i~nhibition and increased miniature end-plate potential frequency. (Abetract) Fed. Proceed. 35:800. 139. Sylianco, C.Y.~., 1978: Some interac~tions affecting the mutagenicity potential of dipyrone, hexachiorophene, thiodan and malathion. Mutation Res. S3:271-272. 140. Wild, D., 1975: ~tagenicity seudies on organophosphon~s in~ecticide~. Hutation Res. 32:133-150. 141. Simmon, V.F., Poole, D.C., and Newell, G.W., 1976: In vitro mutagenic studies of twenty pesticides. (Abetract) Tox. Appl. Pharmacol. 37:109. 142. Griesemer, R.A. and Cueto, C. Jr., 1980: Toward a classification acheme for degrees of experi~ental evidence for the carcinogenicity of che~cals for animale. Molecular and cellualr aspects of carcinogen acreening teste. (Moncoano, R., Bart ach, H. a" Tomatis, ~ . ede) lARC Scientif ic Pu blications, No. 27, Z.yon, France. 143. Krause, W. an Homola, S., 1972: Beeinflusaung der Spermiogenese diurch DI)VP (Dichlonro 8)e Arch. Dermstol. Forech. 244:439-441. 144. Bateman, A.~., 1976: The mutagenic action of urethane. MOtation Re s. 39 :7 5-96 . 145. Tomatis, L., Agthe, C., Bartsch, II., Huff, J., Montesano, R. Saracci, R., Walker, E., and Wilbourn, J., 1978: Evaluation of the carcinogenicity of chemicala: a re~riew of the monograph programe of the International Agency for Research on Cancer. Cancer Research 38: 877-~85. 49

146. Kimbrough, R.D., and Gaines, T.B. 1968: Effect of organic phosphorus compounds and all~ylating agents on the rat fetus O Arch. Environ. Health 16:805008. 147. Dobbins, P.K., 1967: Organic Phosphate ~aecticides as Teratogens in the rat. J. Fla. Hied. Assoc. 54:452-456. 148. Altelason, U. and Hol~berg, As' 1966: The frequency of cataract after Biotic therapy. Acta..Ophthal. 44.421-429. 16~9. Shafer, R.N. and Hetherington, JO, Jr., 1966. Anticholinesterase druge.and cataracts. AD. J. Ophthal. 62:613-618. 150. Latiea, Add., 1969: Localization in cornea and lens of topically-applied irreversible cholineatereae inhibitors. Am. J. Ophthal. 68.848-857. 151. Kaufman, POD. ant Axelason, V., 1975: Induction of aubcapsular cataracts tn aniridio velvet monkeys by echothiophateO InvestO OphthalO 140863-8660 -. 1520 Chamberlain, W., 1975: Anticholinestease miotles in the management of accommodative eso tropia O J. Pedia trio Ophthal . 12: 151-1S6. 153. Van ICaulla, K. and Holmes, J.~.: Changes in blood coagulation following exposure to antlchollnestersse agents (parathion and sari n) Semi-sanual progre so report . Contract DA-18-108 6~05~CML-264. October 1958-March 1959, Supplement 20 1540 Von Kaulla, K. and Hole, J.H., 1961: Changes following anticholinesterase exposures. Arch Environ. Health 2:82-168. 155. Holmes, Jut., Starr, H., Hacisch, R.C. and van Kanalla9 Ko' 1974: Short-term toxlelty of mevInphos. Arch. Environ. Health 29: 84-89. 156. *Sidell, F.R., Groff, W.A., and Vocci, F., 1965: Effects of EA 3148 ado~intstered intravenously to humane (U). CRDL TM 2-31, Etgewood, ha). 157. Research News; 1981: 2lalathlon threat tebunlcet. Science 213: 526-52 7 ~ *Fount only at the Edgewood library. 50

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