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16DEVELOPMENTAL NEUROTOXICITY OFANTICHOLINESTERASE PESTICIDES

JOHN FLASKOS AND MAGDALINI SACHANA

Laboratory of Biochemistry and Toxicology, School of Veterinary Medicine, Aristotle University of Thessaloniki,Thessaloniki 54124, Greece

16.1 Introduction 203

16.2 The Basis for the Increased Vulnerability of theDeveloping Nervous System Compared to the Adult 204

16.3 Developmental Neurotoxicants and AnticholinesterasePesticides 205

16.4 The Developmental Neurotoxicity of AnticholinesterasePesticides as a Result of Pharmacokinetic DifferencesBetween the Young and the Adult 205

16.5 The Developmental Neurotoxicity of AnticholinesterasePesticides as a Result of Interference withNeurodevelopmental Processes 207

16.6 Effects of Anticholinesterase Pesticides on NeuronalCell Replication and Differentiation 20716.6.1 Effects on Synthesis or Levels of DNA,

RNA, and Protein 207

16.6.2 Effects on Cell Signaling, TranscriptionFactors, and Neurotrophic Factors 208

16.7 Effects of Anticholinesterase Pesticides on NeuriteOutgrowth 210

16.8 Effects of Anticholinesterase Pesticides on SynapticDevelopment 212

16.9 Effects of Anticholinesterase Pesticides on GlialDevelopment 213

16.10 Effects of Anticholinesterase Pesticides on Apoptosis 215

16.11 Effects of Anticholinesterase Pesticides onNeurobehavior 215

16.12 Testing for Developmental Neurotoxicity: The Use ofAnticholinesterase Pesticides as Testing Agents 216

16.13 Conclusions 217

References 218

16.1 INTRODUCTION

Any toxicity inflicting damage on a developing organism isan important issue that has severe social, economic, and bio-logic consequences. In humans, the death of a fetus or a dis-abled child can be the source of great distress to the family.The costs arising from the decreased productivity of affectedindividuals who have not been properly educated and trainedand may need treatment for a long time are a big burden tonational economies. In animals, apart from the financiallosses incurred from the sickness or death of young farm ani-mals, developmental toxicity can be biologically important in

the case of rare or endangered species, as death before ani-mals reach their reproductive age can severely compromisespecies survival. Developmental toxicity is, also, importantin quantitative terms. Almost one in six children suffersfrom a developmental disability (Boyle et al., 1994) andapproximately 30% of these defects arise wholly or partlyas a result of exposure to toxic chemicals in the environment(Grandjean and Landrigan, 2006).

Almost all of the developmental abnormalities caused bytoxic agents involve effects on the nervous system. Indeed,the developing nervous system is much more sensitive thanthe other developing systems of the body to toxic injury

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

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and this is mainly due to the complexity of the nervous systemand the much longer period over which this system developscompared to the other systems (Rodier, 1994).

Developmental neurotoxicity can arise as the result ofeither prenatal or postnatal exposure of the developing organ-ism to the neurotoxic insult. Prenatal exposure occurs follow-ing exposure of the mother and the neurotoxic agent gainsaccess to the embryo or fetus after its passage through the pla-centa (Pelkonen et al., 2006). Postnatal exposure occurs eitheras a result of exposure of the mother via breastfeeding orthrough direct exposure of the developing organism itself tothe neurotoxic agent following dermal absorption, ingestion,or inhalation. Developmental neurotoxicity may involvemotor, sensory, and cognitive effects. Neurodevelopmentaleffects vary in intensity, duration, and time of onset. Thus,deficits may be profound or subtle, which are not clinicallyvisible and can only be recognized by applying specialtests, for example, IQ testing. Neurodevelopmental effectscan be reversible or can have permanent, life-long conse-quences. Furthermore, neurotoxic effects can, in someoccasions, manifest themselves only a considerable lengthof time after developmental exposure and sometimes notuntil adulthood. Neurodevelopmental toxicity is complexand includes effects on both the structure and function ofthe developing nervous system. These effects involvechanges in neuroanatomy, neurophysiology, neurochemistry,and behavior. Consequently, the assessment of developmen-tal neurotoxicity can be performed by the application of awide range of different techniques and the use of a largenumber of different endpoints.

16.2 THE BASIS FOR THE INCREASEDVULNERABILITY OF THE DEVELOPINGNERVOUS SYSTEM COMPARED TO THE ADULT

Developmental neurotoxicity is important not only becausethe developing nervous system is more sensitive than theother developing systems and organs but, also, because it ismore vulnerable to toxic injury compared to the mature ner-vous system. This increased susceptibility can be attributedmainly to the fact that during normal development a seriesof unique and complex processes involving the participationof many molecules with a particular developmental functiontake place. These processes, which are not found in the adult,present a sensitive target for a number of exogenous sub-stances. The amazing structural and functional diversity ofthe mature nervous system implies that neurodevelopmentis an extraordinarily complex event. This consists of asequence of distinct developmental processes that are strictlyregulated in both time and space. This sequence includes theprocesses of neuron proliferation, migration, differentiation,synaptogenesis, gliogenesis/myelination, and apoptosis.Both the time of onset and duration of each of these processes

are tightly controlled, with each process emerging at a timedepending on the proper progress or completion of the pre-vious process in the developmental sequence. Although thesame sequence applies to all nervous system areas, it occursat different times. Development of the nervous system as awhole is a long process extending from the embryonicperiod until adolescence. The sequence of developmentalprocesses is generally comparable among animal species,but the time at which each process occurs shows some differ-ences. Thus, in rodents synaptogenesis and gliogenesis arealmost neonatal, whereas in humans these processes arealready under way in the third trimester of pregnancy. Inboth rodents and humans, however, these processes continuethrough adolescence. Importantly, neurodevelopment occurseven during adulthood with the elimination of synapses andother neuronal elements in the course of neuronalremodeling.

The above data have important implications for develop-mental neurotoxicity. Each developmental process is sensi-tive to the toxic action of exogenous agents. As each ofthese processes occurs at a certain time in a given region, itfollows that the toxicity of a substance depends on thetiming of exposure. Thus, an agent that has antiproliferativeproperties will be able to induce neurotoxicity only whenadministered during the period of active cell proliferation.Similarly, a substance affecting myelination will have littleeffect if given to a rodent during pregnancy, since in thesespecies this process is mainly postnatal. Furthermore, sinceeach developmental process occurs at different times indifferent regions and functional domains, the timing ofexposure will also determine which regions and functionsare going to be predominantly affected.

Although the actions of toxic agents may be specific to adevelopmental process, the fact that these processes occurin strict order, however, means that a compound interfer-ing with a developmental process earlier in the sequencewill inevitably disrupt, also, developmental processes thatfollow. Thus, when proliferation is perturbed, migration isalso affected. This has been shown for both methyl mercury(Choi, 1986, 1989) and ethanol (Miller, 1993, 1996). Simi-larly, disruption of proliferation or migration can oftenaffect differentiation (Rice and Barone, 2000). Thus, a toxicinsult inflicted early in neurodevelopment has a greaterimpact than one exerted at a later stage.

The increased vulnerability of the developing nervoussystem compared to that of the adult may also be due to“quantitative” differences in pharmacokinetic and pharmaco-dynamic factors operating both in the young and the adult.Thus, there are notable differences between the developingand the mature organism in the absorption, distribution, bio-transformation, and excretion of the exogenous compounds.The presence of the placenta, excretion into milk and theblood–brain barrier (BBB) are also important in this context.Absorption in the developing organism both prenatally and

204 DEVELOPMENTAL NEUROTOXICITY OF ANTICHOLINESTERASE PESTICIDES

postnatally is greater than in the adult, since keratinization ofthe human epidermis only occurs in late gestation, and alsobecause an increased range of unwanted substances cangain access into babies and children through their normalmouthing and touching activities. Distribution differencesmay also account for the increased vulnerability of the devel-oping organism to neurotoxic insult, as there are loweramounts of serum proteins in the young, giving rise toincreased effective blood concentrations of toxic chemicals(Vidair, 2004). Differential sensitivities between the youngand the adult to neurotoxic damage are also due to differencesin biotransformation, since xenobiotic-metabolizing enzymesare not fully developed until after birth (Cresteil, 1998). In thecase of prenatal exposure, an additional factor affecting thevulnerability of the developing organism to neurotoxicinsult is the presence of the placenta. Although the placentais supposed to act as a barrier protecting the fetus fromunwanted molecules, it does not, in fact, prevent the intrusionof many compounds into the fetus (Andersen et al., 2000;Gupta, 2007; Pelkonen et al., 2006). Furthermore, since pla-cental transfer involves in several cases an active transportmechanism (Myllynen et al., 2005), fetal concentrations oftoxic compounds can be considerably higher than those inthe mother, thus contributing to the increased vulnerabilityof the developing organism to neurotoxic damage. Finally,the enhanced susceptibility of the nervous system of theyoung to toxic impairment can also be accounted for by thelate development of the BBB. Indeed, this structure, whichnormally prevents the entry of many unwanted agents intothe brain, is not completely developed to its fully functionalform until after birth (about 6 months in humans; Rodier,1995). Thus, both prenatal and postnatal exposure to sub-stances that are innocuous in the adult can induce toxicityin the developing brain (Levin and Miller, 1980; Saundersand Dziegilewska, 1998).

16.3 DEVELOPMENTAL NEUROTOXICANTSAND ANTICHOLINESTERASE PESTICIDES

From a very broad perspective, any exogenous substance thatis capable of inducing an abnormal alteration in the develop-ing nervous system of any organism lies within the scope ofdevelopmental neurotoxicology. On this basis, the number ofchemicals that can be regarded as developmental neurotoxi-cants is quite high (Andersen et al., 2000; Goldey et al.,1995; Spencer and Lein, 2005). On the other hand, fromthe narrower perspective of pediatrics, community medicine,and human risk assessment, the focus of developmentalneurotoxicology is restricted to those substances that producetoxic effects on the developing nervous system of the humansand at doses that are not harmful to the adult. In this context,the number of chemicals that, on the basis of the available evi-dence, qualify as developmental neurotoxicants is limited to

only a handful of substances (Grandjean and Landrigan,2006).

Although the anticholinesterase pesticides have been usedon a global scale, in large amounts and over half a century,their potential to cause developmental neurotoxicity hasonly recently been recognized (Slotkin, 2006). Indeed, inthe last few years a rapidly growing body of evidence, bothexperimental and epidemiologic, has been emerging whichindicates that at least some anticholinesterase pesticides arecapable of inducing developmental neurotoxicity. The big-gest part of this evidence concerns the organophosphate(OP) ester pesticides, whereas the number of studies thathave dealt with the developmental neurotoxicity of carbamate(CM) ester pesticides is limited. Even in the case of OPs,however, only a handful of compounds, which form a verysmall part of the tens of different OP pesticides currently inuse worldwide, have been investigated. Over the last fewyears experimental in vivo and in vitro biochemical, molecu-lar and morphological studies have increased in number.These studies offer valuable insights into the mechanismsmediating the developmental neurotoxic effects of OPs andCMs and will be duly described in much more detail in thischapter. On the other hand, human epidemiologic data willnot be covered here and the reader is referred to Chapters17 and 18 of this book. In addition, only data from mamma-lian studies will be reviewed, although the significant contri-bution of nonmammalian models in the screening for OPdevelopmental neurotoxicity is rightly emphasized in a laterpart of this chapter.

16.4 THE DEVELOPMENTAL NEUROTOXICITYOF ANTICHOLINESTERASE PESTICIDES AS ARESULT OF PHARMACOKINETIC DIFFERENCESBETWEEN THE YOUNG AND THE ADULT

The greater sensitivity of the developing nervous system tothe neurotoxic effects of anticholinesterase pesticides com-pared to that of the adult is partly attributed to a number ofpharmacokinetic factors. Developing animals are more sensi-tive than adults to the acute effects of OPs and CMs (Benkeand Murphy, 1975; Brodeur and Dubois, 1963; Harbison,1975; Mendoza, 1976; Moser, 1999) with LD50 valuesmore than 10 times lower in some cases (Zheng et al.,2000). Young animals are also more sensitive to the ChEinhibition produced after exposure to lower, sublethaldoses. This has been shown in a number of studies involvinga total of four OPs and one CM (Vidair, 2004). This increasedsensitivity to the acute lethal and sublethal, ChE-inhibitingeffects of OPs and CMs does not appear to be due to anenhanced sensitivity of the target enzyme to these agents.Indeed, in vitro data from both rats and humans indicatethat the kinetic behavior of ChE is not different between neo-nates and adults. Thus, the affinity of ChE for a number of

16.4 THE DEVELOPMENTAL NEUROTOXICITY OF ANTICHOLINESTERASE PESTICIDES 205

substrates (reflected in the apparent Km values) does notchange during development (Ecobichon and Stephens,1973; Mortensen et al., 1998). Similarly, in vitro inhibitionof brain ChE (reflected in the apparent IC50 values) by theoxon metabolites of parathion (Atterberry et al., 1997;Benke and Murphy, 1975; Mortensen et al., 1998), methylparathion (Benke and Murphy, 1975), and diazinon (DZ;Kousba et al., 2007) as well as by the CMs aldicarb and car-baryl (Mortensen et al., 1998) shows no difference betweenyoung and adults. On the other hand, the data for the oxonmetabolite of chlorpyrifos (CPF) are not as consistent(Atterberry et al., 1997; Kousba et al., 2007).

There is evidence to suggest that the increased sensitivityof the young to the acute lethal and sublethal, ChE-inhibitoryeffects of at least some important OP pesticides is due to thereduced capacity of the neonate to carry out effectively anumber of metabolic processes that result in OP inactivation.The most important of these detoxificating processes involveshydrolysis by A-esterases. Thus, the increases in LD50 valuesoccurring in rats from postnatal day 1 (PND 1) until adult-hood after dosing with parathion and methyl parathion corre-late well with the increases in the activity of A-esterases(oxonases) responsible for the inactivation of the ChE-inhibitory oxon metabolites of the administered OPs(Atterberry et al., 1996; Benke and Murphy, 1975). Oxonaseactivities towards paraoxon (paraoxonase) and methyl para-oxon in liver and plasma of 1-day-old rats are 2 to 10 timeslower than in adults. Even higher (12-fold) differences inserum oxonase activity towards paraoxon between 1-day-old and adult rats have been noted by Li et al. (1997). In7-day-old rats serum paraoxonase activity is sevenfoldlower than in adults (Karanth and Pope, 2000). Similar resultshave been obtained with the oxonase responsible for the inac-tivation of the oxon metabolite of CPF. Thus, compared to theadults, plasma and liver activity of this enzyme has beenfound to be 10 to 20 times lower in 1-day-old rats (Li et al.,1997; Moser et al., 1998), 2 to 11 times lower in 4-day-oldrats (Mortensen et al., 1996) and 5 to 10 times lowerin 7-day-old animals (Karanth and Pope, 2000). Even in17-day-old animals, plasma enzyme activity is considerablyreduced compared to adults (Padilla et al., 2000). A consider-able (30-fold) difference in plasma activity between 1-day-old animals and adults has also been shown for the oxonaseresponsible for the hydrolytic inactivation of diazinon oxon(DZO; Li et al., 1997). It is important that similar data seemto apply to the humans. Thus, serum paraoxonase activity isvery small in newborn humans, with its levels being 2.4-foldlower compared to the adults (Mueller et al., 1983). Infantshave a twofold lower serum paraoxonase activity even com-pared to 2 year olds (Ecobichon and Stephens, 1973).

Decreased catalytic activities of B-esterases (carboxyl-esterases) have also been held responsible for the increasedsusceptibility of the young to OP acute toxicity. Employinga range of substrates, various studies have shown that liver

and plasma B-esterase activity are decreased up to eightfoldin developing animals compared to adults (Vidair, 2004).Developing animals are unable to perform a number ofother detoxification processes, such as glutathione-dependentmetabolism by S-aryl and S-alkyl esterases and oxidativecleavage of parathion and methyl parathion (Benke andMurphy, 1975).

Although the available evidence indicates that the youngare more sensitive to the lethal and sublethal acute effectsof OP and CM pesticides, relevant data only involve a handfulof compounds. Even within the very small group of pesticidesthat have been studied, there are some notable exceptions tothe general rule of the increased sensitivity of the young.Thus, young animals given an acute dose of methamidophos(Moser, 1999) or CPF for 14 or 16 days (Zheng et al., 2000)do not show a greater inhibition of ChE than adults. Thesefindings, together with more recent data showing differencesbetween the oxon metabolites of CPF and DZ in their in vitroinhibition kinetics of brain ChE in neonates (Kousba et al.,2007), indicate that the toxicokinetic and toxicodynamic pro-files of the various OPs in the developing organism may differfrom each other leading to disparities in their capacities toadversely affect the young.

Although the differences in metabolic capacity are animportant reason for the different sensitivities of the youngand the adult to the neurotoxicity of at least some anticholin-esterase pesticides, the role of other pharmacokinetic factorscannot be ignored. OPs and CMs can effectively crossthe placenta (Gupta, 1995; Gupta 2007; Pelkonen et al.,2006). This is inferred from epidemiological studies showingan association between in utero OP exposure and reducedbirth weight and length and abnormal reflexes in neonatesand, also, visuospatial deficits in toddlers (Grandjean andLandrigan, 2006; Grandjean et al., 2006; Whyatt et al.,2004; Young et al., 2005). However, there is also moredirect evidence from experimental studies showing the effec-tive transplacental transfer of parathion, methyl parathion,methamidophos, acephate, CPF, leptophos, and DZ (Abdel-Rahman et al., 2002; Abu-Qare and Abou-Donia, 2001;Abu-Qare et al., 2000; Akhtar et al., 2006; Benjaminovet al., 1992; Piao et al., 1997; Salama et al., 1992a, 1992b).Furthermore, the activity of placental ChE is inhibited byparathion (Benjaminov et al., 1992), methyl parathion andDZ (Abu-Qare and Abou-Donia, 2001) and exposure tomethyl parathion induces abnormal structural changes at thecellular level in the placenta (Levario-Carillo et al., 2004).More worrying from a neurodevelopmental perspective aredata which demonstrate that there is a more rapid inhibitionof fetal brain ChE compared to that of the maternal enzymeafter oral and dermal administration of a subclinical dose ofmethyl parathion to pregnant rats (Abu-Qare and Abou-Donia, 2001; Gupta et al., 1985). Such findings may implyan increased vulnerability of the developing organism toOP neurotoxicity.


An important pharmacokinetic factor contributing to theincreased sensitivity of the developing organism to neuro-toxic injury is the late development of the BBB. Since thedevelopment of this structure is not fully complete duringthe prenatal and the early postnatal periods (Adinolfi, 1985;Saunders and Dziegilewska, 1998), insecticide concen-trations attained in the brain of the fetus and the infant areexpected to be higher than those in the brain of the adult.However, OPs and CMs can also affect the structure and per-meability of the BBB. Thus, a comparative study using adultand 10-day-old rats exposed to acute and subchronic doses ofthe OP quinalphos has shown increased developmental sensi-tivity of the BBB (Gupta et al., 1999). Administration ofparaoxon has also resulted in disruption of BBB permeabilityin young rats (25 to 30 days old), as assessed by determiningthe number of leaky brain capillaries, but has no effect inolder rats (90 days old; Song et al., 2004). In addition,in vitro studies demonstrate that CPF, a well-establishedneurodevelopmental toxicant, is capable of interfering withBBB integrity and stability (Parran et al., 2005; Yang et al.,2001).

With respect to CMs, although pyridostigmine, a knownprophylactic treatment for OP intoxication, has no effect onBBB permeability (Lallement et al., 2001) and its entryinto the brain is normally restricted, its brain penetration, atleast in the mouse, is facilitated under conditions of stress(Friedman et al., 1996). In addition, there is evidence tosuggest that the CM herbicide thiobencarb can induce BBBbreakdown (Pentyala et al., 1993).

16.5 THE DEVELOPMENTAL NEUROTOXICITYOF ANTICHOLINESTERASE PESTICIDES ASA RESULT OF INTERFERENCE WITHNEURODEVELOPMENTAL PROCESSES

Over the last decade a considerable body of biochemical evi-dence has emerged which indicates that anticholinesterasepesticides target specific processes during nervous systemdevelopment, thus causing developmental neurotoxicity.These processes include neuronal proliferation/replication,differentiation, axonogenesis/neurite outgrowth, synapto-genesis, gliogenesis/myelination, and apoptosis. The greatmajority of studies involve CPF, whereas some of the morerecent data also concern DZ. Parathion and CM compoundshave also been studied in a few cases. The evidence is notconfined to neurons, but also extends to glia and glia-relatedevidence, and will be reviewed separately. A significant partof the data derive from studies involving the in vivo adminis-tration of the pesticide to neonatal rats, while prenatal admin-istration has been used less frequently. In addition, in vitrocell culture systems, and particularly cell lines, to which thepesticide has been added, have been employed. Notwith-standing the well-recognized theoretical limitations in the

use of cell lines, these systems have been instrumental inprecisely defining the separate effects of the insecticides onneuronal and glial cells. Apart from constituting a homolo-gous population with respect to cell type (neuronal orglial), cell lines also represent a uniform population inrelation to differentiated state, thus their use has allowed theassessment of the effects of pesticides separately on the pro-cesses of cell replication and differentiation. In addition, asthe metabolic capacity of many cell lines is limited, theiruse has permitted the separate contribution of the parent com-pound and its in vivo metabolites to the obtained effects to beaccurately defined.

16.6 EFFECTS OF ANTICHOLINESTERASEPESTICIDES ON NEURONAL CELLREPLICATION AND DIFFERENTIATION

16.6.1 Effects on Synthesis or Levels of DNA,RNA, and Protein

A considerable number of studies, particularly in the earlierstages, have assessed the impact of OPs on neuronal cellreplication, growth, and differentiation by determining theeffects of these compounds on the synthesis or the levels ofDNA, RNA, and protein. Gupta et al. (1984) determinedin vivo protein synthesis in pregnant rats and their concep-tuses following subchronic administration of methyl para-thion (1 or 1.5 mg/kg body weight, p.o.) from day 6through day 15 or 19 of gestation. The inhibitory effect ofmethyl parathion on net protein synthesis was dose depen-dent, greater on day 19 than day 15 of gestation and morepronounced in fetal than maternal tissues. CPF, given sub-cutaneously to neonatal rats at an acute dose just below thethreshold for systemic toxicity (2 mg/kg), inhibits brain cellDNA and protein synthesis within 4 hours (Whitney et al.,1995). In 1-day-old rats, brain regions lacking substantialcholinergic innervation, for example, the cerebellum showdecreases in DNA and protein synthesis. However, in8-day-old rats, when cholinergic innervation is substantiallyenriched in areas such as the forebrain and the brainstem,these regions become more selectively targeted for effectson DNA synthesis. These data have been taken to indicatethat CPF inhibits cell replication by inducing cholinergichyperactivity, an effect also noted with other cholinergicstimulants (McFarland et al., 1991), but also inhibits DNAsynthesis through a noncholinergic direct action on develop-ing brain cells. That the CPF effects on DNA synthesis are notwholly due to ChE inhibition has been inferred by showingthat CPF introduced directly into the brain by intracisternalinjection, thus bypassing its hepatic conversion to its strongChE-inhibiting metabolite chlorpyrifos oxon (CPO), canstill cause DNA synthesis inhibition in 1-day-old rats(Whitney et al., 1995). CPF, given to 1-day-old rats in fourdaily doses of 1 or 5 mg/kg and to 11-day-old animals in

16.6 EFFECTS OF ANTICHOLINESTERASE PESTICIDES ON NEURONAL CELL REPLICATION AND DIFFERENTIATION 207

four daily doses of 5 or 25 mg/kg, induces changes in bothDNA (an index of cell number) and protein levels after 5days (Campbell et al., 1997), indicating that the deficits inDNA and protein synthesis noted previously after 4 hours(Whitney et al., 1995) translate to subsequent brain loss.CPF does not target only cholinergic cells, as effects arealso noted in the cerebellum. CPF, given to 1-day-old ratsfor 4 days in a daily dose of 1 mg/kg, a dose producing25% AChE inhibition, also affects brain cell differentiation,as indicated by eliciting changes in RNA concentration andcontent (Johnson et al., 1998). These changes appear 1 dayafter the end of treatment and well before the deficits in cellnumber noted in the study of Campbell et al. (1997). ThisCPF dose regimen also induces a decrease in DNA synthesis,but does not affect brain RNA and protein synthesis (Damet al., 1998), indicating a selectivity of CPF effect when, incontrast to the study of Whitney et al. (1995), a low, repeateddose regimen is applied.

The effects of CPF on macromolecular indices of brain cellreplication, growth, and differentiation have also been studiedin developing rats exposed to the OP prenatally. Prenataladministration of CPF, at doses below the threshold for theinhibition of fetal brain ChE has, compared to postnatalexposure, a less pronounced immediate (late gestational)effect on the number of brain cells (Qiao et al., 2002). Instark contrast, however, prenatal CPF administration pro-duces considerable brain cell abnormalities postnatally, byreducing cell density (as indicated by reduced DNA concen-tration) and increasing cell size (as indicated by increasedprotein/DNA ratio; Qiao et al., 2003). Determination ofthese macromolecular parameters up to PND 60 shows thatthe CPF-induced disruption of brain cell proliferation anddifferentiation continues into adolescence and adulthood(Qiao et al., 2003).

In contrast to CPF, whose effects on macromolecular indi-ces of brain cell development following in vivo exposure havebeen well studied, other OPs have not been the focus of simi-lar attention. In a recent study, diazinon (DZ) given to rats onPNDs 1 to 4 at a daily dose of 0.5 mg/kg (causing no ChEinhibition) or 2 mg/kg (causing �20% ChE inhibition) hasbeen found to interfere with brain cell development in adoles-cence and adulthood (PNDs 30 to 100). Specifically, DZexposure increases cell density, increases or decreases(depending on the brain area) cell number, but has no effecton cell size (Slotkin et al., 2008). Importantly, the patternof these changes is strikingly different to that noted afterthe administration of equivalent doses of CPF for the sametime period (Qiao et al., 2003), indicating that the two OPpesticides may interfere with normal brain cell developmentthrough different mechanisms.

In contrast to the above in vivo studies, in which macro-molecule determinations have been invariably made onbrain homogenates and which do not allow precise distinctionof the neuronal and glial effects, investigations that have used

cell culture systems have achieved the assessment of theeffects of OPs on macromolecule synthesis and levels inneuronal and glial cells separately. With respect to OP effectson the development of neuronal cells, CPF, at noncytotoxicmicromolar concentrations, exerts an immediate (within1 hour), direct inhibitory effect on DNA synthesis in culturesof replicating neuronotypic PC12 cells (Qiao et al., 2001).This ability of CPF to interfere with neuronal cell replicationis not related to ChE inhibition, since CPO is less effective ininhibiting DNA synthesis. In addition, the ChE-inhibitingCM compound physostigmine shows no effect, whereas theCPF’s metabolite trichloropyridinol, which does not inhibitChE activity, does have an antimitotic effect. Importantly,under the same cell culture conditions, DNA synthesis isalso rapidly inhibited by equimolar concentrations of DZ,albeit with a lower potency (Qiao et al., 2001). These datahave been expanded more recently by Slotkin et al.(2007a), who have assessed the effects of CPF, DZ, para-thion, and physostigmine on DNA synthesis (cell replica-tion), DNA content (cell number), and protein/DNA ratio(cell size) in both undifferentiated (mitotic) and differentiat-ing cultured PC12 cells. In undifferentiated cells, all com-pounds at noncytotoxic concentrations of 5 and 30 mMinhibit DNA synthesis, with DZ having the greatest effect.After 4 to 6 days, the effects of CPF and DZ remain detect-able, whereas the effects of equimolar concentrations of para-thion disappear. All test agents also elicit after 6 dayssignificant reductions in cell numbers, with parathion andphysostigmine exerting a greater effect compared to CPFand DZ. By contrast, cell size is not suppressed. In differen-tiating cells, differences among the four test compoundsemerge in terms of their effects on cell size after 4 to 6days. These and other data obtained in the same study(Slotkin et al., 2007a) indicate that the various OPs exhibitdifferences from each other and from CMs in their adverseactions on neuronal cell replication and differentiation, apoint that is being illustrated repeatedly in many recentstudies (see below).

16.6.2 Effects on Cell Signaling, TranscriptionFactors, and Neurotrophic Factors

The interference of OPs with the processes of cell replicationand differentiation shown in the above studies, and the invol-vement of noncholinergic mechanisms, have been furtherexplored at the molecular level in a number of studies thathave assessed the effects of OPs on cell signaling pathwaysinvolved in the regulation of brain/neuronal cell replicationand differentiation. CPF administration to neonatal ratsaffects extensively the adenylyl cyclase transduction pathwayby inducing deficits at many different levels, including theexpression and activity of adenylyl cyclase itself, the functionof G-proteins that link cyclase activity to neurotrans-mitter receptors, and the expression and function of


neurotransmitter receptors acting through this cascade (Songet al., 1997). The disruption of the cyclase cascade cannot beattributed to cholinergic hyperactivity, since effects areextended to the sparsely cholinergically innervated cerebel-lum and the greatest effects in the forebrain are obtainedwith a dose regimen that causes only minor ChE inhibition.In addition, CPF effects on signaling and ChE inhibitionare not related in time.

The study of the ability of CPF to target developmentallyrelevant cell signaling pathways in vivo has been extended toinclude its direct effects on the expression and function ofspecific nuclear transcription factors which are downstreameffectors of these pathways and which are involved in thecontrol of genes required in brain cell replication and differ-entiation. Thus, CPF, given to neonatal rats at the dosesapplied by Song et al. (1997), induces changes in theexpression and function (DNA binding) of transcription fac-tors AP-1 and Sp1, which are downstream targets of the ade-nylyl cyclase-cAMP signaling cascade (Crumpton et al.,2000). For AP-1, effects are more pronounced during differ-entiation, whereas for Sp1, they are more pronounced duringreplication. Significantly, effects on Sp1 are less pronouncedwith the dose regimen that produces considerably greater ChEinhibition. The ability of CPF to interfere with the expressionof AP-1 and Sp1 has also been shown in PC12 cells(Crumpton et al., 2000), either when CPF (50 mg/mL) isadded during replication or after initiation of differentiation.Sp1 is reduced in both replicating and differentiating cells,whereas AP-1 is affected only during differentiation, in keep-ing with the greater role of the latter transcription factor indifferentiation. CPF affects the expression of another nucleartranscription factor, namely the Ca2þ/cAMP responseelement binding protein (CREB), which is also phosphory-lated by the adenylyl cyclase pathway and which also playsa major role in cell differentiation during brain development(Schuh et al., 2002). More specifically, in primary cultures ofcortical and hippocampal neurons prepared from embryonicrat pups, CPF treatment increases the levels of the phosphory-lated, active form of CREB. Interestingly, equally significanteffects are induced by the CPF metabolite trichloropyridinol,whereas the oxon metabolite, CPO, evokes increases with apotency that is three orders of magnitude higher than thatof CPF and trichloropyridinol. CPF effects, however, arenot due to its metabolic conversion to CPO, as they persistin the presence of the cytochrome P-450 inhibitor SKF-525A (Schuh et al., 2002). Significantly, all effects onactive CREB levels are attained at doses that do not inhibitChE activity in the cultured neurons.

The advance of gene microarray techniques has recentlyallowed the evaluation and comparison of the effects ofCPF and DZ on the transcription of a family of genes encod-ing various components of signaling cascades and transcrip-tion factors involved in neuronal cell replication anddifferentiation (Slotkin and Seidler, 2007). Administration

of the OPs to 1-day-old rats at doses (1 mg/kg CPF and1 or 2 mg/kg DZ daily for 4 days) producing no or lessthan 20% ChE inhibition has been found to have an extensiveimpact on gene expression, with significant changes noted for51 out of the 95 genes that have been evaluated in total. Inagreement with the studies mentioned above, the dataobtained indicate that the cAMP signaling cascade is amajor target for the adverse effects of both CPF and DZ onbrain development at almost all levels: G-protein-coupledreceptor modulators, G-protein a subunits, adenylyl cyclaseisoforms and their modulators, phosphodiesterases, proteinkinases A, and nuclear transcription factors (AP-1, Sp1 andCREB) known to be downstream targets for protein kinaseA phosphorylation. Only the expression of genes codingfor the b and g subunits of G-proteins is not generallyaffected. In contrast to their profound effects on genes relatedto the cAMP signaling pathway, both OPs have much lessimpact on the transcription of genes coding for proteinkinase C and its modulators (Slotkin and Seidler, 2007).Although CPF and DZ show many similarities in their effectson gene transcription, they also exhibit a number of distinctdifferences. Among these is the much greater effect of DZon the transcription of the gene encoding the receptorkinase that uncouples G-proteins from their ability to signalthrough a subunits. Marked disparities between CPF andDZ are also noted in terms of their effects on the expressionof phosphodiesterase genes. In addition, DZ differs fromCPF in its interference with the transcription of genes encod-ing Sp1 and CREB. The demonstration of these differencesin the effects of CPF and DZ on the transcription of anumber of genes related to the developmentally vital cAMPsignaling pathway further supports the notion that OPs inter-fere with nervous system development through distinctlydifferent molecular mechanisms, leading potentially to themanifestation of distinct ultimate neurobehavioral/clinicalsymptomatologies.

The neurodevelopmental potential of anticholinesterasepesticides has been further studied at the molecular level byassessing the effects of these compounds on the protein andthe mRNA levels of neurotrophic factors known to playimportant roles in neuronal cell development. Oral adminis-tration of CPF to 1-day-old rats for 6 days at a daily dose of1.5 or 3 mg/kg reduces the levels of the nerve growthfactor (NGF) in the forebrain (Betancourt and Carr, 2004).By contrast, levels of the brain derived neurotrophic factor(BDNF) are not affected. The decrease in NGF proteinlevels has been subsequently shown to be due to an effectat the gene transcription level as, under the same dosing con-ditions, mRNA levels of NGF in the forebrain are also signifi-cantly reduced (Betancourt et al., 2006). These effects arenoted under conditions of significant ChE inhibition.Employing a dose regimen of neonatal CPF administrationcausing no inhibition of ChE (a daily dose of 1 mg/kggiven subcutaneously for 4 days to 1-day-old rats), Slotkin

16.6 EFFECTS OF ANTICHOLINESTERASE PESTICIDES ON NEURONAL CELL REPLICATION AND DIFFERENTIATION 209

et al. (2007b) have found significant effects on the levels ofmRNAs coding for a number of different neurotrophic factorsbelonging to the fibroblast growth factor (FGF) superfamily,which is important in neuronal development. Significantly,administration of DZ under the same conditions also affectsthe expression of a number of FGF genes. However, despitesome similarities, there are a number of important differencesbetween CPF and DZ in their effects on FGFs implying,again, differences in their molecular mechanisms of interfer-ence with normal nervous system development.

16.7 EFFECTS OF ANTICHOLINESTERASEPESTICIDES ON NEURITE OUTGROWTH

The effects of anticholinesterase pesticides on the process ofneurite outgrowth have been assessed in a number of studies.In most of these, neurite outgrowth has been measured mor-phometrically with the aid of the microscope. In some cases,the use of specific immunostaining methods, high-resolutioncameras, and sophisticated software has permitted very pre-cise and sensitive determinations of the length and numberof neuritic processes. Alternatively, neurite outgrowth hasbeen determined biochemically by measuring either theratio of membrane protein to DNA or the ratio of membraneprotein to total protein. Both of these ratios increase as a resultof the expansion of the cell membrane surface that occursduring neurite outgrowth. In most studies neurite develop-ment has been determined following addition of the pesti-cides to cultured cells, whereas in some instances theeffects on neurite outgrowth have been assessed after invivo administration of the pesticides to developing animals.In the case of cultured cells, both primary cultures and celllines have been employed.

A number of OP pesticides have been shown to be capableof inhibiting the outgrowth of neurites from cultured cells.The most extensively studied OP pesticide, CPF, can impairneurite outgrowth in both pheochromocytoma and neuroblas-toma cell lines. Thus, exposure of rat PC12 pheochromo-cytoma cells, induced to differentiate by NGF, to 50 mg/mLCPF for 2 days (Song et al., 1998) or to 10 mg/mL CPF for7 days (Das and Barone, 1999) inhibits neurite extension.CPF also interferes with neurite outgrowth in cultures ofmouse N2a neuroblastoma cells induced to differentiate byserum withdrawal and the addition of dibutyryl cAMP. Inthese cells, CPF, at a concentration of 3 mM, is capable ofcausing significant inhibition of neurite outgrowth afteronly 4 hours (Sachana et al., 2001, 2005). When N2a cellsare exposed to CPF for 24 hours, the IC50 value for neuriteoutgrowth inhibition is 13 mM (Axelrad et al., 2003). CPFcan also inhibit neurite outgrowth in the mouse N-18 neuro-blastoma cell line, with an IC50 value of 44 mM (Henschleret al., 1992), although in a subsequent study using almostidentical exposure conditions neurite inhibition has beennoted only at cytotoxic CPF levels (Schmuck and Ahr,

1997). In these studies CPF has been added initially tomitotic N-18 cells for 2 weeks and then for a subsequentperiod of 5 days in the presence of dibutyryl cAMP. CPFeffects have also been studied in primary cultures of embryo-nic rat sympathetic neurons derived from superior cervicalganglia (Howard et al., 2005) as well as in sensory neuronsderived from embryonic rat dorsal root ganglia (Yanget al., 2008). In both systems CPF, at a concentration of0.001 mM, interferes after 24 hours with the outgrowth ofaxonal processes by decreasing axonal length, but has noeffect on the number of axonal processes. The ability ofCPF to impair neurite outgrowth has also been shown inin vivo studies. Administration of the OP to 1-day-old ratsfor 4 days at a daily dose of 1 mg/kg decreases neuriteoutgrowth (Slotkin et al., 2006). Prenatal exposure to CPFat doses of 1 or 5 mg/kg daily on gestational days 17 to 20also induces contraction of neurites (Qiao et al., 2003). Inthese in vivo studies neurite outgrowth has been determinedbiochemically by measuring the ratio of membrane proteinto total protein.

The effects of DZ on neurite outgrowth have also beenstudied in in vivo and in vitro studies. DZ, given to rats onPNDs 1 to 4 at a daily dose of 0.5 mg/kg, which causes noChE inhibition, readily impairs neurite outgrowth, as indi-cated by a decreased membrane protein to total protein ratio(Slotkin et al., 2006). However, this effect is not sustainedand disappears on PNDs 30 to 100 (Slotkin et al., 2008). Incultures of differentiating N2a cells DZ treatment for 24hours inhibits neurite outgrowth, with a concentration of10 mM causing 34.4% inhibition (Flaskos et al., 2007) andan IC50 value of 68 mM (Axelrad et al., 2003). Moreover,the inhibition of neurite extension induced after exposure toDZ for 24 hours is enhanced in N2a cells previously treatedwith this OP for 8 weeks (Axelrad et al., 2003).

The OP mipafox has also been assessed in terms of itseffect on neurite outgrowth in a number of studies usingcell lines. Exposure of mitotic PC12 cells to this pesticidefor 7 days followed by addition of NGF and further exposurefor 5 days inhibits neurite outgrowth with an IC50 value of100 to 200 mM (Li and Casida, 1998). Similarly, in theN-18 cell line exposure of mitotic cells to mipafox for2 weeks followed by the addition of dibutyryl cAMPand further exposure for 5 (Schmuck and Ahr, 1997) or6 (Henschler et al., 1992) days inhibits neurite extensionwith an IC50 value of 68 mM. Significantly, mipafox inter-feres with neurite outgrowth not only in rodent cell linesbut also in the human neuroblastoma SH-SY5Y cell line(Hong et al., 1998).

Leptophos has also been found to affect neurite outgrowthin more than one cell line. Thus, exposure of mitotic N-18cells for 2 weeks to leptophos followed by further exposurefor 5 or 6 days in the presence of dibutyryl cAMP inhibitsneurite outgrowth with an IC50 value of 14 mg/mL(Henschler et al., 1992; Schmuck and Ahr, 1997). Leptophosalso interferes with neurite extension in the N2a cell line,


with a concentration of 3 mM causing almost 50% inhibitionwithin 4 hours (Sachana et al., 2003). On the other hand, OPpesticides that inhibit neurite outgrowth in one cell lineinclude trichlorfon (Flaskos et al., 1999), methyl chlorpyrifos(Sachana et al., 2001), pirimiphos methyl and phosmet(Axelrad et al., 2003), all of which impair neurite extensionin N2a neuroblastoma cells, and dichlorvos, cyanofenphos,haloxon, DEF, and EPN, which inhibit neurite outgrowth inN-18 neuroblastoma cells (Henschler et al., 1992).

The cell lines employed in the neurite outgrowth studiesabove have limited ability to metabolize the OPs.Furthermore, in the animal studies the OPs have been givenvia the subcutaneous route, thus bypassing first-pass hepaticmetabolism. This implies that the neurite-inhibiting effectsobtained are caused by the administered OP compoundsthemselves and are not the result of the action of their metab-olites. However, a number of in vitro studies do indicate thatthe major metabolites of two of the most extensively used OPpesticides, CPF and DZ, also have potent neurite-inhibitingproperties. Thus, CPO, at a concentration as low as10 ng/mL, impairs in the PC12 cell line after 7 days theextension of neurites (Das and Barone, 1999). CPO, at aconcentration as low as 0.001 nM, also decreases within24 hours axonal length in primary neuronal cultures derivedfrom embryonic rat superior cervical ganglia (Howardet al., 2005). Similarly, CPO, at a concentration of 0.01 nM,inhibits after 24 hours axonal outgrowth in primary culturesof neurons derived from embryonic rat dorsal root ganglia(Yang et al., 2008). Recent data show that the oxon metab-olite of DZ has similar neurite-inhibiting effects. Thus,DZO interferes with neurite outgrowth in the N2a cell line,with a concentration of 1 mM causing after 24 hours a greaterthan 50% neurite inhibition (Sidiropoulou et al., 2009).Comparison of the neurite-inhibiting potencies of CPO andDZO with those of the parent compounds CPF and DZ,respectively, assessed under identical conditions, indicatesthat these oxon metabolites are 10 to 1000 times stronger ininhibiting neurite outgrowth (Das and Barone, 1999;Flaskos et al., 2007; Howard et al., 2005; Sidiropoulouet al., 2009; Yang et al., 2008). On the other hand, availabledata on the oxon metabolite of parathion, paraoxon, show thatthis compound has no effect on neurite outgrowth in themouse N-18 (Henschler et al., 1992) and the human SH-SY5Y (Hong et al., 1998) neuroblastoma cell lines andimpairs neurite development only at cytotoxic doses(Santos et al., 2004).

There is a paucity of data on the effects on neurite out-growth of CM pesticides. In the few relevant studies, carbarylhas been shown to be capable of interfering with neurite out-growth in N2a cells (Flaskos et al., 1999; Sachana et al.,2003) with a concentration of 3 mM inducing after 8 hoursa 43.9% inhibition.

In summary, available data suggest that a notable numberof anticholinesterase pesticides can interfere with the out-growth of neurites. These data have mainly derived from

in vitro studies that have used a range of cell culture systems,differentiation-inducing agents, and neurite countingmethods. No matter how valuable these findings undoubt-edly are, a central issue that remains unclear is the extent towhich these cell culture data relate to real in vivo effects.Thus, it is not certain whether inhibition of neurite outgrowthby the test compound is, in fact, indicative of its ability tocause toxicity on the developing nervous system. Indeed, ina number of cell culture studies a range of exogenous sub-stances of widely differing structures have been shown tointerfere with neurite outgrowth, although there is, so far,no evidence of those compounds being developmental neuro-toxicants in vivo. These substances include the excitatoryamino acids b-N-methylaminoalanine and kainate (Abdullaand Campbell, 1993), the antimalarial drug artemisinin(Smith et al., 1997), the insecticide pyrethrum (Axelradet al., 2003), and many others. In fact, in several of the earlierstudies, neurite outgrowth inhibition, instead of reflecting thepotential of an OP to interfere with nervous system develop-ment, has been considered indicative of this compound’s abil-ity to cause neurodegenerative effects in vivo, especially inthe context of the characteristic OP-induced delayed neuro-pathy (OPIDN) known to be elicited by some OPs (Flaskoset al., 1998; Henschler et al., 1992; Li and Casida, 1998;Schmuck and Ahr, 1997). In view of these, it is importantto establish in future studies whether OPs and other com-pounds that have been shown to inhibit neurite outgrowthin vitro are indeed capable of inducing neurodevelopmentaltoxicity in vivo following prenatal or postnatal exposure.Further in vitro neurite-inhibiting studies for OP and CMpesticides are warranted, however, for a number of additionalreasons. Thus, for many common OPs there are no data oravailable data derive only from a single study or from theuse of a single cell culture system. Similarly, more studiesare needed on CMs, since only carbaryl has been assessedand data indicate that this CM can readily inhibit neurite out-growth with a potency similar to that of CPF (Flaskos et al.,1999; Sachana et al., 2003), the OP for which there is thestrongest evidence for being a developmental neurotoxicant.Additional studies are also required on OP metabolites inview of the fact that CPO and DZO are very potent inhibitorsof neurite outgrowth. In fact, evidence suggests that even OPmetabolites generally considered as relatively innocuous caninterfere, at biologically relevant levels, with neurite out-growth in PC12 cells (Das and Barone, 1999) and dendriticoutgrowth in primary cultures of embryonic sympatheticneurons (Howard et al., 2005). Future studies should alsoassess neurite inhibition following exposure to a combinationof different OPs and CMs in light of the data of Axelrad et al.(2003) showing changes in the neurite-inhibiting propertiesof OPs when these are given in combination with otherOP and non-OP pesticides. Such changes may be signifi-cant in vivo, as exposures to combinations of pesticidesare common in agricultural environments. Furthermore,for the purposes of risk assessment, more neurite outgrowth

16.7 EFFECTS OF ANTICHOLINESTERASE PESTICIDES ON NEURITE OUTGROWTH 211

studies using cell cultures of human origin are required, asavailable human data derive only from a single study(Hong et al., 1998).

An additional important reason for conducting furtherstudies on the effects of OPs and CMs on neurite outgrowthis the need to elucidate the underlying molecular mechan-isms. Inhibition of neurite outgrowth by OPs has beenlinked in some studies using cell lines to the inhibition ofneuropathy target esterase (NTE; Henschler et al., 1992; Liand Casida, 1998; Sachana et al., 2001), in line with thealleged role of this enzyme in neuronal development(Glynn, 2000). Although in NGF-induced, differentiatingPC12 cells neurite outgrowth is highly correlated with anincrease in ChE activity (Das and Barone, 1999), a numberof studies involving both neuronal primary cultures and celllines indicate that OP-induced neurite outgrowth inhibitionis not related to inhibition of the enzymatic activity of ChE(Das and Barone, 1999; Hargreaves et al., 2006; Howardet al., 2005; Slotkin et al., 2006). Instead, inhibition of neuriteoutgrowth by OPs may be partly linked to OP interferencewith the known (Bigbee et al., 1999; Brimijoin andKoenigsberger, 1999) normal morphogenic activity of theAChE protein (Howard et al., 2005; Yang et al., 2008).However, the molecular mechanisms by which OPs disturbthe morphogenic function of ChE are not known.Anticholinesterase pesticide-induced neurite outgrowth inhi-bition has also been related to alterations in the levels of anumber of cytoskeletal and axonal-enriched proteins, in linewith the known importance of these molecules in axongrowth and/or stability (Cambray-Deakin, 1991; Skene,1989). Changes in the expression of these proteins havebeen shown to be induced by six different anticholinesterasepesticides including one CM (Flaskos et al., 1999, 2007;Sachana et al., 2003, 2005). These changes are the causerather than the effect of the decreased neurite outgrowth, asthere are distinct differences in the pattern of biochemicaleffects induced by different pesticides, tested under identicalconditions, in the presence of the same morphological effect(Flaskos et al., 2007; Sachana et al., 2003; Sidiropoulouet al., 2009). These data do not necessarily imply that cyto-skeletal and axon-growth-associated proteins are directmolecular targets of the neurite-inhibiting action of anticho-linesterase pesticides, but they do indicate that these agentsinterfere with neurite outgrowth via distinct biochemicalmechanisms.

16.8 EFFECTS OF ANTICHOLINESTERASEPESTICIDES ON SYNAPTIC DEVELOPMENT

Although studies on the effects of anticholinesterase pesti-cides on synaptic development have involved both thecholinergic and the monoaminergic (catecholaminergic andserotonergic) pathways, this review, for reasons of space,

will be restricted exclusively to cholinergic data. The abilityof OPs to interfere with the development of cholinergicneurotransmitter systems has been assessed by determiningthe effects of these agents on two separate parameters: (1)the development of cholinergic synapses and (2) the cholin-ergic synaptic activity. As a biochemical marker for cholin-ergic synaptic development these studies have used theactivity of choline acetyltransferase (ChAT). The activity ofthis enzyme, which is responsible for ACh synthesis,increases during cholinergic synaptogenesis, but does notchange in response to changes in cholinergic neuronalactivity. For cholinergic synaptic activity the biochemicalindex used has been the binding of hemicholinium-3 to thepresynaptic high-affinity choline transporter on the cell mem-brane. This is a measure of the expression of the choline trans-porter and the high-affinity choline uptake and, in contrast toChAT activity, is responsive to neuronal activity. All studieshave been carried out by a single research group, have mainlyinvolved CPF and DZ, and have included the assessment ofboth immediate effects and effects persisting or appearingin adolescence and adulthood.

Rats exposed in utero to methyl parathion (1.5 mg/kgbody weight, p.o.) from day 6 through day 20 of gestationshowed significant reduction in AChE activity and decreasein ChAT activity in brain regions (cortex, brainstem, striatum,and hippocampus) that persisted through postnatal day 28(Gupta et al., 1985). However, administration of CPF to1-day-old rats for 4 days at a daily dose of 1 mg/kg produceswithin 24 hours a decrease in ChAT activity, but does notaffect hemicholinium-3 binding to the choline transporter(Dam et al., 1999). This effect is noted in the absence ofany significant downregulation of m2-muscarinic receptors,indicating no major AChE inhibition. This rapidly expresseddeficit in the development of cholinergic projections persistsinto adolescence and adulthood and leads to the emergence ofsubstantial deficiencies in cholinergic synaptic activity, asindicated by decreases in hemicholinium-3 binding (Slotkinet al., 2001), as well as to behavioral anomalies (Levinet al., 2001). The effects of CPF on cholinergic synapticdevelopment have also been studied in rats exposed to theOP prenatally. Thus, administration of CPF to pregnant ratson gestational days 17 to 20 at a daily dose of 1 mg/kg,which does not inhibit fetal brain ChE, affects the subsequentdevelopment of cholinergic systems in forebrain regionsinvolved in cognition by causing postnatal deficits incholinergic synaptic activity, as indicated by reducedhemicholinium-3 binding (Qiao et al., 2003). These deficits,which are already apparent in the early postnatal period,extend into adolescence and adulthood (PND 60) andcorrespond to the observed long-term defects in memory(Levin et al., 2002). By contrast, CPF does not inducesubstantial deficits in the development of cholinergic nerveterminals, as indicated by only minor changes in ChATactivity. In addition, these deficits are no longer present by


PND 60 (Qiao et al., 2003). The lasting deterioration ofsynaptic activity is not compensated by upregulation ofcholinergic receptors, as m2-muscarinic receptor bindingis not altered.

Studies similar to those mentioned above have been car-ried out more recently with DZ. Administration of DZ to1-day-old rats for 4 days at daily doses of 0.5 to 2 mg/kg,which cause little (�20%) or no AChE inhibition, elicitswithin one day a pattern of effects identical to that notedwith CPF, with deficits in ChAT activity but no effects onhemicholinium-3 and m2-muscarinic receptor binding(Slotkin et al., 2006). On the other hand, parathion has noeffect on the cholinergic parameters assessed. Evaluation ofthe effects of DZ on PNDs 30 to 100 shows profound deficitsin all three cholinergic synaptic markers in adolescence andadulthood (Slotkin et al., 2008). These impairments arenoted in brain regions rich in cholinergic projections andmay account for the late-onset cognitive (Timofeeva et al.,2008a) and emotional (Roegge et al., 2008) deficits noted fol-lowing DZ administration. Although the reductions in ChATactivity and hemicholinium-3 binding are also caused byCPF, there are differences between the two OPs in theirregional profiles and particularly in their effects in thestriatum (Qiao et al., 2003; Slotkin et al., 2001). Such differ-ences may be responsible for dissimilar neurobehavioralpathologies (Levin et al., 2001, 2002; Roegge et al., 2008;Timofeeva et al., 2008a).

The effects of CPF and DZ on the developing cholinergicsystem have also been studied at the transcriptional level. Theuse of gene microarray technology has revealed a number ofeffects on the transcription of genes related to ACh synthesis,storage, degradation, and receptors following OP adminis-tration to neonatal rats at doses causing no or less than 20%ChE inhibition (Slotkin and Seidler, 2007). The most impor-tant effect, which is shared by CPF and DZ, is a reduction inthe expression of the gene coding for ChAT, an effect consist-ent with the reductions in the activity of this enzyme noted inthe previous studies. The reduction in the expression of thegene encoding ChAT is accompanied by changes in theexpression of genes coding for the choline transporter indicat-ing that CPF and DZ both can interfere with the developmentof the ACh phenotype. The latter effect has been suggested tocause miswiring of brain circuits during development and be-havioral abnormalities (Slotkin and Seidler, 2007). Both CPFand DZ also induce large decreases in the expression of thegenes encoding the m2-muscarinic and a7-nicotinic recep-tors. However, DZ differs strongly from CPF in that its effectson the expression of nicotinic receptors are much more exten-sive. Differences between CPF and DZ are also noted inrelation to their effects on the expression of a particularChE splice variant, the ChE-S isoform, which is specificallyrelated to neurotoxicity. Thus, expression of mRNA encodingChE-S increases following neonatal DZ administration, but isnot significantly affected by similar CPF treatment (Jameson

et al., 2007). This induction of the ChE-S isoform by DZ isalso seen in cultures of differentiating PC12 cells. CPO, inthis in vitro system, has no effect implying that the increasein ChE-S expression induced by DZ is not due to ChEinhibition.

16.9 EFFECTS OF ANTICHOLINESTERASEPESTICIDES ON GLIAL DEVELOPMENT

Initial indications of a glial involvement in the developmentalneurotoxicity of OPs have been drawn from the earlier in vivostudies showing that the CPF effects in the intact brain occurat a time of vigorous glial development. Thus, CPF inhibitsDNA synthesis (Whitney et al., 1995; Dam et al., 1998)and causes brain cell loss (Campbell et al., 1997) after theend of neurogenesis and during gliogenesis. CPF effects oncell signaling (Song et al., 1997) and synaptic developmentand activity (Dam et al., 1999) appear during periods ofmaximal glial development. Finally, significant neuro-behavioral effects are induced by CPF when exposureoccurs at times of active gliogenesis and decreased neuro-genesis (Dam et al., 2000; Levin et al., 2001; Moser andPadilla, 1998).

More definitive evidence that OPs specifically targetdeveloping glial cells has been provided by studies involvingthe assessment of glial-specific markers in the brain or in cul-tures of mixed neurons and glia. When CPF is given at dosescausing no systematic toxicity to rats on PNDs 11 to 14(a period at the peak of glial development), there are immedi-ate reductions in the levels of the specific astrocyte markerglial fibrillary acidic protein (GFAP) in all brain areas, aneffect indicative of specific suppression of normal glial devel-opment (Garcia et al., 2002). By contrast, administration ofCPF prenatally (when there is limited gliogenesis) has noeffect on fetal brain GFAP. Glial cell markers are also affectedin cell cultures of mixed neurons and glia. Thus, in aggregat-ing cell cultures of fetal rat telenchephalon, activity levels ofglutamine synthase (a marker of astrocytes) and cyclicnucleotide phosphohydrolase (a marker of oligodendrocytes)are altered by treatment with CPO and paraoxon (Monnet-Tschudi et al., 2000). Levels of GFAP are also affected byOPs in the same cell culture system (Zurich et al., 2000).

Use of gliotypic cell lines, which present a uniform cellpopulation in terms of cell type, has been instrumentalin showing direct effects of OPs specifically on gliotypiccells. As cell lines consist of cells that are all in the samestate of differentiation, their use has also assisted in theevaluation of the effects of OPs separately on glial cell repli-cation and glial cell differentiation. In addition, since celllines have generally little or no metabolic capacity, the sep-arate effects of the parent OP compounds and their in vivometabolites on glial development have been assessed withsome accuracy.

16.9 EFFECTS OF ANTICHOLINESTERASE PESTICIDES ON GLIAL DEVELOPMENT 213

Specific targeting of glial cell replication by anticholin-esterase compounds has been demonstrated in studiesemploying the rat C6 glioma and the human 1321N1 astrocy-toma cell lines. Thus, CPF causes immediate inhibition ofDNA synthesis both in the absence (Qiao et al., 2001) andpresence (Garcia et al., 2001) of serum, an effect independentof cholinergic stimulation. DNA synthesis in the same culturesystem is also inhibited by CPO, DZ, and the CM physostig-mine, albeit to a smaller extent (Qiao et al., 2001). CPF, DZ,and parathion as well as their in vivo oxon metabolites alsoinhibit DNA synthesis in 1321N1 cells (Guizzetti et al.,2005). In this cell line, however, the antiproliferative effectsof these compounds are similar. In both cell lines, the CPFmetabolite trichloropyridinol also inhibits DNA synthesis,but with the lowest potency.

The ability of OPs to interfere with glial cell developmenthas also been indicated by their effects on the adenylylcyclase transduction cascade, a signaling pathway importantin both glial cell proliferation and differentiation. Thus, inreplicating, undifferentiated C6 cells CPF causes consider-able impairment of G-protein signaling, leading to persistentchanges in the catalytic properties of adenylyl cyclase (Garciaet al., 2001). CPF disturbs G-protein functioning also in dif-ferentiating C6 cells, indicating that its adverse effects extendbeyond the phase of glial cell replication and into the glialdifferentiation stage. In line with this, CPF interferes withthe expression of the transcription factor Sp1, a downstreamtarget of the adenylyl cyclase pathway which is necessaryfor C6 cell differentiation (Garcia et al., 2001). On theother hand, in cultured astrocytes CPF does not affect thephosphorylation (activation) of the transcription factorCREB, another target of the adenylyl cyclase pathway thatis important in cell differentiation (Schuh et al., 2002).

More recent studies have assessed the ability of OPs tointerfere with glia development by determining their effectson the transcription of relevant genes. Administration ofCPF to rats on PNDs 1 to 6, at doses causing considerableChE inhibition, decreases the expression of the gene encod-ing the oligodendrocyte marker myelin-associated glyco-protein, but elicits an increase in the expression of the geneencoding GFAP, an effect indicative of increased astrocytereactivity due to the high dosages employed (Betancourtet al., 2006). Treatment of cultures of human fetal astrocyteswith CPF for 7 days has a major impact on the expression ofgenes coding for various developmentally important mol-ecules, with a total of 35 genes being affected (Menseet al., 2006). Administration of CPF to rats on PNDs 1 to 4,at doses causing little or no ChE inhibition, also affects theexpression of a number of genes related to glial development.CPF elicits, among others, a marked decrease in theexpression of the gene encoding GFAP, indicative of adirect, specific effect on glia in the absence of neuronal cellinjury (Slotkin and Seidler, 2007). Administration of DZ torats on PNDs 1 to 4 also affects the expression of a number

of genes related to glial development. In common withCPF, DZ decreases the expression of the gene encodingGFAP (Slotkin and Seidler, 2007). The pattern of the effectsinduced by CPF and DZ on the expression of genes related toastrocyte development is quite similar. On the other hand,there are distinct differences between the two OPs in their pat-terns of effects on genes associated with the development ofoligodendrocytes and the process of myelination.

The ability of OPs to interfere with the differentiation ofglial cells has also been shown at the morphological level,as these compounds can impair the development of exten-sions from cultured C6 cells under differentiation-promotingconditions. Data from earlier studies indicate that several OPpesticides, such as CPF, dichlorvos, leptophos, cyanofen-phos, haloxon, DEF, and EPN, but not paraoxon, inhibitextension outgrowth from differentiating C6 cells exposedto the OPs for a total of 20 days (Henschler et al., 1992).Recent studies, employing much shorter incubation timesand subcytotoxic concentrations of OPs that lie within therange of expected fetal exposures in agricultural environ-ments (Ostrea et al., 2008), also indicate that OP pesticidesinhibit the development of extensions from C6 cells. Thus,CPF suppresses extension outgrowth in differentiating C6cells within 24 hours (Sachana et al., 2008). Equimolar con-centrations of CPO also inhibit outgrowth. In fact, the effectof CPO is stronger than that of CPF and temporally relatedto a significant decrease in the levels of tubulin, a cytoskeletalprotein critically involved in cell differentiation and extensiondevelopment. In contrast, under the same conditions, devel-opment of extensions from C6 cells is not affected bytrichlorphon and the CM carbaryl (Flaskos et al., 1999).Interestingly, DZ also differs from CPF in its lack of aninhibitory effect on C6 cell extension outgrowth (Flaskoset al., 2007). On the other hand, the in vivo metabolite ofDZ, DZO, causes inhibition of the development of C6 cellextensions, an effect related to a reduction in the levels oftubulin as well as the glial-specific cytoskeletal proteinGFAP (Sidiropoulou et al., unpublished data).

Comparison of the effects of anticholinesterase pesticideson glial development with those exerted on the developmentof neuronal cells indicates that in several cases glial cells aremore sensitive. Thus, CPF, CPO, DZ, and physostigmine aremore effective in inhibiting DNA synthesis, and hence cellreplication, in gliotypic C6 cells than in neuronotypic PC12cells (Qiao et al., 2001). This preferential targeting of glioty-pic cells by CPF is noted both in the absence (Qiao et al.,2001) and presence (Garcia et al., 2001) of serum.Furthermore, oral administration of CPF to rats on PNDs 1to 6 affects the expression of genes coding for glial-specificmarkers but not for the neuronal-specific marker b-III tubulin(Betancourt et al., 2006). On the other hand, there areinstances where pesticide effects on developing glial cellsare less pronounced than those exerted on developing neur-onal cells. Thus, CPF affects the expression of transcription


factors AP-1 and Sp1 in replicating PC12 cells (Crumptonet al., 2000), but has no effect on replicating C6 cells(Garcia et al., 2001). Similarly, CPF increases the levels oftranscription factor CREB in cultured cortical and hippocam-pal neurons, but has no effect in cultured astrocytes derivedfrom developing rats (Schuh et al., 2002). Furthermore, CPFas well as CPO and paraoxon all affect with much less potencythe expression of glial-specific markers than the expressionof neuronal-specific markers in aggregating brain cell culturesat various stages of development (Monnet-Tschudi et al.,2000). At a morphological level, DZ impairs the developmentof extensions from neuronotypic N2a cells, but has no effecton the outgrowth of extensions from C6 cells under identicalconditions (Flaskos et al., 2007).

In summary, available data indicate that anticholinesterasepesticides, and particularly OPs, interfere with the normaldevelopment of glial cells. However, certain prominentdifferences among OPs and between CPF and DZ in particu-lar in their biochemical and morphological effects have beennoted, leading potentially to divergent neurobehavioral con-sequences. The use of cell cultures, especially of the gliotypicC6 cell line, has been valuable in establishing that the OPstarget specifically developing glia, that the effects of OPsare exerted during both gliogenesis and glial differentiationand that both the oxon metabolites of CPF and DZ have anincreased capacity to interfere with glial cell differentiation.Glial cells are essential for axonal guidance, synaptogenesis,and architectural modeling of the developing brain (LoPachinand Aschner, 1999; Ullian et al., 2004). As these processescontinue well into childhood, the potent targeting of glialdevelopment by the OPs signifies that humans are vulnerableto the developmental neurotoxicity of these compounds for aprolonged postnatal period, during which, in fact, exposurescan be particularly increased (Gurunathan et al., 1998).This clearly underscores the need for further studies on theglial developmental toxicity of anticholinesterase pesticides.

16.10 EFFECTS OF ANTICHOLINESTERASEPESTICIDES ON APOPTOSIS

Although apoptosis occurs during both prenatal and postnatalneuronal development, affects both mitotic and postmitoticneuronal and glial cells, and is essential for the proper devel-opment of the nervous system, there is a paucity of data on thespecific effects of OPs and CMs on this process. The beststudied OP, CPF, induces apoptosis in the neuroepitheliumof cultured rat embryos exposed to the OP on embryonicday 9.5 (Roy et al., 1999). The ability of CPF to elicit mor-phological changes characteristic of apoptosis has also beenshown in primary cortical neuronal cultures prepared eitheron embryonic day 17 or 18 or from newborn rats. Embryonicneurons are more sensitive than neonatal neurons, with30 mM CPF inducing apoptosis in the former, but having

no effect on the latter. Although CPO has a slightly greaterapoptotic effect than CPF, CPF-induced apoptosis is notlikely to be related to ChE inhibition (Caughlan et al.,2004). The ability of OPs to elicit apoptosis under conditionsof little or no ChE inhibition has been shown recently in vivo.Thus, postnatal administration of CPF or DZ to neonatal ratshas a profound influence on the expression of a number ofgenes related to apoptosis (Slotkin and Seidler, 2007), with8 out of 17 genes being affected in total. Both CPF and DZincrease the transcription of genes coding for caspases 9and 12 and the tumor protein 53 (p53). However, CPF differsfrom DZ in that the latter has a more widespread apoptoticeffect, inducing additional changes in the expression ofgenes encoding caspases 1 and 4 and certain bcl 2-associatedproteins (Slotkin and Seidler, 2007).

16.11 EFFECTS OF ANTICHOLINESTERASEPESTICIDES ON NEUROBEHAVIOR

Since anticholinesterase pesticides cause excessive accumu-lation of ACh in cholinergic receptors in the nervoussystem, they can affect human and animal behaviors relatedto the cholinergic pathway. However, a number of studieshave also assessed the neurobehavioral effects of these com-pounds following exposure to noncholinergic doses, a situ-ation that is, in fact, more frequent in fetuses and children.The emergence of recent biochemical data indicating theinterference of OPs with the development of the serotonergicsystem has also prompted studies on the effects of OPs on5-HT-related behaviors.

In a review of a number of developmental studies asses-sing neurobehavior, CPF has been noted on PNDs 12 and66 to cause alterations in motor activity and auditory startleresponse accompanied by brain structural changes (Phang,2002). On the other hand, dimethoate and malathion induceneurobehavioral changes, but elicit no major neuropathologi-cal effects. However, the changes caused by these two OPsare only noted at doses causing inhibition of ChE.Important differences in the neurobehavioral profiles amongthree CM pesticides are also evident. Thus, carbaryl inducesno behavioral changes, but affects brain morphometry onPNDs 11 and 60 (Phang, 2002). By contrast, aldicarb and car-bofuran elicit behavioral alterations, as recorded by a func-tional observational battery of tests, in the absence of anyneuropathological findings. In addition, aldicarb causes sig-nificant changes in motor activity, whereas carbofuran affectslearning and memory parameters related to Y maze perform-ance (Phang, 2002).

CPF has been the focus of investigations by severalresearch groups in relation to its neurobehavioral effects ondeveloping animals. The assessment of these effects hasbeen performed both in the presence (Chanda and Pope,1996; Maurissen et al., 2000; Moser and Padilla, 1998;

16.11 EFFECTS OF ANTICHOLINESTERASE PESTICIDES ON NEUROBEHAVIOR 215

Stanton et al., 1994) and absence of significant inhibition ofChE activity (Carr et al., 2001; Dam et al., 2000; Jett et al.,2001; Ricceri et al., 2006), as well as under conditions ofaltered hippocampal cholinergic synaptic function (Icenogleet al., 2004; Levin et al., 2001) and altered expression ofmuscarinic receptors (Chanda and Pope, 1996; Jett et al.,2001; Moser and Padilla, 1998; Stanton et al., 1994). Thestudies conducted cover a wide window of brain developmentinvolving both prenatal (Chanda and Pope, 1996; Icenogleet al., 2004; Levin et al., 2002; Maurissen et al., 2000;Ricceri et al., 2006) and early or late postnatal treatment(Carr et al., 2001; Dam et al., 2000; Jett et al., 2001; Levinet al., 2001; Moser and Padilla, 1998; Ricceri et al., 2006;Stanton et al., 1994). Accordingly, behavioral changes eli-cited by CPF have been assessed using motor activitymeasurements (Carr et al., 2001; Dam et al., 2000; Icenogleet al., 2004; Levin et al., 2001, 2002; Maurissen et al.,2000; Moser and Padilla, 1998; Ricceri et al., 2006), reflextests (Chanda and Pope, 1996; Dam et al., 2000) or cognitivefunction endpoints (Icenogle et al., 2004; Jett et al., 2001;Levin et al., 2001, 2002; Maurissen et al., 2000; Ricceriet al., 2006; Stanton et al., 1994). Most of these studieshave demonstrated alterations in neurobehavioral perform-ance in the weanling rodents, with the more pronouncedeffect being the impairment of cognitive function (Icenogleet al., 2004; Jett et al., 2001; Levin et al., 2001, 2002;Ricceri et al., 2006; Stanton et al., 1994), although thereare some exceptions (Maurissen et al., 2000). The neurobeha-vioral deficits elicited by CPF are sex-selective (Dam et al.,2000; Levin et al., 2001, 2002; Ricceri et al., 2006), butthis has not been confirmed in other studies (Carr et al.,2001; Icenogle et al., 2004; Jett et al., 2001; Moser andPadilla, 1998). Comparison of the effects of CPF to thoseof DZ, given at the same developmental period and at equiv-alent doses causing no ChE inhibition, reveals that, althoughboth pesticides induce long-lasting cognitive changes, theyalso exhibit a number of important differences in sensitivityand outcome (Timofeeva et al., 2008a).

Data implicating targeting of the developing 5-HT systemby OP doses causing no ChE inhibition have spurred interestin the effects of these compounds on 5-HT-related behaviors.Thus, parallel experiments have assessed the effects of CPF,DZ, and parathion on 5-HT-associated behaviors in adoles-cence and adulthood after exposure of neonatal rats, using abattery of emotional tests closely related to 5-HT function.Both CPF and DZ have a significant effect on the majorityof emotional responses tested (Roegge et al., 2008).However, these OPs exhibit also substantial differences intheir effects including a difference in sex selectivity. Incontrast to these OPs, parathion has a much smaller effecton key behavioral tests and lacks the sex selectivityshown by CPF and DZ (Timofeeva et al., 2008b). Theabove data indicate that developmental exposure to anumber of OP and CM pesticides induces a range of adverse

neurobehavioral effects. The occurrence of considerabledifferences among these pesticides in their neurobehavioraleffects supports the notion that there are multiple mechanismsthat mediate the developmental neurotoxicity of thesecompounds.

16.12 TESTING FOR DEVELOPMENTALNEUROTOXICITY: THE USE OFANTICHOLINESTERASE PESTICIDES ASTESTING AGENTS

Currently, all departments, agencies, and organizationsworldwide that are in charge of the registration and safetyof chemicals do not require toxicity testing on the developingnervous system. However, there is increasing concern thatprenatal and early postnatal exposure to xenobiotics, includ-ing anticholinesterase pesticides, may result in permanentalterations of brain development leading to neurobehavioraldysfunction, disturbance of learning ability, and attentiondeficit and hyperactivity disorders in children (Jurewicz andHanke, 2008; Szpir, 2006). New pieces of legislation andpolicies aiming to protect this vulnerable population stress,therefore, the urgent need to accumulate neurodevelopmentaltest data, to consider such data in health risk assessment, andto regulate accordingly the use of these chemicals. For theabove reasons, the U.S. Environmental Protection Agency(EPA) has recently required neurotoxicity studies for anumber of already registered neurotoxic pesticides includingChE inhibitors. In the European Union, despite the aims ofthe Scientific Committee for Food to establish criteria toaddress the necessity of collecting neurodevelopmental testdata, the new regulation of chemicals, REACH (Registration,Evaluation, Authorization and Restriction of Chemicals) doesnot include any developmental neurotoxicity endpoints(Hass, 2006). However, lately, efforts have been made toreplace the two-generation reproductive study with a newextended one-generation study that would incorporate devel-opmental neurotoxicity for testing chemicals according to theREACH policy (Spielmann and Vogel, 2007).

The EPA has been the first organization to provide clearand separate developmental neurotoxicity testing guidelinesbased on laboratory animals (OPPTS 870.6300; U.S. EPA,1998). EPA has been followed by the Organization forEconomic Cooperation and Development (OECD), whichhas further developed and revised EPA’s suggestions to itsrecent draft proposal (TG 426; OECD, 2006). Both regulatoryapproaches enable the detection of gross neuropathologicaland neurobehavioral changes, mainly in rats, during postnataldevelopment and adulthood. According to a recent report,114 developmental neurotoxicity studies have been con-ducted on the basis of the EPA or OECD guidelines, leadinggradually to the formation of a database for predicting devel-opmental neurotoxicity (OECD, 2008). These studies


encompass a total of 19 OPs and 5 CMs. Furthermore, thesedata, derived from the current in vivo tests, will be renderedvaluable for the future validation and regulatory use of allalternative approaches mentioned below.

The application of standard neurodevelopmental toxicitytests in animals is prohibiting for the large number of chemi-cals that have to be evaluated, for financial and animal welfarereasons. As a result, there is a common effort worldwide toinitially follow alternative approaches in order to establishan intelligent tiered strategy that could finally determine rec-ommendation of compounds for in vivo neurodevelopmentaltoxicity testing. At the same time, this would contribute to therefinement and, eventually, the reduction of laboratory animaluse (Coecke et al., 2007; Lein et al., 2007). Among in vitrotests, those involving organotypic explants, reaggregatingbrain cell cultures, primary neuronal or glial cell cultures,immortalized neuronotypic or gliotypic cell lines and, morerecently, human or mouse embryonic stem cells are particu-larly important for establishing developmental neurotoxicityendpoints, and valuable for screening various compoundsincluding ChE inhibitors (Coecke et al., 2007; Lein et al.,2007). Throughout the last few years, several separateattempts have been made to develop cell-based screeningmodels that would permit the determination of suitable mor-phometric and biochemical/molecular parameters of devel-opmental neurotoxicity through the use of OPs and CMs astesting substances (Monnet-Tschudi et al., 2000; Qiaoet al., 2001; Slotkin et al., 2007a). However, there is a needfor a more systematic approach towards this direction inorder to establish a successful and widely acceptable predic-tive cell model of neurotoxic responses in the developingnervous system.

Apart from in vitro models, nonmammalian alternativemethods are also available which make use of such organismsas zebrafish, medaka, Caenorhabditis elegans, sea urchin,and chick embryos. These systems have been suggested tobe used for secondary screening following primary assess-ment through a high-throughput cellular system (Coeckeet al., 2007; Lein et al., 2007; Slotkin, 2006). Most of thesemodels express genes similar to humans that are related toneurodevelopmental disorders. In addition, these nonmam-malian systems permit the assessment of more complexendpoints compared to the in vitro models, including neuro-behavioral alterations. In these models, CPF has been themost extensively studied OP pesticide. On the basis ofmeasurements of various neurochemical and neurobehavioralendpoints, these nonmammalian models have been highlypredictive of the neurodevelopmental toxicity properties ofCPF and have been suggested to be suitable for screeningpurposes (Buznikov et al., 2001, 2007; Levin et al., 2003;Slotkin, 2006; Yanai et al., 2004).

Despite their increasing use, the value of the currentalternative methods in hazard characterization has notbeen extensively explored. Additionally, the reliability,

reproducibility, and relevance to neurodevelopmental tox-icity of these methods have been hardly addressed, limitingtheir recommendation for inclusion in any test guidelines.Overall, an ideal alternative approach aiming to successfullypredict neurotoxicity in the developing human nervoussystem should express mechanisms that are considered cru-cial for neurodevelopment and should be tested against asignificant number of reference chemicals including ChEinhibitors. Furthermore, the adaptability of these alternativemethods to a high-throughput screening battery is a crucialissue complemented by cost effectiveness and limited timeconsumption.

16.13 CONCLUSIONS

Biochemical, morphological, and neurobehavioral dataderived from experimental in vivo and in vitro studies indicatethat at least some widely used OP pesticides can cause devel-opmental neurotoxicity. On the other hand, the available evi-dence on CM pesticides is very limited. The preferentialneurotoxicity of OPs on the developing organism is partlydue to pharmacokinetic factors, and mainly to the poor abilityof the young to hydrolytically inactivate these compounds.More importantly, however, the developmental neurotoxicityof OPs is due to their ability to interfere specifically with theunique and intricate process of development. Indeed, the beststudied pesticide, CPF, is capable of disrupting every singlestage of nervous system development including neuronalcell proliferation and differentiation, axonogenesis, synapto-genesis, and apoptosis. The use of cell lines has been instru-mental in establishing the adverse effects of OPs specificallyon the development of glia, a finding of considerable signifi-cance, as it signifies in humans an extension of the period ofvulnerability to late adolescence. Cell lines have also beenvaluable in showing the capacity of several major in vivo pes-ticide metabolites to perturb both neuronal and glial cell pro-liferation and differentiation. These include some hithertounsuspected compounds, such as CPF’s metabolite trichloro-pyridinol, and both the oxon metabolites of CPF and DZ,notable traditionally only for their strong ChE-inhibitingeffects. It now seems clear that the developmental neurotoxi-city of OPs is not related to their ability to inhibit the enzy-matic activity of ChE. However, ChE may still be a target,as OPs may interfere with the morphogenic function of thisprotein during development.

By far the biggest volume of evidence for the developmen-tal neurotoxicity of OPs involves CPF. The rather recentextension of systematic studies to a second OP pesticide,DZ, has indicated several similarities in the effects of thesetwo compounds. Significantly, however, it has also revealeda considerable number of differences. Although some ofthese differences are not very pronounced and may, possibly,be eliminated if OP doses are appropriately adjusted, there

16.13 CONCLUSIONS 217

still remains an impressive number of distinct differences.The few available biochemical data on other OPs, forexample, parathion, further support the notion that each OPinduces developmental neurotoxicity by its own unique setof multiple mechanisms. For the dedicated academic mechan-istic toxicologist, unraveling the complexities of a largenumber of different mechanisms may be a fascinating chal-lenge. For the regulatory toxicologist, however, the existenceof many different mechanisms for the dozens of different OPsin current use worldwide is, indeed, not a very pleasant stateof affairs. Nor is it good news for state and agrochemicalcompany budgets. This calls for the imperative need toemploy high-throughput screening systems enabling therapid primary evaluation of the commercially available anti-cholinesterase pesticides. Despite certain widely acknowl-edged drawbacks, neuronal cell cultures, particularly celllines are, for a number of reasons, well suited for use in thecontext of a high-throughput screening system. For theirpart, the genomic and proteomic technologies are also par-ticularly appropriate for such use. Thus, the application ofthese technologies in combination with the use of cell linescan be especially helpful in successfully addressing theneed for rapidly obtaining comprehensive and reliable neuro-developmental toxicity profiles for the large number of anti-cholinesterase pesticides. In response to these needs, ourresearch group has embarked in the last 3 to 4 years on astudy of the effects of DZ and other pesticides on developingneuronotypic cell lines using proteomic analysis. Meanwhile,the results of a genomic study on the effects of CPF and DZon developing PC12 cells have just been published (Slotkinand Seidler, 2009). It is hoped that these studies will pavethe way for more analogous investigations in order to estab-lish underlying mechanisms and mechanistically relevant,useful endpoints for screening. Progress in our mechanisticknowledge and screening strategies should, in turn, improveprevention and treatment of anticholinesterase-induceddevelopmental neurotoxicity and thwart the grave socioeco-nomic consequences that this toxicity entails.

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