Bull. Org. mond. Sante |1971, 44, 289-307Bull. Wld Hlth Org.

Cholinesterase Inhibition by OrganophosphorusCompounds and its Clinical Effects*

TATSUJI NAMBA1

The clinical manifestations of acute poisoning by organophosphorus compounds in manare in accord with, initially, the stimulation and, later, the blocking of cholinergic trans-mission due to acetylcholinesterase inhibition. The manifestations involve mainly the para-sympathetic nerves, the neuromuscular junctions, and the central nerve synapses, and toa smaller degree the cholinergic sympathetic nerves. Miosis and muscle fasciculationsare useful signs for diagnosis and for the control of therapy. Blood cholinesterase deter-mination is the best diagnostic test. The cause of death is usually respiratory paralysis.Persistent manifestations have not been confirmed. Atropine and pralidoxime are effectivefor treatment and useful for diagnosis. Other oximes are promising but their clinicalvalue has not been established. Poisoning by malathion is characterized by a prolongedcourse and by motor signs. Poisoning by organophosphorus compounds in man differsfrom animal experiments in several ways: in man, exposure may occur by several differentroutes, the manifestations are detected more easily, and therapy is given throughout thecourse of illness.

The introduction during the Second World Warof new insecticides, particularly chlorinated hydro-carbons and organophosphorus compounds, markeda rapid technological advance. DDT is representa-tive of chlorinated hydrocarbons, and it has beenwidely used in agriculture, forestry, vector control,and for other purposes, with little apparent toxiceffect on humans and other vertebrates. On theother hand, organophosphorus insecticides have fre-quently caused poisoning in humans and otheranimals. Many of them are highly toxic; indeed,the organophosphorus compounds were initiallydeveloped as chemical warfare agents.However, the potential and persistent hazards of

DDT and other chlorinated hydrocarbon insecticideshave become evident in recent years. These com-pounds are stable, and are found unchanged ubi-quitously in soil, air, and water (including rain) in

* Supported by US Public Health Service Grant NS 03464from the National Institutes of Neurological Dircases andStroke, National Institutes of Health.

1 Director, Neuromuscular Disease Laboratories, Mainro-nides Medical Center, and Associate Professor of Medicine,State University of New York Downstate Medical Center,Brooklyn, N.Y., USA. Requests for reprints should beaddressed to Tatsuji Namba, M.D., Ph.D., MaimonidesMedical Center, 4802 Tenth Avenue, Brookl3n, N.Y.11219, USA.

almost all areas of the earth (Crosby, 1969). Theyaccumulate particularly in the adipose tissue of manand other animals. DDT has been found in penguinsand seals in the Antarctic, where the compound hasnever been used. Moreover, the concentration ofchlorinated hydrocarbons increases in each step ofthe food chain. The decline of populations of wildanimals, birds, fish, and useful insects has beenattributed to the use of these compounds.

Since they are at the highest end of the food chain,humans also accumulate chlorinated hydrocarboninsecticides. In the USA, the concentration of DDTand its metabolites is about 11 ppm in the adiposetissue of the general population (Durham, 1969),4 ppm in necnates (Zavon et al., 1969), and 0.08-0.13 ppm in human milk (Durham, 1969). Althoughthere is little evidence at present that DDT or otherchlorinated hydrecartons in human tissues is harm-ful, and although scme studies have indicated thatthe concentration has teen ccnstant for years, thereis concern that it may eventually beccme unbearablyhigh as these stable ccmpcunds further accumulateon the earth. Chlorinated hydrocarbcns are neuro-toxic, and high concentraticns in the nervous tissuesmay he attained when the adipose tissue is mobil-ized-fcr example, in disease or starvation states

2641 - 289 -

290 T. NAMBA

(Durham, 1969). In conjunction with the generalawareness of ecological problems, measures havebeen taken to restrict the use of chlorinated hydro-carbon insecticides in Canada, Italy, New Zealand,Sweden, the United Kingdom, the USA, and theUSSR.New methods have been explored in order to find

alternatives to DDT and other chlorinated hydro-carbons for insect control, but the practical methodat present is the use of organophosphorus insecti-cides, which are rapidly hydrolysed after applicationand do not accumulate on the earth or in humantissue. However, organophosphorus insecticides usedas substitutes have already caused poisonings anddeaths.

TOXICITY OF ORGANOPHOSPHORUS COMPOUNDS

The toxicity of organophosphorus compounds ismainly, if not entirely, due to their inhibition ofcholinesterase. These compounds are absorbed intothe human body through all possible routes, includ-ing the skin, lungs, gastrointestinal tract, and con-junctiva; they may also enter the body by injection,although this is of rare occurrence. Some of them areused medically for the treatment of glaucoma and(rarely) myasthenia gravis. The most toxic of theorganophosphorus compounds are the chemical war-fare agents, whose oral lethal dose for man is esti-mated to be in the milligram range or less.Of the organophosphorus insecticides, parathion

and malathion are the most widely used. Parathion(O,O-diethyl-O-p-nitrophenyl phosphorothioate) isone of the most toxic, and most organophosphorus-insecticide poisonings have been caused by thiscompound. In volunteers, an oral intake of 0.07 mgof parathion per kg of body-weight did not causeany manifestations (Eicken, 1954); 0.1 mg/kg pro-duced uneasiness, warmth, tightness of the abdomen,and frequent urination, with a reduction in cholines-terase levels of 3% in plasma and 12% in wholeblood in one subject and 5 % in plasma in anothersubject (who showed no symptoms); and 0.4 mg/kgresulted in increased peristalsis, tightness of thechest, and a reduction in cholinesterase levels of69% in plasma and 47% in whole blood (Takahashi,1956). The last case appeared to be on the vergeof developing manifest poisoning. The dermal appli-cation of 1 g or 2 g of parathion for 4 hours produceda 10-20% reduction in plasma cholinesterase butno symptoms (Ueda, 1957). Death has occurredfollowing the ingestion of 900 mg, 120 mg, and

50 mg of parathion by adults and 2 mg by childreni(Hayes, 1963). The oral lethal dose of parathion isestimated to be 1.43 mg per kg of body-weight-that is, 100 mg for a man weighing 70 kg (DuBois,1958).Malathion (diethyl mercaptosuccinate S-ester with

O,O-dimethyl phosphorodithioate) is one of theleast toxic organophosphorus insecticides. The inges-tion of 16 mg of malathion daily for 47 days byvolunteers did not cause symptoms or affect bloodcholinesterase, while the ingestion of 24 mg dailyfor 56 days caused a 25% decrease in blood cholin-esterase (Moeller & Rider, 1962). There were nosymptoms and there was no decrease in blood cholin-esterase following the dermal application of 1 %,5% or 10% malathion dust 5 times weekly for8-16 weeks (Hayes et al., 1960), or following expo-sure to air containing 0.15 g, 0.6 g, or 2.4 g ofmalathion per 1000 ft3 84 times in 42 consecutivedays (Golz, 1959). Death has occurred followingthe ingestion of about 5 g, 25 g, 35 g, 70 g, or 60-90 g,and severe poisoning following the ingestion of 15 g,25 g (Namba, Greenfield & Grob, 1970), 35 g(Mathewson & Hardy, 1970), 30 g, or 35-50 g(Windsor, 1968). Poisoning probably caused by thedermal absorption of malathion has been described,although it seems exceptional in view of the lowtoxicity of the compound. The estimated oral lethaldose of malathion, 858 mg per kg of body-weight,is less than the lethal dose of DDT, 429 mg/kg(DuBois, 1958), or even aspirin, 400-500 mg/kg(Dreisbach, 1969). If malathion is administeredwith EPN t another organophosphorus insecticide,potentiation occurs in experimental animals, but inman there is an additive effect (Moeller & Rider,1962).

OCCURRENCE OF POISONING BYORGANOPHOSPHORUS INSECTICIDES

Poisoning has frequently resulted from the agri-cultural use of organophosphorus pesticides, thecompounds usually having been absorbed dermallyor by inhalation during application or during sub-sequent work in the fields. A high incidence ofpoisoning was reported among pilots who engagedin the aerial application of these compounds, becauseeven a mild symptom-e.g., blurred vision-resultedin aircraft accidents or because even a minor accidentcaused exposure of the pilot to concentrated prepara-

f Names against which this symbol appears are identifiedin the Glossary on pages 445-446.

CLINICAL EFFECTS OF CHOLINESTERASE INHIBITION BY OP COMPOUNDS

tions of organophosphorus compounds (Reich &Berner, 1968). Industrial poisoning in plants syn-thesizing these compounds occurred during theearly stages of their development, but it is nowgenerally rare since the manufacturing processesare supervised by specialists. Accidents occur morefrequently in the formulation plants where con-centrated preparations of organophosphorus com-pounds are diluted with solvents, emulsifiers, dusts,or other vehicles. Formulation plants are morenumerous than synthesizing plants, the processesinvolve steps that may result in greater exposures,and seasonal insecticide demands may necessitatethe employment of unskilled temporary workers.Poisoning has occurred among industrial or scientificresearch workers, particularly with new compoundsof unknown toxicity. Poisoning during vector controloperations has been rare, since such operations arecarried out under the supervision of experts andcompounds of low toxicity are generally used. Poi-soning of persons and animals not involved in spray-ing operations has been reported occasionally, butonly when highly toxic compounds were involved.The spraying of a field with parathion led to theappearance of this pesticide in a neighbouring drink-ing-water well. In Utah, USA, 6 000 sheep died fromexposure to organophosphorus warfare gas thatdrifted after aerial spraying during a military exercise(Boffey, 1968). Food that had been sprayed withorganophosphorus insecticides immediately beforeharvest caused poisoning in only a few persons livingin the neighbourhood, since hydrolysis of thesecompounds results in a rapid loss of toxicity. How-ever, the contamination of flour or barley with con-centrated parathion during shipment caused out-breaks of poisoning involving hundreds of cases inColombia, India, Mexico, Singapore, and the UnitedArab Republic. Accidental poisoning has occurredmore often in children than adults, particularly infarm communities. The cause has often been un-known. Children have absorbed organophosphorusinsecticide dermally or orally while playing with acontainer or spray machine or while playing in anarea to which the insecticides have been applied.Adults have ingested these insecticides by mistakefor liquor, fruit juice, or remedies for toothache orcough, and have applied them externally for bodylice, fleas, or skin diseases. Clothes contaminated byparathion during shipment have caused poisoningin the wearers. Most household insecticides andthose used by professional exterminators for residen-tial areas contain organophosphorus compounds,

and their use has resulted in poisoning. Such com-pounds have been used for suicide to a considerableextent, in areas where the suicide rate is high andwhere the compounds are readily available, and themortality rate has been high. The use of organo-phosphorus compounds for homicide has also beenreported.The numbers of cases of parathion poisoning in

Japan in 1960-69 were as follows: poisoning result-ing from application of the insecticide, 2059 cases(110 deaths); accidental poisoning, 113 cases (56deaths); and suicidal poisoning, 3 243 cases (3 040deaths). In California, USA, there were 950 casesof poisoning by organophosphorus compounds dur-ing 1957-60 (789 agricultural, 91 industrial, and 70from other causes). In Dade County, Fla., there were90 cases in 1964-67, 24 of them occupational(1 death), 44 of them accidental (10 deaths), and 22of them suicidal (16 deaths) (Reich et al., 1968).In Denmark 273 fatal poisonings by organophos-phorus compounds were recorded from 1957 to1962-6 accidental, 263 suicidal, and 4 homicidal(Frost & Poulsen, 1964).Even malathion, a compound of low toxicity, has

caused substantial numbers of poisonings. In Japanduring 1957-69 there were 100 poisonings with21 deaths resulting from occupational handling oraccidents and 1024 poisonings with 844 deathsresulting from suicidal or homicidal attempts. InBritish Guiana 3 deaths by accident and 43 deathsby suicide occurred during 1959-64 (Mootoo &Singh, 1966). In Denmark 3 of the 273 fatal organo-phosphorus-compound poisonings were cases ofsuicide with malathion (Frost & Poulsen, 1964).

Legislation and campaigns to promote the safeuse of these compounds have effectively reduced thenumbers of poisonings. In Japan during 1953 and1954 there were 3 949 cases of parathion poisoning.Following legislation governing the application andhandling of parathion and a nation-wide campaignon its proper use, the number of cases of poisoningwas reduced to 2 194 in 1960-61, but it still accountedfor 72% of all poisonings by pesticides. Furtherrestriction of the use of parathion reduced thenumber of cases of poisoning to 753 in 1965-66-34% of the total number of poisonings by pesticides.

PHYSIOLOGY OF POISONING BY

ORGANOPHOSPHORUS COMPOUNDS

Organophosphorus compounds are powerful in-hibitors of carboxylic ester hydrolases, including

291

T. NAMBA

acetylcholinesterase (acetylcholine acetyl-hydrolase,3.1.1.7), which is present in human erythrocytes,nerves, synapses, and skeletal muscle; and choline-sterase (acetylcholine acyl-hydrolase, 3.1.1.8), presentin human plasma (serum) and liver. These esterasesare differentiated by (1) substrate specificity (acetyl-cholinesterase hydrolyses acetyl-f-methylcholine butvery little benzoylcholine, propionylcholine, or bu-tyrylcholine, while the opposite occurs with cholin-esterase); (2) selective inhibition; and (3) substrateinhibition (acetylcholinesterase is inhibited by acetyl-choline concentrations equal to or greater than4 mM or higher and cholinesterase by concentra-tions greater than 100 mM). Organophosphorusinsecticides generally inhibit both enzymes.

Non-synaptic acetylcholinesterase (AChE) is pos-tulated to maintain excitability and to initiate andpropagate action potentials in nerve and muscle byregulating passive and active transport of electrolytes.In patients with paroxysmal nocturnal haemoglobin-uria, erythrocyte AChE activity is low, particularlyin erythrocytes that are readily lysed upon the addi-tion of complement (Kunstling & Rasse, 1969).However, no signs or symptoms are found in familialreduction of erythrocyte AChE (Johns, 1962). Cholin-esterase (ChE) is considered to excite cardiac andsmooth muscles locally, and to provide free cholinein acetylcholine synthesis by hydrolysing butyryl-choline (Clitherow et al., 1963) or by dissociatingconjugated choline (Funnel & Oliver, 1965). How-ever, there was no clinical manifestation in twofamily members who had a complete lack of plasmaChE (Hodgkin et al., 1965). Plasma ChE is syn-thesized in the liver, and its activity is a sensitiveindicator of liver function. The syndrome of genetic-ally inherited qualitative change of plasma ChEproduces hypersensitivity to succinylcholine, a cholin-esterase inhibitor used as a muscle relaxant in anaes-thesia, but patients with this syndrome do not haveany other symptoms. Therefore, the effect of organo-phosphorus compounds is primarily, if not entirely,explained by the inhibition of AChE at the cholin-ergic synapses.At the cholinergic synapses, acetylcholine is

released from the nerve ending as the neurohumoraltransmitter in response to the nerve impulse, andinitiates excitation by reacting with the receptor(Fig. 1 and 2). Acetylcholine is then hydrolysed,as follows: (1) the acetylcholine is bound to AChE,the quaternary nitrogen to the anionic site and thecarboxyl group to the esteratic site, forming a sub-strate-enzyme complex; (2) choline is split off,

Fig. 1Reactions of acetylcholinesterase

Ac-Ch + - A jCh I + Ch - J + Ac +Ch

Ph-Or + PAR_ rq - + Or + Ph + Or

PAM

Ph-Or + + Or p + PAM-Ph + Or

First row: acetylcholine hydrolysis by AChE. Second row:inhibition of AChE by organophosphorus compound (the dotted-line arrow indicates the practically negligible progress of thisreaction). Third row: reactivation of phosphorylated AChE bypralidoxime.

Ac = acetic acid. Ac-Ch = acetylcholine. AChE = acetylcho-linesterase. Ch = choline. Or = organic residue of organophosphorus(OP) compound. PAM = pralidoxime. PAM-Ph = phosphorylatedpralidoxime. Ph = phosphate group of OP compound. Ph-Or = OPcompound.

leaving acetylated AChE; and (3) the latter reactswith water, dissociating into acetic acid and activeAChE. This reaction is completed rapidly and thesynapse becomes ready for the next impulse. Incases of poisoning, AChE is bound with the organo-phosphorus compound, and its organic residue dis-sociates, leaving the phosphate group bound with

Fig. 2Transmission across cholinergic synapses

QY B a:3 ZIAc ChZ

N.C

At-ne

~~~ ~~~~~~~~~E~~~~2A C~~CChEPAN

PAM-Ph

First row: normal transmission by acetylcholine. Second row:poisoning by OP compound. Third row: effect of atropine inpoisoning by an OP!,compound. Fourth row: effect of pralidoximein poisoning by an OP compound.

Ac = acetic acid. At-ne = atropine. Ac-Ch = acetylcholine.AChE = acetylcholinesterase. Ch = choline. N nerve ending.PAM = pralidoxime. PAM-Ph = phosphorylated pralidoxime.Ph = phosphate group of OP compound. R = receptor.

292

CLINICAL EFFECTS OF CHOLINESTERASE INHIBITION BY OP COMPOUNDS

Fig. 3Modification of -the toxicity of organophosphorus

compound in vivo

SkinMucosLung

Blooda Tissue

(Li ver)

U

Chol inergicsynapses

I

Black circles:Tmolecules of the OP compound. Broad verticallines: tissue as indicated. Horizontal lines: penetration of the OPcompound into'the tissues, the thickness of the line indicating thetoxicity.

Fig. 4

Cholinesterase activity of erythrocytes and plasmain patients with mild, moderate, and

severe parathion poisoning'

80-

* The activity is expressed as a percentage of the normal level,measured 3 months after poisoning.

the esteratic site of AChE (Fig. 1 and 2). Since therate at which this phosphorylated AChE dissociatesis so slow as to be practically negligible, organo-phosphorus compounds are said to be " irrever-sible " inhibitors. As the result of AChE inhibition,acetylcholine molecules accumulate at the synapse,initially causing excessive excitation and later leadingto the blockage of synaptic transmission. The inhibi-tion of AChE may be considered to be an extremelyslow enzymatic hydrolysis of the organophosphorus-compound molecule.The toxicity of these compounds in vivo does not

always match their AChE-inhibiting activity in vitro,owing to differences in their metabolism in thebody (Fig. 3). The rate at which they are absorbedand transported varies, depending on the nature ofthe compounds, the vehicle, and the characteristicsof the ports of entry. A substantial proportion ofthe absorbed compound does not reach the choliner-gic synapse because it is bound with non-synapticcholinesterase or is detoxified in the liver or othersites. On the other hand, certain organophosphoruscompounds are converted into more toxic derivativesin vivo. For example, parathion, which inhibits50% of cholinesterase at a 2 x 1O-4 M concentrationin vitro and whose intravenous LD50 for the rat is3 mg per kg of body weight, is converted in the liverinto paraoxon, which inhibits 50% of cholinesteraseat a 10-6 M concentration and whose LD50 for the ratis 0.4 mg/kg (Heath, 1961); malathion is similarlyconverted into malaoxon, whose toxicity to themouse is about 20 times that of malathion (Murphyet al., 1968). On the other hand, both malathion andmalaoxon are detoxified in the liver by malathionase(Murphy, 1967).

Since synaptic AChE activity has been measuredonly experimentally (Namba & Grob, 1970), bloodcholinesterase activity is used to assess the degree ofinhibition of synaptic AChE. Signs and symptomsof poisoning by organophosphorus compounds occurwhen more than 50% of the plasma ChE or erythro-cyte AChE is inhibited (Fig. 4), and therefore thethreshold level of synaptic AChE inhibition is prob-ably about 50%. The recovery of blood cholin-esterase takes about 2 weeks in patients with mildpoisoning. However, the recovery of synaptic AChEappears to be very rapid, since signs and symptomsdisappear within 24 hours in patients with mild ormoderately severe poisoning. Since inhibition byorganophosphorus compounds is " irreversible" therecovery of cholinesterase is probably due to replace-ment and not reactivation. Since the recovery of

m m

293

Fig. 5 The cholinergic synapses, in which acetylcholineCholinesterase activity of blood and cholinergic is the transmitter, include the synapses of the central

synapses in repeated exposures to subtoxic doses of nervous system, neuromuscular junctions, sensoryorganophosphorus compounds* nerve endings, ganglionic synapses of both sympa-

thetic and parasympathetic nerves, postganglionicv W _ lw -w +sympathetic nerve terminals that innervate the sweat

100% glands and blood vessels, sympathetic nerve termin-als (without ganglionic synapses) in the adrenal

Blood cholinesterase medulla, and all postganglionic parasympatheticsox 5_ nerve terminals (Fig. 6). Most of the postganglionic

sympathetic nerve terminals are adrenergic, norepi-nephrine serving as the transmitter. The cholinergicand adrenergic manifestations are generally antagon-

Normal istic. Most of the manifestations of poisoning bySynaptic cholinesterase organophosphorus compounds are in agreement with

jin esteras\ excessive cholinergic action (Table 1). Exceptions-Thieshold ,>e.g., tachycardia and increased blood pressure-areexplained by overwhelming cholinergic effects on the

1 2 3 4 5 6 7 central nervous system, sympathetic ganglionic syn-(days) apses, or adrenal medulla (Paulet, 1954; Dirnhuber

*The arrows at the top of the figureindicate the days of exposure. & Cullumbine, 1955; De Burgh-Daly & Wright,1956; Hornykiewicz & Kobinger, 1956; Polet &De Schaepdryver, 1959).

tissue cholinesterase and detoxifying activity takes along time, repeated exposures to organophosphoruscompounds, even at levels below the toxic dose,decrease these protective mechanisms, resulting inincreasingly greater exposure of synaptic AChE tothe compounds and ultimately producing inhibitiongreater than the threshold level (Fig. 5).

Fig. 6Cholinergic synapses'

Centralnervoussystem

Peripheralnerves

Effectors

Motor nerveSkeletal muscles

Sensory nerve

Sensory terminals

Sympathetic nerve

Most sympatheticreceptorsSweat glandsblood vessels

Adrenal medullaParasympathetic nerve

I| -+All parasympatheticreceptors

* The locations of the synapses are indicated by shading.

SIGNS AND SYMPTOMS OF ACUTE POISONING

The interval between exposure to organophos-phorus insecticides and the onset of signs and symp-toms varies from minutes to hours, but is usuallyless than 12 hours. Manifestations that occur morethan 24 hours after the last exposure usually cannotbe attributed directly to the insecticides.The severity of poisoning is classified by clinical

manifestations and the degree of inhibition of plasmaChE (Table 2; see also the Annex). The classifica-tion serves as a guide for prognosis and therapy.In mild poisoning, the manifestations are predomi-nantly due to the stimulation of parasympatheticnerve endings. Manifestations resulting from thestimulation of other nerve endings appear in moder-ate or severe poisoning. Impulse generation at somesensory endings and their synapses in the centralnervous system is cholinergic, but the sensory signsare not clearly identified.The characteristic central nervous system mani-

festation is disturbance of consciousness, whichoccurs in patients with severe poisoning and mayappear without circulatory or respiratory distur-bance. Mental signs such as anxiety, insomnia,excessive dreaming, and difficulty in concentrationmay occur as prodromal manifestations or after thedisappearance of acute somatic manifestations.

294 T. NAMBA

295CLINICAL EFFECTS OF CHOLINESTERASE INHIBITION BY OP COMPOUNDS

Table 1Adrenergic effects, cholinergic effects, and manifestations of poisoning

by organophosphorus compounds

Organ, tissue, or functionaffected

pupil

accommodation

pulse rate

heart contractility

heart conduction

blood pressure

blood vessels of skin and mucosa

sweating

lachrymal secretion

salivary secretion

nasopharyngeal secretion

bronchial secretion

bronchial muscle

gastrointestinal motility

gastrointestinal secretion

urinary bladder

detrusor

sphincter

adrenal medulla

skeletal muscle

Adrenergiceffects

dilatation

far vision

increase

increase

increase

increase

contraction

(increase)

increase(viscous)

decrease

relaxation

decrease

decrease

relaxation

contraction

Cholinergic Manifestations of poisoningeffects by OP compounds

contraction

near vision

decrease

decrease

decrease

decrease

dilatation

increase

increase

increase(profuse,watery)

increase

increase

contraction

increase

increase

contraction

relaxation

adrenalsecretion

contraction

miosis

blurred vision

tachycardia initially,bradycardia later

generally normal

occasional A-V block

high initially, low later

red initially, may becomepale later

increase

increase

increase

increase

increase

dyspnoea

nausea, vomiting, abdominalpain, diarrhoea

urinary incontinence

high blood pressure,tachycardia

muscle fasciculations, cramp

Miosis and muscle fasciculations are valuable objec-tive manifestations, and are found in about 50% ofthe patients. Muscle fasciculations occur in moder-ate or severe poisoning, particularly in the earlystage, and disappear in later stages either becauseof improvement, with the disappearance of neuro-muscular stimulation, or because of the advance ofpoisoning, with depolarizing neuromuscular block.Miosis occurs in patients with poisoning of anyseverity, generally lasts throughout the course of theillness, and is a good indicator of the effectivenessof treatment. However, miosis and muscle fascicula-tions may not be present even in severe poisoning.The most important manifestation and the usualcause of death is respiratory difficulty caused by

weakness of the respiratory muscles, paralysis of therespiratory centre, bronchospasm, and increasedbronchial secretion. Cardiac manifestations-inclu-ding atrial fibrillation, conduction block, and ventri-cular fibrillation and flutter-may occur, but usuallyin the terminal stage. Recently there have been morereports of cardiac manifestations in poisoning byorganophosphorus compounds because of prolongedcourses resulting from improved respiratory care(Namba et al., 1970; Barckow et al., 1969; Harriset al., 1969; Heitmann & Felgenhauer, 1969; Singhet al., 1969). Tachycardia and increased bloodpressure occur in the initial stage and bradycardiaand decreased blood pressure in the later stages(Fig. 7).

T. NAMBA

Table 2Signs and symptoms in patients with parathion poisoning

No. of patients with the following plasmaTotal no. cholinesterase levels (% of normal) showing signsSign or symptom showing signs listed at left

listed at left 0-10% 11-20% 21-50%

weakness 77 27 25 25

nausea and vomiting 69 25 25 19

excessive sweating 62 23 17 22

headache 61 17 21 23

excessive salivation 51 24 15 12

difficulty in walking 49 27 22 0

dyspnoea 41 24 13 4

miosis 40 23 11 6

muscle fasciculations 38 26 12 0

disturbance in speech 34 22 12 0

fever 31 17 14 0

diarrhoea 30 15 6 9

disturbance of consciousness 29 23 6 0

abdominal pain 26 10 9 7

increased blood pressure 24 19 5 0

loss of pupillary reflex 22 17 5 0

bronchopharyngeal secretion 22 14 8 0

cramps 20 15 5 0

cyanosis 15 15 0 0

* In 77 patients who developed poisoning following the application of parathion or parathion-methyl

Patients with moderate or severe poisoning mayhave a low-grade fever, not related to infection, forabout 1 week (Fig. 8). Hyperglycaemia and glyco-suria are often present in severe poisoning (Fig. 9).Judging from the relatively mild hyperglycaemiaaccompanied by glycosuria, a lowered renal thresholdfor glucose excretion is also present. The absenceof acetone bodies differentiates the condition fromdiabetic coma, except for coma in diabetes resultingfrom hyperosmolarity. Urobilinogen is present in theurine of about 50% of the patients on the first day.In moderate or severe poisoning there is leucocytosis,with a white-cell count of up to 20 000 per mm3 andwith an increased number of neutrophils and adecrease in the proportion of lymphocytes andmonocytes. In cases of severe poisoning, eosino-phils are usually not found on the first day, unless

there has been pre-existing eosinophilic leucocytosis(usually resulting from parasitic or allergic con-ditions).

Local exposure to organophosphorus insecticidesproduces comparatively severe local manifestations.Exposure of the eye causes severe miosis andlachrymation. Dermal exposure may cause copioussweating. Absorption from the respiratory tractmay cause tightness of the chest initially and dys-pnoea and bronchial secretions later. The ingestionof organophosphorus compounds is often followedby severe abdominal pain, diarrhoea, and vomiting.Fortunately, the vomiting reduces the amount ofthe compound that is absorbed.The signs and symptoms and prognoses listed in

the Annex are based on observations of poisoningsthat occurred following field spraying with parathion

296

CLINICAL EFFECTS OF CHOLINESTERASE INHIBII'ON BY OP COMPOUNDS

Fig. 7Systolic blood pressure in 5 patients with severe

parathion poisoning

Fig. 8Course of temperature in a patient with severe

parathion poisoning but without infection

101

U. 1i00

Fl°98

Ie 97 r *_ .Z 3 4 5 6 8 Vu(days)

Fig. 9

Blood and urine glucose in 2 patients with severe

parathion poisoning

-140-0: With positive urinary glucose

120-*\ : With negative urinary glucose

120

100

and parathion-methyl. The situation is different inpatients who have ingested (or injected) a largequantity of an organophosphorus compound; insuch patients, the ingested compound in the gastro-intestinal tract or other tissues is continuouslyreleased, and consequently the course of the poison-ing is long, necessitating continuous treatment. Theresults of studies of extremely toxic organophos-phorus compounds (the warfare gases) and of studieson experimental animals may not always be applic-able to the poisoning of man by organophosphorusinsecticides. For example, only a few minutes afterthe inhibition of AChE by warfare gases the enzymecan no longer be reactivated by pralidoxime, althoughit takes hours to reach this condition when AChEis inhibited by organophosphorus insecticides. Manis more sensitive to organophosphorus compoundsthan are experimental animals and shows signs andsymptoms-particularly central nervous system man-ifestations-earlier than do the latter. Therefore,the quantity of absorbed organophosphorus com-

pound necessary to produce a given effect may berelatively smaller in man.

Prognosis in relation to the severity of poisoningis indicated in the Annex. Untreated parathionpoisoning leads to death within 24 hours of theonset of manifestations: if an untreated patient isalive after 24 hours, he will recover. Patients whoare under treatment may die from 24 hours to1 week after the onset of manifestations.

POISONING BY ORGANOPHOSPHORUSINSECTICIDES OF LOW TOXICITY

The organophosphorus compounds of low tox-icitv are expected to be used more widely as sub-stitutes for chlorinated hydrocarbons. However,only limited information is available on compoundsof this group other than malathion.The toxicity of malathion is due mainly to cholin-

esterase inhibition following the conversion of thecompound to malaoxon in the liver. Pure malathionhas little cholinesterase-inhibiting action, but it mayhave an independent toxic activity, since it inhibitsthe growth and respiration of chicken embryo tissuein culture, whereas malaoxon at the same concen-tration does not (Wilson & Stinnett, 1969).The time of onset of the signs and symptoms of

malathion poisoning ranged from 5 minutes afteringestion to a gradual development over a periodof 6 weeks when there were multiple exposures.Patients with malathion poisoning generally devel-

6 10rs(hours)

-1n

297

:3S.-ei011)wS_a

0

11)

T. NAMBA

oped severe signs and symptoms, since 25 of 30 re-ported patients developed poisoning following theingestion of a large dose of malathion with suicidalintent, or by accident (Namba et al., 1970; Windsor,1968; Harris et al., 1969; Mathewson & Hardy,1970). Of 30 patients, 27 (90%) became comatose,8 (27 %) had convulsions, and 7 (23 %) died. Miosiswas present in 27 patients (90%), but 3 patients didnot show this condition. Muscle fasciculations wereobserved in 7 patients (24%) but not observed in3 patients. There was paralysis of the extremitiesin 6 patients (20%), and loss of tendon reflexesoccurred in 14 patients (47 %). A long-lasting poly-neuropathy was reported in 1 patient. Extensorplantar reflexes were observed in 5 patients. Signsof meningeal irritation occurred in 2 children. Thecourse was protracted (up to 3 weeks) in 9 patients,who showed continuous or intermittent respiratorydifficulty or unconsciousness, indicating continuousabsorption of the compound from the gastrointestinaltract.The cholinesterase activity of whole blood, ery-

throcytes, plasma, or serum was reduced to lessthan 20% of the normal value in 7 patients, butwas 50% of the normal level in whole blood of1 patient and 60% of normal in the serum of anotherpatient. Some patients showed a progressive reduc-tion, over a period of days, of both plasma ChE anderythrocyte AChE levels.

DIAGNOSIS OF ACUTE POISONING BY

ORGANOPHOSPHORUS COMPOUNDS

Diagnosis depends on (1) history or evidence ofexposure to organophosphorus compounds withinthe previous 24 hours, (2) signs and symptoms ofpoisoning (see the Annex), (3) inhibition of thecholinesterase activity of the blood or other tissues,and (4) the effectiveness of atropine and pralidoxime.With most patients, a history or evidence of

exposure to organophosphorus compounds within24 hours before the onset of symptoms can beobtained. The patients may retain a characteristicgarlic-like odour for several days. In gastric aspirateor urine and on the skin or clothing organophos-phorus compounds can be identified by means of gasor thin-layer chromatography, or their presence canbe demonstrated by the inhibition of cholinesteraseactivity in vitro. Metabolites of organophosphoruscompounds-e.g., p-nitrophenol, a metabolite ofparathion, parathion-methyl, Chlorthion, t dicap-thon, t and EPN t -may also be detected in urine

Fig. 1 0Excretion of p-nitrophenol in the urine of 4 patients

with severe parathion poisoning

200

100

1 2 3 4 5 6 7(days)

(Fig. 10). However, a history of exposure and thedetection of organophosphorus compounds or theirmetabolites do not always indicate that signs andsymptoms are due to poisoning by such compounds.For example, cerebrovascular accidents may developduring the use of organophosphorus insecticides.Although p-nitrophenol was detected in the urineof 75 (83 %) of 90 farm workers who had sprayedparathion in fields, none of them had any symptoms,and the p-nitrophenol concentration was not parallelwith the degree of inhibition of serum ChE activity(Namba et al., 1971).

In poisoning by organophosphorus compoundsthe determination of erythrocyte AChE is theoreti-cally preferable, but the determination of plasmaChE is advantageous in that it is simpler. Followingthe administration of pralidoxime, the erythrocyteAChE level indicates its effectiveness; plasma ChEindicates the prior presence of cholinesterase inhibi-tion even after the recovery of erythrocyte AChEactivity as a result of pralidoxime treatment (Namba& Hiraki, 1958) (Fig. 1 1). A finding of normal bloodcholinesterase activity excludes systemic poison-ing by organophosphorus compounds. In acutepoisoning, manifestations generally occur only aftermore than 50% of the plasma ChE is inhibited, andthe severity of manifestations parallels the degree ofinhibition (Table 2). However, this correlation holdstrue only in the initial stage of acute poisoning, andinhibition of the activity remains even after thepatient becomes symptom-free. Following severe

298

CLINICAL EFFECTS OF CHOLINESTERASE INHIBITION BY OP COMPOUNDS 299

Fig. 1 1Erythrocyte and plasma cholinesterase activity in apatient with severe parathion poisoning who was given

pralidoxime intravenously*

- 4 Pralidoxime iodide 1 g x 24

Erythrocyte chol inesterase

413......

26.1

Plasma chol i nesterase

0 1 2 4 6 12 18 24 2 3 4 5 6 7(hours) (days)

Parallel solid lines: range of normal erythrocyte AChE activity;parallel dotted lines: range of normal plasma ChE activity.

poisoning, recovery to the normal levels takes about4 weeks for plasma ChE and about 5 weeks forerythrocyte AChE when pralidoxime is not admin-istered (Fig. 4). Since plasma ChE is relativelystable, samples can be shipped to laboratories ofother institutions. Plasma can be refrigerated for aweek without appreciable loss of ChE activity.The effect of pralidoxime and atropine may be of

help in differential diagnosis. The intravenous injec-tion of 1 g of pralidoxime generally results in somerecovery from signs and symptoms, particularly inparathion poisoning. The failure of 1-2 mg ofatropine administered parenterally to produce signsof atropinization (flushing, mydriasis, tachycardia,or dryness of the mouth and nose) indicates poison-ing by organophosphorus compounds; conversely,the occurrence of these signs casts doubt on thediagnosis, or indicates that the poisoning is of milddegree.

TREATMENT OF ACUTE POISONING BY

ORGANOPHOSPHORUS COMPOUNDS

Treatment consists of (1) the maintenance ofrespiration, (2) the administration of atropine andpralidoxime, (3) the removal of organophosphoruscompounds, and (4) other supportive measures (seethe Annex).The therapeutic action of atropine in poisoning by

organophosphorus compounds results from its bind-

ing with the synaptic receptor replacing acetylcholine,thus blocking synaptic transmission (Fig. 2). Atro-pine may also reduce acetylcholine production atthe nerve terminals (Giarman & Pepeu, 1962; Holm-stedt et al., 1963; Milosevic, 1970). Atropine anta-gonizes organophosphorus compounds by blockingtransmission at the parasympathetic nerve terminals,central nerve synapses, and (when in high doses)autonomic ganglia, but not at the neuromuscularjunctions. The dose of atropine should be sufficientto produce slight signs of atropinization. The sizeof the pupils is a good indicator for regulating thedose of atropine, except for patients who havesuffered local exposure of the eye to organophos-phorus compounds. When neither pralidoxime noratropine is available, parasympatholytic drugs maybe used as substitutes (Namba & Takata, 1957).The largest dose of atropine used by the author was40 mg within 24 hours or 65 mg in 17 days, but adose of 1065 mg was administered to a patientdescribed in the literature (Windsor, 1968).

Pralidoxime counteracts organophosphorus com-pounds by reactivating inhibited synaptic AChE(Fig. 2 and 3). It has been questioned whether thedrug counteracts the central nervous system effectsof these compounds, but recent animal experimentsand the prompt recovery of consciousness by manypatients indicate that it has a definite central nervoussystem effect (Namba et al., 1971).1 The effectivenessof pralidoxime has been established for only alimited number of organophosphorus insecticides.The intravenous injection of 1.0 g of pralidoximeproduces effects in 10-14 minutes (Table 3). Prali-doxime is also effective when administered orally orintramuscularly, but the intravenous route is pre-ferred for prompt effect. In the author's experience,when the first 2 intravenous injections of 1.0 g ofpralidoxime are not satisfactory, continuous infu-sion at rates up to 0.5 g per hour is more effectivethan further repeated single injections of 0.5 g or1.0 g, probably because of the effect on continuouslytransported organophosphorus compounds in theblood (Namba et al., 1959b). For infusion 2.5%pralidoxime solution appears to be more effectivethan diluted solutions. Such extended treatment isusually necessary only when large quantities oforganophosphorus compounds have been ingested.The combined use of pralidoxime and atropine is

most desirable, since the actions of these two drugsare complementary. The initial administration of

' Pralidoxime also crosses the placenta (Edery et al., 1966).

T. NAMBA

Table 3Signs and symptoms in patients with parathion poisoning before and 30 min after

intravenous injection of 0.9-2.0 g of pralidoxime iodide

Total no. of patients No. of patients with the following plasma cholinesterase levelsshowing signs listed (% of normal) showing signs listed at left

Sign or symptomatlf0-10% 11-20 % 21-50%

Before After -____ ______- I______________r___ _e Before After Before After Before After

nausea and vomiting 24 4 8 0 14 3 2 1

pallor 18 5 8 1 7 4 3 0

dizziness 16 3 5 0 9 2 2 1

headache 15 7 3 0 10 6 2 1

excessive salivation 15 0 6 0 7 0 2 0

paraesthesia 14 2 6 0 7 2 1 0

muscle fasciculations 13 0 7 0 6 0 0 0

dyspnoea 13 2 6 0 6 2 1 0

miosis 10 3 4 0 5 3 1 0

impairment of speech 10 2 4 0 6 2 0 0

cramps 9 0 4 0 5 0 0 0

bronchopharyngeal secretion 6 0 2 0 4 0 0 0

disturbance of consciousness 5 0 4 0 1 0 0 0

Total no. of patients I 25 J 8 [ 14 [ 3

1.0-2.0 g of pralidoxime, and the subsequent admin-istration of atropine and additional pralidoxime asdescribed above, is the easiest way to regulate doses.The injection of pralidoxime after the administrationof a large dose of atropine may result in severesigns and symptoms of atropinization, although thiscondition rarely affects the prognosis.The administration of cholinesterase from human

plasma or erythrocytes or from animal sources isineffective, since it does not affect the cholinesteraseactivity of synapses (Hiraki et al., 1956).

Treatment with oximesPralidoxime iodide, 2-formyl-1-methylpyridinium

iodide oxime, was developed for the treatment ofpoisoning by organophosphorus compounds on thebasis of the molecular reactions of AChE (Wilson& Ginsburg, 1955), and was the first compound tobe effective in man (Namba & Hiraki, 1958). Pralid-oxime is also used in the forms of the chloride andthe methanesulfonate. Other oximes that have beenused for treatment include obidoxime chloride, 1,1'-

(oxydimethylene)bis(4-formylpyridinium) dichloridedioxime, and trimedoxime bromide, 1,1'-trimethy-lenebis(4-formylpyridinium) dibromide dioxime(Fig. 12).

In addition to the reactivation of inhibited AChE,the actions of oximes may include prevention of theformation of phosphorylated AChE, which cannotbe reactivated by these compounds; direct detoxifi-cation of organophosphorus compounds; and directaction on the synaptic receptor. High, lethal con-centrations of acetylcholine were found in the brainsof animals that had been given pralidoxime orobidoxime and thus saved from death from poison-ing by organophosphorus compounds, indicatingthat reactivation is not the sole action of theseoximes (Milogevi6, 1969, 1970). Dephosphorylationof the esteratic site by oximes, and therefore thereactivation of the enzyme, become difficult afterseveral hours in cases of inhibition by parathion andother organophosphorus insecticides, and withinminutes in cases of inhibition by sarin, a warfaregas. However, the reactivation of blood cholinester-

300

CLINICAL EFFECTS OF CHOLINESTERASE INHIBMIION BY OP COMPOUNDS

Fig. 12Structural formulae of pralidoxime chloride, obidoxime chloride,

and trimedoxime bromide

ase and a reduction of signs and symptoms havebeen observed in clinical practice even 2 or 3 daysafter the onset of poisoning, probably because newlyinhibited cholinesterase is constantly produced asa result of the continuing absorption of organo-phosphorus compounds from the gastrointestinaltract or other tissues.

Pralidoxime chloride (molecular weight, 173),iodide (molecular weight, 264), and methanesul-fonate (molecular weight, 232) are approximatelyequally effective at equimolar doses (Namba et al.,1959d), but the chloride is preferable since it is moresoluble and produces fewer side-effects than theother compounds.The intravenous LD50 of chloride oximes for mice

are as follows (mg per kg of body-weight): pralid-oxime, 93.6 (542 ,tmoles) (Namba et al., 1959d);obidoxime, 70.0 (195 ,tmoles) (Erdmann & Engel-hard, 1964); and trimedoxime, 57.0 (158 timoles)(O'Leary et al., 1961). At high concentration theseoximes have cholinesterase-inhibiting activity invitro, but neither plasma ChE nor erythrocyte AChEwas inhibited in man following the intravenousinjection of therapeutic doses of pralidoxime iodide,and the inhibition of blood cholinesterase was notmore than 20% following the intravenous injectionof lethal doses of pralidoxime iodide in rabbits(Namba et al., 1958a). Obidoxime appears to havea greater cholinesterase-inhibiting activity than pral-idoxime (Zech et al., 1967).When volunteers were given intravenous injec-

tions of 15-20 mg of pralidoxime iodide or methane-sulfonate per kg of body weight, they experienceddizziness, blurred vision, diplopia, tachycardia, head-

ache, impaired accommodation, or nausea lastingfor several minutes (Jager & Stagg, 1958; Sundwall,1960). In another study no ill effects occurred fol-lowing the intramuscular or intravenous administra-tion of 15-20 mg of pralidoxime chloride per kg ofbody weight (Calesnick et al., 1967). The more

frequent side-effects that have been reported may

be due to contamination of the drugs with aldehydecompounds. Following the ingestion of 1-10 g ofpralidoxime iodide, some volunteers experiencedtension and fatigue in the jaw, a bitter taste, andrhinitis, beginning 30 minutes after intake andlasting about 2 hours (Namba et al., 1958a). Thebitter taste was also experienced with the chloride(Lipson et al., 1969). Pralidoxime chloride producedno side-effects when given orally in a single dose ofup to 7 g (Calesnick et al., 1967; Sidell et al., 1969),or a daily dose of 0.25-4 g given for 3 weeks to6 months, a total of up to 400 g (Calesnick et al.,1967; DeRoetth et al., 1965; Lipson et al., 1969),but the administration of a single dose of 8 g or

more, or of doses of 2-5 g every 4 hours for 48 hours,produced anorexia, malaise, nausea, vomiting, or

diarrhoea (Sidell et al., 1969). One person developedmaculopapular rash (Lipson et al., 1969). In theauthor's experience pralidoxime iodide caused no

side-effects when administered, to treat poisoning byorganophosphorus compounds, in intravenous dosesas large as 40.5 g over a 7-day period, 26 g of thistotal being given during the first 54 hours (Namba etal., 1959b). In one patient with malathion poisoning,the administration of pralidoxime was followed byrespiratory difficulty and cyanosis, but these were attri-buted to the withdrawal of atropine (Richards, 1964).

HC = NOH HC = NOH HC = NOH HC = NOH

N C=NOH N ¢N Cl ¢NICH3 CH2- 0-CH2 CH2- CH2- CH2

pralidoxime obidoxime trimedoximechloride chloride bromide

301

302 T. NAMBA

The intravenous injection of obidoxime in dogswas followed by vomiting at doses of 20-50 mg perkg of body-weight and by generalized weakness atdoses of 50-70 mg/kg (Erdmann & Clarmann, 1963).In 22 volunteers 1 or 2 intramuscular injections of250 mg of obidoxime caused pain at the site ofinjection and transitory feelings of cold, heat, ortension in the nasopharynx, face, or head (Erdmannet al., 1965; Boelcke et al., 1970a). Both pralid-oxime and obidoxime are rapidly excreted in theurine.The intravenous injection of 15 mg and 20 mg

of trimedoxime per kg of body-weight in 2 normalindividuals caused marked hypotension, and, in oneof them, tachycardia (Wills, 1959).The effectiveness of oximes for the treatment of

poisoning by organophosphorus compounds in manwas first demonstrated in 1956 by Namba & Hiraki(1958), who used pralidoxime to treat patients withparathion poisoning. It was fortunate that thesefirst patients had parathion poisoning and showedremarkable recovery after treatment with pralid-oxime, a result that stimulated further work onoximes, since later studies showed that pralidoximeis most effective against poisoning with parathionand parathion-methyl (Namba et al., 1971). Fourpatients with EPN t poisoning and a child whoingested diazinon have also been treated successfullywith pralidoxime (Namba et al., 1959a, 1959b,1959c). Pralidoxime was also reported to be effec-tive for the treatment of poisoning with TEPP(2 patients); dicrotophos, carbophenothion, dichlor-vos, and dimethoate (1 patient each), and, to a lesserdegree, mevinphos (9 patients). The effect was notclearly demonstrated in 1 case of phosphamidonpoisoning, 1 case of azinphos-methyl poisoning,2 cases of demeton-methyl poisoning, 13 cases ofmalathion poisoning (Namba et al., 1970, 1971), and1 case of monocrotophos poisoning (Simson et al.,1969). However, these patients may have been givenan insufficient amount of pralidoxime, late in thecourse of poisoning. Pralidoxime has also beenshown to be effective in man against poisoning bynon-insecticide organophosphorus compounds, in-cluding diisopropyl phosphorofluoridate and eco-thiopate 1 (used in the treatment of glaucoma), andby quaternary ammonium anticholinesterase com-pounds, neostigmine, pyridostigmine, and ambeno-nium (used in the treatment of myasthenia gravis).

1 International nonproprietary name for S-(2-diethyl-aminoethyl)-O,O-diethylphosphorothioate methiodide.

The effectiveness of pralidoxime and trimedoximein experimental animals suffering from poisoning bydifferent organophosphorus compounds has beensummarized by Durham & Hayes (1962) and Ellin& Wills (1964). However, the effect of chemo-therapy in man may be different from that in animals,since animal experiments usually involve a singleadministration of both the organophosphorus com-pound and the therapeutic agent, while in man theorganophosphorus compound may be absorbed bydifferent routes and drugs are administered con-tinuously throughout the course of illness.The effective dose of obidoxime and trimedoxime

is smaller, and their therapeutic index greater, thanthose of pralidoxime when used to treat poisoningby organophosphorus compounds in experimentalanimals (Erdmann & Engelhard, 1964). Obidoximewas effective in experimental animals with poisoningby parathion, paraoxon, diisopropyl phosphoro-fluoridate (Bisa et al., 1964; Erdmann & Engelhard,1964), sarin, tabun, soman (Heilbronn & Tolagen,1965), mevinphos, oxydemeton-methyl, or malathion,but was not effective in poisoning by dimethoate orformothion (Zech et al., 1967). The advantages ofobidoxime over pralidoxime are said to be itsstronger and more rapid reactivation of cholinester-ase in animal experiments, the feasibility of intra-muscular injection of a sufficient therapeutic dosein a small volume (1 ml of 25% solution) (Erdmannet al., 1965), and the ease with which it penetratesthe blood-brain barrier (Erdmann & Clarmann,1963; Erdmann, 1965; Falb & Erdmann, 1969).Twenty-two patients suffering from poisoning by

organophosphorus compounds have been given obid-oxime in amounts ranging from a single dose of250 mg to multiple doses totalling 3.25 g over a22-hour period (Erdmann & Clarmann, 1963; Stau-dacher, 1963; Wohlenberg et al., 1965; Clarmann& Geldmacher-v. Mallinckrodt, 1966; Stoeckel &Meinecke, 1966; Himmel & Sterz, 1968; Klemmet al., 1968; Barckow et al., 1969; Heitmann &Felgenhauer, 1969; Prinz, 1969; Wender & Owsian-owski, 1969; Knolle, 1970; Boelcke et al., 1970b).Of these patients, 10 showed some indications oftherapeutic response, either in signs and symptomsor in blood cholinesterase; 6 of the 10 patients hadattempted suicide by ingesting parathion, 1 patienthad been poisoned by an injection of paraoxon and 1by an injection of fenthion, 1 patient had ingestedan unknown organophosphorus compound, and1 patient had been exposed to demeton and otherinsecticides during spraying in the garden. In two

CLINICAL EFFECTS OF CHOLINESTERASE INHIBITION BY OP COMPOUNDS

patients the whole-blood cholinesterase returned tonormal from levels of zero and 18o% of the normalvalue, respectively; erythrocyte AChE in I patientrecovered from less than 10% to 4000 of the normallevel and in another patient from zero to 80%; andserum ChE in 1 patient recovered from I % to 40%of the normal level. Of the 10 patients, 2 ultimatelydied of parathion poisoning, but showed temporaryimprovement of manifestations. Atropine in dosesfrom 1 mg to 332 mg was given to 9 patients, 1 patientreceived 0.5 g of pralidoxime iodide, and anotherpatient received 1.5 g of pralidoxime iodide and 3 gof pralidoxime methanesulfonate. In 1 patient ex-change transfusions of 3-4 litres had a dramaticeffect. Therefore, the improvement in manifestationsor the recovery of cholinesterase activity in thesepatients may not necessarily have been caused byobidoxime. At most only 3 of these patients receivedunequivocal benefit from the administration of obid-oxime. Twelve patients poisoned by parathion, di-methoate, triamiphos, parathion, demeton, mevin-phos, or PFU-26t received no therapeutic benefit fromobidoxime. In at least 4 patients the administrationof obidoxime did not affect the blood cholinesteraseactivity. Obidoxime is believed to cross the blood-brain barrier more easily than pralidoxime, butprompt disappearance of the central nervous systemmanifestations of poisoning by organophosphoruscompounds has not been described. Of the patientsnoted above, 7 developed cholestasis, generally 1 weekafter the administration of obidoxime, accompaniedby jaundice and elevated serum bilirubin, trans-aminases, and lactate dehydrogenase, although stud-ies in experimental animals and in volunteers indi-cated that cholestasis was caused not by obidoximebut by the organophosphorus compound (Boelcke& Erdmann, 1969; Boelcke & Gaaz, 1970; Boelckeet al., 1970a). From these results, obidoxime doesnot appear at present to be superior to pralidoximefor the treatment of poisoning by organophosphorusinsecticides in man. Obidoxime may be superior topralidoxime for treating poisoning with non-insecti-cide organophosphorus compounds, including diiso-propyl phosphorofluoridate, sarin, and tabun. Therecommended dose of obidoxime is 3-6 mg per kg ofbody-weight, linmited to 1 or 2 doses; it should begiven only following the administration of, and incombination with, atropine (Erdmann, 1968).Trimedoxime has not been used widely and

only one report has described beneficial effectsin patients with trichlorfon poisoning (Titova &Badjugin, 1970).

Opinions vary on the usefulness of oximes in thetreatment of poisoning with organophosphorus com-pounds, probably as a result of the experience ofdifferent workers. Thus, investigators who havetreated parathion poisoning in farm workers wouldfind that pralidoxime has dramatic life-saving effects.Individuals who handle only experimental animalsmight not encounter the dramatic recovery of con-sciousness that occurs in man. Others who studypoisoning by nerve gases would be disappointedwith pralidoxime, and might consider it useless oronly an adjunct to atropine, and might prefer obid-oxime, trimedoxime, or 2,3-butanedioxone mono-oxime (DAM). Oximes are like other drugs in thatthey are not equally effective in all stages of poison-ing, nor are they equally effective in the treatmentof poisoning by different organophosphorus com-pounds. There is no doubt that pralidoxime is moreeffective than atropine in the early stage of poison-ing with parathion or parathion-methyl, and it maybe that certain oximes would be ideal for treatingpoisoning by certain organophosphorus compounds.This aspect has not yet been fully explored clinically.The possible usefulness of oximes for preventing

poisoning by organophosphorus compounds wasstudied by administering pralidoxime orally to work-ers who handled such compounds. The results showedan increase in the urinary excretion of metabolites(Namba et al., 1958b) or the prevention, to a slightdegree, of a decrease in erythrocyte AChE activity(Quinby, 1968). However, since the preventive useof oximes requires oral intake every 3-4 hours inorder to maintain an effective blood concentration,and possibly creates overconfidence in the workers,it does not seem to have practical value at present.

PERSISTENT MANIFESTATIONS OF POISONING

Poisoning by organophosphorus insecticides is anacute process, but there have been occasional reportsof persistent manifestations.

PolyneuropathyPolyneuropathy may be a persistent manifestation

of organophosphorus insecticides, since some non-insecticide organophosphorus compounds cause thiscondition. For example, tri-o-tolyl phosphate causedan outbreak of " Ginger Jake " paralysis in the USAin 1930 and 1931 and one of polyneuropathy inMorocco in 1959 in which thousands of peoplewere poisoned by ood contaminated with this com-pound. The use of mipafox as an insecticide was

21

303

304 T. NAMBA

abandoned after neuropathy occurred among work-ers in a pilot plant. In animal experiments persistentneuropathy has been caused by triaryl phosphates,S,S,S-tributyl phosphorotrithioite, diisopropyl phos-phorofluoridate, and mipafox, none of which is usedas an insecticide, and by the insecticide Dursban, twhile Chlorthion, t demeton, diazinon, dichlorvos,parathion, paraoxon, and trichlorfon did not pro-duce paralysis (Namba et al., 1971). The develop-ment of neuropathy is not related to the inhibitionof cholinesterase, and is not prevented by the admin-istration of pralidoxime or atropine. A possiblecause may be the inhibition of other as yet uniden-tified esterases.The present author has not found neuropathy

among patients with acute poisoning by organo-phosphorus insecticides. One patient with peripheralneuropathy was exposed to non-insecticide organo-phosphorus compounds and their intermediates,which were synthesized in a research laboratory(Namba et al., 1971). No neuropathy was foundduring a 5-year follow-up of 398 workers who wereexposed to organophosphorus insecticides, 108 ofwhom had had acute poisoning (Kovarik & Sercle,1966). In the literature there are reports of only7 patients who developed neuropathy that mighthave been caused by these insecticides: 3 patients hadbeen exposed to trichlorfon, 2 to parathion, 1 toparathion, EPN, t and other insecticides, and 1 tomalathion (Namba et al., 1971; Humperdinck, 1951;gutov & Varanhiva, 1969).

CNS manifestationsPersistent central nervous system manifestations

were first reported to include impaired memory,depression, impaired mental concentration, schizo-phrenic reaction, and instability, lasting for 6-12months in 16 subjects who had been exposed toorganophosphorus insecticides for 18 months to10 years (Gershon & Shaw, 1961). However, anepidemiological study showed that admissions to

mental institutions in areas where organophosphorusinsecticides were widely used were no greater thanin areas where they were little used (Stoller et al.,1965). There have been many reports of mental orbehavioural changes, but most of these symptomsare transitory or are caused bynon-insecticide organo-phosphorus compounds (Namba et al., 1971).

Liver functionThere have been isolated reports of patients with

increased serum bilirubin or abnormal liver function,and with histological abnormalities of liver structurewith oedema or mild degeneration of parenchymalcells, hyperaemia, fat infiltration, or lymphoid-cellinfiltration in liver obtained post-mortem or bybiopsy. One month after 70 persons had sufferedacute parathion poisoning, jaundice occurred in 4.3 %of the patients, liver enlargement in 14.3 %, increasedurinary urobilinogen in 30%, and a positive serumTakata reaction in 17.1 % (Maruyama, 1954). Of12 patients with acute poisoning by parathion orother organophosphorus insecticides, 8 showed ab-normal results of liver function tests (Lutterotti,1961). However, our patients with acute poisoningshowed normal results of liver function tests through-out the period of observation, except for increasedurinary urobilinogen limited to the first day ofacute poisoning, and persistent liver enlargement inone patient (Namba et al., 1971). A similar findingwas reported in 15 patients who had been exposed5 or more times to organophosphorus compoundsduring a 2-year period (Kaulla & Holmes, 1961).

Other effectsOther possible persistent effects of organophos-

phorus insecticides included changes in coagulationfactors, effects on the fetus, dermatitis, stomatitis,bronchial asthma, and impotence (Namba et al.,1971). The number of reported patients is small,and no relationship between cause and effect hasbeen established.

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Annex

CLASSIFICATION AND TREATMENT OF POISONING BY ORGANOPHOSPHORUSCOMPOUNDS *

Latent poisoning: no clinical manifestationsDiagnosis: depends on the estimation of serum

cholinesterase activity, which is inhibited but ismore than 50% of normal.

Treatment: unnecessary, but the patient should beobserved for at least 6 hours, since poisoning mayprogress.

Prognosis: good.

Mild poisoning: the patient can walk

Diagnosis: the patient shows fatigue, headache,dizziness, numbness of the extremities, nauseaand vomiting, excessive sweating and salivation,tightness in the chest, abdominal cramps, diar-rhoea. The serum cholinesterase activity is 20-50%of the normal value.

* Reproduced, in modified form, from Namba et al.(1971) by permission of the publisher. Compiled principallyfrom experience with cases of poisoning by parathion andparathion-methyl. The dosages listed are for adults.

Treatment: pralidoxime 1 g intravenously; atropinesulfate 1 mg subcutaneously.

Prognosis: good.

Moderate poisoning: the patient cannot walk

Diagnosis: the patient shows generalized weakness,difficulty in talking, muscular fasciculations,miosis, and the signs listed under Mild poisoningabove. The serum cholinesterase activity is10-20% of the normal value.

Treatment: pralidoxime 1 g intravenously; atropinesulfate 1-2 mg intravenously every 20-30 minutesuntil the signs of atropinization appear (drynessin mouth and nose, flush, and mydriasis).

Prognosis: recovery if treatment is given; withouttreatment, the condition may advance to severepoisoning.

CLINICAL EFFECTS OF CHOLINESTERASE INHIBITION BY OP COMPOUNDS

Severe poisoning: the patient is unconscious

Diagnosis: the patient shows marked miosis andloss of pupillary reflex to light, muscular fascicula-tions, cramp, flaccid paralysis, moist rales in thelungs, respiratory difficulty, secretions from themouth and nose, cyanosis. The serum cholinester-ase activity is less than 10% of the normal value.

Treatment: pralidoxime I g intravenously. If thereis no improvement, an additional intravenousinjection of 1 g. If these injections are not followedby improvement, intravenous infusion of pralid-oxime at rates up to 0.5 g per hour. Atropinesulfate 5 mg intravenously every 20-30 minutesuntil the signs of atropinization appear.

Prognosis: Fatal if not treated.

Other therapeutic measures

(1) Maintenance of respiration by securing openairway by means of oropharyngobronchial suctionand endotracheal tube; assistance of respiration bymeans of respirator, if necessary, using oxygen.

(2) Removal of organophosphorus compound (re-moval of clothing, washing of the skin and conjunc-tivae, gastric lavage).

(3) Other supportive measures, including:(a) intravenous fluids,(b) antibiotics if pulmonary infection is present,(c) diphenylhydantoin and other anticonvulsants

if convulsions are not relieved by atropine andpralidoxime.

DISCUSSION

BOOTH: Can you consistently correlate or measure accu-

rately either synaptic or nonsynaptic cholinesterase in-hibition in man with the symptoms of poisoning? Or isa combination of the two types of inhibition perhaps in-volved ?

NAMBA: It is not practicable to measure synaptic cholin-esterase. In fatal cases in man, in which blood cholin-esterase activity was almost zero, a marked decrease ofcholinesterase activity was detected in the brain.

307