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Hindawi Publishing CorporationJournal of Biomedicine and BiotechnologyVolume 2006, Article ID 70414, Pages 1–13DOI 10.1155/JBB/2006/70414

Mini-Review ArticleScreening Pesticides for Neuropathogenicity

John D. Doherty

Health Effects Division (7509C), Office of Pesticide Programs, United States Environmental Protection Agency,1200 Pennsylvania Avenue NW, Washington DC 20460, USA

Received 1 December 2005; Revised 18 May 2006; Accepted 30 May 2006

Pesticides are routinely screened in studies that follow specific guidelines for possible neuropathogenicity in laboratory animals.These tests will detect chemicals that are by themselves strong inducers of neuropathogenesis if the tested strain is susceptiblerelative to the time of administration and methodology of assessment. Organophosphate induced delayed neuropathy (OPIDN)is the only known human neurodegenerative disease associated with pesticides and the existing study guidelines with hens are astandard for predicting the potential for organophosphates to cause OPIDN. Although recent data have led to the suggestion thatpesticides may be risk factors for Parkinsonism syndrome, there are no specific protocols to evaluate this syndrome in the existingstudy guidelines. Ideally additional animal models for human neurodegenerative diseases need to be developed and incorporatedinto the guidelines to further assure the public that limited exposure to pesticides is not a risk factor for neurodegenerative diseases.

Copyright © 2006 John D. Doherty. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

INTRODUCTION

In the early 1980s, there was an unfortunate human situ-ation in which drug abusers developed Parkinsonism syn-drome [1] following exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, see Figure 1) that is a by-productin an attempt to chemically synthesize heroin. Although ear-lier researchers sought links between manganese exposureand Parkinsonism [2], the MPTP incident greatly increasedthe interest in correlating environmental exposure to con-taminants and human neurodegenerative diseases. Humanexposure to pesticides is essentially unavoidable in modernlife both in the developed nations and more increasingly inthe developing ones. Worldwide pesticide sales and usage inboth 2000 and 2001 were in excess of five billion pounds. Inthe United States alone there were about 1.2 billion poundsof pesticides used including insecticides, herbicides, fungi-cides, rodenticides, but not including wood preservatives,special biocides, and chlorine/hydrochlorides [3]. Humanexposure to pesticides depends upon many factors and of-ten agricultural workers have the highest rates of exposureas they apply pesticides to crops. Spray drift and migra-tion of the pesticides to potable water as well as residuesin food stuffs and residues resulting from home and gar-den applications are also very significant sources of expo-sure. Many insecticides are neurotoxic by design with tar-gets being acetylcholinesterase (organophosphates and car-bamates), the Na+ conductance channel (DDT, pyrethrins,

and pyrethroids), the acetylcholine receptor (nicotinics), theGABA receptor (emamectin), Ca++ channels (ryanodine),and some agents such as rotenone that affects mitochondrialfunction and also may affect the nervous system. If a poi-soned individual recovers from the initial toxicity followinga single dose of anticholinesterase inhibitors (with the excep-tion of some organophosphates) or agents that act on trans-mitter receptors, and when the chemical is rapidly metabo-lized and excreted, there is usually no established patholog-ical or neurodegenerative change although there are manyanecdotal reports of persistent subtle effects (see reference[4]). The trauma of the acute poisoning incident may havesome psychological effects that may not actually be relatedto the neuropharmacology of the agent. The consequencesof chronic exposure to pesticides, whether they are designedto act on the nervous system as are insecticides or are her-bicides designed to be specific for plants, may be causingeffects in humans through their known or yet to be dis-covered effects in the nervous system. Over the past decadethere has been a growing body of literature that suggestspesticides as being risk factors either for possibly initiat-ing or facilitating the progression of neurodegenerative dis-eases (eg, see Table 1). Theoretically humans may have theinitiation of the diseases triggered by exposure to a pes-ticide or a pesticide in combination with other environ-mental contaminants. In some cases, it is possible that in-dividuals with a genetic predisposition for a neurodegen-erative disease may be at an increased risk to exposure to

2 Journal of Biomedicine and Biotechnology

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pesticides that might initiate the disease. In other cases wherethe initiating event in either normal or genetically suscep-tible persons is caused by a spontaneous event or anotherchemical exposure, the progression following its initiationmay be facilitated to various degrees by exposure to pesti-cides.

The potential toxicity of pesticides is evaluated in lab-oratory animals prior to registration and updated in thereregistration process in a series of required or condition-ally required studies that follow specific guidelines [5].Partly as a consequence of the discovery that MPTP causeda neurodegenerative disease as well as the interest in thepossibility that there is increased susceptibility associatedwith prenatal and neonatal exposures, there has been in-creased testing as a part of the registration/reregistration pro-cess to attempt to determine the potential effects of pesti-cides on the nervous system. As a result, a series of spe-cial neurotoxicity study guidelines were developed in theearly 1990s. These guidelines for special neurotoxicity test-ing together with other more general study guidelines thatalso assess for effects on the nervous system are listed inTable 2.

OVERALL GOAL OF THE STUDY GUIDELINESAND RISK ASSESSMENT

In classical terms, the goal of the study guidelines is to char-acterize the toxicity of the pesticide and to identify the mostsensitive endpoint in the most sensitive species. Once thisendpoint is selected from the pesticide’s toxicity databaseincluding the required studies following the guidelines inTable 2, nonguideline studies that are either conducted at theregistrant’s own initiative or as recommended by the USEPAas well as studies from the open literature, a risk assess-ment is performed. Traditionally the risk assessment is basedon the no observable adverse effect level (NOAEL) for thisendpoint coupled with available or estimated exposure data.The NOAEL is adjusted by uncertainty factors to further as-sure the safety of the chemical to humans. First, a factor of10 X for intraspecies variation based on the assumption thatwithin species some individuals may be 10 times more sen-sitive than the tested group is employed. Another 10 X fac-tor for interspecies variation based on the assumption thathumans may be 10 times more sensitive than the most sensi-tive laboratory animal species is also employed. Another 10 X

John D. Doherty 3

Table 1: Selected examples of human neurodegenerative and other neurological diseases both demonstrated and possibly attributed topesticides.

Disease Pesticide (reference) Association with humans Guidelines for assessment

Organophosphate-induced delayedneuropathy (OPIDN)

Organophosphatescholinesterase inhibitors.(8-review)

Strong. Actual associationdemonstrated

Yes—hen studies

Parkinson’s disease

Paraquat [20–25], maneb[26, 27], rotenone [28–30],organochlorines [31–34],also [35]

Not firmly established butcircumstantial evidence

No

Alzheimer’s diseaseNo specific pesticide—agricultural workers [36, 37]

One case study—associationnot proven.Epidemiological study with 68cases—no associationconcluded

No learning andmemory not assessed inolder animals

Amyotrophic lateralsclerosis

2-4-dichlorophenoxy-aceticacid [38, 39]

Report of increased relativerisk among employees inmanufacturing.Agricultural workers havehigher rates

No specific test butseveral tests woulddetect neurological andmuscular degeneration

Autism No specific pesticide[22]

Suggestion that impairedmetabolism of pesticides maybe associated with increasedincidence of autism

No, but certainpatternsin the DNT study maybe an indicator

Psychiatric disordersOrganophosphates[40–42]

Authors claim of positiveassociation in epidemiologicalstudies and EEG changes inhumans and monkeys

No

factor may be applied if it is determined that the database isincomplete or there is no NOAEL for the most sensitive end-point. When there is an evidence of developmental toxicityin fetuses or neonatal animals at lower doses than parental oradult toxicity, an additional FQPA (Food Quality ProtectionAct) 10 X (reducible to 3 X or 1 X depending upon the cir-cumstances) safety factor is applied to assure the protectionof fetuses, newborns, and children. It should be noted thatthe application of the uncertainty factors is to the NOAELand the lowest observable adverse effect level (LOAEL) is al-ways higher than the NOAEL. Thus, a total uncertainty fac-tor of 100 applied to an NOAEL is in reality a factor of 300from a dose where there is an effect when the LOAEL is adose three times higher than the NOAEL. In order to elimi-nate or compensate for some of the limitations of the NOAELand LOAEL approaches, statistical methods have been devel-oped to determine a benchmark dose (BMD) that accountsfor gaps in dose spacing or account for a study not showingan NOAEL [6]. The uncertainty factors as described above(except for not having an NOAEL) are applied to the BMD.

A risk assessment for a chemical with an NOAEL basedon liver toxicity at the LOAEL that has evidence of neu-ropathogenicity in laboratory animals at higher doses than

at the LOAEL will be protective against the neuropathogen-esis although neuropathogenicity was not the basis for theselection of the NOAEL. The protective nature of both theNOAEL and BMD approaches to risk assessment assumesthat most other potential target organs in humans will onlybe affected at higher doses than the most sensitive endpointin laboratory animals. If humans are especially susceptible toneuropathogenesis resulting from exposure to a certain pes-ticide, the endpoint based on animal studies may underesti-mate the risk to humans. However, the minimum 100 X un-certainty factor plus any additional factors would only in rarecases not be protective against such neuropathogenesis in hu-mans having extreme sensitivity to the chemical. To date, al-though this may be debatable by some, there is no knownneuropathological condition caused or facilitated by pesti-cides that should not be protected against by the current ap-proach to risk assessment as outlined above provided that thepesticide does not interact with other environmental con-taminants, drugs, or naturally occurring chemicals to ren-der neuropathogenicity. The principle of selecting the mostsensitive endpoint in the most sensitive test animal speciesand using either the NOAEL or BMD and applying uncer-tainty factors to drive down exposure is still the basis for risk


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assessment although some may consider this principle out-dated. The toxicity database as generated by the requiredstudies is intended to be a thorough screening process andis not intended to be an in-depth assessment of any organincluding the nervous system unless special inclusions aremade. However, when there are justifications to believe thatthe toxicity for a given chemical is being underestimated bythe standard set of required toxicity studies, and validatedmethods for additional testing are available, these additionalstudies can be recommended to further characterize the tox-icity.

An inherent problem with the guidelines for neurotoxic-ity studies is that the rat, dog, mouse, or rabbit may not pro-vide a model for certain types of neurotoxicity that humansmay be especially sensitive to. No matter how much testingis done in animals, such toxicity will not be detected priorto exposure to humans. The case of aplastic anemia is oneexample of there not being an animal model for predictionof a particular type of toxicity. It is estimated that one per-son in 30–40,000 is susceptible to the aplastic anemia causedby the antibiotic chloramphenicol [7]. There might also becases of unusual human susceptibility to a neuropathogen ofsimilar low frequency and there would be no way to detectthem using the current battery of studies. Unlike the chlo-ramphenicol model where the dosage was intentional andmonitored, exposure to pesticides is much smaller and theactual amounts, times, and frequencies of pesticide exposureare not known.

The guidelines (Table 2) for the more general acute(870.1100 for oral, 870.1200 for dermal, and 870.1300for inhalation), subchronic (870.3100 for rodents and870.3150 for nonrodents—usually dogs), prenatal develop-ment (870.6300) and reproductive (870.3800) and chronictoxicity in rats and dogs (870.4100) and carcinogenicity inrats and mice (870.4200 or 870.4300) are nonspecific in theirdescription of methods recommended for evaluation of thehistopathology of the nervous system. The more obviousneurotoxicity would be detected by observation of the be-havior of the animals based on daily cage-side evaluations ifthe technical staff is appropriately trained to look for and de-tect changes in behavior. The only instructions in the nona-cute studies for histopathology preparation apply to all tis-sues and are not specific for nerve tissue: “tissues and organsdesignated for microscopic examination should be fixed in10 percent buffered formalin or a recognized suitable fixativeas soon as necropsy is performed and no less than 48 hoursprior to trimming.” No commentary on the special stainsto be used is provided. Hematoxylin and eosin are routinelyused.

SPECIAL STUDIES FOR ASSESSMENTOF NEUROTOXICITY IN RATS

The studies designed for specific assessment of potentialneurotoxicity in rats include the series 870.6200 for acuteand subchronic screening in adults and 870.6300 for de-velopmental neurotoxicity (DNT). The latter study includesexposure to pups in utero and during lactation either vialactation or by direct gavage exposure to the pups. These

studies include cage-side observations for the more obviousclinical signs and for functional observational battery (FOB)1

which assess the animal for motor and sensory effects. Thetechnical staff making these observations is supposed to beespecially trained to detect subtle changes in clinical signsindicative of neurotoxicity and typically is unaware whetherthe animal was dosed with the test material or otherwise. Forthese studies, the instructions for histopathological evalua-tion of the nervous system are more specific than for the gen-eral screening studies. “Tissues should be prepared for histo-logical analysis using in situ perfusion and paraffin and/orplastic embedding procedures. Paraffin embedding is accept-able for tissues from the central nervous system. Plastic em-bedding of tissue samples from the central nervous system isencouraged, when feasible. Plastic embedding is required fortissue samples from the peripheral nervous system. Subject toprofessional judgment and the type of neuropathological al-terations observed, it is recommended that additional meth-ods such as Bodian’s and Bielchowsly’s(sic) silver methods,and/or glial fibrillary acidic protein (GFAP) immunohisto-chemistry be used in conjunction with more standard stainsto determine the lowest dose level in which neuropathologi-cal alterations are observed.”

In the developmental neurotoxicity study (870.6300),pups (11 or 21 day old depending on the length of lactationalexposure) and adults (about 62 days old) derived from damsexposed to the pesticide from day 6 of gestation throughlactation (at least to day 10 but many laboratories continuedosing up to the time of parturition) via lactation or by di-rect gavage dosing during the lactation period are examinedhistologically. In addition to histopathology, an abbreviatedFOB assessment, learning and memory and motor activityand acoustic startle responses are all evaluated in the pupsat about weaning time and again as young (about 60 days)adults. Histology of pups is different from the 870.6200 stud-ies in that the brain is fixed by immersion rather than in situ.The guidelines for the neurotoxicity screening studies andthe developmental neurotoxicity study provide references formore detailed instructions for histopathology and behaviorassessments. The laboratory conducting the study is respon-sible for selecting the techniques and stains to be used.

Positive control studies such as with trimethyl tin or acry-lamide for neurohistopathology as well as positive controlssuch as amphetamine and haloperidol for motor activity andscopolamine for learning and memory are currently recom-mended to assure the susceptibility of the strain and the com-petence of the laboratory personnel conducting the study.The argument for also considering nonchemical agents toevaluate the proficiency of a laboratory in the use of a test(eg, memory) has been made [8].

These screening studies should detect alterations of thenervous system that occur within the limited time frames oftesting with respect to the age of the rat when tested providedthat the relatively pure strains of rat used are susceptible to

1 The FOB assessment in the series 870.6300 is less detailed than for theseries 870.6200. In particular, there is no requirement to include gripstrength or landing foot splay.

John D. Doherty 7

any neurotoxicity that could be induced by the chemical.Consequently there are limitations with regard to their pre-dictive value for the major neurodegenerative diseases whichare associated with older humans such as Parkinson’s andAlzheimer’s. In particular, only young adults are assessed inthe 870.6200 screening studies and exposure to the rats isin utero and up to the first three weeks of life in the se-ries 870.6300 developmental neurotoxicity study. The ratsare not kept on the study to determine if the in utero expo-sure predisposed them to development of neuropathologicalconditions in the later stages of life or if a challenge by thetest chemical would be worse if the rats were not exposed inutero.

Three other studies (870.6500, scheduled operant be-havior, 870.6850, peripheral nerve function, and 870.6855,neurophysiology: sensory evoked potentials) are rarely con-ducted but can be used to further characterize indicationsof neurotoxicity suggested in either the general or the spe-cial neurotoxicity guideline studies or based on the pesticide’sstructure and predicted activity relationships.

There are no guidelines for studies with monkeys whichmay have a similar level of susceptibility to neurotoxicantsthat may produce or facilitate human neuropathogenesis.Reasons for this are that studies with monkeys are expensiveand only limited numbers of animals can be used.

NONGUIDELINE STUDIES

Studies from the open literature or studies conducted by thepesticide industry that either do not have protocols consis-tent with the guidelines or that are conducted to address aspecific question are grouped together as nonguideline stud-ies. Such nonguideline studies can provide endpoints for riskassessment when peer review determines that they are of ac-ceptable scientific merit. It is, however, difficult to requestthat companies conduct special nonguideline studies with-out sufficient justification that the study is validated to ren-der data useful for risk assessment purposes.

A recurring problem with nonguideline studies is thatthey often use routes of test chemical administration not re-lated to human exposure scenarios. Intraperitoneal, intra-venous, and intramuscular modes of administration maybe very useful in attempting to determine the mode of ac-tion of a chemical. Such data are important in understand-ing the possible molecular basis for the neuropathogenesis.However, extrapolating data from these routes of administra-tion to human exposure by the dietary, dermal, or inhalationroutes is problematic.

The use of nonguideline studies with purposeful dosingof human volunteers to assist in the risk assessment for pesti-cides is done on a case by case basis following both scientificand ethical review. Such studies with human volunteers areoccasionally conducted with pesticides that may cause tran-sitory effects such as cholinesterase inhibition but certainlynot to see if a neurodegenerative condition results. Epidemi-ological studies that attempt to correlate the incidence of cer-tain types of diseases with pesticide exposure to humans de-rived from surveys of the subjects’ personal history provide

insight into the possibility that exposure to the pesticide maybe related to the onset and progression of neuropathogenesis.These studies, however, can only suggest a possible relation-ship because the subjects are also simultaneously exposed tomany other chemicals and there is no real way to determinethe actual extent to which the subjects were exposed to thesuspect pesticide chemical or if exposure occurred during thecritical times to affect the onset or progression of the neu-ropathological condition.

STUDY GUIDELINES FOR ASSESSINGORGANOPHOSPHATE-INDUCEDDELAYED NEUROPATHY

The only established neuropathy in humans associated withpesticides is organophosphate-induced delayed neuropathy(OPIDN) caused by certain but not all organophosphate in-secticides and some other organophosphates not used as in-secticides. A recent review on OPIDN provides more detailedinformation on the history and development of this model[9]. Documentation that OPIDN affects humans dates backto the early part of the last century when a major incident oc-curred during the prohibition years in the USA as a result ofconsumption of a Jamaican ginger alcoholic drink that waslater demonstrated to be contaminated with tolyl phosphateesters. It is estimated that some 20,000 persons were affectedto various degrees with a syndrome that was called Gingerjake paralysis or jake leg. The classical work of M. Smith andR.D. Lillie [10] of the US Public Health Service in the 1920sand 30s demonstrated that the phosphate contaminants wereresponsible for the condition and could reproduce the syn-drome in rabbits, dogs, monkeys, and calves. In human ex-posure to ginger jake, the condition was described as “the ini-tial flaccidity, characterized by muscle weakness in the armsand legs giving rise to a clumsy, shuffling gait, was replacedby spasticity, hypertonicity, hyperreflexia, clonus, and abnor-mal reflexes, indicative of damage to the pyramidal tractsand a permanent upper-motor neuron syndrome. In manypatients, recovery was limited to the arms and hands anddamage to the lower extremities (foot drop spasticity andhyperactive reflexes) was permanent, suggesting damage tothe spinal cord” [4]. Validation that organophosphate insec-ticides cause OPIDN in humans comes partly from an inci-dent concerning workers manufacturing the insecticide mi-pafox following an accident [11]. Domestic animals are alsosusceptible to OPIDN as indicated by the poisoning of waterbuffalo in Egypt [12] by the insecticide leptophos. A reviewof the possible association between leptophos with OPIDN inhumans [13] describes problems in distinguishing betweenleptophos and other contaminants as the cause of OPIDN.

Considerable research on the structure of organophos-phate insecticides that can cause this neuropathy has beendone [9]. Of the tolyl phosphate contaminants in the gingerproduct, it was later determined that only one, the ortho-isomer, was responsible for the toxicity, indicating the highlyspecific chemical structural nature of the induction of thissyndrome. Figure 1 presents some of the chemical struc-tures of organophosphates that are known to cause OPIDN.


Research on biochemical approaches has led to the discoverythat inhibition of “neuropathy target esterase” (NTE) by theorganophosphates that cause the delayed-type neuropathyhas provided a basis for screening of new organophosphatecandidates for development as insecticides [14]. Chemicalsshowing higher levels of inhibition of NTE are reported tohave a good correlation with development of OPIDN.

The hen was determined to be a relatively very suscep-tible species but the laboratory rat and mouse were not ap-preciably susceptible to OPIDN. The hen provides a modelfor assessing the potential for an organophosphate to causeneurotoxicity and is used in the acute and repeat dose studyguidelines (870.6100). It is necessary to use adult domestichens 8–14 months of age since the chick has a lower sensi-tivity [15]. In the acute study, a near lethal dose is admin-istered usually by gavage and the hen may be protected byatropine from the inhibitory effects of the organophosphateon acetylcholinesterase. Following dosing, the hens are ob-served for their gait characteristics including the ability towalk up an incline and after 21 days are sacrificed and ex-amined histologically. The repeat dose study is conductedwhen there is an evidence of OPIDN in the acute study orwhen there is an evidence of inhibition of NTE. The focusof the repeat dose study is to determine the NOAEL andLOAEL for OPIDN and it includes control, low, mid, andhigh (maximum 1 gm/kg) doses. The guidelines provide thefollowing details for histopathological examination of thenervous system. “Tissues should be fixed by whole body per-fusion, with a fixative appropriate for the embedding me-dia. Sections should include medulla oblongata, spinal cord,and peripheral nerves. The spinal cord sections should betaken from the rostral cervical, the midthoracic, and the lum-bosacral regions. Sections of the proximal regions of both ofthe tibial nerves and their branches should be taken. Sectionsshould be stained with appropriate myelin- and axon-specificstains.” The guidelines recommend that TOCP (tri-ortho-cresyl phosphate, Figure 1) be used as a positive control toassure the susceptibility of the hens. Not all hens are equallysusceptible to OPIDN [16].

No new organophosphate insecticides have been intro-duced in recent years and either organophosphate insecti-cides that were demonstrated to cause OPIDN have beenphased out or their uses have been greatly restricted. Neworganophosphates, however, may in the future be needed forcontrol of certain pests that become resistant to currentlyregistered pesticides. OPIDN is not considered to be relatedto another known human neurodegenerative disease. How-ever, the OPIDN model may be very useful in studying theprogressive degeneration of the nervous system followinginitiation of the nerve degeneration that may be applied tohuman neurodegenerative diseases if the underlying mecha-nisms of OPIDN can be elucidated and compared with hu-man diseases.

PESTICIDES AND PARKINSONISM SYNDROME

Parkinson’s disease (PD) is regarded as the second mostcommon neurodegenerative disorder in humans and affects

about 2% of the population over the age of 60 years. Clin-ically, PD is a disorder of motor function characterized bytremor, slow and decreased movement (bradykinesia), mus-cular rigidity, poor balance, and problems in gait [17]. Patho-logically, PD patients show loss of dopaminergic neuronsin the substantia nigra pars compacta and frequently haveLewy bodies, eosinophilic intracellular inclusions composedof amyloid-like fibers and α-synuclein [18]. PD may have agenetic basis for susceptibility for an early onset form but theoccurrence of the more prevalent late onset form does nothave an established genetic basis [19]. The latter form mayresult from a multitude of different factors including insultsfrom xenobiotics and an individual’s inherent sensitivity ordifferences in the metabolism and pharmacokinetics of thexenobiotics. Since the discovery that MPTP [1] could causePD like syndrome, interest in the herbicide paraquat, whichhas some structural similarity to MPTP (Figure 1) led to thepossibility that this herbicide could be a risk factor in the PDsyndrome [20–25]. Factors such as exposure from living inrural areas, farming, drinking water from wells and exposureto agricultural chemicals have been investigated and claimedas support for an association between paraquat and increasedPD. Interest in the herbicide maneb as a possible risk factorfor PD developed because of its reported effects on dopaminewhereas it was demonstrated to enhance the effects of the ac-tive metabolite of MPTP or MPP+ [26, 27]. Rotenone, a pes-ticide that is an inhibitor of mitochondrial Complex I func-tion, has also been implicated for being associated with PDbased partly on work that associates the mode of action ofMPTP or its principal metabolite MPP+ with an effect onmitochondrial Complex I function [28–30].

Organochlorine insecticides as well as tricyclohexyl andtriphenyl tin inhibit various ATPases in nerve membranesincluding one enzyme species that also shows a bell-shapedcurve for activation and then inhibition of activity by Mn++

and it was earlier suggested [31] that inhibition of ATPasesmight be related to an environmental factor in Parkinson’sdisease etiology. DDT and dieldrin persist in the body andonce ingested can remain there indefinitely. Mobilization ofDDT or dieldrin from fat stores as the body ages to critical ar-eas associated with PD might be a factor in its developmentor progression. An association between dieldrin presence andPD syndrome [32] was reported based on a small number ofpatients examined. Heptachlor has also been demonstratedto affect dopamine function [33] in laboratory animals.

The association between PD and pesticides is a contro-versial issue and the USEPA does not currently consider thatpesticides are risk factors in this disease. A recent compre-hensive review of this issue, supported in part by industrybut published in a peer reviewed journal, led the authors toconclude “that animal and epidemiological data reviewed donot provide sufficient evidence to support a causal associa-tion between pesticide exposure and PD” [43].

If a pesticide was causing or affecting a PD like syndromein susceptible laboratory animals, the signs of tremor, slowand decreased movement, muscular rigidity, problems in gaitwould be expected to be detected in the screening process ifall of the appropriate studies were requested and conducted.

John D. Doherty 9

The available studies for paraquat, maneb, or rotenone donot show obvious indications of these signs at least not attheir LOAELs in the strains tested. The histopathological ef-fects would probably not be so obvious within the limited as-sessment for histopathology in the current study guidelinessince the substantia nigra is a relatively small section of thebrain and would require special assessment to determine ifthere were test chemical induced changes in the dopaminedependent cells within it. Chronic exposure for paraquat iscurrently based on “chronic pneumonitis” in dogs with aconventional 100 fold uncertainty factor. Maneb is currentlyregulated for chronic exposure based on its effects on the thy-roid in rats at the LOAEL plus a 1000 fold uncertainty factorincluding an extra 10 X because of an incomplete database.Endpoints for rotenone are currently being reevaluated andthe reports of its association with PD syndrome being con-sidered for future testing but the current LOAEL is not basedon indications of neuropathogenesis.

Historically, the rat has limitations as an in vivo modelfor PD and attempts to study the effects of either Mn++ orMPTP in this species resulted with some but limited data. Adetailed review of the development of animal models for PDand other neurodegenerative diseases is beyond the scope ofthis review and there are no suitable models for incorpora-tion into the guidelines. Reviews of neurotoxicant inducedmodels of PD in the rat have been published recently in 2004[44] and 2005 [45] and provide comprehensive discussionsof the many problems associated with trying to develop ananimal model. A review of the development of animal mod-els in mice has also been presented [46] and limitations ofthis species including genetically engineered strains are dis-cussed [47]. Factors such as the low susceptibility of rodentsto PD like syndrome or a narrow or limited vulnerable ageor the differences in metabolism and access to the criticalsites by the critical form of the toxic agents as well as the cu-mulative effects and the influence of combinations of chem-icals all contribute to problems in developing animal modelsfor predicting a chemical’s potential to be a risk factor forPD.

One important consideration in the development of an-imal models for PD concerns the question: what is the goalof the model? For example, some models are developed tofurther understand the neurochemical events associated withthe initiation and progression of the disease in order to de-velop therapy. Other models may have the goal of establish-ing a basis for risk assessment. One of the criticisms of someof the developing models that they do not mimic the dis-ease in humans closely enough is not necessarily detrimentalto the goal of providing data for risk assessment. This is be-cause if the model shows an effect suggestive at all of neu-ropathogenesis it would be important in the hazard char-acterization of the chemical. Thus, genetically manipulatedmice that spontaneously develop PD like syndrome whetherit mimics the human condition exactly or not would be animportant addition to the guidelines. The suspect chemicalscould be tested in these strains to see if the spontaneous ratesof the syndrome are increased, occur at an earlier onset time,or are worse in the presence of the pesticides.

In vitro data using rat or other animal tissue preparationscan be very useful for providing data on mechanisms but notalways generalize to the in vivo situation. One such exam-ple is the effect of paraquat which was suspected as caus-ing PD like syndrome based on its structural similarity toMPTP/MPP+ that does not have the same affinity for thedopamine transporter or cause inhibition of mitochondrialcomplex I in in vitro studies indicating that paraquat has aseffect on dopamine neurons that is unique from rotenoneand MPTP [48]. It is still possible that an NTE like modelsuch as for predicting OPIDN could be developed based onin vitro studies. Limitations associated with in vitro modelsbased on animal tissue include that in real life, exposure isnot just to the single chemical but to complex mixtures, invitro studies do not reflect the cumulative effects of the pes-ticide or the temporal aspects of the initiation or progressionof the disease.

Another animal model for induction of PD involves miceand their early exposure and later challenge based on workwith paraquat and maneb [49]. The mice exposed as fetusesduring pregnancy were reported to be more susceptible toindications of PD when challenged later in life by these pes-ticides. This implied that an initial injury predisposed theanimals to susceptibility in later stages of life. This model isbased on the “Barker hypothesis” or its expanded form forParkinsonism where it is postulated [50] that early exposureto chemicals destroys certain critical cells in the substantianigra to levels below those needed to sustain function asso-ciated with advancing age. In these studies, combinations ofparaquat and maneb were used assessing the mutual influ-ence of each. The role of early life environmental risk factorsin PD has been independently reviewed [51].

As indicated above, a problem with attempting to assessfor the effects of pesticides as risk factors of neuropatho-genesis by the study guidelines is that some of the literaturereports associating pesticides with PD imply that combina-tions of pesticides or other agents rather than the individualpesticides are the risk factors. Extensive justification wouldbe needed before studies with combinations of xenobioticscould be requested to provide data for risk assessment. Es-tablishing what combinations of chemicals should be tested,how long the tests should run for and what relative doses ofeach chemical to be tested would be a task in itself and inter-preting the data with regard to which chemical is really thecontributing factor would be problematic.

OTHER NEUROLOGICAL DISORDERS

Table 1 lists Alzheimer’s disease (the most common neurode-generative disease), amyotrophic lateral sclerosis, autism andpsychiatric disorders as possibly being related to pesticideexposure. Tests for learning and memory through the lifecycle including the later months near study termination inchronic or cancer studies might be considered for incorpo-ration into the guidelines to attempt to assess for at leastsome aspects of Alzheimer’s disease. Alzheimer’s disease mayhave both genetic and environmental factors [52] and ani-mal models of Alzheimer’s disease are being developed [53]


but their usefulness for evaluating risk associated with pes-ticide exposure has not been established. Many factors mayinfluence the progression of Alzheimer’s disease and a veryrecent report indicates that persons with higher levels of ed-ucation have faster rates of cognitive decline [54]. Animalmodels of autism are being developed [55] and the endpointsof (i) lower sensitivity to pain and higher sensitivity to non-painful stimuli, (ii) diminished acoustic prepulse inhibition,(iii) locomotor and repetitive/stereotypic-like hyperactivitycombined with lower exploratory activity, and (iv) decreasednumber of social behaviors and increased latency to socialbehaviors are considered possible indicators of a drugs as-sociation with autism based on studies with valproic acid.The developmental neurotoxicity study (DNT, 870.6300) canassess for some of these parameters but there are no inclu-sions in the current guidelines for DNT studies for assessingsocial behaviors. If a pesticide caused neurological or mus-cular degeneration, it could possibly aggravate amyotrophiclateral sclerosis but would be regulated based on its NOAELto be protective. Although neuropsychiatric disorders maynot be strictly within the description of neurodegenerativedisease, there has been a continuous debate over the pos-sibility that organophosphate poisoning causes neuropsy-chiatric sequella [40]. A review of this topic is beyond thescope of this manuscript. Nonguideline studies with mon-keys [41] with sarin have been reported to produce long-lasting EEG changes that are claimed to confirm an earlierobservation of changes in the human EEG patterns [42] fol-lowing organophosphate exposure. The persons exposed tosarin (a potent cholinesterase inhibitor) gas in the Tokyosubway incident in the mid 1990s have been assessed peri-odically and reports indicate possible neurological effects ei-ther related to the gas itself of post traumatic stress disorder[56–58].

OVERALL ASSESSMENT OF NEUROTOXICITY STUDYGUIDELINES FOR CHARACTERIZING RISKFOR NEUROPATHOGENICITY

Strength of the neurotoxicity study guidelines. The neurotox-icity study guidelines provide a screening procedure thatshould detect pesticides that are strong inducers of neu-ropathogenesis in the animal strains and species tested rel-ative to the time of administration and ages of the testedanimals. The guidelines are adaptable and as more and bet-ter techniques and models (such as genetically manipulatedstrains) are developed these models can be incorporated intothe guidelines. Humans would have to be inherently espe-cially more sensitive to a neuropathological response to a pes-ticide or there would have to be other contributing factorsfrom the real world if they were to develop neuropathologicalconditions as a result of the low level of exposure that is set bythe selection of the most sensitive endpoint in the most sen-sitive species as determined by the battery of required studiesand other available data and the application of uncertaintyfactors to drive down exposure.

Weaknesses or limitations of the study guidelines. Severalinherent weaknesses in the study guidelines can be identified.

Most of these reflect a disparity between the stringent con-ditions of laboratory testing and real world exposure. Theseinclude the following.

(i) Pesticides are tested individually. Thus, the interactionand cumulative effects of the individual pesticide with themany other pesticides, xenobiotics, drugs, and natural food-stuffs are not assessed.

(ii) Relatively pure strains of standardized laboratory an-imals are tested meaning that a neuropathological conditionwill be detected only if that particular strain is sensitive to thechemical. The human population is very diverse with varyingdegrees of sensitivity to a given chemical. The standardizedstrains do not have a predisposition to develop neuropatho-genesis such that it cannot be assessed if there is a poten-tial for the pesticide to accelerate the progression of a neu-ropathological condition once started.

(iii) Healthy young animals on diets optimized for theirhealth would be the least susceptible to a toxic insult are usedfor testing. There is a wide variation in diet and disparity inages and in the level of health that makes humans possiblymore susceptible.

(iv) Temporal conditions are not fully evaluated such asearly in utero and fetal exposure affecting the animal to bemore sensitive to an insult by the chemical in the later phasesof its life.

(v) Neuropathogenesis may result from the destructionof only a very small structure of the brain (ie, the pars com-pacta of the substantia nigra) and such changes in structuremay be missed in routine histopathological assessment of thebrain. This is an important concept since the laboratory an-imal may have a higher tolerance to destruction of the brainarea than the human and the animal may not show clinicalsigns until there is a major destruction but the human mayshow clinical signs after only minimal or moderate destruc-tion.

(vi) Laboratory animals are not the same as humans. De-tection of neuropathogenesis in animals does not mean thatthe human will develop the same lesion. Conversely, failureof animals to develop a neuropathological condition does notmean that the human will.

All of the factors above for weaknesses or limitations ap-ply in to the study guidelines in general such as for assessingfor cancer and developmental toxicity and these limitationsare well recognized. In essence, studies conducted followingthe guidelines, as imperfect as they may be, plus other avail-able data are what risk assessments are based on. Epidemio-logical data come later.

SUMMARY

Pesticides are individually tested in a series of studies withestablished guidelines with laboratory animals to determineif they have the potential for neuropathogenicity. Thus, theneuropathological effects of chemicals that are strong induc-ers of neuropathogenesis in the species of animals tested andif tested at the critical susceptible times will be detected inthe battery of studies required for registration and reregis-tration. Additional testing in animals can be conducted based

John D. Doherty 11

on suggestions of neuropathogenesis from existing studies orbased on structure activity relationships to further charac-terize a neuropathological condition possibly associated withthe pesticide. Currently, the endpoints determined by thecompleted battery of required and other studies and the useof uncertainty factors in risk assessment are designed to pro-vide a reasonable protection against possible neuropathogen-esis of pesticides to humans. If humans are uniquely suscep-tible or the timing for test chemical administration in theanimal studies is not appropriate, or if the pesticide mustinteract with other chemicals, potential effects in humanscould be missed but the inclusion of the uncertainty factorsis designed to protect against such possibilities by drivingdown exposure. The discovery that humans are susceptible toOPIDN resulted from accidental exposure to an organophos-phate led to the development of the hen model for OPIDNtesting which is the only model for neuropathy that is pur-posefully assessed for in routine screening studies. Had thisaccident not happened, there might be an occasional incidentof persons developing the OPIDN syndrome today withoutknowing its cause. The OPIDN model is unlike the majorhuman neurodegenerative diseases since OPIDN starts soonafter exposure while Parkinson’s and Alzheimer’s may requirelong intervals between exposures and onset or they may re-quire a natural onset before pesticides can facilitate their pro-gression. Therefore, the possibility that pesticide exposurescan be risk factors for neurodegenerative diseases needs tobe considered in epidemiological models, whether alone orin combination with other factors. Development of animalmodels to more completely assess for possible relationshipsbetween pesticide exposure and neurodegeneration in hu-mans need to be developed and validated to render data use-ful for risk assessment. Animal models with strains genet-ically engineered to be susceptible to known human neu-rodegenerative diseases may eventually be developed and val-idated and be important additions to the guidelines for neu-rotoxicity assessment.

ACKNOWLEDGMENTS

This manuscript reflects the perspective of the author anddoes not imply policy of the US Environmental ProtectionAgency. The author greatly appreciates the editorial com-ments and stimulating discussion provided by Dr LouisScarano.

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