PART I METABOLISM AND MECHANISMS · 2020. 2. 21. · innervated by cholinergic interneurons that...

PART I

METABOLISM AND MECHANISMSCO

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1ACETYLCHOLINESTERASE AND ACETYLCHOLINERECEPTORS: BRAIN REGIONAL HETEROGENEITY

HARUO KOBAYASHI AND TADAHIKO SUZUKI

Faculty of Agriculture, Department of Veterinary Medicine, Iwate University, Morioka, Japan

FUMIAKI AKAHORI

Faculty of Veterinary Medicine, Azabu University, Japan

TETSUO SATOH

Department of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, andHAB Research Institute Cornea Center, Ichikawa, Chiba, Japan

1.1 Introduction 3

1.2 Role of Acetylcholinesterase and Mechanism ofCholinergic Neurotransmission 4

1.3 Effects of Organophosphates and Carbamates onAcetylcholinesterase 51.3.1 Determination of Acetylcholinesterase Activity 51.3.2 Effects of Organophosphates on Regional

Brain Acetylcholinesterase Activity 51.3.3 Effects of Carbamates on Brain Regional

Acetylcholinesterase Activity 7

1.4 Effects on Release, Synthesis, and Storage ofAcetylcholine 9

1.5 Effects on Acetylcholine Receptors 111.5.1 Effects on Muscarinic Receptors 111.5.2 Effects on Nicotinic Acetylcholine Receptors 13

1.6 Effects on Neuroactivities Other than the CholinergicSystem 13

1.7 Conclusions 14

Acknowledgments 14

References 14

1.1 INTRODUCTION

Acetylcholinesterase (AChE) inhibitors are used throughoutthe world for many purposes. Probably the best known arethe pesticides that are used to control the insects that affectpublic health (e.g., mosquitoes, flies, cockroaches, ticks,fleas, and bedbugs) as well as those that affect agricultureand gardening (e.g., grasshoppers, aphids, caterpillars, riceinsects, and stinkbugs). Although compounds with compara-tively low toxicity, such as pyrethroids and novel insecticidesincluding fipronil and neonicotinoids, have been developed

and are widely used, carbamates and organophosphorus com-pounds (organophosphates) are still commonly used through-out the world for the control of these various insects.

In addition to their use as pesticides, AChE inhibitors suchas sarin, tabun, and VX, highly toxic organophosphates, havealso been used as nerve gases. These compounds were usedin the Iran–Iraq War (1980–1988), and in the terrorist attackson the Tokyo subway (1995) and in Matsumoto (1994),Japan (Okumura et al., 2003). There continues to be a realthreat that these types of nerve agents can be misused in thefuture.

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

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On the other hand, since carbamates and organophos-phates are highly effective inhibitors of AChE, they canalso be used for the treatment of diseases where cholinergicfunction is inadequate (Giacobini, 2000). By the inhibitionof AChE, acetylcholine concentrations are increased withinthe synapses and clinical improvement can be produced inperipheral and central disorders such as myasthenia gravisand Alzheimer’s disease, respectively. The AChE inhibitorsthat have been used either clinically for therapeutic purposesor experimentally for research purposes include organophos-phates such as diisopropylfluorophosphate (DFP) and tetra-isopropyl pyrophosphoramide (iso-OMPA), carbamates suchas neostigmine, physostigmine, and rivastigmine, and antide-mentia drugs or candidates, such as 2-[(1-benzyl-4-piperi-dyl)methyl]-5,6-dimethoxy-2,3-dihydroinden-1-one (done-pezil), tetrahydroamino acridine (THA), galantamine, andmethanesulfonyl fluoride.

In spite of the toxicological or therapeutic importance ofAChE inhibitors, the regional effects of these compoundson various cholinergic systems of the brain are not adequatelyunderstood. Many of the prior studies have focused ongrossly defined changes in whole brain or major subdivisionssuch as the forebrain or hindbrain. Since higher brain func-tions involve the integration of information from severaldifferent regions, neurochemical, biochemical, physiological,pharmacological, immunochemical, and electrophysiologicalinvestigation of brain activities can probably be best under-stood by detailed study of specific structures or areas. Forexample, very small changes that might be observed indetailed studies of specific areas such as the striatum andhippocampus may be lost when analyzed as a part of thewhole brain, forebrain, and hindbrain.

The study of specific areas of the brain is particularlyimportant because the cholinergic system has major functionsin the brain, especially in the cerebral cortex (cortex), limbicsystem, and hippocampus. In addition, the striatum is denselyinnervated by cholinergic interneurons that are crucial formotor behavior (Pisani et al., 2001), and this structure ishighly enriched in cholinergic markers such as AChEactivity, choline acetyltransferase (ChAT) activity, andacetylcholine (ACh) content.

Carbamates and organophosphates produce toxicitythrough the inhibition of AChE whether by acute, repetitive,or chronic exposure. Changes in cholinergic neurons pro-duced by AChE inhibition also have secondary effects ondopaminergic, g-aminobutyric acid (GABAergic), and gluta-matergic neurons, especially in the central nervous system(CNS). The two cholinergic receptors, muscarinic (mAChR)and nicotinic (nAChR), are located postsynaptically orpresynaptically on cholinergic neurons and these noncholi-nergic neurons, such as dopaminergic, GABAergic andglutamatergic neurons, and are involved in mediating theeffects of AChE inhibitors. Therefore, the cholinergic regu-lation and modulation of the brain can be determined by the

density of innervation in brain regions, cholinergic activities,such as AChE activity, ChAT activity, and ACh level, and thedistribution or sensitivity of AChRs.

In this chapter, the toxicological effects of cholinesteraseinhibitors, mainly organophosphates and carbamate insec-ticides, on brain cholinergic mechanisms are described. Thefocus of this chapter is on the effects of AChE inhibitorson ACh dynamics such as the synthesis, storage, and releaseof ACh, changes in ACh receptor density and function, andAChE activity in discrete brain regions of experimental ani-mals. For further reading on brain regional heterogeneity inrelation to the cholinergic system, readers are referred torecent publications elsewhere (Gupta, 2004, 2006b).

1.2 ROLE OF ACETYLCHOLINESTERASEAND MECHANISM OF CHOLINERGICNEUROTRANSMISSION

ACh is stored, in part, in synaptic vesicles (about 50 nm indiameter) in the cytoplasm of cholinergic nerve terminals(Bloom, 2002; Dani and Bertrand, 2007; Deutch and Roth,2004). When impulses arrive at the terminal membrane (8to 10 nm thick) causing depolarization, a portion of theACh-containing vesicles fuse with membrane and undergoexocytosis and release ACh into the fluid of the synapticcleft. Since the clearance of the cleft between the pre- andpostsynaptic membranes is about 20 nm, the extremelyhydrophilic ACh molecules diffuse and bind to AChRs onthe postsynaptic and/or presynaptic membranes almostinstantly (0.1 to 0.2 msec). Immediately after binding onthe AChRs, the ACh is hydrolyzed by AChE into cholineand acetic acid, destroying it within a few milliseconds afterexocytosis (Taylor, 2002). This rapid destruction of AChis required for normal cholinergic function. Any delayin the hydrolysis of ACh causes the accumulation of ACharound AChRs and prolongs excitation or transmission.There are several different mechanisms for terminating theactions of other neurotransmitters, such as dopamine,noradrenaline, GABA, histamine, and 5-hydroxytriptamine(serotonin). The biggest difference in inactivation of releasedneurotransmitter between ACh and other neurotransmittersis that most noncholinergic neurotransmitters are inactivatedby the reuptake into presynaptic nerve terminals, enzymaticcatabolism, and diffusion away from the receptor sites fol-lowed by dilution in extracellular fluid or plasma to subthres-hold concentration (Bloom, 2002; Deutch and Roth, 2004).Therefore, inactivation of noncholinergic transmitters maytake much longer than the hydrolysis of ACh by AChE.

Because the inactivation of ACh depends critically on theaction of AChE, increases in the synaptic effects of ACh canbe produced through the action of AChE inhibitors. The pop-ular AChE inhibitors used to affect cholinergic activity areorganophosphates and carbamates (Gupta, 2006a, 2006b).

4 ACETYLCHOLINESTERASE AND ACETYLCHOLINE RECEPTORS: BRAIN REGIONAL HETEROGENEITY

1.3 EFFECTS OF ORGANOPHOSPHATES ANDCARBAMATES ON ACETYLCHOLINESTERASE

The primary mechanism of action and the most acutelylife-threatening effect of exposure to carbamates and organo-phosphates result from the inhibition of AChE. Organophos-phates are grossly classified into oxon-type compounds,which directly inhibit AChE, such as dichlorvos, DFP, tri-chlorphon, and sarin, and thion-type compounds, such aschlorpyrifos, parathion, fenitrothion, and malathion. Thelatter organophosphates do not inhibit AChE directly butrequire the replacement of a sulfur atom with oxygen tobecome oxon-type compounds. It is well known that the inhi-bition of AChE by organophosphates is persistent, lastinghours to days, and potentially may not be reversible if anonenzymatically mediated dealkylation, termed “aging,”occurs. The phosphorylated aged AChE is refractory toreactivation.

Inhibition occurs as a result of phosphorylation of theserine (S200) included in the catalytic triad of the activecenter by the organophosphate (Aldridge, 1950; Fukuto,1990). Although this inhibition is usually considered irrevers-ible, some reactivation of phosphorylated acetylcholinester-ase can occur slowly as a result of hydrolytic cleavage, ifthe process of aging is not complete (Sultatos, 1994, 2006).Aging is a poorly understood mechanism in which onealkoxy group is hydrolyzed leaving the monoalkoxy phos-phate bound essentially irreversibly to the active site ofAChE (Sultatos, 1994, 2006).

The inhibition of AChE by an organophosphate is a func-tion of both binding affinity at the active site and the rate ofphosphorylation (Main, 1964). As a result, the bimolecularrate constant that can be determined experimentally continuesto be considered the single best approach to compare theinhibitory power of various organophosphates (Fukuto,1990; Mortensen et al., 1998). This strategy, however,depends upon the assumption that these in vitro reactionsapproximate first-order conditions because the oxon concen-trations are much higher than the uninhibited enzyme.However, a new approach based on continuous systems mod-eling to determine the apparent inhibition rate constant ofparaoxon and methyl paraoxon towards mouse brain AChEhas challenged the validity of that assumption. Theseexperiments have shown that the bimolecular rate constantsfor organophosphate-induced inhibition appear to change asa function of oxon concentrations, indicating that theefficiency of phosphorylation appeared to decrease as theparaoxon concentration increased (Kardos and Sultatos,2000).

In addition, in studies of the direct effects of AChE inhibi-tors, AChE knockout animals may be useful as a model forinvestigating the effects of selective, complete, and chroni-cally diminished AChE activity on AChRs, other cholinergicactivities and functions. AChE knockout mice were

developed recently and have provided a valuable tool forexamining the effects of long-term complete and selectiveabolition of AChE activity in brain regions (Duysen et al.,2002; Volpicelli-Daley et al., 2003; Xie et al., 2000). AChEknockout mice showed dramatic and selective reduction inmAChR, a marked redistribution of mAChRs to intracellularcompartments, upregulation of the high-affinity cholinetransporter, and altered behavior induced by mAChR antag-onists. In contrast, there was no change in the activity ofChAT, the levels of vesicular ACh transporter, and the b2subunit of nAChRs.

1.3.1 Determination of Acetylcholinesterase Activity

Excessive inhibition of AChE, a critical enzyme involved inboth peripheral and central cholinergic functions, by organo-phosphates or carbamates produces a cholinergic crisis that isthe mechanism of both acute and chronic toxicity. Themeasurement of AChE inhibition produced by these com-pounds is, therefore, important for evaluating and predictingmammalian toxicity. Studies of brain AChE are traditionallybased on biochemical assays, immunoreactivity, and histo-chemistry. A variety of methods have been developed forquantifying AChE activity, including detection of the thiolgroup formed by hydrolysis of acetylthiocholine as a sub-strate (Ellman et al., 1961) or another colorimetric methodof Hestrin (1949), or determination of radiolabeled acetatefollowing hydrolysis of radiolabeled ACh (Johnson andRussell, 1975). Several additional histochemical methodsdeveloped in recent years, including microdensitometry,microphotometry, and video-based histochemistry, are effec-tive in quantitative and detailed study of AChE in tissuesections (Ma et al., 2001; Sun et al., 2003).

1.3.2 Effects of Organophosphates on RegionalBrain Acetylcholinesterase Activity

Many studies of the effects of organophosphates on the brainhave focused on the effects of chlorpyrifos, dichlorvos,malathion, ethyl parathion (parathion), and methyl parathion.These pesticides have been used extensively as agriculturaland commercial insecticides throughout the world.

Most studies of the effects of AChE inhibitors on the CNShave reported AChE activity in gross neuroanatomical areassuch as the whole brain or forebrain and they have focusedon the consequences of high levels of organophosphateswith either acute or repeated exposures in either animals orpostmortem human victims. Relatively little attention hasbeen given to the effects of low-level organophosphateexposure that is not associated with acute cholinergic symp-toms (Ray and Richards, 2001). There are only a few reportsfocused on persistent effects of repeated and low-levelexposures to organophosphates on brain regional AChEactivity and other neurochemical and behavioral parameters

1.3 EFFECTS OF ORGANOPHOSPHATES AND CARBAMATES ON ACETYLCHOLINESTERASE 5

(Karanth et al., 2007; Kobayashi et al., 2007; Ma et al., 2001;Sun et al., 2003; Terry et al., 2007).

Terry et al. (2007) injected rats with chlorpyrifos at dosesof 2.5, 10.0, and 18.0 mg/kg, subcutaneously (s.c.) everyother day over the course of 30 days and then gave a two-week washout period. The activity of acetylcholinesterasewas measured in six brain regions, including prefrontalcortex, cortex, anterior hippocampus, posterior hippocampus,basal forebrain, and striatum at the end of the washout period(Fig. 1.1). For five of the six brain regions, AChE activity wassignificantly decreased even 14 days after the 10 and 18 mg/kgregimens. For the striatum, AChE inhibition was significant14 days after all three dose regimens. Across the six brainregions, 14 days after the final 18 mg/kg dose of chlorpyri-fos, AChE activity was still inhibited by 55%. The authorsalso reported that the brain/plasma ratio of cholinesteraseactivity varied from a low of 0.67 in the striatum to a highof 1.04 in the anterior hippocampus and averaged 0.82across all three dose regimens and all six brain regions.They also determined the levels of chlorpyrifos and its metab-olite 3,5,6-trichloro-2-pyridinol in brain tissue. Although thelevels of chlorpyrifos and its metabolite were low or nearlyundetectable 14 days after the 10 and 18 mg/kg regimens,AChE activity continued to be inhibited by at least 50%.

In another study, Kobayashi et al. (2007) measured AChEactivity in the striatum, hippocampus, and cerebral cortex ofrats after they were treated with dichlorvos (DDVP) at 3 mg/kg/day, s.c., for 7 and 14 days. AChE activity was assayed 1,6, and 11 days after the last treatment with DDVP (Kobayashi

et al., 2007). AChE activity was markedly decreased inthe three brain regions 1 day after treatment over both 7 and14 days. AChE activity showed gradual recovery at 6 and11 days (Fig. 1.2a). The depression of AChE activity in thethree brain regions appears to be more severe in groupsadministered for 14 days than for 7 days. Although theactivity increased depending on days after withdrawal, therecovery was about 20% for 10 days in all brain regions.The irreversibility and slow recovery from DDVP treatmentare considered to be due to an aging and a new synthesis ofAChE (Taylor, 2002).

Sun et al. (2003) treated rats repeatedly with either vehicleor methyl parathion at a dose of 3 mg/kg/day, s.c. for oneweek or three weeks. The animals were sacrificed 24 h afterthe last treatment and AChE activity was measured histo-chemically in different brain regions, including striatum, hip-pocampus, frontal cortex, thalamus, and midbrain (Fig. 1.3,Table 1.1). The activity of AChE in the striatum, cortex, thala-mus, and midbrain was reduced to about 40%, 45%, 35%,33%, and 25% of respective controls after 1 week of treatmentand 20%, 20%, 13%, 13%, and 15% of respective controlsafter three weeks of treatment. As shown in Figure 1.3 andTable 1.1, AChE is distributed heterogeneously throughoutthe brain, and the relative regional preponderance is in thestriatum and thalamus. This study demonstrated that repeatedexposures to methyl parathion inhibit AChE to a similardegree in different brain regions.

The effects of organophosphate exposure on the brain alsoseems to depend to some degree on age. For example,Karanth et al. (2007) compared the effect of acute exposureto ethyl parathion on striatal AChE levels in adult (3-month-old) and aged (18-month-old) male Sprague-Dawleyrats. The activity of AChE was determined 3 and 7 daysafter a single subcutaneous treatment with a range of dosagesof ethyl parathion (adult: 1.8, 3.4, 6.0, 9.0, 18, and 27 mg/kg;aged: 1.8, 3.4, 6.0, and 9.0 mg/kg). It is interesting to notethat in this experiment, striatal AChE activity was signifi-cantly lower in control aged rats compared to control adultrats. Comparison of identical dosages in adult and aged rats(i.e., 9 mg/kg versus 9 mg/kg) showed that AChE inhibitionwas significantly higher in aged rats than in adults. Similarresults were reported by Scali et al. (1997) who foundhigher levels of brain AChE inhibition in aged rats than inadults following acute exposure to metrifonate (trichlorfon).Karanth et al. (2007), however, found that similar maximaldegrees of inhibition of 92% to 94% were noted in adultsand aged rats treated at the highest dose used for each agegroup, 27 and 9 mg/kg, respectively (Karanth et al., 2007).AChE inhibition in the striatum peaked at 3 days afterthe single injection and there was little evidence of recoveryby 7 days in either age group. Although it remains unclearwhy striatal AChE is more sensitive to ethyl parathion-induced inhibition in aged rats than adults, the authors pro-posed that the higher sensitivity in aged rats could be due

Figure 1.1 Brain acetylcholinesterase (AChE) activities measuredin six brain regions 14 days after the last chlorpyrifos administration.Each value represents the mean+SEM derived from five to six rats.�, p , 0.05 with respect to the vehicle control mean. Adapted fromTerry et al. (2007).


to lower tissue AChE (about 14% significantly lowercompared to adult rats).

It seems that the inhibition of AChE activity and itsirreversibility after repeated administrations of the respectiveorganophosphates described above is not specific to variousbrain areas and is not selective for specific regions.

1.3.3 Effects of Carbamates on Brain RegionalAcetylcholinesterase Activity

Although carbamates react with the same serine moiety in thecatalytic site of AChE as do the organophosphates, the effectsof carbamates may be very different because of the duration

Figure 1.2 The activity of acetylcholinesterase in the striatum, hippocampus, and cortex of rats 1, 6, and 11 days after withdrawal of repeatedadministration of DDVP (a), propoxur (b), or oxotremorine (c) for 7 and 14 days. The activity of brain regional AChE was determined 1, 6, and11 days after withdrawal of repeated administration of DDVP, propoxur, or oxotremorine for 7 days (†) or 14 days (O) and expressed aspercent of control (W). Data are expressed as mean+SEM (n ¼ 4–6). Asterisks indicate values that are significantly different from controlvalue (�p , 0.05, ��P , 0.01). Adapted from Kobayashi et al. (2007).

1.3 EFFECTS OF ORGANOPHOSPHATES AND CARBAMATES ON ACETYLCHOLINESTERASE 7

of the inhibition. In contrast to the organophosphates, recov-ery of AChE activity after a carbamate (e.g., carbofuran)-induced inhibition is quite rapid since recovery simplyrequires the spontaneous hydrolysis of the covalent bondbetween the methyl carbamyl moiety and the enzyme(Ferguson et al., 1984; Gupta and Kadel, 1989). This obser-vation is also true for several other carbamate pesticides,including aldicarb, methomyl, and propoxur (Gupta, 1994,2004; Gupta and Kadel, 1990, 1991a, 1991b).

In an experiment conducted by Kamboj et al. (2006),carbofuran was administered to rats orally (in corn oil) at adose of 1 mg/kg/day for 28 days. The activity of acetylchol-inesterase was measured in three brain regions (cerebral

cortex, cerebellum, brain stem) one day after the finaladministration (Table 1.2). Carbofuran treatment resulted ina significant decrease in AChE activity in cortex (66.9%),cerebellum (71.7%), and brain stem (66.6%) compared tothe control animals.

Kobayashi et al. (2007) measured AChE activity in thestriatum, hippocampus, and cortex of rats 1, 6, and 11 daysafter the last treatment with propoxur at a dose of 10 mg/kg/day, s.c. for 7 and 14 days. As shown in Figure 1.2b,repeated injections of propoxur did not produce a uniformchange in the activity of AChE in every brain region 1, 6,and 11 days after withdrawal from repeated treatments withpropoxur across 7 or 14 days. The administration of propoxur

Figure 1.3 Typical images of AChE histochemistry staining from control and treated rats. Rats were treated with either vehicle or methylparathion (3 mg/kg) daily for 1 week or 3 weeks. Adapted from Sun et al. (2003).


for 7 days suppressed the activity in the three brain regions 1,6, and 11 days after withdrawal. Surprisingly, the activity ofAChE in brain regions was generally higher in rats treatedwith propoxur for 14 days than for 7 days.

Other experiments have shown the complexity of theeffects of repeated exposures to carbamates. For example,Costa et al. (1981a) found that administering propoxur indrinking water decreased the activity of forebrain AChEonly in rats treated for 5 weeks but not for 2, 3, 4, and 6weeks. Kobayashi et al. (1988) also noted that a single injec-tion of propoxur (10 mg/kg, s.c.) resulted in a significantdecrease in the activity of AChE in the forebrain of micefor 180 min, but the repeated administration (5 mg/kg/day,s.c.) did not produce a significant change in activity. It hasalso been shown that chronic administration of rivastigmine, acarbamate that is used to ameliorate dementia in Alzheimer’sdisease, did not decrease the activity of AChE in the ratbrain regions studied (frontal cortex, hippocampus, striatum,and thalamus þ midbrain) (Tanaka et al., 1994). However,rivastigmine with an acute dose (0.35 mg/kg, intraperitone-ally, i.p.) reduced 40 to 50% AChE activity in the cortex andhippocampus of rats. Furthermore, rivastigmine at double thedose (0.7 mg/kg, i.p.), produced about 80% AChE inhibition

(Gupta and Dekundy, 2005, 2007). It is possible, therefore,that chronic administration of a carbamate may not producea uniform and predictable effect on brain regional AChEactivity even after recurring episodes of inhibition causedby repeated administrations. Chronic exposure to carbamatescauses reversible but recurring inhibition of AChE and mayinduce alterations in metabolic kinetics of the compounds(Tang et al., 2006), which may result in different activity ofAChE independent of the dose and frequency of exposure.These factors may explain the reason for inconsistent effectsof some carbamates on brain regional AChE activity follow-ing chronic exposure, while a carbamate compound like aldi-carb produces consistent effect on AChE activity (Gupta andKadel, 1991b).

1.4 EFFECTS ON RELEASE, SYNTHESIS, ANDSTORAGE OF ACETYLCHOLINE

As described previously, ACh released from the presynapticcholinergic nerve terminal is hydrolyzed into choline andacetic acid very rapidly. Since choline is a quaternaryammonium compound, its membrane permeability through

TABLE 1.2 Effect of Carbofuran Administration on the Activity of Acetylcholinesterase in Rat Brain

Acetylcholinesterase (nmol AcetylthiocholineHydrolyzed/min/mg Protein)

Lipid Peroxidation(nmol MDA/mg Protein)

Cerebral Cortex Cerebellum Brainstem Cerebral Cortex Cerebellum Brainstem

Control 148.97+ 6.62 125.66+ 10.94 107.46+10.48 2.06+0.22 2.59+0.22 2.02+0.21CF treated 49.26+ 4.10a 35.62+ 2.43a 35.88+2.86a 3.40+0.20a 3.45+0.35a 2.68+0.42a

NAC treated 154.83+ 8.43 131.10+ 4.44 116.75+3.84 2.11+0.09 2.70+0.12 2.13+0.10CF þ NAC treated 101.29+ 5.09a,b 75.83+ 10.06a,b 66.29+2.72a,b 2.16+0.23a,b 2.55+0.21b 2.16+0.12b

Note: CF, carbofuran; NAC, N-acetylcysteine; Values are expressed as mean+S.D., n ¼ 6.aSignificantly different from control group ( p , 0.05).bSignificantly different from carbofuran treated group ( p , 0.05).

Source: Adapted from Kamboj et al. (2006).

TABLE 1.1 Intensity of AChE Staining in Rat Brain Regions After 1 Week and 3 Weeks of Methyl ParathionRepeated Treatment

Brain Region

AChE Staining (O.D.)

1 Week Treatment 3 Week Treatment

Control MP3 (mg/kg) Control MP3 (mg/kg)

Striatum 0.55+0.03 0.21+0.02 (263%) 0.53+0.03 0.11+0.01 (280%)Frontal cortex 0.05+0.01 0.02+0.00 (266%) 0.02+0.00 0.00+0.00 (287%)Hippocampus 0.08+0.01 0.04+0.01 (254%) 0.05+0.00 0.01+0.00 (280%)Thalamus 0.14+0.01 0.05+0.01 (267%) 0.11+0.01 0.01+0.00 (287%)Midbrain 0.15+0.01 0.04+0.01 (274%) 0.11+0.01 0.02+0.00 (285%)

Note: Images of AChE histochemical staining (Figure 1.3) were analyzed with a digital scanning densitometer. Values are the Mean O.D.+SEM (n ¼ 7 or 8).Values in parentheses represent difference to control value in percentage at same region.

Source: Adapted from Sun et al. (2003).

1.4 EFFECTS ON RELEASE, SYNTHESIS, AND STORAGE OF ACETYLCHOLINE 9

the blood–brain barrier is very low, indicating that bloodis not the main source of choline for ACh synthesis.Therefore, choline derived from the hydrolysis of ACh inthe synaptic cleft is transported into the presynaptic nerveterminal by high-affinity choline uptake (or transport)(HACU or HACT). This high-affinity choline transporter(Apparsundaram et al., 2001; Okuda et al., 2000) is asodium ion- and chloride ion-dependent process with a Kmvalue of 1 to 2 mM for choline and it is the rate-limitingstep in the biosynthesis of ACh (Kuhar and Zarbin, 1978).Following reuptake, choline and acetylcoenzyme A (pro-vided by mitochondria) are transformed into ACh by cholineacetyltransferase (ChAT) in the cytoplasm of the nerve term-inal. Although ChAT and HACU are specific markers ofcholinergic innervation in the CNS, the enzyme is notaccepted to be a rate-limiting step in the synthesis of ACh(Apparsundaram et al., 2001; Kuhar and Zarbin, 1978).

The newly synthesized ACh is subsequently incorporatedinto synaptic vesicles via the vesicular ACh transporter(VAChT) that is located in the vesicular membrane (Prioret al., 1992). VAChT exchanges luminal protons for cyto-plasmic ACh and concentrates ACh inside the synapticvesicles. It is well known that this action of VAChT is specifi-cally blocked by 2-(4-phenyl piperidino)-cyclohexanol(vesamicol) (Prior et al., 1992; Schuldiner et al., 1995).

Organophosphates and carbamates exert acute toxicityprimarily through persistent inhibition of AChE at cholin-ergic junctions, resulting in prolonged residence time ofACh within the synaptic cleft. Because of this persistentAChE inhibition, certain compensatory mechanisms arise tocombat excess ACh present in the synaptic cleft. Thesehomeostatic mechanisms involve modulation of both presyn-aptic and/or postsynaptic components of the cholinergicsynapse. These may involve changes in HACU, ChAT, andthe vesicular VAChT in the presynaptic terminal and modifi-cations of mAChRs and nAChRs for both the postsynapticand presynaptic components (Costa et al., 1981a, 1981b,1982a, 1982b; Kobayashi et al., 1986, 1997; Padilla, 1995;Richardson and Chambers, 2003, 2004, 2005; Russell andOverstreet, 1987; Schwab et al., 1981, 1983; Whalley andShih, 1988).

Several studies have reported consistent inhibition ofAChE by organophosphates. However, the effects of organo-phosphates on ChAT are shown to be variable, ranging fromno effects (Kobayashi et al., 1986; Sivam et al., 1984), someinhibition in PC cells (Jameson et al., 2006), to activation invitro (Brooks and Goldberg, 1979) or in vivo (Gupta et al.,1985; Khan et al., 2000). Furthermore, the effects of organo-phosphates on the cholinergic elements, such as AChE, ACh,ChAT, HACU, and VAChT, depend, at least in part, on devel-opmental level. Rats exposed in utero to methyl parathion(1.5 mg/kg, perorally, p.o. daily from day 6 through day 20of gestation) showed significant reduction in AChE activityand increase in ChAT activity in brain regions (cortex,

brainstem, striatum, and hippocampus) that persisted throughpostnatal day 28. Some studies have also shown that repeatedpostnatal exposure of rats to chlorpyrifos resulted in persistentbrain AChE inhibition and a decrease in ChAT activity in ratsexposed to the organophosphate on postnatal day 1 through 4.These changes persisted through postnatal day 30 and wereaccompanied by decreases in HACU levels (Dam et al.,2000; Slotkin et al., 2001). It was also reported that gesta-tional exposure to chlorpyrifos results in persistent reductionsin ChAT activity, HACU levels, and also vesicular AChtransporter (VAChT) levels (Richardson and Chambers,2003, 2004). Therefore, it is suggested that presynapticcholinergic neurons may be especially vulnerable to earlypostnatal and gestational exposure to organophosphates likechlorpyrifos.

A variety of acute neurotoxic effects of sarin, in addition tothe AChE inhibition, have also been studied in rats (Khanet al., 2000). The animals were treated with a single intramus-cular injection of sarin at 0.01, 0.1, 0.5, or 1 � LD50 andsacrificed 0.5, 1, 3, 6, 15, or 20 h later. Brain regionalAChE activity was inhibited (44% to 55% of control)30 min after the LD50 dose and it remained inhibited for upto 20 h. ChAT activity was increased in the cortex, brainstem,and midbrain 6 h after the LD50 dose and the elevated activitypersisted up to 20 h after treatment. Midbrain and brainstemseemed to be most responsive to sarin treatment at lowerdoses as these regions exhibited inhibition (�49% and10%, respectively) in AChE activity 20 h following 0.1 �LD50 treatment. Cortical ChAT activity was significantlyincreased following a 0.1 � LD50 dose, whereas the activityin the brainstem and midbrain did not show any effect atthis lower dose. The authors speculate from various reportsand their previous findings that increased ChAT activity fol-lowing sarin exposure may be a consequence of protease-mediated activation of the enzyme (Khan et al., 2000).

Microdialysis is a useful tool with which to investigatethe extracellular release of neurotransmitters successivelyevery 10 min or so in the brains of awake freely moving ani-mals (Bradberry, 2000; Chang et al., 2006; Mas et al., 1996;Westerink, 1995; Young, 1993). Microdialysis methods havebeen used frequently to investigate the effect of drugs onACh, especially those that have been proposed for the treat-ment of Alzheimer’s disease. However, there is a paucity ofstudies that have utilized this method to evaluate the effectsof toxicological exposure to organophosphates and carba-mates on the release or extracellular level of ACh (Bueterset al., 2002; Karanth et al., 2007). Intracerebral microdialysisstudies in rats have shown that sarin (0.144 mg/kg, intramus-cularly, i.m.) and VX (0.024 mg/kg, i.m.) produced an11-fold and 18-fold increase in ACh levels, respectively, 10minutes after exposure (Bueters et al., 2002). Parathion atdoses of 6, 9, 18, and 27 mg/kg, s.c. dose-dependently elev-ated extracellular ACh levels in the striatum of adult (three-month-old) and aged (18- to 19-month-old) rats 3 and 7 days


after treatment (Karanth et al., 2007). These results show thatorganophosphates induce increases in extracellular ACh.

Head-focused microwave irradiation to rapidly (up to 1 secor slightly longer) inactivate enzymes in brain has also beenused to determine the level of ACh in brain regions of rats andmice. Stavinoha et al. (1976) measured the accumulationof ACh in rat brain regions after administration of dichlorvosand demonstrated that the striatum had the highest rate ofaccumulation of ACh followed by cerebral cortex andhippocampus, and the cerebellum had the lowest.

1.5 EFFECTS ON ACETYLCHOLINERECEPTORS

AChRs are classified into mAChRs and nAChRs. In themammalian brain, both mAChRs and nAChRs are presenton neurons (Caulfield and Birdsall, 1998; Dani andBertrand, 2007). These receptors mediate diverse cellularresponses by distinct signaling mechanisms (Bakry et al.,1988; Caulfield and Birdsall, 1998; Dani and Bertrand,2007; Huff et al., 1994; Jett et al., 1991; Katz et al., 1997;Khan et al., 2000).

The mAChR is a member of the superfamily of G-proteincoupled receptors (Caulfield and Birdsall, 1998; Matsuiet al., 2004). It is comprised of five receptor subtypes thathave distinct molecular and functional properties (Bymasteret al., 2003). The M1, M3, and M5 mAChRs are coupledto a Gq protein and stimulation of these receptors promotesneurotransmission by activating phospholipase C to hydro-lyze phosphatidyl inositol 4,5-2 phosphate (PIP2) into inosi-tol 4,5-3 phosphate (IP3) and diacylglycerol. Both IP3 anddiacylglycerol stimulate signal transduction by releasingCa2þ and activating protein kinase C, respectively.Stimulation of M2 and M4 receptors inactivates adenylatecyclase activity via Gi protein to inhibit synthesis of cyclicadenosine monophosphate (cAMP) from ATP. Studies innonhuman primates, rats, and mice have provided evidencethat primarily the M2 subtype in brain areas, such as hippo-campus and cerebral cortex, and predominantly M4 subtypein striatum, act as cholinergic autoreceptors (Douglas et al.,2001; Levey, 1996; Zhang et al., 2002). Agonists and antag-onists of M2 subtype decrease and increase ACh release,respectively (Billard et al., 1995; Douglas et al., 2002).

Peripheral and central nAChRs belong to the superfamilyof ligand-gated ion channels. nAChRs in the CNS have pen-tameric structures, which form the ligand-gated ion channel.Some consist of ab combinations (heteromeric receptors)which include a2-a6 subunits and b2-b4 subunits.However, others form homomeric nAChRs consisting ofthe same a subunits including either of a7-a9 and a hetero-mer of a10 with a9 (Dani and Bertrand, 2007; Gotti andClementi, 2004). Nine ligand-binding subunits (a2-a10)and three structural subunits (b2-b4) have so far been

cloned from different species. The a8 subunit has beenfound in avian tissue but not in mammals. Of the homomericnAChRs, only the a7 pentameric structure is widely distrib-uted in the mammalian brain. The different combinations ofa and b subunits can form different heteromeric receptorshaving distinct pharmacological and biophysical character-istics. The different subtypes of nAChRs present in differentbrain regions are yet to be completely identified and charac-terized. Interestingly, however, it has been shown that recep-tors containing the a6 subunit are concentrated in the visualand catecholaminergic pathways, including the dopaminergicnigrostriatal pathway (Quik and McIntosh, 2006).

As described above, inhibition of AChE activity leads tothe accumulation of ACh in the synaptic cleft. The accumu-lation of abnormal levels of ACh can cause excessiveactivation of mAChRs and nAChRs (Ecobichon, 1996;Huff et al., 1994; Katz et al., 1997) and can alter the pharma-codynamics patterns of receptor binding kinetics (Padilla,1995). It is commonly accepted that many symptoms ofAChE inhibitor-induced toxic effects are mediated throughthe stimulation of both mAChRs and nAChRs by ACh.Notwithstanding, studies on the specific involvement ofnAChRs in poisonings induced by inhibitors of AChE arerelatively few. It has generally been found that numbers ofmAChRs and nAChRs are reduced (downregulated) inAChE-deficient mice (Adler et al., 2004; Li et al., 2003;Volpicelli-Daley et al., 2003). However, there are someexceptions to the expectation that reduced or absent AChEactivity will produce downregulation. For example, increasedlevels (upregulation) of mAChRs and nAChRs have beenfound in some cases where animals were exposed to lowdoses of organophosphates and some carbamates (Abou-Donia et al., 2003; Kobayashi et al., 2007).

Several reports have described a range of neurobehavioraland neuropsychiatric disorders after chronic exposure oforganophosphates at levels that cause no symptoms of acuteantiAChE poisoning (Salvi et al., 2003; Stephens et al.,1995). Therefore, it is suggested that exposure to low-leveldoses of organophosphates, as well as acute poisoning, canlead to several neurological and neurobehavioral changesthat cannot be accounted for on the basis of AChE inhibitionalone. Other, more sensitive brain proteins, such as receptors,may be involved (Ray and Richards, 2001). In addition toinhibition of AChE activity, some of these compounds havebeen shown to interact directly with mAChRs (Lockhartet al., 2001; Van den Beukel et al., 1997) and nAChRs(Nagata et al., 1997; Smulders et al., 2004a, 2004b; Storchet al., 1995; Van den Beukel et al., 1998; Zwart et al., 2000).

1.5.1 Effects on Muscarinic Receptors

By far the most common and consistent finding among thereports on chronic exposure to organophosphate compoundshas been a reduction in the number of muscarinic AChRs

1.5 EFFECTS ON ACETYLCHOLINE RECEPTORS 11

(Churchill et al., 1984a, 1984b; Costa et al., 1982a, 1982b;Gupta et al., 1985; Kobayashi et al., 2007; Russell andOverstreet, 1987; Schiller, 1979; Sun et al., 2003; Yamadaet al., 1983). Previous studies have shown that repeated post-natal exposure of rats to organophosphates resulted in persist-ent inhibition of brain AChE and transient reductions of totaland M2/M4 mAChR (Liu et al., 1999; Tang et al., 1999).

Sun et al. (2003) treated rats repeatedly with either vehicleor methyl parathion at a dose of 3 mg/kg/day, s.c. for 1 weekor 3 weeks. The rats were then sacrificed 24 h after the lasttreatment for the autoradiographic measurement of[3H]quinuclidinyl benzilate (QNB) (nonselective or total),[3H]pirenzepine (M1-selective), and [3H]AF-DX384 (M2-selective) binding to mAChRs in different brain regions,including striatum, hippocampus, frontal cortex, thalamus,and midbrain. Three weeks of treatment with methyl para-thion significantly reduced [3H]QNB binding in all fivebrain regions, with the greatest reduction (70% of the controllevel) in the striatum (Fig. 1.4a). However, at the end of thefirst week of treatment, only the striatum showed a reductionin mAChR binding. On the other hand, [3H]pirenzepine bind-ing was not affected in the first week of methyl parathiontreatment but it was decreased by 21%, 9%, and 22% inthe frontal cortex, hippocampus, and striatum, respectively,at the end of 3 weeks of treatments (Fig. 1.4b). [3H]AF-DX384 binding was reduced a little but significantly in thefirst week in the brain regions of frontal cortex, striatum,and thalamus (Fig. 1.4c). The binding was decreased by22%, 11%, 38%, 6%, and 10% in the frontal cortex, hippo-campus, striatum, thalamus, and midbrain, respectively, atthe end of 3 weeks of treatment with methyl parathion. Theauthors suggest that the downregulation of mAChRsdepended on differential sensitivity of each receptor subtypeto methyl parathion and differential distribution of receptorsubtype proteins in brain regions.

In another study, Kobayashi et al. (2007) studied the bind-ing of [3H]QNB, [3H]pirenzepine and [3H]AF-DX384 tomAChRs in the striatum, hippocampus, and cerebral cortexof rats at 1, 6, and 11 days after the last treatment with dichlor-vos (DDVP) at a dose of 3 mg/kg/day, s.c., for 7 and 14 days.As measured at one day after the last treatment, the binding ofall three radiolabeled ligands in the three brain regions wasgenerally decreased by the DDVP treatment given for both7 and 14 days. Such downregulation of the receptors was gen-erally absent at 6 or 11 days if the rats had been treated foronly 7 days but it was still evident at these time points if theyhad been treated for 14 days. As described above, repeatedadministration of DDVP for 7 or 14 days induced a remark-able decrease in the activity of AChE in the three brainregions of rats, with gradual recovery until 11 days after with-drawal or almost without recovery, respectively. Therefore, itis possible that repeated administration of DDVP producesmore intensive and prolonged direct exposure of mAChRsto ACh by more effective inhibition of its hydrolysis.

Figure 1.4 (a) [3H]QNB binding levels, (b) [3H]pirenzepine bind-ing levels, and (c) [3H]AF-DX384 binding levels (percentage ofcontrol value at the same region) in 1-week methyl parathion treatedand 3-week methyl parathion treated rats (3 mg/kg/day). Data arepresented as percentage of control values. Each bar representsmean+SEM (n ¼ 5). Data are analyzed by one-way ANOVA, fol-lowed by Tukey test. #, Significantly different from 1-week control;�, Significantly different from 3-week control; ‡, Significantly differ-ent from 1-week methyl parathion treated ( p , 0.05). Adapted fromSun et al. (2003).


In a similar experiment on the effects of a carbamate, thebinding of [3H]QNB, [3H]pirenzepine, and [3H]AF-DX384to mAChRs in the striatum, hippocampus, and cerebralcortex of rats was also determined 1, 6, and 11 days afterthe last treatment with propoxur at a dose of 10 mg/kg/day, s.c. for 7 and 14 days (Kobayashi et al., 2007). Bothdownregulation and upregulation as measured by [3H]QNB,[3H]pirenzepine, and [3H]AF-DX384 binding was observed1, 6, or 11 days after treatment with propoxur for 7 daysand/or 14 days. These results are consistent with other reportsthat chronic administration of carbamates induced a decrease(Costa et al., 1981a; Kobayashi et al., 1988), increase (Costaet al., 1981a), or no change (Costa et al., 1981a) in the bindingability of [3H]QNB. It may be concluded that chronicexposure to carbamate pesticides does not induce consistentand predictable changes in mAChRs in the brain. It wouldbe, however, interesting to clarify if some type of repeatedtreatment with selected carbamates might produce enoughprolonged exposure to ACh to induce downregulation ofmAChRs.

1.5.2 Effects on Nicotinic Acetylcholine Receptors

There have also been several in vivo and in vitro studiesfocused on the neurotoxicological effects of organophos-phates and carbamates on brain nAChRs. In one of these,Khan et al. (2000) treated rats with a single LD50 dose(0.1 mg/kg, i.m.) of sarin. This treatment caused a biphasicresponse in cortical nAChR ligand binding as measuredwith [3H]cytisine. This exposure resulted in a decrease(�25%) and an increase (�132%) in binding of[3H]cytisine at 1 and 6 h after treatment, respectively. Theseresults suggest that sarin exerts acute effects on nAChRligand binding.

Terry et al. (2007) injected rats with chlorpyrifos (2.5 �18.0 mg/kg, s.c.) every other day over the course of30 days and then gave the animals a 2-week chlorpyrifos-washout period. At the end of the 2-week washout, therelative levels of a7-nAChR protein were measured(ELISA) in tissue lysates from four brain regions (basalforebrain, hippocampus, prefrontal cortex, and cortex). Inthe prefrontal cortex and hippocampus a significant decreasein a7-nAChR was found.

In a study of the differences, neonatal and adult rats weregiven chlorpyrifos at 7 doses (0.15 to 15 mg/kg/day, p.o.) for14 days and sacrificed 4 h after either the first or 14th dose(Zheng et al., 2000). [3H]epibatidine was used to measurethe nAChR ligand binding in the front cortex. Epibatidinebinding did not change significantly in either age group fol-lowing the first dose. Four hours after the 14th dose, however,chlorpyrifos (7.5 mg/kg/day) reduced cortical epibatidinebinding 32% in adults and 25% in neonates. Therefore, asimilar reduction in cortical nAChR was noted betweenadults and neonates after repeated exposure to chlorpyrifos.

The specific mechanism of action of carbamate and orga-nophosphate pesticides remains to be elucidated. Smulderset al. (2003, 2004a, 2004b) studied the effects of commonlyused carbamate pesticides and organophosphate insecticideson rat neuronal nAChR expressed in Xenopus laevis oocytesusing the two-electrode voltage camp technique. The potencyorder of six carbamates to inhibit a4b4 nAChRs wasfenoxycarb . S-ethyl N,N-dipropylthiocrbamate (EPTC) .

carbaryl, bendiocarb . propoxur . aldicarb with IC50

values ranging from 3 mM for fenoxycarb to 165 mM forpropoxur and .1 mM for aldicarb (Smulders et al., 2003).The a4b2, a3b4, and a3b2 nAChRs are inhibited by fen-oxycarb, EPTC, and carbaryl with potency orders similar tothat fora4b4 nAChRs. Comparing the potencies of inhibitionof the distinct subtypes of nAChRs shows that the a3b2nAChR is less sensitive to inhibition by fenoxycarb andEPTC. The potency of inhibition depends on the carbamateas well as on a combination of a and b subunit properties.The authors concluded that carbamate pesticides affect differ-ent subtypes of neuronal nAChRs independently of AChEinhibition (Smulders et al., 2003). Several organophosphates,such as chlorpyrifos, ethyl parathion, and disulfoton,inhibited a4b2 nAChRs with potencies in the micromolarrange (Smulders et al., 2004a). Binding experiments ona4b2 nAChRs showed that the organophosphates interactnoncompetitively with nAChRs. The inhibition is accountedfor by a two-step mechanism resulting in receptor desensitiza-tion. The authors concluded that organophosphates interactdirectly with a4b2 nAChRs to inhibit the agonist (ACh)-induced response and a4b2 nAChRs are additional targetsfor some organophosphates (Smulders et al., 2004a).

1.6 EFFECTS ON NEUROACTIVITIES OTHERTHAN THE CHOLINERGIC SYSTEM

Although the main mechanism for organophosphates and car-bamates is cholinesterase inhibition leading to accumulationof ACh at synaptic clefts, recent studies indicate that this isnot the sole mechanism underlying the toxicity of AChEinhibitors (Gupta, 2004; Gupta et al., 2000; Rocha et al.,1996; Weinbroum, 2004). Of the numerous receptor systemsimplicated in the lethal and convulsant effects of AChEinhibitors, the excitatory amino acid system has gainedthe most interest (Lallement et al., 1992; Raveh et al.,2003; Shih and McDonough, 1997).

Excitatory amino acids such as N-methyl-D-aspartate(NMDA), other endogenous neurotransmitters and neuro-modulators in the CNS have been implicated in neurodegen-erative and epileptogenic processes. It has been shown thatantagonists such as dizocilpine (MK-801) exert neuroprotec-tive and anticonvulsant effects in various experimentalmodels. The coadministration of MK-801 with atropine(muscarinic antagonist) has been shown to counteract the

1.6 EFFECTS ON NEUROACTIVITIES OTHER THAN THE CHOLINERGIC SYSTEM 13

lethality and seizures produced by organophosphate insecti-cides (chlorfenvinfos, dichlorvos) and carbamate compounds(methomyl, physostigmine) (Dekundy et al., 2001, 2003,2007). These authors suggested that mAChRs and nAChRs,as well as NMDA receptors, seem to play major roles inanticholinesterase-induced neurotoxicity and that combinedtreatment with cholinergic and NMDA antagonists mightbe beneficial in anticholinesterase-induced poisonings(Dekundy et al., 2001, 2003, 2007). In similar studies,another NMDA receptor antagonist memantine, in combi-nation with atropine, has been found equally effectiveagainst organophosphate and carbamate poisoning (Gupta,1994; Gupta and Kadel, 1989, 1990, 1991a, 1991b;McLean et al., 1992).

Inhibitory amino acids may also be involved in themediation of organophosphate-induced neurotoxicity. Forexample, the effects of organophosphates on GABA havealso been investigated. Paraoxon at low doses (1029 to1026 M) increased and at high doses (1025 to 1023 M) inhib-ited GABA uptake of rat cerebral cortex synaptosomes withdecreasing Vmax value but without affecting Km values(Ghasemi et al., 2007).

The above studies indicate that many noncholinergiceffects may be important in mechanisms underlying thetoxicity of AChE inhibitors.

1.7 CONCLUSIONS

Clarifying the mechanisms by which organophosphates andcarbamates induce neurotoxicity is very important for the pre-diction of neurotoxicity from unknown compounds andto characterize the mode of actions in more detail and moreprecisely. Many studies have indicated that neurotoxicity inbrain induced by exposure to organophosphates and carba-mates has multiple cholinergic targets, such as mAChRs,nAChRs, HACU, ACh release, and ChAT, in addition toAChE. It is clear that these effects shown in animals (gener-ally rats and mice) may be dependent or independent of thedirect effects of AChE inhibition and the resulting changesin ACh levels.

Relative to many published studies concerning the effectsof organophosphates and carbamates on total, M1, and M2mAChRs, few studies have examined the effects on other sub-types of mAChRs. Although highly selective antagonists ofM4 and M5 mAChRs are still lacking, five mAChR geneshave been characterized and the understanding of their coup-ling characteristics is increasing. It seems worthwhile toinvestigate the effects of exposure to organophosphates andcarbamates on all subtypes of mAChRs in brain regions.

It is also generally accepted that mAChRs are the predomi-nant AChRs in mammalian brain. But is it true? It is knownthat nAChRs participate in fundamental aspects of the synap-tic plasticity that are involved in attention, learning, memory,

and development. Loss of nAChRs as well as disruption andalterations of nicotinic cholinergic mechanisms have beenimplicated in various disorders such as schizophrenia, epi-lepsy, autism, Alzheimer disease, and addiction (Dani andBertrand, 2007). As described above, nine ligand-bindingsubunits (a2 to a10) and three structural subunits (b2, b3,and b4) have so far been cloned from the brains of differentspecies. Further studies are needed to evaluate the effect oforganophosphates and carbamates on the different subtypesof nAChRs in vivo or in vitro.

It remains to be clarified that cholinergic presynaptic nerveterminals are identical or individual to make synapses withmuscarinic postsynaptic neurons and nicotinic postsynapticneurons in brain. Research with organophosphates and carba-mates in this respect will be expected to intensify the furtherunderstanding on their effects toxicologically, pharmaco-logically, and neurologically.

Although the effects of drugs used for the treatmentof dementia in Alzheimer disease on the release of ACh inthe brain have been extensively studied by using microdialy-sis in freely moving animals, the effects of other organophos-phates and carbamates used as pesticides remain to be fullyevaluated. There is an obvious lack of similar microdialysisstudies on the effects of organophosphates and carbamateson the release of other neurotransmitters, such as dopamine,GABA, glutamate, and serotonin.

ACKNOWLEDGMENTS

We are thankful to Dr. Donald E. Moss, Department of Psychology,University of Texas at El Paso, for editing the manuscript andvaluable suggestions.

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