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8 TOXICOLOGY OF ORGANOPHOSPHATE NERVE AGENTS

Timothy C. Marrs

Edentox Associates, Edenbridge, UK

INTRODUCTION very high mammalian toxicity, were synthesized by Schrader in 1937 and a small pilot produc- The organophosphate (OP) anticholinesterases tion plant was set up at M¬unster-Lager. Later, include the chemical warfare nerve agents, a va- at D¬uhernfurt near Breslau in Prussian Silesia riety of OP pesticides (Ballantyne and Marrs, (now Bzerg Dolny and Wroclaw in Poland), a 1992) and, less well-known, a natural compound, production plant for these agents was established anatoxin-As (Dittmann and Wiegand, 2005). The and both tabun and sarin were manufactured in active ingredients of OP pesticides are used as quantity: in the case of tabun, 12 000 tonnes were drugs in human medicine, e.g. malathion to treat produced. Soman, another nerve agent, was also head-lice and metrifonate/trichlorfon in tropi- synthesized in Germany during the war, but only cal medicines. OPs are also used in veterinary manufactured in small quantities. Strangely, per- medicine notably as ectoparasiticides (Beesley, haps in view of the large stocks held by Ger- 1994). The chemical warfare nerve agents have a many, the nerve agents were not used in World much higher mammalian acute toxicity, particu- War II (UK Ministry of Defence, 1972) and ap- larly via the percutaneous and inhalation routes, pear to have been dumped in the Baltic sea. The than the OP pesticides. Additionally many chem- V agents similarly arose out of studies of puta- ical warfare agents are phosphonofluoridates and tive insecticides and VX has been variously re- phosphonothioates, while many pesticides are ported as being first synthesized at the Chemical = S type-phosphorothioates. Qualitatively, the Defence Experimental Establishment (CDEE) at anticholinesterase toxicology of the OP nerve Porton Down, UK (now Dstl, Porton Down) agents and pesticides is similar and in general or by Ghosh at Imperial Chemical Industries in treatment strategies are alike so that much infor- 1952 (SIPRI, 1971). Other nerve agents were de- mation on OP pesticides is relevant to nerve agent veloped subsequently and stocks have been held toxicology. by a number of countries, including USA, the for- mer USSR and successor states, the UK, France and Iraq. However, nerve agents have rarely been History used in warfare; the only notable instance of use OP compounds were intensively investigated being by Iraq against that country’s own Kur- in Germany in the 1930s. The German con- dish population (le Chˆene, 1989). There have glomerate IG Farbenindustrie looked at a num- also been allegations of use of OP nerve agents ber of these compounds for use as insecticides during the Iran/Iraq war. The nerve agent sarin, and a programme of synthesis of a large num- in an impure form, was used in two terrorist at- ber of compounds was undertaken. Tabun and tacks in Japan, respectively, in Matsumoto 1994 sarin, OPs of little use as insecticides but of (Okudera, 2002) and Tokyo in 1995 (Nagao et al.,

Chemical Warfare Agents: Toxicology and Treatment (2nd Edition) Edited by Timothy C. Marrs, Robert L. Maynard and Frederick R. Sidell C 2007 John Wiley & Sons, Ltd

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Table 1. Possible targets for nerve agents in warfare.a Adapted and reproduced from Chapter 34, ‘Organophosphorus compounds as chemical warfare agents’, by Maynard RL and Beswick F, in Clinical and Experimental Toxicology of Organophosphates and Carbamates (B Ballantyne and TC Marrs, eds), 1992, with the kind permission of the publishers, Elsevier, and the authors

Type of agent Target Sarin Soman VX Delivery system Rear areas Airports/airfields — L L Aircraft (bombs, cluster spray bombs, spray tanks, missiles) Seaports — L L Aircraft (bombs, cluster spray bombs, spray tanks, missiles) Railways, especially — L L Aircraft (bombs, cluster) junctions Headquarters and — L L Aircraft (bombs, missiles) communication centres Storage sites — L L Aircraft (bombs, cluster missiles) Troop concentrations — L L Aircraft (bombs, spray tanks)

Forward areas Nuclear delivery weapons, L — L Multiple rocket launchers, aircraft other key weapons (bombs, rockets) and systems Defence positions L — L Multiple rocket launchers, artillery, mortars, aircraft (bombs), rockets Own flanks L — L Mines Own defence L — L Artillery, mortars, mines front generally To produce casualties, L — L Multiple rocket launchers, artillery, to harass and reduce mortars, aircraft (bombs, rockets) combat efficiency To deny ground — L L Aircraft (spray, mines) Harass civilian L — — Aircraft (bombs, rockets, sprays) populations

Note: a L, likely use.

1997), as was, almost certainly, VX, but this tabun is easier than the other G agents, so that agent was used for assassination of individuals tabun is more likely to be used in terrorist sce- (Nozaki et al., 1995). narios (see below). The reason is that tabun has no fluorine in its structure. Incorporation of the fluorine leaving group requires the use of hy- Use drofluoric acid during the synthesis and this is, Possible roles of OP nerve agents in warfare are of course, corrosive to glass. Early bulk synthe- outlined in Table 1. Until the use of sarin in Mat- sis of nerve agents with fluorine leaving groups sumoto and Tokyo, the use of chemical weapons was carried out using special apparatus made or as terrorists’ tools had been considered unlikely. lined with pure silver. Such a process is inevitably Since then, and the emergence of al-Qaida, this costly, although the difficulties did not deter a has been reconsidered and Table 2 gives some Japanese terrorist group (the Aum Shinrikyo). It of the roles whereby terrorists might make use is of interest that the nerve agent likely to have of chemical weapons. Note that the synthesis of been used by Iraq against the Kurds was tabun, FYX FYX

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TOXICOLOGY OF ORGANOPHOSPHATE NERVE AGENTS 193

Table 2. Possible terrorist targets for nerve agents while tabun does not contain a fluorine atom and is a cyanidate. The G agents include GB Major target Specific target (sarin, isopropyl methylphosphonofluoridate), Air transport Airport terminals/interiors GD (soman, pinacolyl methylphosphonofluori- of planes date), GA (tabun, ethyl N,N-dimethylphospho- Railways/subway Stations, ramidocyanidate) and GF (cyclosarin, cyclo- systems interiors of carriages hexyl methylphosphonofluoridate). The V agents Road transport Freeways/motorways, are phosphonothioates of the PÐO type in which especially intersections the leaving group is linked to phosphorus through and service stations a sulphur atom, except for VG which is a Public meeting places Concert halls, phosphorothioate. The V agents are exemplified major sporting events, political meetings, by VX (O-ethyl S- (2-(diisopropylamino)ethyl) churches and synagogues methylphosphonothioate). Financial centres Headquarters of financial All of the nerve agents are colourless liq- institutions, uids, although impure agents may be yellow to exchanges, e.g. trading brown in colour. However, these compounds dif- floors fer amongst themselves in physical properties Energy supply Power stations, (Table 4), for example, the V agents are much gas terminals, less volatile than the G agents, the latter being oil terminals volatile liquids (tabun less volatile than sarin or Communications Television and radio broadcasting stations, soman). Soman may be thickened to increase per- telephone exchanges sistence (Marrs and Maynard, 2001). VX is a non-volatile liquid with the result that the VX (unless aerosolized) is not an inhalation hazard, the first nerve agent to be synthesized on a large a fact that may significantly affect its role in war- scale. fare. Tabun is said to have a fruity odor, while the other agents are said to be odorless. Sarin may be mixed with tributylamine (sarin type I) or di- Structure and physical properties isopropylcarbodiimide (sarin type II), to prevent The nerve agents comprise a group of OPs of high spontaneous hydrolysis. acute mammalian toxicity. They are derivatives As is discussed below, more information of phosphoric or phosphonic acids (more often on cyclosarin (GF, cyclohexyl methylphospho- the latter) and contain two alkyl groups (R and R) nofluoridate) has accumulated recently, because, and a leaving group. The general formulae of the during operations ‘Desert Shield’ and ‘Desert OP nerve agents is similar to the OP pesticides: Storm’, it was discovered that cyclosarin was among the nerve agents that Iraq possessed. O Thus, some limited information is now avail- able on the physical properties of this nerve agent ′ RRP (gulflink, 2005). The material is a liquid at room ◦ X temperature with a boiling point of 239 C and a freezing point of −30◦C. The vapour pres- The nerve agents are traditionally divided into sure is 0.044 mmHg at 20◦C and the volatility the G agents and V agents (Table 3). Accord- is 438 mgm−3 at 20◦C. ing to Watson et al. (2005), G stands for Ger- man and V for venom. In the case of the G agents the leaving group is often a fluorine atom TOXICOLOGY and, exceptionally in GF (cyclosarin), one of the alkyl groups is replaced by a cyclohexyl Until recently, attention has almost entirely been group. Soman is distinguished by the fact that given to the acute toxicity of nerve agents and, one of its alkyl groups is a bulky pinacolyl group, in the military context, this is still the most FYX FYX

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Table 3. Formulae of nerve agents. Adapted and reproduced from Chapter 34, ‘Organophosphorus compounds as chemical warfare agents’, by Maynard RL and Beswick F, in Clinical and Experimental Toxicology of Organophosphates (B Ballantyne and TC Marrs, eds), 1992, with the kind permission of the publishers, Elsevier, and the authors

Abbreviation Common name Proper name GA Tabun Ethyl N, N-dimethylphosphoramidocyanidate

CH3CH2O O P

(CH3)2N CN GB Sarin Isopropyl methylphosphonofluoridate

CH3

CH3CHO O P

CH3 F GD Soman Pinacolyl methylphosphonofluoridate

CH3

(CH3)3C CHO O P

CH3 F GE — Isopropyl ethylphosphonofluoridate

CH3 CHO O CH3 P

C2H5 F GF Cyclosarin Cyclohexyl methylphosphonofluoridate

CH2 CH2 CH2 CHO O

CH2 CH2 P

CH3 F VX — O-Ethyl-S-[2(diisopropylamino)ethyl]methylphosphonothioate

C2H5O O P CH(CH3)2 CH3 SCH2CH2N CH(CH3)2 VE — O-Ethyl-S-[2-(diethylamino)ethyl]ethylphosphonothioate

C2H5O O P

C2H5 SCH2CH2N(C2H5)2 VG — O,O-Diethyl-S-[2-(diethylamino)ethyl] phosphorothioate

C2H5O O P

C2H5O SCH2CH2N(C2H5)2 VM — O-Ethyl-S-[2-(diethylamino)ethyl] methylphosphonothioate

C2H5O O P

CH3 SCH2CH2N(C2H5)2

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JWBK130-08 JWBK130-Marrs et al February 28, 2007 16:15 Char Count= ) 3 − d a 12 − 0.044 438 ) (mm Hg) (mg m 3 − c 20 − (B Ballantyne and TC Marrs, eds), 1992, ) (mmHg) (mm m 3 − 035 . 16 . × A nRT MW = × PV 80 VP − = ) (mm Hg) (mg m A 3 MW − × × 3143 . 8 101 325 × × 760 VP = 56 − Vol ) (mm Hg) (mg m 3 − . 3 − Clinical and Experimental Toxicology of Organophosphates with the kind permission of the publishers, Elsevier, and the authors 49 − C (mmHg) (mg m ◦ 01020 0.004 0.01325 0.03630 38 0.0740 119.5 0.09450 319.8 0.23 0.56 611.3 1.07 807.4 0.52 2.10 1912.4 2.9 4512.0 3.93 8 494 7.1 4 16 279 101 12.3 21 29 862 138 0.11 0.044 0.27 60 959 83 548 0.40 0.61 1 470.9 135.5 2 692.1 — 2.60 3 5 921.4 881.4 0.00044 — — 23 5.85 516.0 0.0007 — — — 10.07 — — — — 0.068 0.017 — 0.006 — 659 0.104 173 63 0.501 991 0.234 4480 2159 C 1.073 1.0087 1.022 1.0083 1.133 ◦ C. ◦ C C 246 147 167 300 — ◦ ◦ by Maynard RL and Beswick F, in concentration of saturated vapour at specified temperature. Volatility calculated from Physico-chemical properties of nerve agents. Adapted and reproduced from Chapter 34, ‘Organophosphorus compounds as chemical warfare agents’, = is the Absolute temperature. A b : Table 4. Where a range is given, this may be because materials of different purity have been studied. Temperature, 20 where Volatility Some authorities quote values as low as 0.1Ð1.0 mg m PropertyMolecular weight, MW (Da)Specific gravity at 25 162.3Boiling point Tabun, GAVapour pressure (VP) and Volatility(Vol) 140.1 Sarin, GB VP 182.18 VolNotes Soman, GDb VP 267.36 VXc Vold VP 180.14 GF Vol VP Vol VP Vol Melting point a

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important consideration. It is also the case when with nerve agents, do not suggest any major dif- considering casualties from terrorist use. Two ferences between man and other animals in clin- other exposure patterns have also to be consid- ical response to nerve agents. ered, (1) the long-term effects of low dose ex- Acute toxicity figures for nerve agents other posure, particularly in relation to manufacturing than tabun, sarin, soman and VX are scanty, workers and individuals involved in clean-up of with the exception of cyclosarin. Since the 1st contaminated sites, and (2) the delayed and long- Gulf War, a considerable amount of work has term effects after recovery from acute exposure. been carried out on the treatment of poisoning It has been pointed out (Reutter, 1999) that the with cyclosarin and data have also become avail- toxicity of chemical weapons is unchanged but able on the acute toxicity of this compound. In our perception of toxicity has since these com- studies by Anthony et al. (2004), the LCt50s in pounds were developed in the 1940s and 1950s. the rat for inhaled cyclosarin were of a simi- Other scenarios that need to be considered in- lar order of magnitude to the LCt50s for sarin clude contamination of food and public places (Table 6). Although cyclosarin is a powerful and, in such situations, levels to which it is nec- acetylcholinesterase inhibitor, rapid ageing of essary to decontaminate to ensure public safety. the inhibited enzyme as seen with soman, does A further consideration of importance is the tox- not occur, while spontaneous reactivation does icity of nerve agent degradation products (see (Worek et al., 1998) (see below). Qualitatively, review by Munro et al., 1999) and, particu- the toxic effects of cyclosarin seem similar to larly in the case of terrorist use, manufacturing other nerve agents (Young and Koplovitz, 1995). contaminants. It should be noted that the nerve agents have one or more chiral centres and the toxicity of the enantiomers may differ dramatically from one to Acute toxicity another and the racemic mixtures (Spruit et al., The toxic actions of all of the nerve agents are 2000). very similar, although some differences have been observed. There are differences between the Mechanism of toxicological actions OPs with respect to their relative central and pe- ripheral effects (Ligtenstein, 1984; Misulis et al., The action of OP nerve agents on the nervous 1987) and, within the central nervous system system results from their effects on enzymes, par- (CNS), some differences between the OPs may ticularly esterases. The most notable of these es- exist. For example, in the case of soman, there terases is acetylcholinesterase. The active site of is some indication that there are differences in acetylcholinesterase comprises a catalytic triad the sensitivity of acetylcholinesterase within the of serine, histidine and glutamic acid residues nervous system (Sellstr¬om et al., 1985). Acute and other important features of the enzyme are a toxicity figures are available for the nerve agents ‘gorge’ connecting the active site to the surface of tabun, sarin, soman and VX in many species (Ta- the protein and a peripheral anionic site (Bourne ble 5). The acute toxic dose in humans in not et al., 1995,1999; Sussman et al., 1991; Thomp- known with any exactitude as poisoning with son and Richardson, 2004), The OPs phosphy- nerve agents has only rarely been observed in late1 the serine hydroxyl group in the active site man (Sidell, 1974; Maynard and Beswick, 1992). of the enzyme. Where poisoning has been observed in man, de- The binding to acetylcholinesterase of acetyl- tailed information on dose has usually not been choline (the natural substrate of the enzyme) re- available and this was the case with the sarin ca- sults in acetylation of the serine at the active sualties in Japan, where the material was, in any site of the enzyme, with loss of the choline case, impure. Therefore, lethal doses and likely moiety. The reaction for acetyl choline can clinical effects in man have to be inferred from be envisaged as shown below, with E being the experimental poisoning in animals and from OP pesticide poisoning. The cases that have been 1 Phosphylation includes phophorylation and phosphonyla- studied, together with low-dose volunteer studies tion, the latter being more common with nerve agents. FYX FYX

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Table 5. Comparative acute toxicity of nerve agents. Adapted and reproduced from Chapter 34, ‘Organophosphorus compounds as chemical warfare agents’, by Maynard RL and Beswick F, in Clinical and Experimental Toxicology of Organophosphates (B Ballantyne and TC Marrs, eds), 1992, with the kind permission of the publishers, Elsevier, and the authors

Species Routea Term Unit (duration) Tabun Sarin Soman VX

−1 b Man pc LD50 mg kg — 28 — — pc LCLO μgkg−1 — — — 86c pc LDLO mg kg−1 23b — 18b — inhal LDLO mg m−3 150b — 70b — −3 b inhal LD50 mg m — 70 — — inhal ECLO μgm−3 — 90d — — iv TDLO μgkg−1 14b — — — iv TDLO μgkg−1 — — — 1.5e oral TDLO μgkg−1 — 2 f — 4e sc LDLO μgkg−1 — — — 30g im TDLO μgkg−1 — — — 3.2g

−1 h Rat pc LD50 mg kg 18 — — — −3 h d inhal LC50 mg m (10 min) 304 150 — — −1 h i j iv LD50 μgkg 66 39 44.5 — −1 h h m oral LD50 μgkg 3700 550 — 12 −1 j k l sc LD50 μgkg 193 103 75 — −1 f n n im LD50 μgkg 800 108 62 — −1 G im LD50 μgkg 130 — — — −1 i o ip LD50 μgkg — 218 98 —

−1 h h Mouse pc LD50 mg kg 1 1.08 — — −3 h p p inhal LC50 mg m (30 min) 15 5 1 — −1 h q r iv LD50 μgkg 150 113 35 — −1 s p p s sc LD50 μgkg 250 60 40 22 −1 q sc LD50 μgkg — 319 — — −1 E sc LD50 μgkg — 172 — — −1 q q im LD50 μgkg 440 222 — — −1 u u c ip LD50 μgkg — 420 393 50

−1 h Dog pc LD50 mg kg 30 — — — −3 h h inhal LC50 mg m (10 min) 400 100 — — −1 v v iv LD50 μgkg 84 19 — — −1 p oral LD50 μgkg 200 — — — −1 l w sc LD50 μgkg 284 — 12 — −1 H H H sc LD50 μgkg — 120 14 10

−1 h Monkey pc LD50 μgkg 9300 — — — −3 h h inhal LC50 mg m (10 min) 250 100 — — −1 x sc LD50 μgkg — — 13 — −1 y z im LD50 μgkg — 22.3 9.5 —

−3 h v Cat inhal LC50 mg m (10 min) 250 100 — — −1 h iv LD50 μgkg — 22 — —

−1 h h Rabbit pc LD5 μgkg 2500 925 — — −3 h h inhal LC50 mg m (10 min) 840 120 — — −1 h A iv LD50 μgkg 63 15 — — −1 h oral LD50 μgkg 16300 — — — −1 B C w D sc LD50 μgkg 375 30 20 14 −1 D ip LD50 μgkg — — — 66 (cont.) FYX FYX

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Table 5. (cont.)

Species Routea Term Unit Tabun Sarin Soman VX

−1 h Guinea Pig pc LD50 mg kg 35 — — — −3 h inhal LC50 mg m (2 min) 393 — — — −1 C sc LD50 μgkg 120 — — — −1 B C C sc LD50 μgkg — 30 24 8.4

−1 F B Hamster sc LD50 μgkg 245 95 —— −1 h Farm pc LD50 μgkg 1100 — — — −3 h Animal inhal LC50 mg m (14 min) 400 — — —

−1 w Chickens sc LD50 μgkg — — 50 — −1 o ip LD50 μgkg — — 71 —

−1 o Frog ip LD50 μgkg — — 251 —

Notes: a pc, percutaneous; inhal, inhalation; iv, intravenous; sc, subcutaneous; im, intramuscular; ip, intraperitoneal. b Robinson (1967). c WHO (1970). d Rengstorff (1985). e Sidell (1974). f Grob and Harvey (1958). g National Academy of Sciences (1982). h Gates and Renshaw (1946). i Fleisher et al. (1963). j Pazdernik et al. (1983). k Brimblecombe et al. (1970). l Boskovi«c et al. (1984). m Jovanovic (1982). n Schoene et al. (1985). o Chattopadhyay et al. (1986). p Lotts (1960). q Schoene and Oldiges (1973). r Brezenoff et al. (1984). s Maksimovi«c et al. (1980). t Fredriksson (1957). u Clement (1984). v O’Leary et al. (1961). w Berry and Davies (1970). x Clement et al. (1981). y D’Mello and Duffy (1985). z Lipp (1972). A Wills (1961). B Coleman et al. (1968). C Gordon and Leadbeater (1977). D Leblic et al. (1984). E Inns et al. (1992). F Coleman et al. (1966). G Cabal et al. (2004). H Weger and Scinicz (1981).

enzyme, AX acetylcholine, EAX is a reversible Table 6. Acute toxicity of cyclosarin (GF)

MichaelisÐMenten complex and A is acetate: c −1 Species Sex Route LD50 (μg kg )or −3 k+1 k2 EA k3 LCt50 (mg min m ) E + AX−−→ EAX−−→ −→ E + A +X Rat Male inhal 371 (10 min)b b where k+ , k− , k and k are rate constants. In- 396 (1 h) 1 1 2 3 585 (4 h)b hibition with OPs takes place by a process anal- b ogous to the reaction of acetylcholine with the Rat Female inhal 253 (10 min) 334 (1 h)b enzyme, by a reaction in which the leaving group 533 (4 h)b of the OP is lost so that the esterase is phosphy- Rat Male im 80c lated at the hydroxyl group of the serine residue Mouse Male sc 243d instead of acetylated. In the above equation, AX would be the OP nerve agent, EAX a reversible Guinea pig Male sc 44e MichaelisÐMenten complex of the enzyme and Monkey, nerve agent, EA the phosphylated enzyme, X the rhesus Male im 46.6 f leaving group and A, with nerve agents, (typi- cally) an alkoxy alkylphosphonate. Reactivation Notes: a Inhal, inhalation; sc, subcutaneous; im, intramuscular. of nerve agent-inhibited acetylcholinesterase oc- b Anthony et al. (2004). curs by hydrolysis of the alkoxy alkylphopho- c Kassa and Cabal (1999). nyl or phosphoryl enzyme, resulting in dephos- d Clement (1992). e Lundy et al. (1992). phylation and the rates of phosphylation are f Koplovitz et al. (1992). very variable, which partly accounts for dif- ferences in acute toxicity between the nerve agents. to acetylcholinesterase is described by the dis- The affinity with which a substrate such as sociation constant for the complex EAX, KD. acetylcholine or an inhibitor such as an OP binds This is equal to k−1/k+1. For inhibitors whose FYX FYX

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TOXICOLOGY OF ORGANOPHOSPHATE NERVE AGENTS 199

complexes with acetylcholinesterase reactivate the enzyme has been described as irreversible slowly, such as OPs, k3 can be ignored and the re- (WHO, 1986) (for a tabulation of the t1/2s for action with acetylcholinesterase can be described spontanous reactivation of various phosphy- by a bimolecular rate constant, ki as follows: lated acetylcholinesterases, see Wilson et al., 1992). While the active site of the enzyme is E + AX −−→ X + EA ki phosphylated it is of course unavailable for hydrolysis of acetylcholine. With some Some useful relationships can then be derived, organophosphorylated and organophosphony- e.g. k = k /K (Main and Iverson, 1966). In i 2 D lated acetylcholinesterases (but notably not addition, k = ln 2/I (Aldridge, 1950) which i 50 most OP insecticides), hydrolysis is very slow. allows easy estimation of k (The I is the con- i 50 With soman, the situation is further complicated centration of inhibitor, which inhibits the en- by an additional reaction known as ageing. zyme by 50%). These constants have been mea- This consists of monodealkylation of the di- sured for many OP chemical warfare agents and alkylphosphyl enzyme, creating a much more also pesticides (e.g. Gray and Dawson, 1987). stable monoalkylphosphyl enzyme, the reacti- The hydrolysis reaction for acetylated acetyl- vation rate of which is negligible (see review by cholinesterase is fast (Koelle, 1992), in the re- Curtil and Masson, 1993). On the basis of studies gion of 100 μs (Lawler, 1961; O’Brien, 1976). using acetylcholinesterase from Torpedo califor- The key to the powerful anticholinesterase ef- nica, Millard et al. (1999) suggested a number fects of OPs is what happens after inhibition by of non-covalent forces that might stabilize the these compounds. In the case of OPs, hydroly- aged enzyme, thereby preventing reactivation. sis of the phosphylated serine residue is much Rates of ageing depend on the structure of the slower2 than the acetylated analogue. inhibited acetylcholinesterase produced with Rates of phophorylation or phosphonylation each nerve agent. Soman produces an inhibited (inhibition) are a function of the whole OP acetylcholinesterase, where the active site serine molecule [see Maxwell and Lenz (1992) for a is phosphylated with a pinacoloxy methylphos- discussion of the structure-activity effects that phonyl structure. This ages very rapidly by loss underlie cholinesterase inhibition]. Rates of of the large pinacolyl group, with the result that reactivation (hydrolysis) are determined by the reactivation of inhibited acetylcholinesterase structure left attached to the active-site serine does not occur to any clinically significant residue after loss of the leaving group. Reactiva- extent. Talbot et al. (1988) studied in vitro and tion by hydrolysis of the phosphylated enzyme, in vivo ageing rates of soman-inhibited erythro- which is a nucleophilic displacement reaction, cyte cholinesterase in several animal species. In occurs at a clinically significant rate, with nearly vitro, t / s were (mean ± sem) 8.0 ± 0.82 min all important OPs but always much slower than 1 2 for guinea pigs, 1.1 ± 0.08 min for marmosets, when the enzyme is acetylated by its natural 1.4 ± 0.11 min for cynomolgus monkeys and substrate: thus the enzyme deacetylates in μs, but 0.88 ± 0.03 min for squirrel monkeys. In vivo dephosphorylates in a matter of hours to days, t / s were 8.6 ± 0.94 min for the rat, 7.5 ± 1.7 In general, spontaneous reactivation is faster, 1 2 min for the guinea pig and 0.99 ± 0.10 min the smaller the alkyl groups; thus dimethyl for the marmoset. The ageing t / in human phosphoryl acetylcholinesterase reactivates 1 2 erythrocytes is known to be rapid (1.3 min) for faster than the diethyl analogue and diisopropyl soman-inhibited enzyme (Harris et al., 1978). phosphorfluoridate (DFP)-inhibited enzyme Incidentally, this suggests that the guinea pig hydrolyses extremely slowly, in reality so slowly would not be a good model for humans in that the binding of the organophosphate to antidotal experiments in soman poisoning and that such experiments could only be undertaken 2 The term irreversible is often used for this reaction. This realistically in primates. Because of the rapid should not be taken to mean that reactivation of acetyl- cholinesterase by hydrolysis does not occur. The term is rate of ageing of the soman-inhibited acetyl- used because the OP is not recovered intact upon reactiva- cholinesterase, recovery of function depends tion of the enzyme (Chambers, 1992). on resynthesis of acetylcholinesterase (Gray, FYX FYX

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1984). Substances which produce complexes transmission of a nerve impulse: failure of with acetylcholinesterase which age virtually acetylcholinesterase activity results in accumu- instantaneously have been synthesized, for lation of acetylcholine (Burgen and Hobbiger, example crotylsarin, O-(2-butenyl) methylphos- 1951), which in turn causes enhancement and phonofluoridate, which has been used as an prolongation of cholinergic effects and also de- experimental tool in the investigation of the polarization blockade. At the neuromuscular action of oximes (van Helden et al., 1994). With junction, where accumulation of acetylcholine most other OPs, including most other nerve initially causes fasciculation, continued accumu- agents, ageing occurs, by the same mechanism lation produces flaccid type paralysis due to de- as soman (monodealkylation or, in the case polarization blockade. of tabun, possibly PÐN scission (Barak et al., 2000)), but more slowly and some spontaneous CHOLINERGIC NEUROTRANSMISSION reactivation may take place. Where treatment is instituted late, or where there has been repeated Fully to undertand the effects of esterase in- exposure, significant amounts of enzyme may be hibitors, such as OPs, it is necessary to discuss the in the aged state (for tabulations of ageing t1/2s cholinergic neurotransmission system. The es- of various phosphylated acetylcholinesterases, sential features in this system are a synthetic en- see Wilson et al., 1992 and Maynard, 1999). zyme, choline acetyltransferase (ChAT),the neu- The slow ageing process which occurs in rotransmitter itself (acetylcholine), a hydrolytic regard to tabun and sarin has been taken by enzyme, acetylcholinesterase and specialized re- some to indicate that treatment with oxime ceptors, with which the acetylcholine interacts. acetylcholinesterase reactivators can be safely Acetylcholine is one of a number of neurotrans- delayed. Nothing could be further from the truth. mitters in the nervous systems of mammals. It is Ageing has little to do with the toxic effects synthesized from acetyl coenzyme A and choline of organophosphates: these are, of course, by the enzyme ChAT in the perikaryon of cholin- dependent on the accumulation of acetylcholine ergic neurones and transported to nerve termi- at essential sites and the patient will always nals, with ChAT existing in both soluble and benefit from treatment being instituted as soon membrane-bound forms. The ChAT gene also as possible. encodes another protein besides ChAT, the vesic- Tabun-inhibited enzyme (an O-ethyl N,N- ular acetylcholine transporter. This transporter is dimethylamidophosphoro derivative) is slow responsible for transporting acetylcholine from to reactivate and Heilbronn (1963) found no the neuronal cytoplasm to the synaptic vesi- detectable spontaneous reactivation with hu- cles (Oda, 1999). At the nerve terminals, acetyl- man acetylcholinesterase. The reasons for this choline is released in response to an action are becoming more clear. Tabun binding of potential, thereby triggering opening of voltage- mouse acetylcholinesterase causes conforma- gated calcium channels in the presynaptic termi- tional changes in the enzyme that may stabilize nal. The acetylcholine thus released crosses the the enzymeÐinhibitor complex even without age- synaptic cleft. Contact between the acetylcholine ing of the complex (Ekstrom et al., 2006). and specialized receptors (see below) at the prox- imal end of the post-ganglionic nerve fibre results in localized depolarization of the postsynaptic ACCUMULATION OF ACETYLCHOLINE membrane and generation of a nerve impulse In normal circumstances, acetylcholine is in the postganglionic nerve fibre. Transmission hydrolyzed almost immediately by acetyl- is similar at parasympathetic nerve endings ex- cholinesterase close to receptors in the synap- cept that the acetylcholine stimulates receptors tic cleft at sites of action (see Bowman, 1993). in parasympathetic effector organs. At the neu- The normal function of acetylcholinesterase romuscular junction, muscle fibre depolarization is to hydrolyze acetylcholine in the synap- is initiated at the motor end plate following a tic cleft, parasympathetic effector organ or similar sequence of events. In the cases of both neuromuscular junction, in order to terminate autonomic ganglia and muscle, it is necessary for FYX FYX

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the depolarization to exceed a threshold in order parasympathetic activity via the vagus nerve. to produce postjunctional activity, e.g. initiate an The distribution and some functions of the other action potential in a postsynaptic nerve cell or sub-types have also been characterized (Eglen muscle fibre. and Whiting, 1990; Jones, 1993). M3 receptors are found in glandular tissue and M4 receptors are found in the striatum while the M subtype is CHOLINERGIC RECEPTORS 5 present in the hippocampus and brainstem. Cholinergic receptors are divided into mus- carinic and nicotinic on the basis of their sensitiv- NICOTINIC RECEPTORS ity to pharmacological stimulation by muscarine and nicotine, respectively; muscarinic receptors Nicotinic receptors, found at all autonomic sys- are found in parasympathetic effector organs, tem ganglia and at the skeletal neuromuscu- and, prejunctionally at the neuromuscular junc- lar junction have been well characterized at a tion, whereas nicotinic receptors are found at au- molecular level largely because the same type tonomic ganglia and the neuromuscular junction. of receptor is found in the fish (Torpedo califor- More or less specific antagonists are available; nica) electric organ. The structure of the nico- thus, atropine antagonizes muscarinic agonists tinic acetylcholine receptor has been reviewed at muscarinic sites but has little effect at the neu- (Kaminski and Ruff, 1999). These receptors are romuscular junction, whereas the reverse is true part of a ‘superfamily’ of ligand-gated ion chan- of tubocurarine. The two types of receptor are nels with γ-aminobutyric acid A (GABAA) re- fundamentally different Ð muscarinic receptors ceptors and glycine receptors (Ortells and Lunt, using a second messenger system, whereas nico- 1995). Each receptor comprises five sub-units ar- tinic receptors are ligand-gated ion channels. ranged around a channel that passes through the cell membrane, but there are some differences be- tween the receptors found at the neuromuscular MUSCARINIC RECEPTORS junction and those in neuronal tissue, in struc- Five sub-types of muscarinic receptor, desig- ture and in inhibition characteristics (the former nated M1 to M5, have been cloned. Each re- are inhibited by α-bungarotoxin). In neuronal tis- ceptor has a serpentine structure that spans the sue, the receptors are composed of α and β sub- cell membrane seven times. The receptor is cou- units and a number of each type of sub-unit have pled via guanosine triphosphate-binding proteins been cloned (Boyd, 1997). The muscle receptor (G-proteins) to the enzymes adenylate cyclase in adult innervated muscle contains 5 sub-units, or phospholipase C. MI receptors, activation 2 α,1β 1 δ and 1 ε; in denervated or embryonic of which leads to an increase in Ca2+ con- muscle the ε is replaced by a γ sub-unit. Binding ductance of local ligand-gated Ca2+ channels, of acetylcholine leads to a widening of the chan- are found in neuronal tissue. These calcium nel and an increase in Na+ conductance. Influx channels are ligand-gated, unlike those in the of Na+ ions leads to depolarization of the cell presynaptic or prejunctional terminals which membrane (see Lefkowitz et al., 1996). are voltage-gated. Influx of calcium ions via open channels is both electrically driven by Clinical Effects the negative intracellular potential and concen- tration driven as the intracellular free Ca2+ The clinical effects of nerve agents are, to a concentration is about 100 nmol l−1 while large extent, those of acetylcholine accumula- the extracellular concentration is about 1.2 × tion and the effects of all of the nerve agents 6 −1 10 nmol l (Ganong, 1991). Activation of M2 are similar. Those differences that have been receptors leads to a decrease in Ca2+ conduc- observed are presumably due to a combination tance. M2 receptors may play a prejunctional of different rates of inactivation and reactiva- role in modulating the activity at the neuromus- tion of the enzymes, together with different rates cular junction. M2 receptors are also found in of ageing of the inhibited enzyme and differ- the heart and mediate the depressor effects of ences in absorption, distribution, metabolism and FYX FYX

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Table 7. Main effects of nerve agents at various sites in the body

Receptor Target organ Symptoms and signs Central Central nervous Giddiness, anxiety, restlessness, system headache, tremor, confusion, failure to concentrate, convulsions, respiratory depression, respiratory arrest

Muscarinic Glands Nasal mucosa Rhinorrhea Bronchial mucosa Bronchorrhea Sweat Sweating Lachrymal Lachrymation Salivary Salivation Smooth muscle Iris Miosis Ciliary muscle Failure of accommodation Gut Abdominal cramp, diarrhoea, involuntary defecation Bladder Frequency, involuntary micturition Heart Bradycardia

Nicotinic Autonomic ganglia Sympathetic effects, including pallor, tachycardia, hypertension Skeletal muscle Weakness, fasciculation

excretion of the nerve agent. It is noteworthy that produce pallor, tachycardia and hypertension. in the case of soman, kinetic differences have The clinical effects in the cardiovascular system been recorded between different stereoisomers depend on whether muscarinic or nicotinic (Benschop et al., 1987). effects predominate; bradycardia or tachycardia The symptoms and signs of nerve agent may occur and, in some cases arrhythmias (see poisoning may be divided into three groups, below). At the neuromuscular junction, nicotinic muscarinic, mediated by muscarinic receptors signs include muscle fasciculation and later in parasympathetic effector organs, nicotinic, paralysis. If the patient survives the acute cholin- mediated by nicotinic receptors in autonomic ergic syndrome, the effects of nerve agents are ganglia and the neuromuscular junction, and largely reversible, although, as discussed above, effects in the central nervous system, which with soman recovery may be very slow and in are mediated by receptors of both types. Acute certain circumstances there may be long-term effects of OP nerve agents are given in Table 7. changes in the CNS (see below). Where death The muscarinic symptoms and signs result from occurs, it is caused by respiratory paralysis, increased activity of the parasympathetic system which may be central or due to the anticholineste- and include bronchorrhoea, salivation, constric- rase action at the neuromuscular junction tion of the pupil of the eye (miosis),3 abdominal (Chang et al., 1990). colic and bradycardia (Grob and Harvey, 1953). Nicotinic effects at autonomic ganglia can NON-ANTICHOLINESTERASE EFFECTS

3 Miosis is the term used to describe the constriction of the Clinically, the most important effects of OP nerve pupil. The term is often assumed to be derived from the agents are anticholinesterase actions. However, same Greek root as meiosis, i.e. a diminution. This is in it should not be forgotten that OPs bind to a fact not the case. Miosis, and the earlier variant myosis, are derived from the Greek root myein (or muein): to close, variety of enzymes, including esterases other blink (Shorter Oxford English Dictionary, Webster’s Third than acetylcholinesterase, e.g. carboxylesterase, New International Dictionary). (long-chain fatty acid hydrolase), serine FYX FYX

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peptidases, amidases and proteases and others Kovacic (2003) suggested that oxidative stress (see Lockridge and Schopfer, 2005). Moreover, might play a part in the toxic manifestations of there is some evidence that some OP anti- OPs. cholinesterases can act directly on muscarinic It is possible that some of the CNS effects and nicotinic receptors (Bakry et al., 1988; of nerve agents are secondary to changes in Silveira et al., 1990; Mobley, 1990; Eldafrawi the blood brain barrier. There is little evidence et al., 1992). More complex direct effects at that nerve agents will cause OP-induced delayed cholinergic receptors may be important: Rocha polyneuropathy at doses likely to be encountered et al. (1999) showed that VX, at concentra- and survived by man (see below). tions that had little effect on cholinesterase, interacted directly with presynaptic muscarinic receptors, causing a selective inhibition of the EFFECTS ON SPECIFIC ORGANS evoked release of GABA. This would increase excitatory neurotransmission and these workers The eye hypothesized that this might account for the ability of VX to induce convulsions. Unlike OP insecticides, the G-type nerve agents It has also been demonstrated that OPs, in- are most likely to be encountered as vapours and cluding nerve agents, can affect neurotransmis- eye effects occur early. Nerve agents produce sion pathways other than cholinergic ones, for miosis (constriction of the pupil); this produces example γ-aminobutyric acid (GABAergic) re- a feeling that the surroundings are dim, or that il- ceptors as well as glutamatergic, dopaminergic, lumination has been reduced. The onset is rapid somatostatinergic and noradrenergic systems and may last for several days. Miosis is a very (Lau et al., 1988; Fletcher et al., 1989; Fosbraey sensitive indicator of exposure to nerve agents et al., 1990; Naseem, 1990; Smallbridge et al., and van Helden et al. (2004a) concluded, on the 1991; Chechabo et al., 1999; Tonduli et al., 1999; basis of studies in guinea pigs and marmosets Rocha et al., 1999). It is likely that most if not with sarin, that miosis would occur during low- all such perturbations are secondary to effects on level exposure at levels that would not be de- cholinergic systems, but there is evidence that re- tectable by the currently fielded alarm systems, cruitment of non-cholinergic systems is respon- assuming that humans are of similar sensitivity sible, at least in part, for seizure activity (Solberg to these experimental species. and Belkin, 1997). Furthermore, the efficacy of Spasm of the ciliary muscle may impair some experimental treatments not directed at the accommodation and is associated with severe cholinergic neurotransmission system suggests a headache. Long-lasting miosis, associated with role for other neurotransmission systems. Thus, eye pain, was a notable clinical sign in the Tokyo Braitman and Sparenborg (1989) found that a Subway (underground railway) terrorist attack potent antagonist at the N-methyl-d-aspartate with sarin and the same was true of the sarin at- (NMDA)-type glutamate receptor (MK-801), to- tack at Matsumoto (Nohara and Segawa, 1996). gether with other antidotes, protected against Dilatation of subconjunctival blood vessels oc- seizures induced in guinea pigs by soman. More- curs and the eye becomes bloodshot. After ex- over, Filliat et al. (1999) found that learning posure to high concentrations of nerve agent, the deficits produced in rats by soman were re- eyes take on a glassy appearance: the appearance duced by antagonists at α-amino-3-hydroxy-5- is sometimes compared to that of a glass marble. methyl-4-isoxazolepropionic acid (AMPA)-type The lachrymal glands do not seem to be much glutamate receptors and NMDA-type glutamate affected by exposure to nerve agent vapour and receptors, together with atropine. It has also tearing is not a reliable early sign of exposure. been suggested that the protective action of caramiphen aginst OP poisoning may be, in part, The Respiratory Tract due to the ability of this drug, which is also an anticholinergic agent, to modulate the NMDA The upper respiratory tract contains two compo- receptor (Raveh et al., 1999) nents that are under cholinergic control. These FYX FYX

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are the mucous glands and smooth muscle. The INTERMEDIATE SYNDROME (IMS) response to nerve agents by the former is an Senanayake and Karalliedde (1987) described increase of secretions resulting in bronchorrhea a new form of neurotoxicity following intoxi- and rhinorrhea (runny nose) and, in severe cases, cation by organophosphorus insecticides. As it foaming around the nose may occur. The effect occurred after the acute syndrome and before on smooth muscle is to produce bronchospasm. the onset of classical OPIDN, they called it the Respiratory function may also be affected by ‘Intermediate Syndrome’. This phenomenon the action of nerve agents on the neuromuscular consists of marked weakness of the proximal junction of the (striated) muscles of respiration skeletal musculature (including the muscles of and by central effects on the respiratory centre. respiration and neck muscles) and cranial nerve palsies. IMS comes on 1Ð4 days after acute poi- Heart soning; respiratory support is often necessary and, if it is provided, recovery occurs within The acute effect on the heart depends on 4Ð18 days. Although the IMS has not been de- the relative predominance of muscarinic or scribed in cases of accidental nerve agent poi- nicotinic effects. Bradycardia or tachycardia may soning, it is likely that it would occur, at least occur, as well as arrhythmias, including atrioven- in some cases. The pathogenesis of IMS is not tricular block and various ventricular arrhyth- known with certainty, although it appears to be a mias. An arrhythmia characteristic of poisoning post-junctional non-depolarizing blockade. IMS with OP pesticides is Torsade de Pointes is probably a consequence of cholinergic overac- (Ludomirski et al., 1982), while soman and other tivity at the neuromuscular junction (NMJ), per- OPs have been reported to produce histopatho- haps through down-regulation of post-junctional logical changes in the myocardium in both exper- nicotinic receptors (i.e. a reduced density of imental animals and humans (McDonough et al., functioning nicotinic receptors), with a possible 1989; Singer et al., 1987; Pimentel and Carring- contribution from prejunctional muscarinic re- ton da Costa, 1992; Koplovitz et al., 1992; Britt ceptors (Marrs et al., 2005). It has also been et al., 2000). A few patients in the Matsumoto suggested that failure to hydrolyze acetylcholine nerve agent attack had arrhythmias (Okudera, may reduce the supply of choline, which is a 2002). substrate for ChAT, the enzyme that synthesizes acetylcholine. Furthermore, connection has been Nervous system and skeletal muscle suggested between the IMS and OP-induced my- opathy (Senanayake and Karalliedde, 1992). My- Systemically, nerve agents produce fascicula- opathy associated with OPs was first observed tion and then blockade at the neuromuscular many years ago and has been observed histo- junction with weakness and paralysis. Paralysis logically in experimental animals with the nerve of the muscles of respiration (see above) may agents tabun, soman, and sarin (Preusser, 1967; interfere with respiration and is potentially life- Ariens et al., 1969; Gupta et al., 1987a,b; Bright threatening. Some of these effects may be et al., 1991; Hughes et al., 1991; Koplovitz et al., mediated by direct actions at the receptor-ion 1992; Britt et al., 2000). The changes characteris- channel complex. Soman and sarin were reported tic of OP-induced myopathy seem to be initiated by Goldstein et al. (1987) to alter muscle spindle by calcium influx (Leonard and Salpeter, 1979) function. as a consequence of acetylcholine accumulation Separate from the acute effects of anti- at the neuromuscular junction (Marrs et al., 1990; cholinesterases upon the neuromuscular junc- Inns et al., 1990). In general the distribution of tion (discussed previously), two further syn- the myopathy parallels the distribution of mus- dromes involving the neuromuscular system cles affected by IMS, although that was not true have been associated with OP poisoning. These of the study in rhesus macaques by Britt et al. are (1) the intermediate syndrome (IMS), and (2) (2000). If it is these histological changes that organophosphate-induced delayed polyneuropa- underlie IMS and, as they have been observed thy (OPIDP). FYX FYX

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with chemical warfare nerve agents, the devel- of soman, Johnson et al. (1985) showed that opment of the syndrome can be anticipated in only a tiny proportion of inhibited NTE from the recovery phase of nerve agent poisoning in hen brain and spinal cord underwent ageing. at least some cases. However, it should be noted Crowell et al. (1989) studied the potential for that oximes are reported to protect against the sarin and soman to cause OPIDP. Sarin type I4 myopathy, whereas there have been reports that, was given by gavage at single doses of 61, 200 in insecticide poisoning, oximes have not always and 400 μgkg−1, sarin type II at doses of 70, protected against IMS. Furthermore, the time- 140 and 280 μgkg−1 and soman at doses of 3.5, course of the myopathy observed in experimental 7.1 and 14.2 μgkg−1 to groups of five Leghorn animals is not similar to the time-course for the hens protected from acute cholinergic toxicity development of IMS. Karalliedde et al. (2006) with atropine. There were appropriate vehicle concluded that IMS arose from down-regulation controls and tri-o-cresyl phosphate was used as of the nicotinic acetylcholine receptor, caused by a positive control. NTE was measured in brain, acetylcholine accumulation, and that myopathy spinal cord and lymphocytes. With sarin type I, and IMS are not causally related, but have a com- the highest dose was lethal within 24 h and the mon origin in acetylcholine accumulation. treatment did not significantly depress NTE in any tissue. The highest dose of sarin II decreased lymphocyte NTE to 33% of the control value. In NERVE AGENT-INDUCED DELAYED the brain and spinal cord, significant depression POLYNEUROPATHY of NTE was not seen at any dose. Soman did Organophosphate-induced delayed polyneu- not produce significant depression of NTE. ropathy (OPIDP) is a symmetrical sensory-motor In all cases tri-o-cresyl phosphate produced neuropathy, with both central and peripheral significant depression of NTE. Despite this, components, tending to be most severe in long there have been reports that sarin may produce axons and occurring 7Ð14 days after exposure to histopathological changes in rodents similar to OPs. In severe cases, it is an extremely disabling those found in OPIDP (see Somani and Husain, condition, the central component being irre- 2001). versible. Inhibition of neuropathy target esterase Studies in vitro also suggest that the develop- (NTE), an esterase of unknown function present ment of OPIDP is unlikely to be a clinical prob- at several sites, including neurones, where it is lem in humans. Thus, Gordon et al. (1983) com- an integral membrane protein (Glynn, 2000), pared the inhibitory potency of the nerve agents, appears to be necessary for OPIDP to develop sarin, soman, tabun and VX, and some other (see reviews by Somani and Husein, 2000 and OP compounds, including diisopropyl phospho- Senanayake, 2001). This is followed by an rofluoridate (DFP), mipafox and related com- ageing reaction similar to that described for so- pounds, against NTE and AChE. The ratio I50 man with acetylcholinesterase above (Johnson, for inhibition of acetylcholinesterase/I50 for in- 1975). By contrast with IMS, it is unlikely that hibition of NTE was 0.0056 for sarin, 0.0012 for nerve agents possess the capability to cause soman, 0.0005 for tabun and 10−6 for VX. In the OPIDP. In experimental studies, nerve agents do case of the neuropathic OPs, the ratio was 1.13 for not usually bring about OPIDP (Anderson and DFP and 1.8 for mipafox. Moreover, structureÐ Dunham, 1985; Crowell et al., 1989; Johnson activity considerations lend no support to sug- et al., 1985; Goldstein et al., 1987; Parker et al., gestions that nerve agents would be neuropathic 1988; Henderson et al., 1992). Gordon et al. (Aldridge et al., 1969). (1983) reported studies in hens (Ross white or Thus, the reason for the expectation that nerve Sussex) in vivo, protected with physostigmine, agents would be non-neuropathic in man is that atropine sulphate and pralidoxime mesilate concentrations of nerve agent required to pro- (P2S): OPIDP (as assessed clinically) with duce AChE inhibition are low, by comparison NTE inhibition was only seen at 30Ð60 × LD50 with concentrations required for inhibition of for sarin, but not at 38 × LD50 for soman or 4 82 × LD50 for tabun. Furthermore, in the case Types I and II refer to the stabilizers used. FYX FYX

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NTE. Nevertheless, OPIDP was one hypothesis patterns of exposure, and these have been re- for Gulf War Syndrome (Institute of Medicine, viewed (Ray, 1998a; Committee on Toxicity of 1996). Products in Food, Consumer Products and the Environment, 1999; Karalliedde et al., 2000; Romano et al., 2001). Many of the studies re- CENTRAL NERVOUS SYSTEM viewed refer to OP insecticides. It is difficult OPIDP, which has a central component, was dis- to summarize this work as the patterns of expo- cussed above. The nerve agents produce a wide sure have been very varied. Of course, long-term range of effects upon the central nervous sys- low dose exposure and acute high dose exposure tem, ranging from anxiety and emotional labil- are two ends of a spectrum of different expo- ity at low doses, to convulsions and respiratory sure scenarios and many intermediate patterns paralysis at higher ones. It is important to note of exposure are possible. Studies have also been that doses considerably below lethal doses can undertaken in experimental animals (including markedly degrade performance of tasks in be- primates) and some of these are discussed below. havioral studies (Brimblecomb, 1974; Wolthuis and Vanwersch, 1984; D’Mello and Duffy, 1985; Delayed effects of high dose exposure D’Mello, 1993; DiGiovanni, 1999) and there is evidence that the decremental effects of expo- The anticholinesterase effects induced by nerve sure to single doses of nerve agents may be pro- agents in the CNS are reversible and histopatho- longed (McDonough et al., 1986). Clearly, this is logical changes are usually exiguous in fatalities of importance as it is likely that military perfor- and in animals dying during acute toxicity stud- mance of personnel would be impaired: affected ies. Thus Anzueto et al. (1986) found that, in servicemen might not only lose the motivation baboons, given intravenous infusions of soman, to fight but also lose the ability to defend them- only minimal CNS histopathological changes selves and be unable to carry out the complex were observed. The absence of specific changes tasks frequently required in the modern armed hinders diagnosis at autopsy; moreover, it implies forces. Effects on skilled personnel, such as pilots that complete recovery after successful treatment and navigators, would be particularly disabling. is probable. However, the situation may be differ- In a terrorist attack, rescuers may be affected and ent after survival of near-lethal doses and where their skills impaired. humans survive, it is probable that functional deficits would be observed. It is also likely that were humans to survive for an appreciable period Long-term and delayed effects before dying, histopathological changes would Previously, experimental work with nerve agents be seen in the CNS. In animals, after substan- has been concentrated on the acute effects. How- tial doses of soman, the initial histopathological ever with the possibility of mass casualties from changes seen are edema, particularly astrocytic terrorist outrages, the possibility of persistent and perivascular hemorrhages. Neuronal degen- neurobehavioral and psychiatric effects, such eration and necrosis, sometimes diffuse, may be have sometimes been observed with excessive observed, together with more discrete infarcts, exposure to OP pesticides, needs to be con- with necrosis of all cell types. Such changes sidered. Such effects have included headache, may be detected particularly in the hippocam- anxiety, agitation, insomnia, irritability, impaired pus and piriform cortex (McLeod, 1985). This memory and difficulty in concentrating (Annau, picture, which does not seem to correlate with 1992; Jamal, 1995; Lader, 2001; Feldman, 1999). areas in which the blood-brain barrier is compro- The long-term effects of low dose exposure mised, may proceed to an encephalopathy. Most and the delayed effects of acute exposure are commonly, this affects the cortex, hippocam- often conflated. The latter are easier to under- pus and thalamic nuclei, a distribution that sug- stand. A number of studies have been performed gests anoxia is a likely cause (McLeod, 1985; to investigate subtle changes in humans ex- McDonough et al., 1986,1989). The hypothe- posed to various sorts of OPs, and with differing sis that hypoxia secondary to convulsions is the FYX FYX

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cause, is supported by the fact that the nerve prolonged compared to matched controls, al- agent-induced effects have been correlated with though no subject had obvious clinical abnor- seizure activity (Anzueto et al., 1986; Britt et al., mality (Murata et al., 1997). A case-report from 2000) and that anticonvulsant γ-aminobutyric Japan noted specific interference with memory in acid (GABAA) agonists, such as diazepam or a victim of the sarin exposure in Tokyo: this per- midazolam, alleviate the effects (Martin et al., sisted to 6 months after exposure and extended to 1985; Anderson et al., 1997). Nevertheless, the the period in a retrograde fashion to 70 days be- attribution of nerve-agent-induced pathological fore exposure (Hatta et al., 1996). In a suspected changes to hypoxia and/or convulsions is not the case of VX poisoning, antegrade and retrograde view of all (Petras, 1981) and in an in vitro ultra- amnesia was observed (Nozaki et al., 1995). In a structural study using rat rat hippocampal slices, study of members of a rescue team, who attended Lebeda et al. (1988) found morphological dif- the Matsumoto sarin exposure, all symptoms of ferences between the effects produced by hy- the affected personnel had resolved a year af- poxia and soman. OP poisoning is indeed some- ter exposure (Nakajima et al., 1997). In a cross- times associated with long-term CNS changes, sectional study of rescue staff and policemen ap- both in experimental animals and in humans proximately 3 years after they were exposed to (Holmes and Gaon, 1956; Korsak and Sato, 1977; sarin on the Tokyo subway, effects were observed Rosenstock et al., 1991) and this is likely to be on memory (Nishiwaki et al., 2001). Yokoyama the case both with pesticides and nerve agents et al. (1998) reported vestibulocerebellar effects (see reviews by Marrs and Maynard, 1994 and 6Ð8 months after exposure to sarin in the Tokyo Ray, 1998a). incident. This comprised low-frequency sway, Human data on nerve agent exposure is scanty which was more severe in females. Of course, in compared to that on OP pesticides. A notable the aftermath of a terrorist use of a nerve agent, study (on workers) involving nerve agents was psychological effects are to be expected in ad- carried out by Duffy et al. (1979). Behavioral dition to any effect from damage to the CNS signs and subtle EEG changes were noted after (DiGiovanni, 1999). exposure to sarin, severe enough to cause symp- The implications for treatment are, at this toms and clinical signs. Burchfiel et al. (1976) time, uncertain; the most logical course would be described changes, which persisted for a year to avoid convulsions and/or anoxia as much as in the EEGs of rhesus monkeys after a single possible during treatment of acute nerve agent large dose or repeated small doses of sarin. The poisoning. lower dose used was 1 μg, given by ten weekly injections (total dose 10 μg = 7% LD50). This Low dose exposure result, which is somewhat surprising, must be in- terpreted cautiously as the group size was small A variety of effects in human and animals have (three per group) and similar but not identical been attributed to OP exposure but it is less clear electroencephalographic changes were seen after whether low doses, particularly subconvulsive the organochlorine cyclodiene pesticide, dield- ones, can bring about long-term changes in CNS rin, namely a relative increase in § activity. Sub- function. If that were the case, it is clearly desir- jects from volunteer programmes have been stud- able to know if there is a threshold dose below ied in the USA (National Academy of Sciences, which effects are not observed. Effects observed 1982) and here there was no clear evidence of after long-term low dose exposure to OPs are CNS effects or any effect on mortality. In a 1- and less easy to explain than delayed effects of high 2-year follow-up of survivors of the Matsumoto dose exposure, but it should be noted that OPs, sarin exposure, epileptiform EEG abnormalities including nerve agents, can react with targets were found in four out of six severely poisoned other than cholinesterases (see above). Richards subjects (Sekijima et al., 1997). In a study of et al. (1999) found two novel OP-reactive sites in 18 victims of the Tokyo subway sarin incident, rat brain homogenates by inhibition of 3H-DFP 6Ð8 months after exposure, the event-related labeling using several OP insecticides or their and visual evoked potentials were significantly oxons. One of the sites had a molecular mass FYX FYX

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of 85 kDa and Richards et al. (2000) purified the which produced red cell acetylcholinesterase de- 85 kDa site from porcine brain and characterized pressions of 20Ð30%, and 2.5 μgl−1, which pro- it as an acylpeptide hydrolase (see reviews by duced red cell acetylcholinesterase depression of Ray, 1998a,b). 40Ð50%. Group sizes were 10. Exposures were After reviewing the available data, both in hu- on single occasions, except that exposure to the mans and in experimental animals, Moore (1998) middle concentration was carried out repeatedly concluded that the available data indicated that and additionally in a separate group of animals. exposure to nerve agents at doses producing no A functional observational test battery (FOB) of clinical signs or symptoms of acute toxicity did behaviour was carried out at 3 months. Signifi- not produce chronic illness (see also Brown and cant abnormality was seen after single exposure Brix, 1998). Pearce et al. (1999) were unable at the highest concentration in respect of a num- to find significant persisting effects of single im ber of observations (gait disorder and score, mo- doses of sarin producing 36.4 to 67.1% ery- bility score, activity and stereotypy and at the throcyte acetylcholinesterase inhibition in mar- mid-concentration only in respect of the last Ð mosets on EEG or the CANTAB automated test more changes were seen after repeated expo- battery. There was a marginal increase of beta 2 sure). Changes in spatial discrimination in the EEG activity attributable to an affect in one sub- Y-maze were seen at all doses shortly after ex- ject: Haley (2000) speculated that this might be posure at all test concentrations, but at the high- due to a polymorphism for the enzyme paraox- est concentration this persisted for 3 weeks. This onase 1 (PON1) in one marmoset. van Helden study could be interpreted as suggesting a single et al. (2004b) have shown effects on the EEG exposure threshold for long-term effects at 20Ð from sarin vapor at concentration × time values 30% erythrocyte acetylcholinesterase depression (Cts) of 0.2 and 0.1 mg min m−3 (time of ex- in the rat. posure was 5 h) in vehicle and pyridostigmine- Sleep disruption was seen in mice during a treated marmosets; these effects were persistent. study on soman by Crouzier et al. (2004). The Furthermore, Bajgar et al. (2004) found persis- dose used was 50 μgkg−1 bw sc. tent (4 weeks) effects on a few behavioural pa- Visual field defects persisting for 1 year, which rameters in guinea pigs given single inhalation had disappeared 17 months after exposure, were exposures to soman. The concentrations used found in a single subject exposed to sarin in were 1.2, 1.5 and 2.7 mg m−3 and exposure was Matsumoto (Sekijima et al., 1997). for 1 h. Only butyrylcholinesterase was inhib- ited at the lowest concentration at 1 day, while Reproductive toxicity, developmental red cell cholinesterase was 66% control at the toxicity and developmental neurotoxicity mid-concentration and 28% control at the high- est concentration, both at 1 day. Brain acetyl- On the basis of a study with VX (Goldman et al., cholinesterase measured at 4 weeks was only in- 1988), there was little evidence that OP nerve hibited > 20% at the highest concentration and agents presented a reproductive hazard at doses then only in the basal ganglia, not the other areas below those toxic to the parents. On the basis in which enzyme activity was estimated. Signif- of studies with sarin (Denk, 1975; La Borde and icant increases in neuroexcitability score were Bates, 1986), there is little evidence that OP nerve seen at all test concentrations, compared to con- agents have a potential for teratogenesis or fe- trols at 4 weeks, although the differences were toxicity at doses below those toxic to the dams. small in magnitude. Unsurprisingly, larger dif- There is no direct evidence that exposure to nerve ferences between test groups and controls were agents has produced developmental toxicity in seen at 1 day. The same group (Kassa et al., 2004) humans; data on exposure of pregnant women to reported a study in rats, in which exposure to nerve agents are unsurprisingly extremely scanty. sarin by inhalation was studied. Exposure was Four pregnant women were exposed to sarin in for 1 h at concentrations of 0.8 μgl−1, which the Tokyo exposure at 9Ð36 weeks’ gestation produced red cell acetylcholinesterase depres- (Ohbu et al., 1997). All had normal offspring. sion of < 20% compared to controls, 1.25 μgl−1, Nevertheless, because of the profound effects of FYX FYX

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OPs on neurotransmission, there is a strong the- did not produce unscheduled DNA synthesis in oretical basis for concern that effects which, in isolated rat hepatocytes but decreased repair syn- adults would be reversible, would in the embryo, thesis was seen with sarin (Klein et al., 1987). fetus and neonate be irreversible. With pestici- Decreased excision repair capacity has also been dal OPs, there are data in animals to suggest a seen with the insecticide diazinon, and so this potential for developmental neurotoxicity. Thus, may be a more general effect of OPs. studies have suggested the possibility that chlor- pyrofos may have effects on developing organ- isms (Tang et al., 1999; Qiao et al., 2001) and EFFECT OF ROUTE OF EXPOSURE a body of work is accumulating on OP pesti- cides in response to the development of a stan- Vapour dardized developmental neurotoxicity test in the rat (US EPA, 1998; OECD, 2003; Fenner-Crisp Onset is rapid and the eyes and respiratory sys- et al., 2005). However, formal developmental tem are most affected. Low-level exposure causes neurotoxicity tests on OP nerve agents have not tightness of the chest, rhinorrhea and salivation. been done and the effects have to be inferred Dimming of vision due to miosis, eye pain and from knowledge on OP pesticides [see reviews by headache then follow. On examination, the pupils Kitos and Suntornwat (1992), Slotkin (2005) and are constricted and the conjunctivae hyperaemic. Makris (2005)]. There is little doubt that, with These effects may last several hours after ces- the appropriate pattern of exposure to pregnant sation of exposure and the headache and visual animals, including humans, nerve agents would problems several days. In severe cases, salivation be developmental neurotoxicants. Further infants and rhinorrhea are more marked, and wheezing and children also have developing nervous sys- and dyspnea are prominent. Other effects, such tems and these individuals may be at greater risk as abdominal pain, vomiting, involuntary defe- of long-term effects on the nervous system than cation and micturition, weakness, fasciculation adults (Guzelian et al., 1992). Nevertheless, with and convulsion, follow depending on the degree pesticides, it has been suggested that most expo- of systemic absorption. Death may occur from sures of pregnant women have not shown adverse respiratory failure. effect on the offspring, presumably because ex- The attack with sarin on the Tokyo Subway posure is of short duration and occurs only once (underground railway) has added considerably (McElhatton, 1987; Minton and Murray, 1988) to our knowledge of the clinical effects of sarin and it seems likely that that would be the case vapor. The symptoms observed were largely as with nerve agents. expected, namely cough, difficulty in breathing and tightness of the chest, bradycardia and eye pain (Masuda et al., 1995; Nozaki et al., 1995; Delayed effects outside the CNS Ohbu et al., 1997) (see Chapter 13). The nerve agents are generally considered to be acute toxicants and their delayed effects have been relatively little investigated. Li et al. (1998) Skin contamination reported an increase in sister chromosome ex- Local effects at the site of contamination include change (SCE) in the lymphocytes of victims of sweating and local fasciculation. Fasciculation the Tokyo sarin disaster. Because of the probabil- may spread to involve whole muscle groups, ity that the victims were exposed to by-products while the onset of systemic symptoms and signs of sarin synthesis, diisopropyl methylphospho- is generally slower than after vapour exposure. nate, diethyl methylphosphonate and isopropyl ethyl methylphosphonate, these were also stud- ied. The frequency of SCE was determined in hu- Ingestion man lymphocytes exposed to these by-products: all three compounds increased the frequency of Ingestion of nerve agent may occur from con- SCE compared with controls. Sarin and soman taminated food or water. Colicky pain occurs, FYX FYX

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together with nausea, vomiting, diarrhoea and (see reviews by Costa et al., 2002,2003,2005). involuntary defecation, would be expected. A connection between this polymorphism and Gulf war syndrome has been suggested based on epidemiological studies (Haley et al., 1999) as it has with ill-health in UK sheep farmers SUB-GROUPS PARTICULARLY and shepherds, using OP sheep dips (Cherry LIABLE TO NERVE AGENTS et al., 2005). It should be noted that the PON1 192 polymorphism (and the 55 polymorphism) Most work on nerve agents has been directed have been associated with ill-health outcomes at management of poisoning in military person- independent of OP exposure, including coro- nel, particularly soldiers; soldiers are generally nary heart disease (Shih et al., 2002); this physically fit and young. With the possibility may confound some of the studies mentioned of terrorist use of chemical weapons in public above. There are several variants of butyryl- places, other groups need to be considered. De- cholinesterase, and individuals are known who velopmental neurotoxicity and nerve agents has completely lack plasma butyrylcholinesterase already been discussed (see above) and in this re- activity (¯stergaard, 1992). It has been sug- spect (and possibly others) the fetus, infant and gested that in nerve agent poisoning butyryl- young child might represent a susceptible sub- cholinesterase acts as a ‘sink’ for nerve agents. group. It has already been noted that four preg- If that is the case it would be expected that nant women were exposed to sarin in the Tokyo those with low or absent butyrylcholinesterase disaster and that all had normal offspring. Nev- activity would be more susceptible to nerve ertheless, it must be recognized that OP pesti- agents. cides are embryo- and fetocidal and OP pesti- cide exposure of mothers has been associated with effects including cardiac defects in offspring LABORATORY INVESTIGATIONS (see review by Pelkonin et al., 2005). In a mass- casualty situation with a single high-dose expo- Measurement of cholinesterase activity sure, it is likely that the outcome for the em- bryo/fetus would be largely determined by the The cornerstone of laboratory diagnosis of nerve outcome for the mother. agent poisoning is measurement of enzyme The elderly also represent a potentially suscep- activity. Numerous methods are available for tible sub-group to toxicants (Stevenson, 1990) determination of both acetylcholinesterase and including nerve agents; the reasons for this are butyrylcholinesterase, most of which measure numerous but may include impaired organ func- catalytic activity (see St Omer and Rottinghaus tion, most notably, in acute intoxication, poor car- (1992) and Swaminathan and Widdop (2001). Of diovascular and respiratory status. Furthermore, these, the Ellman method (Ellman et al., 1961) decreased hepatic and renal function may impair and its subsequent modifications are most widely metabolism and excretion of nerve agents (and used. Plasma butyrylcholinesterase activity cor- also antidotes). Other groups, those with chronic relates badly with brain acetylcholinesterase conditions such as diabetes, may represent chal- activity and is best thought of as a marker lenges particularly in management. of poisoning rather than a prognostic indica- In healthy people, variation in activity of tor, although in certain circumstances it may metabolizing enzymes, including that due to have a role in cholinergic neurotransmission polymorphisms, may cause differences in sus- (See review by Casida and Quistad, 2004). ceptibility. One enzyme that has received a lot of Even activity of the preferred red cell acetyl- attention is paraoxonase 1 (PON1). The Q-type cholinesterase correlates poorly with central allozyme (Gln192) of PON1 hydrolyzes sarin and nervous acetylcholinesterase activity, seriously soman more effectively than type R (Arg192) limiting the use of the former to assess the (Costa et al., 1999,2005). The clinical signifi- severity of poisoning (Jimmerson et al., 1989; cance of the PON1 192 polymorphism remains Karalliedde, 2002). The reasons for this are to be established in respect of nerve agents complex. Butyrylcholinesterase is a different FYX FYX

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enzyme than acetylcholinesterase, with different acetylcholinesterase. Hydrolysis of the reaction substrate specificities and with different rates of product produces an alkylphosphate or alkyl- inhibition, reactivation and ageing from acetyl- phosphonate. Methods have been developed for cholinesterase. Butyrylcholinesterase is predom- measuring the VXÐacetylcholinesterase hydrol- inantly synthesized in the liver and activity is af- ysis product, O-ethyl methylphosphonic acid, fected by conditions such as liver disease and and the sarinÐacetylcholinesterase hydrolysis a large number of other factors, including the product, O-isopropoxy methylphosphonic acid existence of inherited variants with different or (IMPA)(Noort et al., 1998) (for review, see Noort even no enzymatic activity (¯stergaard et al., et al., 2002). IMPA and methylphosphonic acid 1992; Swaminathan and Widdop, 2001). These were detected in patients from the Matsumoto factors present difficulties with using butyryl- sarin exposure (Nakajima et al., 1998). An al- cholinesterase in the diagnosis and management ternative is to release isopropyl methylphos- of nerve agent poisoning (see discussion by phonylserine or methylphosphonoserine from Brock and Brock, 1993). The activity of butyryl- the active site of inhibited erythrocytic acetyl- cholinesterase in an individual poisoned with cholinesterase and measure the sarin hydrolysis an OP is, at any time, a function of inhibition, product and this was done on some victims of the spontaneous reactivation and resynthesis, with Tokyo sarin exposure (Nagao et al., 1997). An- the added possibility of ageing, all occurring other approach is to release the inhibitor bound concurrently. It should be noted that the rate to butyrylcholinesterase within the enzymeÐ constants for these processes are different for inhibitor complex, using fluoride and measure butyrylcholinesterase and acetylcholinesterase the resulting phosphofluoridate using gas chro- in any organism, as the two enzymes are not the matography (van der Schans et al., 2004). same gene product. Acetylcholinesterase activity Black et al. (1999) found that sarin and so- is the sum of the same processes, but there are two man bind to a tyrosine residue in human plasma important additional considerations, i.e. (1) it is and suggested that this could form the basis of a the same gene product as acetylcholinesterase in biomarker of exposure to these agents. the nervous system (the enzyme of interest), and (2) resynthesis cannot occur in the erythrocyte and the t1/2 for recovery here is consequently the DIAGNOSIS POST-MORTEM same as the half-life of the red blood cell, while resynthesis can occur at other sites, and may not There may be signs of the acute cholinergic cri- be insubstantial (Wehner et al., 1985). Addition- sis preceding death, e.g. excess secretions in the ally, the enzymes in the plasma and red blood form of foam around the nose. Furthermore, de- cell may be more accessible to inhibitor than vices may have detected nerve agents in the atmo- that in the nervous system, especially the cen- sphere or on surfaces. Diagnosis of acute nerve tral nervous system (for discussion, see Marrs, agent poisoning at autopsy would be made dif- 2001 and Karalliedde, (2002). It is unlikely that ficult because of the paucity of histopathologi- a peak erythrocyte acetylcholinesterase depres- cal changes that would be present in the nervous sion of 30% or less would produce clinical signs system. Cerebral edema may be present. One of or symptoms, and below 40% any signs would the few histopathological changes to be expected be expected to be very mild. post-mortem would be changes in skeletal mus- George et al. (2003) have studied the use of an- cle, sometimes called ‘segmental myopathy’. If tisera to distinguish between native and inhibited biological fluids could be obtained, marked in- acetylcholinesterase, providing a basis for mea- hibition of acetyl- and butyrylcholinesterase ac- suring biomarkers of OP, including nerve agent, tivity would be present and, depending on the exposure. nerve agent involved, urinary alkylphosphates might be present if the patient has survived any length of time (see above). Histochemistry has Alkylphosphates been used to diagnose OP pesticide poisoning OPs, both pesticides and nerve agents, lose (Petty, 1958) and could doubtless be used to de- their leaving groups when they react with tect nerve agent poisoning, although it is, at most, FYX FYX

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semiquantitative (Marrs and Bright, 1992). Important measures in prevention of military Methods may be available to measure the nerve casualties include adequate detection measures, agent itself in body fluids. Measurement of physical protection, chemical prophylaxis and cholinesterase activity and demonstration of in- proper treatment. In the terrorist context, proper hibition is not, of course, specific for particular emergency planning is key. Treatment of poison- types of OP, whereas alkylphosphates may be ing by organophosphorus nerve agents is dealt (see review by Ballantyne, 1992). with in other chapters in this volume.

REFERENCE REFERENCES DOSES/HEALTH-BASED GUIDANCE VALUES FOR NERVE AGENTS Aldridge WN (1950). Some properties of specific cholinesterase with particular reference to the In the past, the notion of reference doses for mechanism of inhibition of diethyl p-nitrophenyl nerve agents would have been thought a lit- thiophosphate (E605) and analogues. Biochem J, tle preposterous. However, chronic reference 46, 451Ð460. doses/acceptable daily intakes have been estab- Aldridge WN, Barnes JM and Johnson MK (1969). lished for chemical warfare agents, inter alia the Studies on delayed neuropathy produced by some nerve agents, soman, sarin, tabun and VX for ex- organophosphorus compounds. Ann NY Acad Sci, posure by the oral route. This was at the behest 160, 314Ð322. of the US Army, which asked the National Re- Anderson RJ and Dunham CB (1985). Electrophys- iologic changes in peripheral nerve following re- search Council to review the US Army’s interim peated exposure to organophosphorus agents. Arch reference doses (Bakshi et al., 2000; Subcom- Toxicol, 58, 97Ð101. mittee on Chronic Reference Doses for Selected Anderson DR, Harris LW, Chang F-C et al. (1997). Chemical-Warfare Agents, 2000aÐg). This was Antagonism of soman-induced convulsions by mi- chiefly a result of the consideration of environ- dazolam, diazepam and scopolamine. Drug Chem mental contamination at various sites in the USA, Toxicol, 20, 115Ð131. but such reference doses could also be useful in Annau Z (1992). Neurobehavioral effects of deciding detection limits that would be needed organophosphorus compounds. Organophos- where food was contaminated with nerve agents, phates, Chemistry, Fate and Effects (JE Chambers either accidentally or deliberately. As many ef- and PE Levi, eds), pp. 419Ð432. San Diego, CA, fects of nerve agents are not route- or pathway- USA: Academic Press. Anthony JS, Haley M, Manthei J et al. (2004). In- specific, these reference doses could probably be halation toxicity of cyclosarin (GF) vapour in rats applied to other routes of exposure. The data used as a function of exposure concentration and dura- to calculate these reference doses, in comparison tion: potency comparison to sarin (GB). Inhalation with dossiers available for regulated substances, Toxicol, 16, 103Ð111. such as pesticides, are deficient. There was a Anzueto A, Berdine GG, Moore GT et al. (1986). lack of long-term studies, and with some nerve Pathophysiology of soman intoxication in primates. agents, multigeneration and developmental tox- Toxicol Appl Pharmacol, 86, 56Ð68. icity studies. Furthermore, there were problems Ariens AT, Wolthuis OL and van Bentham RMJ with study design, in terms of numbers of an- (1969). Reversible necrosis at the end plate re- imals and good laboratory practice. Allowance gion in striated muscles of the rat poisoned with was made for these problems by the use of extra cholinesterase inhibitors. Experientia, 1, 57Ð59. Bajgar J, Sevelov«ˇ a L, Krejˇcov«aGet al. (2004). Bio- uncertainty factors. chemical and behavioural effects of soman va- pors in low concentrations. Inhal Toxicol, 16, 497Ð507. CONCLUSIONS Bakri NMS, El-Rashidi AH, Eldefrawi AT et al. (1988). Direct actions of organophosphate anti- The organophosphate nerve agents continue cholinesterases on nicotinic and muscarinic actyl- to pose major problems in chemical defence. choline receptors. J Biochem Toxicol, 3, 235Ð239. FYX FYX

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Bakshi KS, Pang SNJ and Snyder R (2000). Introduc- Braitman DJ and Sparenborg S (1989). MK-801 pro- tion to the special issue. J Toxicol Environ Health tects against seizures induced by the cholinesterase A, 59, 283Ð288. inhibitor soman. Brain Res Bull, 23, 145Ð148. Ballantyne B (1992). Forensic aspects of acute an- Brezenoff HE, McGee J and Knight V (1984). The ticholinesterase poisoning. In: Clinical and Ex- hypertensive response to soman and its relation to perimental Toxicology of Organophosphates and brain acetylcholinesterase inhibition. Acta Phar- Carbamates (B Ballantyne and TC Marrs, eds), macol Toxicol, 55, 270Ð277. pp. 618Ð622. Oxford: Butterworth-Heinemann. Bright JE, Inns RH, Tuckwell NJ et al. (1991). A histo- Ballantyne B and Marrs TC (1992). Overview of the chemical study of changes observed in the mouse biological and clinical aspects of organophosphates diaphragm after organophosphate poisoning. and carbamates. In: Clinical and Experimental Human Exp Toxicol, 10, 9Ð14. Toxicology of Organophosphates and Carbamates Brimblecombe RW (1974). Drug Actions in Cholin- (B Ballantyne and TC Marrs, eds), pp. 1Ð14. ergic System, pp. 64Ð132. New York: MacMillan. Oxford: Butterworth-heinemann. Brimblecombe RW, Green DM, Stratton JA et al. Barak D, Ordentlich A, Kaplan D et al. (2000). Ev- (1970). The protective actions of some anticholin- idence for PÐN bond scission in phosphoramidate ergic drugs in sarin poisoning. Brit J Pharmacol, nerve agent adducts of human acetylcholinesterase. 39, 822Ð830. Biochemistry, 8, 1156Ð1161. Britt JO, Martin JL, Okerberg CV et al. (2000). Beesley WN (1994). Sheep dipping, with special ref- Histopathologic changes in the brain, heart and erence to the UK. Pesticides Outlook, February, skeletal muscle of rhesus macaques ten days af- 16Ð29. ter exposure to soman (an organophsophorus nerve Benschop HP,Bijleveld EC, de Jong LPA et al. (1987). agent). Comp Med, 50, 133Ð139. Toxicokinetics of the four stereoisomers of the Brock A and Brock V (1993). Factors affect- nerve agent soman in atropinized rats Ð influence ing interindividual variation in human plasma of a soman simulator. Toxicol Appl Pharmacol, 90, cholinesterase activity: body weight, height, sex, 490Ð500. genetic polymorphisms and age. Arch Environ Berry WK and Davies DR (1970). Use of car- Contam Toxicol, 24, 93Ð99. bamates and atropine in the protection of ani- Brown MA and Brix KA (1998). Review of health mals against poisoning by 1,2,2-trimethylpropyl consequences from high-, intermediate- and low- phosphonofluoridate. Biochem Pharmacol, 19, level exposure to organophosphorus nerve agents. 927Ð934. J Appl Toxicol, 18, 393Ð408. Black RM, Harrison JM and Read RW (1999). The in- Burchfiel JL, Duffy FH and Sim van M (1976). Per- teraction of sarin and soman with plasma proteins: sistent effects of sarin and dieldrin upon the pri- the identification of a novel phosphonylation site. mate electroencephalogram. Toxicol Appl Pharma- Arch Toxicol, 73, 123Ð126. col, 35, 365Ð379. Boskovi«c B, Kovacevi«c V and Jovanovi«c D (1984). Burgen ASV and Hobbiger F (1951). The in- 2-PAM chloride, H16 and HGG 12 in soman hibition of cholinesterases by alkylphosphates and tabun poisoning. Fund Appl Toxicol, 4, and alkylphenophosphates. Br J Pharmacol 106Ð115. Chemother, 6, 593Ð605. Bourne Y, Taylor P and Marchot P (1995). Acetyl- Cabal J, Kuca K and Kassa J (2004). Specification cholinesterase inhibition by fasciculin, crystal of the structure of oximes able to reactivate tabun- structure of the complex. Cell, 83, 503Ð512. inhibited acetylcholinesterase. Basic Clin Pharma- Bourne Y, Taylor P, Bougis PE et al. (1999). Crystal col Toxicol, 95, 81Ð86. structure of mouse acetylcholinesterase. A periph- Casida JE and Quistad GB (2004). Organophos- eral site-occluding loop in a tetrameric assembly. phate toxicology: safety aspects of nonacetyl- J Biol Chem, 274, 2963Ð2970. cholinesterase secondary targets. Chem Res Toxi- Bowman WC (1993). Physiology and pharmacol- col, 17, 983Ð997. ogy of neuromuscular transmission, with spe- Chambers HW (1992). Organophosphorus com- cial reference to the possible consequences of pounds: an overview. In: Organophosphates, prolonged blockade. Intensive Care Med, 19, Chemistry, Fate and Effects. (JE Chambers and PE S45ÐS53. Levi, eds), pp. 3Ð18. San Diego, CA, USA: Aca- Boyd RT (1997). The molecular biology of neuronal demic Press. nicotinic acetylcholine receptors. Crit Rev Toxicol, Chang F-CT, Foster RE, Beers ET et al. (1990). Neu- 27, 299Ð318. rophysiological concomitants of soman-induced FYX FYX

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