8LONG-TERM NEUROTOXICOLOGICAL EFFECTS OFANTICHOLINESTERASES AFTER EITHER ACUTE ORCHRONIC EXPOSURE

ANGELO MORETTO

Department of Occupational and Environmental Health, University of Milano, Italy, and International Centre for Pesticides and HealthRisks Prevention (ICPS), “Luigi Sacco” Hospital, Milan, Italy

MANUELA TIRAMANI

European Food Safety Authority, Parma, Italy

CLAUDIO COLOSIO

Department of Occupational and Environmental Health, University of Milan, Italy, and International Centre for Rural Health (ICRH),“San Paolo” Hospital, Milan, Italy

8.1 Introduction 97

8.2 Long-Term Effects After Acute Poisoning 988.2.1 Experimental Data 988.2.2 Epidemiological Data 988.2.3 Organophosphates and Parkinsonism 100

8.3 Long-Term Effects After Long-Term RepeatedExposure 100

8.3.1 Experimental Data 1008.3.2 Epidemiological Data 1028.3.3 Organophosphates, Depression, and Suicide 103

8.4 Conclusions 104

References 105

8.1 INTRODUCTION

One of the neurological functions for which an adverse effectof neurotoxic pesticides, including organophosphorous (OP),has been repeatedly hypothesized is behavior. Behavior is theproduct of various sensory, motor, and associative functionsof the nervous system, and the hypothesis is that neurotoxicsubstances can adversely affect one or more of these func-tions, disrupt learning and memory processes, or cause detri-mental behavioral effects (IPCS, 2001). Since behavior is avery complex system, made of several different functionsand biochemical activities, it can be studied only based ona very complex approach in which different tests are

performed, addressed at a large spectrum of functions, insome cases with different approaches for various populationsubgroups (Anger et al., 2000; Costa et al., 2008; Fiedleret al., 1996; McCauley et al., 2006), and conclusion can bedrawn only from an integrated evaluation of the availabledata. Because of this complexity, a harmonized assessmentis still lacking also because different approaches have beenchosen by different researchers. As a consequence it is verydifficult to make comparisons between different studiesand, therefore, many doubts are still unresolved, and severalquestion marks are still pending.

Long-term effects might be expected as a consequence ofeither a previous acute poisoning or a long-lasting chronic

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

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exposure. This chapter discusses the existing experimentaland epidemiological evidence for these two outcomes.Two well-known consequences of acute OP poisoning,organophosphate-induced delayed polyneuropathy (OPIDP)and the intermediate syndrome, that occur after severe, life-threatening poisonings will not be discussed since they aredescribed in other chapters of this book. Also, developmentalneurotoxicity will not be discussed here, since it is addressedin other parts of this book.

8.2 LONG-TERM EFFECTS AFTER ACUTEPOISONING

8.2.1 Experimental Data

There are a number of studies on delayed, long-term effectsafter acute OP poisoning. Several of these papers deal withOP nerve agents, but since their mode of action is not quali-tatively different from that of OP pesticides, they are summar-ized here, where relevant.

Filliat et al. (1999, 2007) investigated the consequences ofsoman poisoning on brain morphology, motor performances,and mnemonic cognitive processes in mice and rats. In mice,they found a correlation between the extent of body weightloss and hippocampal neuronal damage. Animals exhibitinghigh weight loss and hippocampal neuropathology duringthe acute phase showed strong mnemonic impairment andreduced motor performance when assessed one and threemonths after poisoning. By contrast, animals exhibitingonly mild weight loss and no morphologically evident hippo-campal damage showed complete recovery from the acutephase. Three months later, they showed a slightly decreasedmnemonic cognitive performance. It should be noted, how-ever, that in these animals the acute phase was severeenough to cause body weight loss, and that neuron countingis not a very sensitive measure of neurological damage. Theseauthors did not report on clinical signs of acute poisoning(Filliat et al., 2007). However, Filliat et al. (1999) observedthat memory impairment after soman poisoning was eitherpartially or totally prevented by anticholinergic and antigluta-matergic treatment. Also Raveh et al. (2002, 2003) showed inrats that antidotal treatment with anticholinergic and anti-glutamatergic drugs offered an adequate protection againstlong-term neurological and neurobehavioral sequelae ofsoman poisoning (1.2 � LD50).

This supports the hypothesis that long-term sequelae arenot a direct consequence of acetylcholinesterase (AChE) inhi-bition, but secondary to hyperactivation of the cholinergicsystem that alters the function of various glutamate receptors.These may underlie brain damage and some of the sub-sequent long-term neurological and neurobehavioral effects.

Kassa et al. (2001) also showed some alterations on motoractivity and stereotyped behavior in rats exposed three

months earlier by inhalation to sarin that caused cholinergicsyndrome although without convulsions.

Grauer et al. (2008) exposed rats for 10 minutes to a singlehigh-level air concentration (whole body) of sarin. There washigh mortality (about 35%) and among surviving rats, a group(75%) showed clear signs of toxicity, including convulsions,and another group (25%) did not show overt signs of toxicity.Convulsing animals showed increased prostaglandin (PGE2)levels in the brain, an index of inflammation, and increasedperipheral benzodiazepine receptor levels (a marker of braindamage) in the cortex up to six months after exposure, aneffect that was also shown by Chapman et al. (2006).Unfortunately, neurobehavioral tests were not performedseparately in convulsing and nonconvulsing animals and,therefore, the results cannot be compared. Histologicaldamage especially in hippocampus (CA1 and CA3 areas)and in other brain regions correlated with the severity of theinitial clinical signs.

Moser et al. (2005) did not find long-lasting neurobeha-vioral effects in rats treated for one year with chlorpyrifosand with spikes of higher doses every two months. Thechronic dose caused AChE inhibition but no clinical signs,whereas the spikes caused mild cholinergic syndrome.Using the same treatment schedule, Samsam et al. (2005)found that acute doses of chlorpyrifos, causing signs ofcholinergic toxicity, impaired learning and sustainedattention.

Sanchez-Santed et al. (2004) did not find alteration in aneurobehavioral test involving memory 5 to 12 monthsafter two doses, 22 weeks apart, of either paraoxon or chlor-pyrifos that caused typical, not severe, cholinergic syndrome(paraoxon) or significant AChE inhibition, but no overtclinical signs (chlorpyrifos).

In conclusion, there is substantial evidence that acute OPpoisoning, especially by nerve agents, leads to some long-term neurological and neurobehavioral effects that might beaccompanied by morphological lesions. These effects canbe partially or totally prevented by an appropriate treatmentthat prevents cholinergic overstimulation and its secondaryeffects on the glutamatergic system, and convulsions. Theevidence is less firm on the possibility of having such long-term effects after less severe poisoning causing a mildercholinergic syndrome without convulsions. No experimentalevidence is available to demonstrate that a single dose of anOP causing AChE inhibition but no overt cholinergic syn-drome is associated with long-term neurotoxicity.

8.2.2 Epidemiological Data

Reports on possible neuropsychiatric and behavioral conse-quences following OP poisoning can be found in the literaturesince the early 1970s, as summarized by Stallones andBeseler (2002a). Most of these were anecdotal and testshave not been performed under controlled situations.

98 LONG-TERM NEUROTOXICOLOGICAL EFFECTS OF ANTICHOLINESTERASES

The first well-identified cohort was that studied byWhorton and Obrinsky (1983), who reported that in 19farm workers, poisoned after reentering a field treated withOPs too early, some neurological symptoms such asnausea, dizziness, headache, and weakness still persistedfour months after poisoning. However, since neither neuro-behavioral tests nor any statistical analysis have been per-formed, this anecdotal report did not provide strong evidencebut raised the issue that was later addressed by other studies.

Savage et al. (1988) studied 100 individuals previouslypoisoned with OP and 100 matched controls. Poisoningoccurred on average nine years before examination. Signifi-cant differences were found on tests of widely varyingabilities such as intellectual functioning, academic skills,abstraction and flexibility of thinking, and simple motorskills such as speed and coordination. However, these seque-lae proved to be sufficiently subtle that the clinical neuro-logical examination, electroencephalograms, and severallaboratory tests failed to discriminate between poisoned andcontrol subjects.

Rosenstock et al. (1991) found that subjects with previousoccupational OP poisoning had a lower performance whencompared to control subjects in a number of neurobehavioraltests, including five out of six subtests of the WHO neuro-psychological test battery, and in three of six tests that assessedverbal and visual attention, visual memory, visuomotor speed,sequencing and problem solving, and motor steadiness anddexterity. The subjects were tested on average two yearsafter the poisoning episode, indicating that these effects weremost likely permanent. These effects were not related toduration of exposure to OPs or other pesticides. Despitesome drawbacks that have been pointed out by others (Lotti,2001; Ray, 1998), such as the lack of identification of thespecific OP involved in the poisoning and the impossibilityof assessing the premorbid capacity, the findings point to along-term neurobehavioral effect of acute OP poisoning that,although asymptomatic, warrants consideration.

Steenland et al. (1994) studied 128 subjects who soughtmedical attention for OP poisoning; of these, 28% were hos-pitalized for at least one night. Poisoning occurred 3 to 11years (average 6 years) before examination. The authorsfound some deficits in neurobehavioral tests in more severelypoisoned patients (i.e., those requiring hospitalization orrequiring more days off work). They also found some indi-cation of deficits in the peripheral nervous system, such asreduced vibrotactile sensitivity. However, none of the sub-jects reported or displayed symptomatic damage to eitherperipheral or central nervous system.

Stephens et al. (1996) indicated no correlation betweenself-reported overexposure to OPs and alteration of neuro-psychological tests, general health, and subjective memoryquestionnaires in chronically exposed subjects. However, itshould be noted that self-reporting is not a reliable indicationof cholinergic poisoning, and that in any case, if medical

assistance was not required, poisoning should have beenquite mild. Therefore, this paper does not provide additionalinformation on the occurrence of long-term effects after OPpoisoning.

Wesseling et al. (2002) conducted a cross-sectional studyto evaluate the neurobehavioral performance of 81 bananaworkers in Central America who had received medical atten-tion but not hospitalization for mild occupational poisoningby OPs or carbamates, on average 27 months earlier. OP-poisoned patients performed worse than controls in thecoding skills on the Digit-Symbol test and in two tests forneuropsychiatric symptoms, but not in many other tests.

Delgado et al. (2004) followed up patients hospitalized foracute OP poisoning in Nicaragua, for two years. Theyassessed immediate verbal memory, visuomotor perform-ance, and neuropsychiatric symptoms (Q-16) seven weeksand two years after poisoning. They divided the poisonedpatients into low, medium, and high exposure, the latterbeing mainly suicidal attempts. Although some differenceswere observed in certain neurobehavioral tests, none of thedifferences were statistically significant especially at thetwo-year time-point. There was an excess of neuropsychiatricsymptoms at two years but not earlier. It cannot be concludedwhether there is an association between this finding and theprevious poisoning episode.

Miranda et al. (2004) evaluated the association betweenacute OP poisoning with chronic motor and sensory neuro-logical impairment, updating the follow-up reported inMiranda et al. (2002a, 2002b). They confirmed thatimpairment, mainly motor, of the peripheral nervous systempersisted after severe poisoning with neuropathic OPs, evenin the absence of frank clinical signs of peripheral neuro-pathy, as also previously suggested by the same group(McConnell et al., 1994).

Roldan-Tapia et al. (2005a) reported on two cases ofsevere poisoning by carbamates who were followed up 3and 15 months after poisoning. One subject had been poi-soned by OPs or carbamates six times before; these episodesresolved in 24 hours and the patient did not suffer from con-vulsion or hypoxia. The other patient was of low educationlevel, not having finished primary school. The authorsfound scores of attention, memory, motor skills, and con-structional abilities lower than the cut off at both 3 and 15months. However, in the absence of pre-poisoning data andclinically evident symptoms, these anecdotal reports cannotbe interpreted.

Roldan-Tapia et al. (2006) reported that in 24 patients pre-viously (three months or more earlier) poisoned with OP orcarbamate there were lower scores in memory, perception,and visuomotor tests when compared to controls. All poi-soned subjects required treatment (atropine and shower) atthe hospital but no attempt was made to quantify the severityof poisoning. Only plasma cholinesterase (ChE) activity wasmeasured but this parameter has a different relation with

8.2 LONG-TERM EFFECTS AFTER ACUTE POISONING 99

AChE inhibition, the target of toxicity, depending on thecompound to which the subject has been exposed.

In addition, Steenland et al. (2000) observed a pattern ofworse performance on several neurophysiological and neuro-behavioral tests in subjects who reported chlorpyrifos poison-ing. However, due to the low number of subjects (eight) andthe self-reported nature of the poisoning, these data appearuninformative on the possible long-term effects of acute OPpoisoning.

Stallones and Beseler (2002a, 2002b), Beseler et al.(2006), and Beseler and Stallones (2008) reported a lowerscore for neuropsychiatric symptoms indicative of depression(odds ratios 5.87 and 2.57 in the two cohorts) in subjects whoself-reported pesticide poisoning, including OPs and carba-mates, but also many other non anticholinergic compounds.Also in this case, self-reporting of poisoning represents asignificant bias for the understanding of the results thatadds to the lack of pre-poisoning data, the lack of identifi-cation of the causative agent, and the lack of information onthe severity of poisoning. Similarly, London et al. (1998)found that self-reported previous OP pesticide poisoningwas associated with the neurological symptom scores.

Yokoyama et al. (1998) also reported that survivors of thesarin accident in the Tokio subway, besides indication of apost-traumatic stress disorder (PTSD), also showed alterationin neurobehavioral and neuropsychological tests. These canbe considered indicative of chronic central nervous systemeffects that were possibly but not certainly associated withPTSD. On the same episode, Nishiwaki et al. (2001) reportedthat rescue staff performed worse than controls in onememory test (backward digit span test) but not in othermemory tests or in other neurobehavioral tests 34 to 45months after the accident. However, it is difficult to discrimi-nate between PTSD and sequelae of sarin poisoning(Yokoyama, 2007).

In conclusion, some epidemiological data appears consist-ent with the experimental evidence when reporting increasedincidence of altered neurophysiological or neurobehavioraltests in subjects previously poisoned with OPs. When takentogether, experimental and clinical/epidemiological datastrongly indicate that severe poisoning is likely to result inlong-term effects if the therapy is not timely and sufficientlyaggressive. For instance, McDonough and Shih (1997)showed that in the early phase of the cholinergic syndrome,seizures are a cholinergic phenomenon, whereas at laterstages seizures per se perturb other neurotransmitter systemsand become responsive to benzodiazepines and N-methyl-D-aspartate antagonists, and less so to certain anticholinergicagents. There is less evidence and the data is more controver-sial for properly treated poisonings where the most severesigns such as convulsions are prevented, and for milderpoisonings not associated with overt cholinergic signs andsymptoms. However, although in most cases there is noobvious overt neurological impairment that can be detected

by routine neurological examination, the findings are to beconsidered relevant.

8.2.3 Organophosphates and Parkinsonism

A survey of the literature identified 16 papers from 1978 to2008 that report extrapyramidal signs or parkinsonism as acomplication of organophosphate poisoning (Arima et al.,2003; Aubeneau et al., 2008; Bhatt et al., 1999; Brahmiet al., 2004; Davis et al., 1978; Goel et al., 2006; Hsiehet al., 2001; Joubert et al., 1984; Joubert and Joubert, 1988;Kventsel et al., 2005; Montoya-Cabrera et al., 1999;Mueller-Vahl et al., 1999; Senanayake and Sunmuganathanet al., 1995; Shahar and Andraws, 2001; Shahar et al.,2005; Tafur et al., 2005). In a total of 31 reported cases, 26showed clear evidence of acute OP poisoning (clinical, clini-cal and circumstantial, and/or biochemical). Most of thecases were severe organophosphate poisoning since 20 ofthe 26 subjects required assisted ventilation, the duration ofantidotal treatment was substantial (2 to 14 days), 5 of the26 subjects also presented the intermediate syndrome. Thetypical signs of parkinsonism were present: tremors in allsubjects, dystonia (15 of 25), cogwheel rigidity (14 of 26),choreoathetosis (10 of 26), facial mask (5 of 26). Observationof extrapyramidal signs usually occurred before completerecovery from the cholinergic or intermediate syndrome. Inaddition, these signs were transient and recovery occurredwithin a median of 23 (range 4 to 60) days from the firstobservation. The incidence of parkinsonism appears to bequite low, as reported by Hsieh et al. (2001), who foundthat only 3 patients out of 633 admitted to their hospital forOP poisoning developed extrapyramidal signs. There is noexperimental data or firm hypothesis for a mechanism ofOP-induced extrapyramidal signs or to explain the reasononly a small percentage of poisoned patients are affected.There is some experimental evidence that after acute OP poi-soning the striatal dopaminergic pathway may be disrupted.However, the effects are slight and not entirely consistentwith Parkinson disease (Karen et al., 2001; Moreno et al.,2008).

8.3 LONG-TERM EFFECTS AFTER LONG-TERMREPEATED EXPOSURE

8.3.1 Experimental Data

The number of experimental studies addressing the issueof long-term effects of repeated administration of OPs isnot particularly high if compared to the number of studiesaddressing acute effects. Moser (2007) identified only 33studies where duration of exposure to anticholinesterase pes-ticides was 30 days or longer. Since then, three more studieshave been identified (Terry et al., 2007; Verma et al., 2009a,

100 LONG-TERM NEUROTOXICOLOGICAL EFFECTS OF ANTICHOLINESTERASES

2009b); only Terry et al. (2007) will be discussed below,since the papers by Verma and coworkers (2009a, 2009b)report the results of the same experiment where dichlorvoswas administered repeatedly at doses causing brain AChEinhibition. In the studies reviewed by Moser (2007), theobservation and testing of the animals was performed at theend of the exposure period, whereas only in very few cases(five) it was done also weeks or months after the end of theexposure. Therefore, the outcomes are essentially relatedto ongoing exposure, rather than long-lasting effects. End-points that were evaluated included observation of uncondi-tioned and conditioned behavior. The former include generalobservations, neuromotor tests of vestibular, motor, and/orsensory functions, and peripheral and central electrophysio-logical tests. The latter are studied using a variety of tests,including conditioned avoidance, spatial learning, and work-ing memory. In most cases there are human correlates and thisfacilitates the extrapolations. However, since different testsand end-points have been used, these studies are difficultto compare, as in the case of the epidemiological studies(see below).

The most important question is whether alteration of theseneurobehavioral tests is associated with AChE inhibition.Studies that demonstrated significant changes in the presenceof actual AChE inhibition will not be discussed in detail here,because either they can be attributed to AChE inhibition itselfor other alternative mechanisms or pathways cannot be dis-sected in such condition.

For conditioned behavior, only parathion, methamido-phos, chlorpyrifos, and the carbamates carbaryl and arprocarbwere tested in the absence of AChE inhibition (Desi et al.,1974; Ivens et al., 1998; Samsam et al., 2005; Temerowskiand van der Staay, 2005; Terry et al., 2007). Chlorpyrifosdid not affect neurobehavior, learning, or sustained attentiontwo months after one year of exposure that caused significantbrain AChE inhibition (about 50%) but no clinical signs(Moser et al., 2005; Samsam et al., 2005). In another exper-iment (Terry et al., 2007), rats treated with chlorpyrifos for30 days showed about 50% AChE inhibition two weeksafter the end of the treatment. Spatial learning and memorywere slightly impaired in these animals, but not in animalsgiven a lower dose that still caused significant AChE inhi-bition two weeks after the end of treatment. In rats treatedwith methamidofos for 16 weeks at doses causing up to36% AChE inhibition in brain, no effects were observed inworking memory and spatial learning when tested 33 and55 weeks after the end of treatment (Temerowski and vander Staay, 2005). Rats treated for 13 weeks with doses of para-thion causing borderline AChE inhibition also showed noimpairments in working memory, avoidance, and spatiallearning tests during treatment and up to 34 weeks after endof treatment (Ivens et al., 1998). In the case of the two carba-mates, some changes were observed in learning and memoryin treated rats in the absence of AChE inhibition in brain.

However, it should be noted that AChE inhibition was onlymeasured after the tests had been performed, that is, longafter the end of carbamate intake, and that the methodrequired dilution of the sample. Therefore, due to the rapidreactivation of AChE inhibited by carbamates, its actual inhi-bition was substantially underestimated in this study; despitethis, the animals treated with the highest dose, which was justtwice the lowest, showed 15% to 44% inhibition of AChE indifferent areas of the brain. Consequently, it is likely that thebehavioral tests have been conducted while AChE in the ner-vous system was substantially inhibited (Desi et al., 1974).

For unconditioned behavior, the OPs malathion, chlorpyr-ifos, methamidophos, and parathion, and the carbamatesarprocarb and carbaryl have been tested in the absence ofAChE inhibition. Only rats treated with methamidofos for16 weeks were tested after high exposure when AChE activitywas back to normal values (Temerowski and van der Staay,2005) and neuromotor, sensory, and vestibular functionswere found to be normal. Rats treated for 13 weeks withdoses of parathion causing borderline AChE inhibition alsoshowed no impairments in the same tests during treatmentand up to 34 weeks after the end of treatment (Ivens et al.,1998). Malathion and chlorpyrifos were found to impair neu-romotor and vestibular functions during and soon after theend of treatment (Abdel-Rahman et al., 2004; Abou-Doniaet al., 2003). In the case of malathion, the results of thestudy are difficult to interpret because only one low doselevel via dermal administration was used, which caused noAChE inhibition. The results of sensorimotor performance(beam walk score and time, inclined plane and grip time)showed similar deficits in rats treated with malathion orpermethrin or N,N-diethyl-m-toluamide (DEET) alone or incombination, which casts some doubts on the meaning ofthe findings. Regarding chlorpyrifos, the study also includedtreatment with nicotine either alone or in combination withchlorpyrifos, using again only one dose administered der-mally (Abou-Donia et al., 2003). The results of AChE activityand of sensorimotor performance (beam walk score and time,inclined plane and grip time) showed inconsistent patternsthat make the data not interpretable. In addition, the resultsfor chlorpyrifos are in contrast with those of Moser et al.(2005) and Terry et al. (2007) who, in the same experimentsdescribed above, did not show any significant change onsensorimotor and vestibular performance in treated rats,even in the presence of significant residual AChE inhibition.

In conclusion, available data shows that neurobehavioraleffects are observed only in the presence of significantAChE inhibition. There is no experimental evidence thatrepeated exposure to OPs at doses not causing AChE inhi-bition is associated with neurobehavioral adverse effects.There is also evidence that the adverse behavioral effectsafter asymptomatic exposure with AChE inhibition orexposure causing mild cholinergic syndrome with moremarked AChE inhibition do not last beyond the period of

8.3 LONG-TERM EFFECTS AFTER LONG-TERM REPEATED EXPOSURE 101

AChE inhibition. In fact, available data shows that recoveryof AChE activity is associated with recovery from the neuro-behavioral effects. It should be noted that the duration ofthese studies was generally shorter than three months andthat tests addressing neurobehavioral aspects such as anxietyand affect have not been carried out, despite the fact thattests exists and are used in behavioral pharmacology(Moser, 2007).

8.3.2 Epidemiological Data

Many studies have been published on possible neuro-behavioral effects of reported exposure to OPs and severalreviews have been published (Colosio et al., 2003; COT,1999; ECETOC, 1998; Kamel and Hoppin, 2004; Lotti,2001, 2002; McCauley et al., 2006; Ray, 1998; Ray andRichards, 2001).

Twenty-five original papers (Ames et al., 1995;Bazylewicz-Walczak et al., 1999; Beach et al., 1996;Daniell et al., 1992; Farahat et al., 2003; Fiedler et al.,1997; Kaplan et al., 1993; Levin et al., 1976; London et al.,1997; Maizlish et al., 1987; McDonnell et al., 1994;Pilkington et al., 2001; Rodnitzky, 1975; Roldan-Tapiaet al., 2005b, 2006; Rosenstock et al., 1991, Rothlein et al.,2006; Savage et al., 1988; Srivastava et al., 2000;Steenland et al., 1994, 2000; Stephens et al., 1995; Stokeset al., 1995; Whorton et al., 1983; Yokoyama et al., 1998)that addressed this issue, which also include papers thatreported studies on previously acutely poisoned subjects(see previous section), have been evaluated according to anevaluation grid to collect information on compound(s),number of subjects studied, exposure measurement or esti-mation, criteria for selection of control groups and possiblepresence of confounding factors, and type of observed altera-tion(s). Four functional areas (cognitive, sensory-motor,psychological, psychomotor) have been identified asdescribed in Table 8.1, considered as covering the spectrumof neurobehavioral effects. For each of the functions investi-gated, the results have been reported based on the authors’assessment as “positive” (showing significant evidence ofeffect), “negative” (not showing significant evidence ofeffect), “limited evidence” (showing either marginal signifi-cance or on the bases of an overall evaluation).

Table 8.2 reports the summary of the results of this analy-sis. Since, as described earlier, previous acute poisoningsappears to be relevant to the development of long-term neuro-behavioral effects, a distinction has been made between thosereporting or not reporting previous acute poisoning. The totalnumber of functional areas is higher than the number of col-lected papers because some of the papers dealt with more thanone functional area. It can be observed that 29% to 47% of thestudies, depending on functional area, indicated the presenceof adverse effects in the exposed population. On the otherhand, 29% to 44% gave negative results and 10% to 29%

were equivocal. However, it should be noted that subjectswho had suffered from a previous episode of acute poisoningwere present in 8 out of 24 of the studies, and that most of thepositive results have been obtained in these studies. Animportant issue in considering the results of these studies,in particular of those not including previously poisonedpatients, is the definition or estimation of the exposurelevels. These were estimated by means of biological monitor-ing in 11 studies; in 6 of these studies a questionnaire wasalso used; only a questionnaire was used in 12 studies and

TABLE 8.2 Neurobehavioral Effects of OPs: Summaryof the Results per Functional Area

Numberof Papers(Out of 25Available)

NoEffectsFound

(n)

EffectsFound

(n)

Limited/EquivocalEvidenceof Effects

(n)

Cognitive (C) 20 8 10 2(6) (14) (1) (7) (4) (6) (1) (1)

Psychomotor(PM)

14 6 4 4(5) (9) (1) (5) (1) (3) (3) (1)

Sensorimotor(SM)

17 7 7 3(7) (10) (2) (5) (4) (3) (1) (2)

Psychological(P)

15 5 7 3(6) (9) (1) (4) (4) (3) (1) (2)

Data in parentheses refer to studies that (included) or (did not include)subjects who suffered from acute poisoning.

TABLE 8.1 Functional Areas ConsideredRepresentative of Neurobehavioral Effects

Functional Area Examples of Test

Cognitive AlertnessAttentionMemoryPerceptionSymbol digitDigit spanVisual retentionTrail making

Sensory-motor Motor coordinationBalancePosturalVisual acuityAuditoryOlfactory

Psychological SymptomsPsychomotor Vigilance

Reaction timeStabilitySanta AnaPursuit aimingTapping

102 LONG-TERM NEUROTOXICOLOGICAL EFFECTS OF ANTICHOLINESTERASES

in one study a job exposure matrix was applied. However,only in a few cases could the exposure be quantified; gener-ally discrete classes (e.g., , or .10 years of exposure)have been used.

Table 8.3 reports the results of the nine studies where thesame three functional areas were investigated. Since an epi-sode of acute poisoning prior to the onset of neurobehavioralchanges appears to be relevant (see above) for the develop-ment of long-term effects, the presence of such an event inthe personal history of the studied subjects needs to be ident-ified. Interestingly, three studies in which no subject with pre-vious acute poisoning was identified showed no effect (Ameset al., 1995; Maizlish et al., 1987; Rodnitzki et al., 1975).

From this relatively low number of studies carried out withthe aim of identifying possible neurobehavioral effects ofprolonged, low-dose exposure to organophosphorous com-pounds, it can be concluded that the functional areas investi-gated varied significantly among studies, and that there waslow consistency in the results. This leads to uncertainty inthe conclusion and results should be interpreted with caution.In any case, besides the lack of consistency, the observedchanges appear to be small. It should be noted that in most

cases the changes have been observed by using specializedtechniques and did not appear to be correlated with frankclinical signs.

8.3.3 Organophosphates, Depression, and Suicide

Some authors reported that farmers have higher rates ofdepression and suicide compared to other occupations andthis was attributed to use of or poisoning by pesticides(Beseler et al., 2006; Beseler et al., 2008; London et al.,2005; Salvi et al., 2003; Scarth et al., 2000; Stallones andBeseler, 2002b). In some instances pesticides were identifiedas OPs or prevalently OPs. A literature review of mortalityand morbidity studies related to suicide among pesticide-exposed populations, and of human and animal studies ofcentral nervous system toxicity related to OP pesticides,was performed by London et al. (2005). The authors con-sidered that epidemiological studies may lead to the hypoth-esis that acute OP poisoning might be associated withaffective disorders that may lead to an increased rate ofsuicides. No convincing evidence was found regarding thepossible influence of long-term, low-dose exposure to OPs.

TABLE 8.3 Distribution of the Positive/Negative Results in the Nine Papers Investigating Cognitive, Sensorimotorand Psychological Functions

ReferenceCognitiveFunctions

SensorimotorFunctions

PsychologicalFunctions

PreviousEpisodes of

AcutePoisoning Comments

Ames et al. 1995 2 2 2 NoFarahat et al. 2003 þ þ 2 NoMaizlish et al. 1987 2 2 2 No No controls; subjects tested before and

after work shiftRoldan-Tapia et al. 2005b þ 2 2 NoRoldan-Tapia et al. 2006 þ þ þ NoRosenstock et al. 1991 þ þ + Yes Poisoned subjects needed hospitalization;

the authors could not fully assess thecontribution of baseline characteristicsbetween cohorts

Savage et al. 1988 þ þ þ Yes No information on hospitalizationSteenland et al. 1994 þ þ þ Yes 28% of the subjects reporting poisoning

were hospitalized for at least 1 nightSteenland et al. 2000 2 + þ Yes No information on hospitalization; 1

group of 100 “friend” controls,indicated by same age and sex, weresupposed to be similar in lifestyle anddemographic variables; a second groupwas recruited among volunteer bluecollar state workers, matching for age,race, sex, and distribution

Yokoyama et al. 1998 þ 2 2 Yes Poisoned subjects needed hospitalization;8 male and 7 female controls

Total positive 6/9 4/9 4/9

þ, area/test affected; 2, area/test not affected; +, limited evidence.

8.3 LONG-TERM EFFECTS AFTER LONG-TERM REPEATED EXPOSURE 103

Based on some experimental data showing effects of OPs onneurotransmitters, especially serotonin, OPs were hypoth-esized as being not only agents for suicide, but possibly apart of the causal pathway. However, experimental datashows that clear effects on serotonin and on behavioral testsfor anxiety occur at doses causing AChE inhibition(Christin et al., 2008; Sanchez-Amate et al., 2001).

Other studies have been conducted after the review byLondon et al. (2005). Six years after having hypothesizedthe occurrence of anxiety and depression as sequelae of OPpoisoning (Stallones and Beseler, 2002b), the same authorsenrolled workers from the same cohort of subjects exposedto pesticides, and concluded that a self-reported history ofpesticide poisoning was supportive of a state of prolongedirritability and depression (Beseler and Stallones, 2008; seeabove). This was based on 761 individuals enrolled in across-sectional study who showed an odds ratio fordepression associated with self-reported pesticide, mainlyOP, poisoning symptoms of 5.87.

Salvi et al. (2003) evaluated 37 workers from southernBrazil involved in family agriculture of tobacco who hadbeen exposed to OPs for three months, and 25 of themwere also evaluated after three months without exposure.Eighteen of the 37 subjects (48%) had a current psychiatricdiagnosis in the first interview, which included generalizedanxiety disorder and major depression. Among the 25 sub-jects who completed both evaluations, the total number ofcurrent psychiatric diagnoses and the number of affectedindividuals, after three months without using OP, droppedby 45% and 36%, respectively.

In 2006, Beseler and coworkers reported a case-controlstudy evaluating the association between depression and pes-ticide exposure among 29,074 female spouses of privatepesticide applicators enrolled in the study between 1993and 1997. Cases were women who had physician-diagnoseddepression requiring medication. The cohort included asubset of spouses who might have had pesticide exposuresquite similar to those of their applicator husbands. Afteradjustment for state, age, race, off-farm work, alcohol, ciga-rette smoking, physician visits, and solvent exposure,depression was significantly associated with a history ofself-reported pesticide poisoning (Beseler et al., 2006). Twoyears later the same authors evaluated the relationshipbetween diagnosed depression and pesticide exposure usinginformation from the pesticide applicators enrolled in thesame study. There were 534 cases who self-reported phys-ician-diagnosed depression and 17,051 controls who reportednever having been diagnosed with depression and did not feeldepressed more than once a week in the past year. Afteradjusting for several variables, acute pesticide poisoningwas associated with depression (as described above). Inaddition, a minimal increased odds ratio (1.54) was foundin a subgroup without a history of acute poisoning and alifetime exposure to pesticides of more than 752 days, the

association being mainly due to exposure to insecticides,including OPs, and herbicides.

In conclusion, acute OP poisoning might be associatedwith depression, whereas in the case of long-term, low-doseexposure such association is unlikely given the inconsistentepidemiological and experimental data. Whether depressionis part of the cause of the suicidal attempt or a consequenceof the poisoning is not yet clear.

8.4 CONCLUSIONS

Concerns regarding the impact on human health of exposureto OPs either occupationally or environmentally have longbeen raised. These were prompted especially by the factthat OPs do indeed affect the peripheral and central nervoussystem because of inhibition of AChE. Therefore, studieshave been conducted to ascertain whether, besides the acutecholinergic syndrome, OPs might cause other neurologicaleffects, either as a consequence of such inhibition or as aconsequence of other mechanisms of toxicity.

Despite the number of epidemiological and experimentalstudies that have been conducted in the last decades, thereis still controversy over possible long-term adverse effectsof exposure to OPs. Available data, both experimental andepidemiological, indicates that indeed there might be long-term neurological consequences of acute OP poisoning,which are not limited to the well-known OPIDP and inter-mediate syndrome. In particular, such neurobehavioral effectsare consistently found in animals that suffered from severeacute cholinergic toxicity and also in most epidemiologicalstudies. Although in many of the latter studies there is no pre-cise quantification of the severity of the poisoning, and thereis uncertainty regarding the appropriateness of controls sub-jects and on control of confounding factors, the overall evalu-ation is consistent with the positive findings in animals. In thelast decade, some attention has been put on extrapyramidalsigns appearing in poisoned patients linked to the fact thatsome epidemiological studies point to an excess ofParkinson disease in agricultural workers. There are anumber of case reports on extrapyramidal signs appearingduring or soon after a severe OP poisoning; however, theseoccur in a small minority of cases and are reversible, pointingto a pharmacological rather than morphological effect. Inaddition, experimental data is not consistent in this respect.

More controversial is the outcome of long-term, low-doseexposure to OPs. Experimental data gives little support to theappearance of neurobehavioral effects occurring withoutactual or previous AChE inhibition. Epidemiological datagives inconsistent results for cognitive, psycho-/sensori-motor, psychological, and psychiatric effects. In addition,these studies suffer from a number of limitations that precludeany firm conclusions in this respect. In any case, it is of notethat the alterations have been observed by using specialized

104 LONG-TERM NEUROTOXICOLOGICAL EFFECTS OF ANTICHOLINESTERASES

techniques and did not appear to be correlated with frankclinical signs.

In conclusion, although there is a trend to a reduction ofuse of OP insecticides, at least in developed countries,these data indicate the need to take appropriate measures toprevent even minor poisoning or any excessive exposure toOPs, and timely and adequate treatment of poisoning andfollow-up of poisoned patients.

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