Cholinesterases

Chapter 68

Cholinesterases Barry W. Wilson University of California, Davis, California

68.1  Introduction Cholinesterases (ChEs) are specialized carboxylic ester hydrolases that break down esters of choline. Two of spe­ cial concern to the pesticide toxicologist are acetylcholin­ esterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) and butyrylcholinesterase (BuChE; acylcholine acylhydro­ lase, EC 3.1.1.8), also known as nonspecific cholinester­ ase or pseudocholinesterase. The preferred substrate for AChE is acetylcholine (ACh). Nonspecific cholinesterases prefer butyrylcholine and/or propionylcholine, depend­ ing on the species (Silver, 1974). This chapter discusses these enzymes, their importance in understanding the toxi­ city of organophosphate ester (OP) and carbamate (CB) pesticides, and their application to risk assessment (Taylor, 1999). ChEs are classed among the B-esterases, enzymes inhibited by OPs and possessing a serine catalytic site (Aldridge and Reiner, 1972; Ballantyne and Marrs, 1992; Chambers and Levi, 1992; Ecobichon, 1996; Gallo and Lawryk, 1991). Other B-esterases include the carboxyl­ esterases (CarbE, EC 3.1.1.1.), one of which is neuropathy target esterase (NTE), the enzyme associated with organo­ phosphate-induced delayed neuropathy (OPIDN) discussed in other chapters. The A-esterases are a different group of enzymes (e.g., arylesterases, paraoxonases, and DFPases) that actively hydrolyze OPs, providing an important means of detoxification (Furlong et al., 2000; Haley et al., 1999; La Du et al., 1999). There has been a great amount of research on ChEs since 1914 when Sir Henry Dale (Dale, 1914) proposed an esterase capable of hydrolyzing ACh in blood, and when Abderhalden and Paffrath (1925) and Loewi and Navratil (1926) prepared tissue extracts that broke down the chemical. In the past 30 years, the tertiary structure, amino acid, and DNA sequences of several ChEs have been elucidated. From 2000 to the present, approximately 14,000 research reports on ChEs have been listed in SciFinder Scholar, an ACS online database. Hayes’ Handbook of Pesticide Toxicology Copyright © 2010 Elsevier Inc. All rights reserved

Today, techniques such as site-directed mutagenesis, knockout mutants, and high-resolution x-ray crystallogra­ phy enable investigators to dissect the form and function of these proteins literally one amino acid at a time (Faerman et al., 1996; Gnatt et al., 1994; Silman and Sussman, 2008). In the future, this knowledge will help researchers design chemicals specifically targeted for the tertiary struc­ ture of these proteins and their genes. Specific reviews and conferences (Doctor et al., 1998; Massoulie et al., 1999; Reiner et al., 1999; Silman and Sussman, 2008; Taylor, 1994, 1996) and even full-color molecular structures dis­ played on the Internet help bring the reader the latest information from this rapidly growing research area. [For brevity, only selected references to a topic are cited here. The reader is referred to these references and to articles (e.g., Gallo and Lawryk, 1991) in previous editions of this book for citations to earlier work.] OPs with high toxicity were developed as chemical war­ fare agents in the late 1930s and early 1940s (Ecobichon, 1996; Holmstedt, 1963; Koelle, 1963). Since the 1950s, their offspring have been adapted as pesticides for agri­ cultural use (Ecobichon, 1996). Because of their potential as weapons, much research has focused on antidotes (e.g., oximes) and prophylactics to OP chemical warfare agents (National Academy of Sciences, 1999; Romano et al., 2008).

68.2  Distribution ChEs are widely distributed across animal species (Ecobichon, 1996). Their presence in insects and other inver­ tebrates has made anti-ChE agents popular and effective pesticides. Molecular forms of ChE similar to those in verte­ brates have been studied in animals as varied as nematodes (e.g., Caenorhabditis; Culetto et al., 1999), squid (Talesa et al., 1999), and Amphioxus, a protochordate (Pezzementi et al., 1998). A vertebrate-like AChE form has been reported in Paramecium, a ciliated protozoan (Corrado et al., 1999, 2005). 1457

Hayes’ Handbook of Pesticide Toxicology

1458

AChEs in the nervous system regulate excitation by destroying the neurotransmitter ACh. They are found at synapses and neuromuscular and myotendinous junctions and in cerebrospinal fluid, the central nervous system (CNS) neuron cell bodies, axons, and skeletal and smooth muscles (Silver, 1974). AChEs also are present in erythro­ cytes [red blood cells (RBCs)] of mammals, megakaryo­ cytes, lymphocytes, and platelets (Husain, 1994; Paulus et al., 1981; Zajicek, 1957). Although, there has been some interest in ChE in saliva (Ryhanen et al., 1983; Yamalik et al., 1990), the activity reported is low (Henn et al., 2006). Blood ChE forms are often used as surrogates for CNS ChEs in studies of toxicants. The AChE activity of human blood is restricted to its formed elements; most of it is vested in the RBCs (Wills, 1972). Plasma ChEs of other vertebrates often also hydrolyze ACh (Augustinsson, 1948, 1959a,b), and specific AChE enzyme activity is present in the plasma of some mammals. For example, the plasma ChE activity of rodents such as the laboratory rat is high in both AChE and BuChE (Traina and Serpietri, 1984). Neglecting this plasma AChE activity, as is often done, may lead to misinterpreting the extent of ChE inhibition in animals used for pesticide research and in the setting of regulations for food safety and human exposure (Wilson et al., 1996). AChE activity also has been found in the serum of mammalian embryos and birds; it decreases to adult levels after birth. AChE activity in fetal calf serum is high enough to be a source for purifying the enzyme (De la Hoz et al., 1986). In contrast, adult bovine blood has rel­ atively high RBC AChE and very low plasma ChE levels (Zajicek, 1957). Other species have a mixture of ChEs, first established by studies of substrate and inhibitor specifici­ ties and, recently, by DNA analyses (Bartels et al., 2000). Table 68.1 compares adult levels of RBC AChE from several species. The human has the highest activity. The rat has one of the lowest RBC AChE levels even though it is often the species used in biomedical research on anti-ChE agents. BuChEs and AChEs are also found together at syn­ apses, motor endplates, and muscle fibers (Silver, 1974). BuChE activity in blood is restricted to serum. The physiological functions of RBC and serum ChEs are unclear. The primary structure of ChEs is homologous to proteases and lipases (Taylor, 1996). One possibility is that blood ChEs evolved to protect the body from natural anti-ChE agents. A number of plant toxins have anti-ChE activity, including the solanaceous glycoalkaloids, which are naturally occurring steroids in potatoes and related plants (Krasowski et al., 1997; McGehee et al., 2000), and the fungal territrems (Chen and Ling, 1996). The Calabar bean, Physostigma venenosum, was once used by West Africans in a “trial by ordeal” (O’Brien, 1967). Study of the action of its active anticholinergic ingredient, the CB physostigmine (eserine), helped to establish the roles of ACh and AChE in the nervous system (Engelhart and

Table 68.1  Relative RBC AChE Levels of Adults of Selected Speciesa Species

Sex

AChE level (%)

Human

Male/female

100  8.7

Cow

Female

87.6  1.9

Guinea pig

Male/female

32.7  3.5

Horse

Male/female

28.8  9.0

Rabbit

Male/female

21.7  5.3

Rat

Male

12.6  3.0

Cat

Male/female

30b

a Manometric assay at N  3. Mean human AChE was 2180 l CO2/ 30 min/mg nitrogen. ACh substrate. b N  2. Adapted from Zajicek (1957).

Loewi, 1930). Other examples of naturally occurring anti-ChE agents are fasciculin from elapsid snake venom (Marchot et al., 1998), chaconine and solancine from tubers and nightshades (Nigg et al., 1996), and huperzine from moss (Patocka, 1998). The association of cholinergic transmission with Alzheimer’s disease served as a stimu­ lus for modern studies of natural anti-ChE agents (Francis et al., 1999; Nordberg and Svensson, 1998), part of the search for treatments of this common disorder. Tissue ChEs may have specific but still unknown roles in addition to their regulation of neural transmis­ sion. For example, there is evidence (Anderson and Key, 1999; Chiappa and Brimijoin, 1998; Layer et al., 1998; Robitzki et al., 1997; Sharma and Bigbee, 1998; Sperling et al., 2008) for a developmental function for ChEs based on studies of neurite outgrowth in retinal and dorsal root ganglion cultures and embryos using immunological, sense and antisense oligonucleotides and inhibitors. The functions of AChE and BuChE may overlap. The suc­ cessful development and survival of a knockout mouse mutant lacking AChE indicates that other enzymes such as BuChEs can function in its stead (Li et al., 2000; Xie et al., 2000). There have been persistent reports concerning pesticideinduced ocular damage (recognized clinically as Saku dis­ ease in Japan). These studies, reviewed by Dementi (1994) and Jaga and Dharmani (2006), have stimulated stud­ ies by the U.S. Environmental Protection Agency (EPA) (Atkinson et al., 1994; Boyes et al., 1994), although with­ out striking results. However, there are reports of visual changes and damage during growth and development (Geller et al., 1998; Wyttenbach and Thompson, 1985). Hamm et al. (1998) found that diazinon, a widely used pesticide, damaged the development of the neural retina in Medaka, a fish.

Chapter | 68  Cholinesterases

1459

Table 68.2  Plasma Hydrolysis of Choline Esters of Selected Species of Mammalsa Species

ACh

PrCh

BuCh

MeCh

BzCh

Human

135

310

360

 2

60

Cow

  3

  4

  2

 0

 0

Guinea pig

  50

130

170

 5

20

Horse

130

225

365

 2

40

Rabbit

  16

  16

  10

 4

 2

Rat

  20

  30

  15

15

10

Dog

  70

115

180

 6

30

Cat

  50

  75

150

 5

12

ACh, acetylcholine; BuCh, butyrylcholine; BzCh, benzoylcholine; MeCh, methylcholine; PrCh, propionylcholine. a Manometric assay.  lCO2/0.1 ml plasma/30 min. N  3 or more animals. Source: Augustinsson (1959a).

Table 68.3  Plasma Hydrolysis of Choline Esters of Selected Species of Birds, Reptiles, Amphibians, and Fisha Species

ACh

PrCh

BuCh

MeCh

BzCh

Chicken

37

  71

36

19

 2

Duck

43

  74

67

 7

 8

Turtle

14

103

27

 7

 1

Rana

40

  90

87

 2

10

Xenopus

 2

  5

 9

—

—

Pike

12

  2

 1

 1

 1

ACh, acetylcholine; BuCh, butyrylcholine; BzCh, benzoylcholine; MeCh, methylcholine; PrCh, propionylcholine. a Manometric assay.  lCO2/0.1 ml plasma/30 min. N  3 or more animals. Source: Augustinsson (1959b).

68.3  Substrate preferences and selective inhibitors AChEs prefer ACh as a substrate. Substrate preferences and activities of BuChEs vary with the species. For exam­ ple, rat plasma BuChE activity has been reported to favor propionyl rather than butyryl substrates, and cows have been shown to have hardly any plasma ChE activity at all (Tables 68.2 and 68.3). An important distinction between the AChEs and BuChEs is their response to substrate concentration. AChEs are inhibited by substrate in excess of a few millimoles; BuChEs are less sensitive (Hoffmann et al., 1989; Wilson, 1999). In general, mammalian and avian AChEs rapidly hydrolyze ACh and its thiocholine analog acetylthiocholine (ATCh). Mammalian, but not avian, AChEs preferentially hydrolyze acetyl--methylcholine. AChEs are selectively inhibited by several agents, including the CB BW284c51 (1,5-bis(4-allyl-dimethylammoniumphenyl)pentan-3-one

dibromide) (Austin and Berry, 1953; Holmstedt, 1957; Silver, 1974) and ARA 1327 (Augustinsson et al., 1978). BuChEs are preferentially inhibited by iso-OMPA (tetraisopropylpyrophosphoramide; Austin and Berry, 1953), ethopropazine (Mikalsen et al., 1986), and quinidine (Wright and Sabine, 1948). Effective concentrations for these selective inhibitors may vary by species. Useful start­ ing points for testing are 0.1–0.01 mM.

68.4  Multiple molecular forms   and life history ChEs are polymorphic proteins, occurring in multiple mole­ cular asymmetric and globular forms (Figures 68.1 and 68.2). The asymmetric forms tend to be localized at synapses and motor endplates. They have glycosylated heads joined by sulfhydryl groups and collagen tails. The heads contain the active sites; the collagen tails attach the enzymes to cell

Hayes’ Handbook of Pesticide Toxicology

1460

A I2

s s ss

Hs

H

s s

G1 s s

s s s s

s s

A8

s ss s

H

s s s s

s s

s s

s s

G2

s s

s s ss

s s

H

s s

s s s s ss s s

s s

H

G4

H H

A4

Figure 68.1  Organization of subunits in the molecular forms of ChEs. Each circle represents a catalytic subunit. Globular forms are represented by “G.” Asymmetric forms are designated by “A.” Linear elements rep­ resent collagen-like tails. Disulfide bridge locations from studies of eel electroplax AChE, assumed to reflect ChEs from many sources (after Brimijoin, 1992).

Nucleus

surfaces. The globular forms make up the catalytic subunits (Taylor, 1996). These forms have similar kinetic properties but differ in their ionic and hydrophobic interactions. ChE forms (Massoulie et al., 1993, 1999) are synthe­ sized as catalytic globular monomers (G1) that oligomer­ ize via disulfide bonds into multiple G2 and G4 forms (see Figure 68.1). The G1 subunits are synthesized within cells (e.g., nerve, muscle, and liver), glycosylated, and then secreted. Collagen tails are attached to one, two, or three catalytic tetramers to yield A4, A8, and A12 asymmetric forms. Collagen-tailed forms become attached to the cell surface at specific binding sites. Globular forms are released into body fluids or bind to cell surfaces through hydrophobic amino acid sequences or glycophospholipids (Taylor, 1996). Antibodies have been prepared to several purified AChEs and BuChEs; specific protein and nucleic acid sequences have been determined and altered by site-directed mutagenesis (Doctor et al., 1998). AChE and BuChE forms are each coded by single genes. The gene coding sequence for the collagen-tailed forms con­ tains a C-terminal extension of 40 amino acids, the T-peptide, that interacts by a short proline-rich attachment domain with the collagen. A single collagen gene (ColQ) is associated

Synthesis, sequestering glycosylation, assembly of globular forms, degradation

AChE mRNA RER 20–30%

Degradation 70–80%

Processing, assembly of asymmetric forms

Golgi Transport Vesicles Plasma membrane

Membrane bound

Extracellular matrix

Secretion, membrane incorporation and turnover

Binding in basal lamina

Extracellular fluids Figure 68.2  Life history of ChEs. The enzyme is synthesized as a monomeric globular form (G1). Up to 80% of the enzyme is degraded by intra­ cellular proteases after ribosomal translation and before transit of the Golgi. In the Golgi, secretory forms (black triangles) are sequestered from membrane-bound forms (open triangles), collagen-like tails are added to asymmetric forms, the peptide backbone is glycosylated, and the ChE becomes enzymatically active. Globular secretory forms may escape the synaptic cleft to enter extracellular fluids, blood, or external secretions. Asymmetric forms are probably bound quantitatively by adsorption or entrapment in the polyanion-rich, fibrous matrix of the extracellular synaptic basal lamina (after Brimijoin, 1992).

Chapter | 68  Cholinesterases

1461

with both the AChE and the BuChE collagen-tailed forms. The globular forms are synthesized by an alternative splice variant, the H-exon, that is expressed instead of the T-exon [see Krejci (1998) for a model and Massoulie et al. (1999) for further information]. The three-dimensional structures (Figure 68.3) of ChEs are subjects of intense investigation (Doctor et al., 1998; Massoulie et al., 1999). In the past, the active site of AChE was loosely described by models based on the work of Nachmansohn and Wilson (1951), in which there was a negatively charged “anionic” site and an “esteratic” site of catalytic residues. The positively charged choline moiety of ACh was hypothesized to bind to the negatively charged anionic site. A nucleophilic group, assumed to be a serine residue at the esteratic (acylation) site, was proposed to catalyze the hydrolysis [see Silman and Sussman (1998) for a more detailed historical perspective]. The crystallization of ChE proteins such as the dimeric AChE form of Torpedo californica has led to a more detailed understanding of the form/function relationships of the enzyme (Doctor et al., 1998). An important feature is the embedding of the active site of AChE in a “gorge” lined with 14 aromatic residues, approximately 20 Å from the surface of the protein (see Figure 68.3). The quaternary nitrogen of cho­ line binds through interactions with  electrons of tryptophan residues (Sussman et al., 1991) at the “peripheral site” at the mouth of the gorge, a region conceptually corresponding to the historical concept of an “anionic site.” One current view of the molecular mechanism has the ester substrate led down

the gorge by molecular interactions to become hydrolyzed at the bottom by a catalytic triad of glutamate 334, histidine 447, and serine 203 residues (Doctor et al., 1998; Taylor, 1996). How the products escape from the gorge is a matter of specu­ lation; one hypothesis is that they are ejected via a “side” or “back” door, making room for the next substrate molecule; another is that they are rapidly moved to the entrance of the gorge. However, Silman and Sussman (2008), in an excel­ lent review of the subject, note that “the current view of most kineticists working on AChE would be that they could model the kinetic behavior of the enzyme without requiring a back door” and that “experimental evidence is thin” (p. 7). The amino acid sequences around the entrance to the gorge that comprise the “peripheral site” may be important in deter­ mining the differences in substrate specificity and inhibition by excess substrate between AChE and BuChE forms (Doctor et al., 1998; Reiner et al., 1999). Figure 68.3 provides a threedimensional diagram of an AChE molecule. [Other depictions may be found in Silmann and Sussman (2008) and on the Internet by searching for “acetylcholinesterase.”]

68.5  Mechanism of hydrolysis 1

124

72 124

74

74

286

86

86 282

282 297

297

283

203

341 447 295



where E is the enzyme, AX is the substrate (ACh) or inhibitor, EAX is the reversible enzyme complex, X is Ch

72 286

K

K

3 2 E  AX ←kk1 → EAX   → EA  X   →E  A

341 449

447

449

295 334

334

Figure 68.3  Three-dimensional view of the active center of AChE. Modeled from Torpedo AChE with the addition of amino acid side chains of the mammalian enzyme. Amide backbone shown by the ribbons. The catalytic triad of the enzyme is Glu334, His447, Ser203, with hydrogen bonds indicated by dotted lines. The acyl pocket is Phe295 and Phe297; the choline subsite is Trp86, Glu202, and Tyr337; the peripheral site is Trp286, Tyr72, Tyr124, and Asp74. Tyrosines 341 and 449 may help to stabilize some ligands. The catalytic triad, choline subsite, and acyl pocket are at the base of the 18–20 Å gorge; the peripheral site is at its lip. (One way to see the diagram in three dimensions is to use 3-D glasses; another is to move the page toward your eyes until the images superimpose, keeping your head level. If there appear to be three images, concentrate on the middle one.) (After Taylor, 1996.)

Hayes’ Handbook of Pesticide Toxicology

1462

(choline), and ks are reaction rate constants (Rosenberry et al., 1998; Taylor, 1996). The kinetics of ACh hydrolysis is a complicated multi­ step process (Figure 68.4 is one depiction) discussed here in abbreviated form. The first step is a nucleophilic attack of the carbonyl carbon resulting in the formation of a reversible enzyme–substrate complex (EAX), acylation of the catalytic site (EA), and liberation of choline. This is followed by a rapid hydrolysis of the acylated enzyme, pro­ ducing acetic acid and regenerating the enzyme (E  A). A similar reaction scheme applies to BuChEs (Taylor, 1996). The real-time kinetics of the enzyme reactions has been described by one biochemist (Quinn, personal com­ munication) as “approaching catalytic perfection.” The rate of ACh turnover (kcat/Km) is extremely rapid, where kcat  k2k3/(k2  k3) (the geometric mean of the two rate constants). AChE is capable of hydrolyzing 6  105 mol­ ecules of ACh per molecule of enzyme per minute, a turn­ over time of 150 s (Taylor, 1996). The upshot of this rapid rate of hydrolysis is that k1 becomes the rate-limiting step for hydrolysis of ACh and its analog ATCh. Perhaps this

Glu 334

Glu 334

Ser 203 His 447

represents the movement of substrate to the active center. The deacylation step (k3) is considered rate limiting to car­ bamoylating and phosphorylating agents. A major distinction between AChEs and BuChEs is the inhibition of AChE activity with increasing substrate concen­ tration [S]. A plot of activity versus [S] for ACh, ATCh, and, in mammals, acetyl--methylcholine, yields curves with a maximum at 1–3 mM, whereas BuChE activity increases with [S] to at least 10 mM. These effects are illustrated in Figure 68.5 from data of Wilson et al. (1997) for the human. The late Dr. A. R. Main (Hoffmann et al., 1989) noted, “Because of this inhibition, methods for determining AChE activities should employ substrate concentrations at or below [S]opt.” Unfortunately, his advice has not always been followed. The phenomenon of inhibition with excess sub­ strate may be due to interactions at the peripheral site. Rosenberry et al. (1998) present evidence that excess sub­ strate inhibition and the action of peripheral site inhibitors such as propidium are brought about by the imposition of a steric blockade in the catalytic pathway. Figuratively, a chemical cork blocks the gorge.

Glu 334

Ser 203 His 447

Glu 334

Ser 203 His 447

Ser 203 His 447

Gly 122 Gly 121

A Enzyme-substrate complex Glu 334

B Tetrahedral intermediate

Trp 86

Glu 334

Ser 203 His 447

Trp 86

Trp 86

C Acetyl enzyme Glu 334

Ser 203 His 447

Glu 334

Ser 203

Trp 86

Trp 86

F Neostigmine

Glu 334

Glu 334

Ser 203

Glu 334

His 447

His 447

Trp 86

H Dimethyl carbamoyl enzyme Glu 334

Ser 203

His 447 Gly 122 Gly 121

Ser 203

Trp 86

G Hydrolysis of dimethyl carbamoyl enzyme

Ser 203

Trp 86

His 447

His 447

E Edrophonium

D Hydrolysis of acyl enzyme

Ser 203

His 447 Gly 122 Gly 121

Gly 122 Gly 121 2PAM

l Diisopropylfluorophosphate

MOLECULE

Trp 86

J Diisopropyl phosphoryl enzyme

carbon

oxygen

Trp 86

K Aged phosphoryl enzyme

nitrogen

hydrogen

Trp 86

L Reactivation of phosphoryl enzyme

phosphorus

Trp 86

fluorine

Figure 68.4  Hydrolysis of ACh by AChE and inhibition and reactivation of the enzyme. (A) Binding of ACh. (B) Attack by the serine hydroxyl and formation of a transient tetrahedral intermediate. (C) Loss of choline and formation of the acylated enzyme. (D) Deacylation of the enzyme by H2O attack. (E) Binding of the reversible inhibitor edrophonium. (F) Binding of neostigmine. (G) Formation of the carbamoylated enzyme. (H) Hydrolysis of the carbamoylated enzyme. (I) Binding of diisopropyl fluorophosphate. (J) Formation of the phosphoryl enzyme. (K) Formation of the aged form of the phosphoryl enzyme. (L) Attack by pralidoxime (2-PAM) to regenerate active enzyme (after Taylor, 1996).

Chapter | 68  Cholinesterases

1463

The knowledge of the three-dimensional structure of the ChEs has led to a better understanding of the mecha­ nisms of action of drugs and chemical agents that inhibit the hydrolysis of choline esters. In general, there are three major domains for inhibitors to bind: the acyl and choline pockets of the active center and the peripheral anionic site. Taylor (1996) uses edrophonium and tacrine as examples of reversible inhibitors that bind to the choline subsite near tryptophan 86 and glutamate 202; other reversible inhibitors, such as fasciculin and propidium, bind to the peripheral anionic site on AChE at the lip of the gorge encompassed by tryptophan 286 and tyrosines 72 and 124. CBs and OP pesticides inhibit enzyme activity by acting as alternate substrates to ACh. CBs give rise to a carbamoylated enzyme that is more stable than the acylated enzyme, taking minutes instead of milliseconds to rehydrolyze. OPs are true hemisubstrates; they covalently bind with the ser­ ine at the active center, forming a tetrahedral configuration that resembles the transition state formed during hydrolysis of ACh. If the alkyl groups on the OP are methyl or ethyl, spontaneous regeneration may require hours or even lon­ ger if tertiary alkyl groups are involved. Loss of one of the alkyl groups, a phenomenon known as aging, further stabi­ lizes the phosphorylated enzyme, permanently inhibiting its catalytic ability. Thus, it is not appropriate to use the terms “reversible” and “irreversible” to refer to the inhibitions brought about by CBs and OPs, respectively. Both classes of chemicals react covalently with the active center of the enzyme, and at some stage of the reaction sequences both enzyme-inhibitor complexes are rehydrolyzable.

The toxicities of OPs and CBs are often correlated with the extent of their inhibitions of brain AChE. Figure 68.6 depicts the relationship between the toxicity in vivo of 30 directly acting OPs and their inhibition of AChE in vitro. However, such relationships do not necessarily mean there are simple relationships between inhibition of AChE activity in an organ or tissue and in the test tube. For example, Mortinsen et al. (1998) measured IC50 values for chlorpyrifos-oxon with tissues from 4-day-old and adult rats (Table 68.4). The IC50s from young and adult brains were similar; the IC50s from the other tissues were not, dif­ fering by 5:5- (liver) and 20- (plasma) fold even though the IC50s of immunoprecipitated purified AChEs were the same, regardless of tissue or age. Possible interference of BuChE activities was excluded by using the specific

1,000 500

1

200 2

100 50

LD50 (mg/kg)

68.6  Toxicities of anticholinesterases

20

3

31

4

10

5

6, 7, 8

5

10

9 11

2 32

1

12 13

14

15

17,18

0.5

19

20 22

6

21 23

0.2 25

ChE activity (µmol/min/ml)

5

0.1

1 2

4

6

16

8

24 26

10

12

Pl50 4

3

2 pH 8.0 pH 7.2

1

0 0.1

1

10

[Acetylthiocholine] (mM) Figure 68.5  Effect of substrate concentration on activity of human blood AChE at pH 8.0 and 7.2. Ellman assay, pooled blood (after Wilson et al., 1997).

Figure 68.6  Toxicity in vivo of directly acting OPs versus their inhibi­ tion of AChE in vitro. (1) Dipterex. (2) O,O-diethyl-4-chlorophenylphos­ phate. (3) O,O-diethyl-bis-dimethyl pyrophosphorodiamide (sym). (4) TIPP. (5) O,O-diethylphosphostigmine. (6) Isodemeton sulfoxide. (7) Isodemeton. (8) Isodemeton sulfone. (9) DFP. (10) Diethylamidoethoxyphosphoryl cya­ nide. (11) O,O-dimethyl-O,O-diisopropyl pyrophosphate (asy). (12) Diethyl; amido-methoxy-phosphoryl cyanide. (13) Tetramethyl pyrophosphate. (14) O,O-diethyl phosphorocyanidate. (15) O,O-dimethyl-O,O-diethyl pyrophos­ phate (asym). (16) Soman. (17) TEPP. (18) O-isopropyl-ethylphosphone-flu­ oridate. (19) Tabun. (20) Amiton. (21) Diethylamido-isopropoxy-phosphoryl cyanide. (22) O,O-diethyl-S-(2-diethylaminoethyl)phosphorothioate. (23) Sarin. (24) O,O-diethyl-S-(2-triethylammoniumethyl)thiophosphate iodide. (25) Echothiophate. (26) Methylfluorophosphorylcholine iodide. (27) Methylfluorophosphoryl--methylcholine iodide. (28) O-ethyl-methylphos­ phorylthiocholine iodide. (29) Methylfluorophosphoryl-homo-choline iodide. (27), (28), and (29) with LD50 values of 0.03–0.07 mg/kg are not shown. (31) Schradan and (32) dimefox (shown in the graph) were not used to calculate the regression (after Holmstedt, 1963 as shown in Gallo and Lawryk, 1991).

Hayes’ Handbook of Pesticide Toxicology

1464

Table 68.4  Tissue IC50 Values for 4-Day-Old and Adult Mice after Chlorpyrifos-Oxon

Table 68.5  Necrosis of Rat Muscle after DFP and Botulinum Toxina

Tissue

Neonate (nM; mean  SE)

Adult (nM; mean  SE)

Treatment

EDL

SOL

Saline

0

0

Brain

9.6  0.1

10  0.2

DFP

85.5

28

Liver

96  3

530  50

Btx

0

0

Plasma

18  1.5

330  28

DFP  Btx

0

1.59

Purified

3 nM for all tissues

Source: Mortinsen et al. (1998).

BuChE inhibitor iso-OMPA. A-esterase destruction, car­ boxyesterase binding, and sequestration of the lipophilic OP were considered possible factors accounting for the dif­ ferences between the in vitro and in vivo findings. One example of a carboxyesterase is serum paraox­ onase (PON1), an A-esterase associated with high-­density plasma lipoproteins; PON1 destroys OPs such as the oxon analogs of parathion and chlorpyrifos. Direct evidence for its role in detoxifying OPs was provided by the demon­ stration that mice exposed to chlorpyrifos were protected against cholinesterase inhibition and toxicity by admin­ istration of purified PON1 (Li et al., 1995). Shih et al. (1998) demonstrated that knockout PON1-deficient mice were more sensitive to chlorpyrifos and chlorpyrifos-oxon than were genetically unaltered mice. Blood ChEs also have been shown to protect animals from OP toxicity. Studies on chemical warfare agents, led by the initial report of Wolfe et al. (1987), have shown that injection with puri­ fied AChE can protect mice and other animals from expo­ sure to OPs (Doctor et al., 1991). Many physiological actions of anti-ChEs are those expected from an excess of ACh caused by the inhibition of its catalysis. Specific symptoms depend on the chemi­ cals and the receptors concerned (discussed elsewhere in this handbook). Early signs of cholinergic poisoning likely involve stimulation of muscarinic neuroeffectors of the parasympathetic system. Symptoms include slowing of the heart (bradycardia), constriction of the pupil of the eye (miosis), diarrhea, urination, lacrimation, and saliva­ tion (Spencer et al., 2000; Taylor, 1996). Overstimulation at skeletal nicotinic neuromuscular junctions (motor end­ plates) causes muscle fasciculation (disorganized twitch­ ing) and, at higher doses, muscle paralysis. Increased ACh at cholinergic junctions of the sympathetic and ­parasympathetic autonomic ganglia affects the eye, blad­ der, heart, and salivary glands. Finally, anti-ChEs affect junctions of the CNS, producing hypothermia, tremors, headache, anxiety, convulsions, coma, and death. Whether or not there are consistent behavioral effects at low dose levels of OPs and CBs, such as deficits in learning and memory, is a matter of ongoing research.

Btx, botulinum toxin; DFP, diisopropyl fluorophosphate; EDL, extensor digitorum longus; SOL, soleus muscle. a Necroses/1000 fibers. DFP injected 48 h after Btx; sampled 24 h later – 1.5 mg/kg sc. Source: Sket et al. (1991).

In addition to affecting the nervous system, the excess ACh brought about by anticholinergic agents can cause a transient myopathy (Dettbarn, 1984). In vivo studies of Meshul (1989) and in vitro studies of the late Miriam Salpeter and colleagues using ACh receptor antagonists (Leonard and Salpeter, 1979) show that this is due to an influx of Ca2 and other cations into the postsynaptic cell. For exam­ ple, necrosis due to diisopropyl fluorophosphate (DFP) was prevented in vivo by treatment with -bungarotoxin, a snake venom agent that binds irreversibly to the nicotonic ACh receptor (Kasprzak and Salpeter, 1985). Further evi­ dence that the necrosis is ACh mediated was provided by Sket et al. (1991), who demonstrated that botulinum toxin (a presynaptic inhibitor of ACh release) prevented muscle necrosis induced by DFP in the rat (Table 68.5). ACh-induced myopathy may cause necrosis in 10–30% of the muscle fibers around the motor endplates (Dettbarn, 1984). Prolonged muscle weakness and muscle damage lasting several weeks or longer may occur. A similar tran­ sient muscle damage in humans has been termed interme­ diate syndrome (Senanayake and Karalliedde, 1987). Although it is generally accepted that most of the effects of OPs and CBs are due to inhibition of AChE, there is evi­ dence for other modes of action of these agents. Anti-ChE pesticides have been shown to directly affect pre- and post­ synaptic events (Pope, 1999). Electrophysiological studies suggest that choline may act as a regulator of nicotinic recep­ tors in the CNS (Albuquerque et al., 1998; Alkondon et al., 1997). Malathion and other OPs have been shown to affect the immune responses of mammals and fish (Beaman et al., 1999; Rodgers and Ellefson, 1990; Rodgers and Xiong, 1997). A few OPs, including some pesticides (e.g., isofenphos and chlorpyri­ fos) and at least one chemical warfare agent (sarin), have been shown to cause OPIDN, a distal axonopathy. Inhibition of ChEs plays a role in drug interactions. For example, cocaine is both detoxified by and is itself a reversible inhibitor of BuChEs. Studies on experimental animals indicate that depressing ChEs with anti-ChE treatments intensifies the toxic effect of cocaine (Hoffman et al., 1992).

Chapter | 68  Cholinesterases

Genetic variation between individuals can play an impor­ tant role in the toxicity of anti-ChEs. One example is humans with inherited low levels of plasma BuChEs. Although usu­ ally symptomless, patients with genetically low BuChE given succinylcholine (or a similar drug) during surgery to induce relaxation of muscles are unable to speedily destroy the drug, intensifying and prolonging its activity, sometimes with seri­ ous consequences. People with such a genetic makeup can be detected by assays using dibucaine and fluoride (Silk et al., 1979). At least two genetic polymorphisms, those of low BuChE and PON1, are considered risk factors for OP and CB pesticide exposures (Shih et al., 1998).

68.7  Assay techniques The history of ChE assays has been reviewed (Hoffmann et al., 1989; Silver, 1974; Wills, 1972; Wilson, 1999; Witter, 1963). Some techniques are “endpoint” assays; others record the time course of hydrolyses. The colorimetric and radiomet­ ric techniques currently used in my laboratory are based on those of Ellman et al. (1961) and Johnson and Russell (1975) and found in Current Protocols in Toxicology (Wilson and Henderson, 2007). One early assay to determine the hydro­ lysis of acetylcholine used a Warburg manometer to mea­ sure the CO2 released from a bicarbonate containing buffer (Ammon, 1933). The particulars of this method are outlined by Wills (1972). Although accurate, it is little used today. Following World War II, there was widespread develop­ ment of OPs for pest control. A veritable OP race ensued. By 1950, scientists were seeking rapid, accurate, and convenient clinical assays for blood ChE levels. Metcalf (1951) expressed the rationale for monitoring: “Since the cholinesterases of human blood are very sensitive to the presence of cholinesterase inhibitors, it appears that peri­ odic estimation of blood cholinesterase levels may pro­ vide an indication of dangerous levels of overexposure to these toxicants.” Several of the methods developed a half century ago are still in use today. Hestrin (1949) determined the ACh remaining after incubation by reacting it with hydroxyl­amine under alkaline conditions to form a reddishpurple complex read at 515 nm. Metcalf (1951) adapted the method for drops of blood obtained with a spring-loaded lancet. Okabe et al. (1977) oxidized the choline released during ACh hydrolysis; the hydrogen peroxide produced was determined with an indicator reaction at 500 nm (Abernathy et al., 1988). Three kinds of ChE assays are commonly used today utilizing electrometric (pH), radiometric, and colorimetric techniques. Specific examples are listed in Table 68.6.

68.7.1  Radiometric An example of a radiometric technique is that of Johnson and Russell (1975). It is based on the differential solubility of ACh and its hydrolysis products in organic and ­aqueous

1465

Table 68.6  Common ChE Assays Test

Basis

Conditions

Analysis

Johnson and Russell (1975)

Radiometric [3H]ACh

Endpoint

Micro, fast, costly disposal

Michel (1949)

pH

Rate, ACh

Simple, slow, cheap

Ellman et al. (1961)

Colorimetric

Rate, ATCh

Micro, rapid

Adapted from Wilson and Henderson (1992).

media. Sample and tritiated ACh are reacted together; an organic solvent is added when the assay is completed. The unhydrolyzed radioactive ACh substrate remains in the aque­ ous phase, quenching its scintillation. The hydrolyzed radio­ active acetate moves into the organic phase and is counted. The degree of substrate hydrolysis is determined by compar­ ison to a nonhydrolyzed ACh blank and a totally hydrolyzed ACh sample (usually accomplished by incubation with an excess of electric eel AChE). This and similar assays have high sensitivity, problems with dilution of samples encoun­ tered with “reversible” carbamate chemicals are minimized, many tubes may be measured at once, the sample size is small, and readouts may be computerized. However, radio­ active assays involve a high initial investment and costly dis­ posal of radioactive waste. Being an endpoint assay, many duplicate samples are needed to run a kinetic analysis. Potter et al. (1993) applied a radioactive assay similar to that of Thomsen et al. (1988, 1989) in a field study of applicators of OPs and fumigants. In this radiometric endpoint method, 14 C-labeled ACh hydrolysis was stopped by ethanol/glacial acetic acid, the labeled acetic acid was evaporated, and the unhydrolyzed ACh was counted.

68.7.2  pH The modified Michel method (Michel, 1949) directly deter­ mines the change in pH due to ACh hydrolysis with a pH meter or by titrating the acetic acid produced with NaOH while keeping the pH constant (Groff et al., 1976; Nabb and Whitfield, 1967). Potentiometric methods are reliable, they use simple reagents, and they are relatively inexpen­ sive. However, they are limited by their relative insensitiv­ ity; they often have larger sample requirements and lower outputs than radiometric methods. Several micro-pH meth­ ods have been described (Gage, 1967); one using 10 l of capillary blood was described by Mosca et al. (1995). pH assays can have relatively low variability. Results from an early pH assay study by Rider et al. (1957) of 12 males and 12 females aged 40 years, taken from a study of 800 donors at a San Francisco blood bank, are shown in Table 68.7. Whether or not there is a difference between male and female subjects (as suggested in Table 68.7) is

Hayes’ Handbook of Pesticide Toxicology

1466

Table 68.7  Plasma and RBC ACh Hydrolysis Levels for 40-Year-Old Blood Donorsa Men

Women

RBC ChE

0.766  0.081

0.750  0.082

Plasma ChE

0.953  0.157

0.817  0.187

a

pH/h/0.02 ml RBCs or plasma, 25°C, mean  SD. Source: Rider et al. (1957).

not clear. Many studies have noted a higher variability of plasma than RBC ChE activities. Gage (1967) points out that “population averages obtained by different investiga­ tors … depend to some extent upon the method of assay used.” Even so, Gage concluded on the basis of the studies available to him that “an individual in good health with a plasma ChE 33% below the population average, or a red cell cholinesterase more than 20% below, has an abnor­ mally low value and has probably been exposed to a ChE inhibitor.” Such “red-alert level” estimates have changed little in more than 30 years.

68.7.3  Thiol Substrates and the Ellman Assay Thiocholine substrate assays based on the work of Ellman et al. (1961) may be the most popular of ChE assays, replac­ ing the pH methods. Many variations of the original Ellman assay have been published. The conditions of three popular commercial assays are shown in Table 68.8. Several auto­ mated versions (e.g., those of Technicon and COBIAS) are no longer marketed. The basis for the thiocholine assays is the hydrolysis of ATCh or related substrates by the enzyme, producing a thiol group that reacts with a sulfhydryl-sensitive chromogen such as dithiobisnitrobenzoate (DTNB). In this case, peak absorption of the thionitrobenzoate produced is at 410–412 nm. The original Ellman assay used cuvettes; some laboratories have modified the assay for a 96-well microplate reader in a manner similar to that described by

Doctor et al. (1987). The Boehringer–Mannheim (Roche) Diagnostics kits (1981), Nos. 124117 and 450035, use ATCh as a substrate; the Sigma Diagnostic Cholinesterase (PTC) kit Procedure No. 422 uses propionylthiocholine (Sigma Diagnostics, 1989) for both AChE and BuChE. The Boehringer–Mannheim version used with a Hitachi automated spectrophotometer reads the reaction at 480 nm. This avoids possible interference of the Soret band of hemoglobin (Hb), but at the expense of reducing the sen­ sitivity of the assay (Wilson and Henderson, 1992). The manual instrument kits recommend 405 nm. Instructions for both Boehringer–Mannheim and Sigma kits are for human blood. The reader is cautioned that the kits may not be suited to the needs of clinical veterinary laboratories or researchers without modification. In the case of the human, Wilson et al. (1997) showed that the high substrate concen­ tration and low pH of the Boehringer–Mannheim kit intro­ duce a difference of 40% in the manual assay compared to the Ellman assay run under optimum conditions (see Figure 68.5). This is because the optimum concentration of sub­ strate for human AChE is 1 or 2 mM and the optimum pH for the assay is 8.0, whereas the kit uses a substrate con­ centration of 5.4 mM and a pH of 7.2. Absorbances would have been even more reduced if the measurements for the Boehringer–Mannheim substrate and pH conditions were read at 480 nm (Wilson and Henderson, 1992). There have been recommendations that the Ellman assay be modified to use dithionicotinic acid, a chromo­ gen that absorbs in the near-ultraviolet at 340 nm, to avoid the interference of Hb at 410 nm, thus divorcing the wave­ length of the assay from the Soret band of Hb (Christenson et al., 1994; Loof, 1992; Willig et al., 1996). However, instruments reading in the near-ultraviolet are often more costly than those that register in the visual range. The sta­ bility of modern instruments should be sufficient to over­ come the increased noise level of the assay at 410 nm, providing activity levels are sufficiently high. The sub­ strate and pH of the Sigma Diagnostics Kit are optimal for neither RBC nor plasma BuChE. Augustinsson et al. (1978) proposed using propionylthiocholine, the Sigma

Table 68.8  Common Thiocholine-Based Assays Boehringer–Mannheim (Roche) Parameter

Ellman

Manual

Auto

Sigma

Wavelength (nm)

412

405

480

405

Substrate (mM)

ATCh, 0.5–1.0

ATCh, 5.4

ATCh, 5.4

PrTh, 5.0

pH

8.0

7.2

7.2

7.2

DTNB (mM)

0.32

0.24

0.24

0.25

PrTh, propionylthiocholine. Source: Wilson (1999).

Chapter | 68  Cholinesterases

1467

kit substrate, as a compromise substrate for both enzymes. Under the conditions of their assay, it was no better a sub­ strate for one than it was for the other. They recommended measuring the reaction in the ultraviolet, avoiding Hb interference that might well play a role, given the reduced sensitivity of the assay. The Sigma kit is no doubt excel­ lent as a screen for patients with reduced BuChE activities before they undergo surgery and treatment with muscle relaxants such as succinylcholine and mivacurium, but its use may be more difficult to justify, given the excellence of today’s instrumentation, for the determination of RBC AChE activities to detect exposures to pesticides because the conditions are not optimum for the enzyme. What to do? With blood enzyme assays, no one size seems to fit all. One approach is to focus on ATCh hydrolysis, and spe­ cific inhibitors such as iso-OMPA or quinidine to inhibit BuChE, establishing an estimate of BuChE by difference, accepting, for convenience, the use of an inappropriate substrate and substrate concentration. The problems of using commercial kits with condi­ tions designed for the human may be exacerbated when the kits are applied to other species. Many studies (some of which are summarized in Tables 68.9 and 68.10) indi­ cate that, with other species, the relative activities of AChE and BuChEs, and even the properties of BuChEs, may dif­ fer from those of human enzymes. Lack of consideration for this may result in possible discrepancies and misinfor­ mation. For example, Harlin and Ross (1990) used adult bovine blood to establish conditions for the determination of cholinesterase activities in the only approved AOAC assay with ATCh as substrate and bovine blood. This excellent round-robin study did not discuss the report of Augustinsson (1959a, Table 51.2) that bovines had hardly any plasma cholinesterase activity. Further study is needed to establish whether the assay was determining only RBC AChE and is useful for assaying it when serum cholinester­ ase is low or absent. Another problem arises when studies are undertaken with species that lack RBC AChE; the liter­ ature examined by the author suggests that only mammals have RBC AChE and that the properties of serum ChEs may vary with the species (see Table 68.9). A further problem arises when, as is common in studies of laboratory animals that play a role in pesticide registration,

the investigators assume AChE activity is restricted to the RBCs. Table 68.10 illustrates the wide differences between AChE activity in blood for humans and for two species often used in registration-geared research – the dog and the rat. Of the three, only the human has a relatively low ATCh hydrolysis rate in the plasma. Indeed, the AChE activity of rat plasma (Table 68.11) may exceed the activity of plasma BuChE. Another source of plasma AChE is the platelets (Table 68.12). Although the AChE activity per platelet is high, its relative contribution to blood ChE is low because the plate­ let content of blood is several orders of magnitude less than its content of RBCs. Studies of the particular AChE forms in the plasma of many species are lacking. An important and often unrecognized problem with thiocholine-based assays is the presence of a transient nonlinear “thiol oxidase” reaction with the color reagent

Table 68.9  Relative Substrate Specificity of Plasma ChEsa Species

Propionyl

Butyryl

Benzoyl

Dog

150

253

60

Horse

161

231

28

Cat

111

211

27

Human

155

192

36

Duck

139

153

25

Squirrel

122

144

14

Ferret

122

139

28

Hamster

153

128

24

Rat

211

119

17

Chicken

147

83

6

Mouse

139

75

11

Butyryl favoring

Propionyl favoring

a

Normalized to plasma ChE hydrolysis of ACh  100%. Adapted from Hoffmann et al. (1989, Table 11.4) and Myers (1953).

Table 68.10  Relative Acetyl Ester Hydrolysis Levels of Several Speciesa Species

RBC (mean  SD)

No. of trials

Plasma (mean  SD)

No. of trials

Human

135  29

60

37  9.3

56

Dog

17.9  3.5

18

25.4  5.5

18

Male rat

9.0  1.3

24

4.3  1.0

45

a

ATCh 0.8 mM (RBC); 7 mM (plasma); Autotechnicon analyzer. Adapted from Humiston and Wright (1967, Table 4).

Hayes’ Handbook of Pesticide Toxicology

1468

Table 68.11  AChE/BuChE Activity of Rat Plasmaa Total ChE (mU/ml, mean  SE)

BuChE (mU/ml, mean  SE)

AchE (mU/ml, mean  SE)

452  17

175  12

210  8

a

Eighteen rats, Ellman assay. Total with acetylthiocholine; BuChE with butyrylthiocholine; AChE with acetylthiocholine  0.1 mM iso-OMPA. Source: Traina and Serpietri (1984).

Table 68.12  Comparison of RBC and Platelet Activities of Several Speciesa Species

Sex

Red blood cell Platelet

Human

Male/female

2180  189

0

Cow

Female

1910  42

53  21

Guinea pig

Male/female

713  76

493  119

Horse

Male/female

627  200

1500  252

Rabbit

Male/female

473  116

3930  208

Rat

Male

274  64

4240  1070

30

5450

Cat

b

Male/female

a

Manometric, lCO2/30 min/mgN; N  3. N  2. Adapted from Zajicek (1957). b

DTNB in RBCs of some species. It is necessary that assays with species such as the rat that have such high “tissue blanks” be designed to either circumvent or correct for them because they can lead to indeterminate errors greater than 70% (Table 68.13). One way is to include the appro­ priate blank in the assay. Another is to preincubate the samples for a few minutes before adding substrate until the nonlinear first 5–10 min of the assay is over (Chancy et al., 2000). Lack of consideration of such problems in clinical assays of experimental animals submitted for regu­ latory purposes may have played a role in the difficulties encountered when the U.S. EPA tried to compare data on the rat from different laboratories (U.S. EPA, 1992). Errors may be compounded. For example, a large and indetermi­ nate error will occur when the Boehringer–Mannheim kit assays are applied to rats without preincubating the sam­ ples. Such an error could be exacerbated by the low RBC activities and the misinterpreted plasma “BuChE” activities that are actually due to a mixture of the AChE and BuChE activities. The lesson is that whatever the assay, it is critical that its conditions be validated for each species, tissue, and chemical under study. CBs represent a special problem because of the ease of rehydrolysis of carbamate–ChE complexes. The sensitiv­ ity of the assay requires dilution of the enzyme samples, but the act of dilution itself promotes rehydrolysis of the

Table 68.13  RBC DTNB Background Reaction in Various Speciesa Species

% Total activity

Human

10

Monkey

18–27

Dog

30–60

Rabbit

50–70

Rat

60–75

Mouse

4

Brain (all species)

N/A

Source: Loof (1992). a Transient activity, first 5–10 min, Ellman method.

enzyme. Several investigators have suggested ways to mini­ mize the problem, including Thomsen et al. (1988) for the radiometric assays and Nostrandt et al. (1993) for Ellmantype thiocholine-based assays. Regardless of the assay used, samples should be kept iced from the time of their collection to their assay. It is unfortunate that some are under the impression that OP exposures lead to “irreversible” inhibitions, and that icing a blood sample is unnecessary as long as an anticlotting agent such as EDTA or heparin has been used. To the con­ trary, as discussed elsewhere in this handbook, ChE inhi­ bitions by methyl-OPs such as azinphosmethyl (Guthion) have relatively rapid rates of spontaneous reactivation at room temperature; in this case, the half-life of recovery of activity for azinphos-methyl is approximately 2.5 h (Wilson et al., 1992b). The lack of a requirement to keep samples on ice as a part of assay protocols makes it difficult to inter­ pret the results of otherwise excellently designed and exe­ cuted studies such as that of Yeary et al. (1993). Although the instructions that accompany the commercial ChE mon­ itoring kits discussed in this chapter recommend storing samples under refrigeration [4°C (Boehringer–Mannheim) and 2–6 or 20°C (Sigma Diagnostics)], several clinical laboratories I contacted said they did not specify that sam­ ples be delivered to them on ice.

68.7.4  Variability Regarding studies using the Ellman assay, a paraphrase from George Orwell’s classic “Animal Farm” (1945) might be that “all Ellman thiocholine assays are created equal, but some are more equal than others.” The variety of condi­ tions used in field studies and laboratory experiments with thiocholine substrates, and the lack of an accepted standard assay and enzyme, make it difficult to compare the activi­ ties obtained from one experiment to another (Carakostas

Chapter | 68  Cholinesterases

and Landis, 1991; Wilson et al., 1992a). Perhaps this is what has led to the idea that thiocholine-based ChE studies are “too variable” to be relied on for population exposure research and regulatory decisions, even though a number of carefully performed studies such as Sanz et al. (1991) indicate that ChE assays of populations can be performed with satisfactory results. For example, Sidell and Kamiskis (1975) measured RBC and plasma ChEs of a group of 22 subjects biweekly for a year using the Technicon autoana­ lyzer. They found that RBC AChE levels varied less than hematocrit, Hb, or RBC counts. The annual average range of AChE values was 8% for men and 12% for women. The corresponding plasma ChE values were 25% for men and 24% for women. In general, as is true for the pH meth­ ods discussed previously, plasma ChE values appear more variable than RBC AChE activities, whether the data are expressed on a per cell or a per Hb basis (Table 68.14). Bellino et al. (1978) used fingersticks and saponinhemolyzed human blood to carefully determine the optimum conditions for the Ellman assay for human RBCs and plasma, demonstrating inhibition of ATCh hydrolysis with excess substrate and an S-shaped substrate–concentration curve with butyrylthiocholine. They established that 0.01 mM eser­ ine inhibited AChE and 0.3 mM eserine inhibited BuChE. Similarly, they found that 7.0 mM totally inhibited both AChE and BuChE activity, and 2.8 mM of sodium dodecyl sulfate inhibited AChE but not BuChE. Their study of healthy men and women showed relatively low variability (Table 68.15). Large sample numbers were obtained in a study of farm worker families from migrant housing centers in California by Wilson et al. (1998), in which almost 900 volunteers contributed fingersticks of blood. Ten micro­liters of blood was hemolyzed at the site, transported on ice to the labo­ ratory, stored at 70°C, analyzed under optimum assay conditions for RBC AChE using quinidine to inhibit plasma BuCh, and expressed on an Hb basis. Mean activ­ ity of the migrant housing center families (n  894) was 14.6  2.6 nmol/min/mg Hb – virtually the same as those from fingersticks and venous blood draws of approxi­ mately 12 University of California, Davis, volunteers (Figures 68.7 and 68.8). One problem to circumvent is the possible contamina­ tion of the sample with pesticide on the skin (Yuknavage et al., 1997).

68.8  Standards Several companies provide AChE standards for using human and other species ChE preparations. Wilson et al. (2000, 2009) have been using an AChE standard prepared by hemolyzing washed bovine RBCs. These RBC ghosts show low variability when stored refrigerated (4°C) for 60 days or at low-temperature (75°C) freezer tempera­ tures for more than 250 days. Its latest use is by a clinical

1469

Table 68.14  Average Values of Cholinesterase Activity in Fasting Healthy Humansa Subjects

Men

Women

N

40

38

BuChE (U/l, mean  SD)

8920  2500

7490  1950

AChE (nU/RBC, mean  SD)

1210  200

1220  123

AChE (Hb) (U/g, mean  SD)

37.6  4.91

39.3  4.49

a

BuChE with butyrylthiocholine, Boehringer–Mannheim (Roche) kit; AChE with acetylthiocholine according to Ellman. Source: Sidell and Kamiskis (1975).

Table 68.15  ChE Activities of Healthy Human Subjects Mean  SEa

n

Male

4.99  0.14

72

Female

5.18  0.18

71

Pooled

5.08  0.11

143

Male

2.26  0.04

101

Female

2.46  0.09

71

Pooled

2.34  0.05

172

Enzyme AChE

BuChE

a

IU/ml whole blood; Ellman assay. Modified from Bellino et al. (1978).

laboratory (Wilson et al., 2009) conducting court-ordered monitoring of blood of orchard workers in the state of Washington.

68.9  Field kits Well-designed, reliable field kits for cholinesterase deter­ minations would be valuable for monitoring the health of those who apply pesticides and those who work in agricultural workplaces subject to pesticide spraying. Enzyme-impregnated filter paper, potentiometric sensors, and colorimetric comparisons have been used in the past (Collombel and Perrot, 1970; Dahlgren, 1983; Gamson et al., 1973; Rogers et al., 1991). A relatively new device is the EQM Test-Mate kit (Magnotti and Eberly, 1996; Magnotti et al., 1988). It uses a solid-state device and the Ellman method to measure ChE activity in a drop of whole

Hayes’ Handbook of Pesticide Toxicology

1470

6

Number of subjects

5 4 3 2 1 0 1

3

5

7

9

11

13

15

17

19

21

23

25

27

29

31

AChE activity (nmol/min/mg Hb) Figure 68.7  Fingerstick AChE activities of University of California, Davis, volunteers. Whole blood hemolyzed with Triton X-100 and assayed according to Ellman modified for a multiple plate reader. Activity is 14.6  1.2 nmol/min/mg hemoglobin. N  13 (reproduced from Wilson et al., © 1998, Plenum).

200 180

Number of subjects

160 140 120 100 80 60 40 20 0

1

3

5

7 9 11 13 15 17 19 AChE activity (nmol/min/mg Hb)

21

23

25

27

29

31

Figure 68.8  Fingerstick AChE activities of migrant family center residents. Blood-filled capillaries kept on ice before returning to the laboratory. Whole blood assayed as in Figure 68.7. Activity is 14.6  2.6 nmol/min/mg hemoglobin. N  894 (reproduced from Wilson et al., © 1998, Plenum).

blood obtained by a fingerstick. Several models have been marketed. The Test-Mate has the advantages of portability, relative low cost, conveniently prepared reagents, and small sample volumes. Field and laboratory studies have been conducted with it (Keifer et al., 1996; Prall et al., 1998). Nevertheless, the current model (Model ChE) is not recom­ mended by the manufacturer for field use; the instructions advise that it be operated in the laboratory by a trained technician. One difficulty with using whole blood concerns sensitivity; blood samples are not diluted as much as they would be using a larger, more sensitive device. Under these conditions, readings may be affected by the relatively high

absorption of the Soret band of Hb at 412 nm, the optimum absorbance of DTNB, which is the Ellman colorimetric reagent. The Test-Mate models have attempted to circum­ vent this by using higher wavelengths, sacrificing sensitiv­ ity of the chromogen for a lower noise level (Wilson and Henderson, 1992). A second problem with the device is that the Test-Mate does not display the raw absorbance val­ ues of the reaction. Instead, it displays values normalized to those expected at 25°C, employing a temperature sensor and a built-in algorithm that has been criticized (Amaya et al., 1996; London et al., 1995; Oliveira et al., 2002; Wilson et al., 1998).

Chapter | 68  Cholinesterases

68.10  Regulatory matters: are ChE inhibitions adverse effects? Soon after the introduction of OP pesticides, the experi­ ences of University of California scientists led to a re­commendation that “individuals showing a 20% or more depletion from normal preexposure plasma ChE levels should discontinue participation in the work … until cho­ linesterase levels have returned to normal” (Metcalf, 1951). More than 50 years and many studies and task forces later, similar guidelines are still used. The reason for choos­ ing one specific decrease in ChE level over another as constituting a health hazard is not clear. One rationale is that because most clinical laboratories should be able to detect a 20% decrease in ChE activity, and because dose–response curves for many anti-ChEs tend to be steep, this “slippery slope” provides a realistic statistically signif­ icant difference between test groups suitable for regulatory purposes.

68.11  Blood ChEs and detection   of exposure Whether or not a decrease in ChE activity in the blood constitutes an “adverse effect” raises questions concern­ ing the short- and long-term health of individuals, popula­ tions, and their progeny that have yet to be answered to the satisfaction of most investigators. Detecting a statistically significant decrease in blood ChE levels between a puta­ tive exposed group and an unexposed group, or compared to an accepted “normal” range, is often accepted as indi­ cating that a potentially hazardous exposure to an anti-ChE chemical has occurred. Anti-ChE chemicals are not usu­ ally long-lasting within the body, and blood ChE activity can be expected to recover relatively rapidly from inhibi­ tion by an OP or CB. In the human, RBCs (and their AChE activities) are replaced at approximately 0.9% per day (a 120-day life span); plasma BuChE is replaced even more rapidly (Boyer et al., 1977).

68.12  Reactivation of inhibited AChE The discovery by Wilson and Ginsburg (1955) that oximes could displace OPs from the active site of ChEs, restor­ ing enzyme activity, provided an important treatment for OP poisonings. It also opened the door to using reactiva­ tion of inhibited enzymes to establish that exposure had occurred when reliable unexposed control data or normal ranges of activity were lacking (Hansen and Wilson, 1999; reviewed by Wilson et al., 1992b). Useful as it may be, the application of reactivation techniques is not currently per­ formed on a routine basis by clinical laboratories known to the author. Benschop and colleagues treated OP-inhibited serum ChE with potassium fluoride at pH 4 to chemically

1471

detect the presence of the inhibited OP-ChE. The fluoride ions reactivated the inhibited enzyme, converting the OP moiety into the corresponding phosphofluoridate and per­ mitting its subsequent identification. Benschop’s group applied the technique to obtain direct evidence of exposure to sarin in several victims of the release of the nerve gas in a Tokyo subway in 1986 and from an earlier incident in Matsumoto, Japan (Polhuijs et al., 1997, 1999).

68.13  Significance of blood ChEs Even if ChEs were not the targets of a multibillion-dollar, worldwide pesticide industry, they would still be of great interest to physiologists, cell biologists, and biochemists. ChEs have important roles in regulating neural transmis­ sion, and they are targets for pharmacological interven­ tions in disorders such as glaucoma, myasthenia gravis, and Alzheimer’s disease (Taylor, 1996). The widespread use of OP and CB pesticides and the dangers attendant upon their applications have resulted in ChE enzymes being used as biomarkers of both exposure and effect when assessing the risks to workers in agriculture and to consumers. ChEs have several roles. One is in the use of experimental animals – most often rats but also dogs, rabbits, and other species – to determine no-effect levels that can be extrapolated to humans. Another is in the recommendation of safe residue levels in food. A third role is to help provide a safe agricul­ tural workplace by monitoring those who could be exposed to dangerous levels of anti-ChE chemicals (i.e., mixer load­ ers and applicators). A fourth role is to decide whether poi­ soning episodes have involved ChE-inhibiting agents. One factor in favor of using ChEs for such clinical and regula­ tory ends is the ease with which they may be rapidly and inexpensively assayed compared to analyzing for the cho­ linergic chemicals. Generations of toxicologists and public officials have worked to establish ChE assays as a simple way to help provide answers to complicated questions of health effects, exposures, and risk. Today, agencies require submission of blood and brain ChE levels from experimen­ tal animals after short- and long-term experiments as part of the registration process for pesticides. Most agree with the position that statistically significant decreases in brain ChE activities, when accompanied by knowledge of the doses involved, are useful for establishing quantitative toxicity indices. However, issues such as the significance of ChE levels in specific parts of the brain and the applicability of one animal model over another are unresolved. There is continuing discussion of the significance of monitoring blood ChEs of humans and other animals. One issue is the role of no-observable-adverse-effect levels (NOAELs; the highest dose levels at which no important effect of a drug is observed) in assigning safe levels for toxic chemicals. One way to establish NOAELs is to per­ form batteries of behavioral tests under controlled laboratory

1472

conditions. Another is to measure residues on skin and clothing, urinary metabolites of agricultural workers, and fecal metabolites of laboratory and wild animals. Blood ChE levels represent standardized, relatively inexpensive measurements of a biochemical effect due to an exposure to a toxic chemical, in addition to providing evidence of the exposure (Nigg and Knaak, 2000). However, some do not agree. For example, an industry “Acute Cholinesterase Risk Assessment Work Group” published a review of RBC AChE, plasma ChE, and brain AChE activities focusing on their use in risk assessment (Carlock et al., 1999). The review drew upon the literature and a compilation of toxi­ city data from previously unpublished industry sources in which the chemicals were listed by code and category. The work group concluded in part that (1) plasma ChE should not be used in risk assessments because it is not an adverse effect; (2) RBC inhibition is not per se an adverse effect; and (3) when available, cholinergic effects or brain AChE levels should be used before RBC AChE values in setting NOAELs, and human data should take precedence over animal-derived results. In its discussion of NOAELs, the group did not take into account that much of the data were derived from studies of rats and dogs and were not cor­ rected for blood AChE levels or thiol oxidase activities of the RBCs, as discussed previously. A moderate position might be to use blood ChE values as biomarkers of exposure to anti-ChE inhibitors rather than to insist they be considered quantitative indicators of physiological effects, thus supporting their use as early warning signs and as important weight-of-evidence factors.

68.14  Direct effects Although much attention of neurotoxicologists has been directed toward understanding the nature of the inhibition of ChEs and their toxicological consequences, a number of neuroscientists have studied direct effects of anti-ChE agents on the presynaptic release of ACh (Rocha et al., 1996) and on the postsynaptic target receptors of ACh. For example, van den Beukel et al. (1998) found that micromolar levels of physostigmine, parathion, paraoxon, and phenyl saligenin cyclic phosphate blocked ACh-induced transient nicotinic inward currents in mouse N1E-115 and human neuroblastoma and locust thoracic ganglion cells. Eldefrawis and colleagues (Katz et al., 1997) demonstrated that chlorpyrifos, parathion, and their oxons bind to and desensitize the nicotinic receptor (nAChR) of Torpedo, the electric ray. Narahashi’s group (Nagata et al., 1997) used rat clonal pheochromocytoma (PC12) cells to demonstrate that neostigmine and carbaryl blocked nAChR channels. In con­ trast, Albuquerque’s group (Camara et al., 1997) found that methamidophos did not affect neurotransmitter release or act directly on rat nAChR but that choline affected the response of the receptors (Albuquerque et al., 1998). The low levels

Hayes’ Handbook of Pesticide Toxicology

at which these electrophysiological effects occur strongly suggest they should be taken into account when consider­ ing the effects of ChE-inhibiting OPs and CBs, such as the report of Burruel et al. (2000) of effects of methamidophos on sperm in mice.

68.15  Antidotes ChE inhibitions by OPs and CBs are examples of enzyme inhibitions for which there are specific antidotes. Two drugs in use are atropine and pralidoxime (2-PAM). Doses depend on the extent of exposure and species. Atropine binds to the muscarinic ACh receptor (mAChR), reduc­ ing the effectiveness of the excess ACh generated by the inhibition of AChE. It is given intravenously as required to relieve the symptoms of excess ACh in cases of pesticide poisoning. Oximes directly reactivate OP-inhibited AChEs [Wilson and Ginsburg (1955) reviewed in Wilson et al. (1992b)]. 2-PAM Cl (Protopam) is the oxime registered for use in the United States; its methanesulfonate salt (P2S) is used in Europe. Reactivation of the enzyme involves trans­ fer of the substituted phosphate or phosphonate residue from the catalytic site of the enzyme to the oxime. There has been much research on the treatment of exposure to ChE inhibitors focusing on chemical war­ fare agents (Hille et al., 1995; Raveh et al., 1996). The U.S. Department of Defense kit “Convulsant Antidote for Nerve Agents (CANA)” has 2 ml of the anticonvulsant diazepam, and the “Nerve Agent Antidote Kit (NAAK)” contains autoinjectors with 2 mg of atropine and 600 mg of pralidoxime. A third kit, “Nerve Agent Pre-treatment Sets (NAPS),” contains 30-mg tablets of pyridostigmine bro­ mide to be taken orally three times a day. A Swedish auto­ injector contains HI-6 (500 mg), an oxime not available in the United States, and atropine (2 mg). The logic behind the use of pyridostigmine bromide is that this CB AChE inhibitor will reduce the extent to which AChE becomes irreversibly inhibited by rapidly aging chemical warfare agents by virtue of its ability to temporarily occupy the catalytic site of AChE, interfering with its more perma­ nent phosphorylation by soman and other OPs (Tuovinen et al., 1999). Three 30-mg doses of pyridostigmine bro­ mide per day were given to the Allied Forces during the first Gulf War under an experimental-drug U.S. Food and Drug Administration license. This led to speculation that the interaction of this CB with other neuroactive chemicals present in the sector was a factor in the cluster of symp­ toms termed the Gulf War syndrome (Abou-Donia et al., 1996a,b; Kurt, 1998). An alternative approach to OP poi­ soning is to destroy the agent within the body. Two of the most successful treatments in experimental animals are to inject purified ChEs to bind the agents and to inject phos­ photriesterases to destroy them (Tuovinen et al., 1999; Wolfe et al., 1987).

Chapter | 68  Cholinesterases

Experimental evidence of nerve damage (in this case to chickens) was reported by Abou-Donia and co-workers after combined treatments of pyridostigmine bromide, DEET (an insect repellent), chlorpyrifos (Abou-Donia et al., 1996a), or permethrin (Abou-Donia et al., 1996b).

68.16  Risk assessment and ChEs ChE activity has been used for many years as a biomarker of exposure and effect for setting regulations for anti-ChE pesticide use and for safe levels of such pesticides in foods and in the environment. Almost 75 years of research has provided tools to qualitatively establish whether exposure has occurred to humans and other animals in the labora­ tory, the clinic, and the environment. Nevertheless, prob­ lems arise when data are used quantitatively, such as in setting NOAELs of a pesticide in food or in estimating lifetime exposure levels of a chemical warfare agent. The lack of universally acceptable standards for ChE assays and the difficulty in deciding whether ChE levels are, in and of themselves, an adverse effect are two of the diffi­ culties encountered when using ChE activities for regula­ tory purposes. However, cholinergic mechanisms became a criterion for creating a category of aggregate pesticide use in the Food Quality and Protection Act of 1996 (FQPA; Mileson et al., 1998). It created a single health-based safety standard for pesticide residues in food and removed the regulation that specified zero tolerance from pesticides that may be concentrated in processed commodities. FQPA required a “reasonable certainty” that no harm will result from aggregate exposures from chemicals with a common mode of action. Exposures from diet, including drinking water, and nonfood exposures (e.g., residential, lawn, gar­ den, indoor, institutional, and industrial uses) were all to be considered. Children received special treatment; up to 10 additional uncertainty factors could be added for them. The FQPA specified the use of sound science in making the determinations and required a focus on health-based approaches to food safety and the promotion of safer, effective pest control methods. Applying the policies delineated by the FQPA may occupy the attention of the agricultural community, gov­ ernment regulators, and toxicologists specializing in agri­ cultural chemicals for some time to come. It is safe to predict that there will continue to be important issues at each step of the risk assessment process in the assessments of hazard, dose–response, and exposure and in the charac­ terization of risk of a toxic chemical. A special problem involves determining aggregate exposures, deciding which chemicals should be included as part of a common “risk cup.” ChE-inhibiting chemicals played a leading role in this venture when a panel of dis­ tinguished toxicologists asserted that inhibition of AChE was a “common mechanism of toxicity” for OPs. They

1473

concluded that “OP pesticides act by a common mecha­ nism of toxicity if they inhibit phosphorylation and elicit any spectrum of cholinergic effects” (Mileson et al., 1998, p. 8). It may be too soon to state whether such a pharma­ cologically simplistic, but regulatory valuable, view will win the day. Pope (1999), one of the authors of the “com­ mon mechanism” review, concluded in a separate article that “additional macromolecular targets for some OP pesticides … may alter the cascade of events following AChE phosphorylation and thereby modify that common mechanism” (p. 16). His review analyzed the comparative toxicity of 38 OP AChE inhibitors currently in use in pesti­ cides. Demonstrated direct effects of anticholinesterases on receptors and transmitter release discussed previously, and the evidence of groups such as Albuquerque’s that choline may have an effect on ACh receptors (Albuquerque et al., 1998; Alkondon et al., 1997), suggest that there may be holes in the risk cup. Whether the leaks are large enough to lead to a modification of the “common mechanism” policy is a matter for the future.

Conclusion The specific course of research on ChEs parallels, and indeed has often been at the cutting edge of, some of the advances of the mid-20th and early 21st century in biochemistry, pharmacology, physiology, cell biology, and toxicology. Many of today’s disciplines had not yet been christened when ChEs were first recognized as special proteins intimately associated with the development and regulation of the nervous system. Although some anticho­ linergic agricultural chemicals have been replaced by other agents with different modes of action (e.g., imidacloprid) (Thyssen and Machemer, 1999), OP and CB use is likely to continue in the United States and abroad for the fore­ seeable future, if only because some countries may not be able to afford the new generations of chemicals. The rapid development of cDNA microarrays suggests that stud­ ies of the impact of anticholinergic chemicals will soon be routinely conducted on the level of responsive genes (Gupta et al., 1999). The advent of probabilistic methods of assessing risk of pesticides to human health and wildlife will create opportunities to apply sophisticated methods of determining risk from anticholinergic agents based on the population distributions of exposures and effects (Boyce, 1998). Research on the molecular bases of pharmaceuti­ cals, enzyme action, development of the nervous system, and many other basic features of living systems will ben­ efit from studies of ChEs for many years to come. Also, dismayingly, the simplicity of synthesizing and deploying anti-ChEs as weapons of war and terror is likely to be as tempting to governments and to terrorists of the 21st cen­ tury as it was to some in the 20th century. Today’s scien­ tists leave a legacy of knowledge for those who come after,

1474

but the gift of wisdom is not ours to bestow, much as we may wish to do so.

Acknowledgment I am grateful for the assistance of John D. Henderson in the preparation of this chapter.

References Abderhalden, E., and Paffrath, H. (1925). Beitrag der Frage der Inkret(Hormon)-Wirkung aud die motorischen Funktionen des Ver-dauungskanales V. Uber die synthese von cholinestern aus Cholin und Fettsauren mittels Fermenten des Dunndarm. Fermentforsch 8, 299. Abernathy, M. H., Fitzgerald, H. P., and Ahern, K. M. (1988). An enzy­ matic method for erythrocyte acetylcholinesterase. Clin. Chem. 34, 1055–1057. Abou-Donia, M. B., Wilmarth, K. R., Abdel-Rahman, A. A., Jensen, K. F., Oehme, F. W., and Kurt, T. L. (1996a). Increased neurotoxi­city following concurrent exposure to pyridostigmine bromide, DEET, and chlorpyrifos. Fundam. Appl. Toxicol. 34, 201–222. Abou-Donia, M. B., Wilmarth, K. R., Jensen, K. F., Oehme, F. W., and Kurt, T. L. (1996b). Neurotoxicity resulting from coexposure to pyr­ idostigmine bromide, DEET, and permethrin; Implications of Gulf War chemical exposures. J. Toxicol. Environ. Health 48, 35–56. Albuquerque, E. X., Pereira, E. F., Braga, M. F., and Alkondon, M. (1998). Contribution of nicotinic receptors to the function of synapses in the central nervous system, the action of choline as a selective ago­ nist of alpha 7 receptors. J. Physiol. (Paris) 92, 309–316. Aldridge, W. N., and Reiner, E. (1972). “Enzyme Inhibitors as Substrates”. North-Holland/Elsevier, Amsterdam. Alkondon, M., Pereira, E. F., Cortes, W. S., Maelicke, A., and Albuquerque, E. X. (1997). Choline is a selective agonist of alpha7 nicotinic acetylcho­ line receptors in the rat brain neurons. Eur. J. Neurosci. 12, 2734–2742. Amaya, A., Keifer, M., and McConnell, R. (1996). Comment on EQM Testmate OP cholinesterase kit. Occup. Environ. Med. 53, 358. Ammon, R. (1933). Die fermentative spaltung des acetylcholins. Arch. ges. Physiol. Menschen Tiere, 233, 486–491. Anderson, R. B., and Key, B. (1999). Role of acetylcholinesterase in the development of axon tracts within the embryonic vertebrate brain. Int. J. Dev. Neurosci. 17, 787–793. Atkinson, J. E., Bolte, H. F., Rubin, L. F., and Sonawane, M. (1994). Assessment of ocular toxicity in dogs during 6 months’ exposure to a potent organophosphate. J. Appl. Toxicol. 14, 145–152. Augustinsson, K.-B. (1948). Cholinesterases, a study in comparative enzymology. Acta Physiol. Scand. 15(Suppl 52), 1–182. Augustinsson, K.-B. (1959a). Electrophoresis studies on blood plasma esterases: I. Mammalian plasmata. Acta Chem. Scand. 13, 571–592. Augustinsson, K.-B. (1959b). Electrophoresis studies on blood plasma esterases: II. Avian, reptilian, amphibian and piscine plasmata. Acta Chem. Scand. 13, 1081–1096. Augustinsson, K.-B., Eriksson, H., and Faijersson, Y. (1978). A new approach to determining cholinesterase activities in samples of whole blood. Clin. Chim. Acta 89, 239–252. Austin, L., and Berry, W. K. (1953). Two selective inhibitors of cholines­ terase. Biochem. J. 54, 695–700. Ballantyne, B., and Marrs, T. C. (1992). “Clinical and Experimental Toxicology of Organophosphates and Carbamates”. ButterworthHeinemann, Oxford.

Hayes’ Handbook of Pesticide Toxicology

Bartels, C. F., Xie, W., Miller-Lindholm, A. K., Schopfer, L. M., and Lockridge, O. (2000). Determination of the DNA sequences of ace­ tylcholinesterase and butyrylcholinesterase from cat and demonstra­ tion of the existence of both in cat plasma. Biochem. Pharmacol. 60(4), 479–487. Beaman, J. R., Finch, R., Gardner, H., Hoffmann, F., Rosencrance, A., and Zelikoff, J. T. (1999). Mammalian immunoassays for predict­ ing the toxicity of malathion in a laboratory fish model. J. Toxicol. Environ. Health 56, 523–542. Bellino, M., Ficarra, M., Frontali, N., Ghezzo, F., Guarcini, A. M., Orecchio, F., Serpietri, L. A., and Traina, M. E. (1978). A quick and simple method for the routine determination of acetyl- and butyryl­ cholinesterase in blood. Br. J. Ind. Med. 35, 161–167. Boehringer Mannheim Diagnostics (1981). “Reagentet cholinesterase 124117”. Boehringer Mannheim Diagnostics Division, Indianapolis, IN. Boyce, C. P. (1998). Comparison of approaches for developing distribu­ tions for carcinogenic slope factors. Hum. Ecol. Risk Assess. 4(2), 527–577. Boyer, A. C., Brown, L. J., Slomka, M. B., and Hine, C. H. (1977). Inhibition of human plasma cholinesterase by ingested dichlorvos, effect of formulation vehicle. Toxicol. Appl. Pharmacol. 41, 389–394. Boyes, W. K., Tandon, P., Barone, S. Jr., and Padilla, S. (1994). Effects of organophosphates on the visual system of rats. J. Appl. Toxicol. 14, 135–143. Brimijoin, S. (1992). Enzymology and biology of cholinesterases. In “Proceedings of the U.S. EPA Workshop on Cholinesterase Methodologies,” p. 30. U.S. Environmental Protection Agency, Washington, DC. Burruel, V. R., Raabe, O. G., Overstreet, J. W., Wilson, B. W., and Wiley, L. M. (2000). Paternal effects from methamidophos administration in mice. Toxicol. Appl. Pharmacol. 165, 148–157. Camara, A. L., Braga, M. F., Rocha, E. S., Santos, M. D., Cortes, W. S., Cintra, W. M., Aracava, Y., Maelicke, A., and Albuquerque, E. X. (1997). Methamidophos, an anticholinesterase without sig­ nificant effects on postsynaptic receptors or transmitter release. Neurotoxicology 18, 589–602. Carakostas, M. C., and Landis, M. A. (1991). Modification of an auto­ mated method for determining plasma and erythrocyte cholinesterase activity in laboratory animals. Vet. Hum. Toxicol. 33, 450–456. Carlock, L. L., Chen, W. L., Gordon, E. B., Killeen, J. C., Manley, A., Meyer, L. S., Mullin, L. S., Pendino, K. J., Percy, A., Sargent, D. E., Seaman, L. R., Svanborg, N. K., Stanton, R. H., Tellone, C. I., and Van Goethem, D. L. (1999). Regulating and assessing risks of cho­ linesterase-inhibiting pesticides, divergent approaches and interpreta­ tions. J. Toxicol. Environ. Health B 2, 105–160. Chambers, J. E., and Levi, P. E. (eds) (1992). “Organophosphates: Chemistry, Fate, and Effects”. Academic Press, New York. Chancy, L. A., Wineman, R. W., Rockhold, R. W., and Hume, R. S. (2000). Acute effects of an insect repellant, N,N-diethyl-m-toluamide, on cholinesterase inhibition induced by pyridostigmine bromide in rats. Toxicol. Appl. Pharmacol. 165, 107–114. Chen, J.-W., and Ling, K.-H. (1996). Territrems, naturally occurring specific irreversible inhibitors of acetylcholinesterase. J. Biomed. Sci. 3, 54–58. Chiappa, S., and Brimijoin, S. (1998). Pharmacologic tests of a role for acetylcholinesterase in promoting neurite outgrowth by dorsal root ganglia. In “Structure and Function of Cholinesterases and Related Proteins” (B. P. Doctor et al, eds.), pp. 585–592. Plenum, New York. Christenson, W. R., Van Goethem, D. L., Schroeder, R. S., Wahle, B. S., Dass, P. D., Sangha, G. K., and Thyssen, J. H. (1994). Interlaboratory comparison determinations and the effect on the results of statistical evaluation of cholinesterase inhibition. Toxicol. Lett. 71(2), 139–150.

Chapter | 68  Cholinesterases

Collombel, C., and Perrot, L. (1970). Determination of the cholinesterase activity of whole blood removed by pricking the skin and deposited on filter paper. Application to the detection of organophosphorus insecticide poisonings. J. Eur. Toxicol. 3, 368–372. Corrado, M. U. D., Politi, H., Trielli, F., Angelini, C., and Falugi, C. (1999). Evidence for the presence of a mammalian-like cholinester­ ase in Paramecium primaurelia (Protista, Ciliophora) developmental cycle. J. Exp. Zool. 283(1), 102–105. Corrado, M. U. D., Trielli, F., Amaroli, A., Ognibene, M., and Falugi, C. (2005). Protists as tools for environmental biomonitoring: Importance of cholinesterase enzyme activities. In “Water Pollution: New Research” (F. Columbus, ed.), pp. 1–20. Nova Science, Hauppauge, NY. Culetto, E., Combes, D., Fedon, Y., Roig, A., Toutant, J. P., and Arpagaus, M. (1999). Structure and promoter activity of the 5 flanking region of ace-1, the gene encoding acetylcholinesterase of class A in Caenorhabditis elegans. J. Mol. Biol. 290, 951–966. Dahlgren, E. (1983). Recent research in Sweden on chemical detection. In “Proceedings of the International Symposium on Protection against Chemical Warfare Agents,” FOA report No. C 40171-C2, C3, pp. 77–82. National Defense Research Institute, Umea, Sweden. Dale, H. H. (1914). The action of certain esters and ethers of choline and their relation to muscarine. J. Pharmacol. Exp. Ther. 6, 147–190. De la Hoz, D., Doctor, B. P., Ralston, J. S., Rush, R. S., and Wolfe, A. D. (1986). A simplified procedure for the purification of large quantities of fetal bovine serum acetylcholinesterase. Life Sci. 39, 195–199. Dementi, B. (1994). Ocular effects of organophosphates, a historical per­ spective of Saku disease. J. Appl. Toxicol. 14, 119–129. Dettbarn, W. D. (1984). Pesticide induced muscle necrosis: Mechanisms and prevention. Fundam. Appl. Toxicol. 4(2 Pt 2), S18–S26. Doctor, B. P., Raveh, L., Wolfe, A. D., Maxwell, D. M., and Ashani, Y. (1991). Enzymes as pretreatment drugs for organophosphate toxicity. Neurosci. Biobehav. Rev. 15, 123–128. Doctor, B. P., Taylor, P., Quinn, D. M., Rotundo, R. L., and Gentry, M. K. (1998). “Structure arid Function of Cholinesterases and Related Proteins”. Plenum, New York. Doctor, B. P., Toker, L., Roth, E., and Silman, I. (1987). Microtiter assay for acetylcholinesterase. Anal. Biochem. 166, 399–403. Ecobichon, D. J. (1996). Toxic effects of pesticides. In “Cassarett and Doull’s Toxicology” (C. D. Klassen, ed.), 5th ed., pp. 655–666. McGraw-Hill, New York. Ellman, G. L., Courtney, K. D., Andres, V. Jr., and Featherstone, R. M. (1961). A new rapid colorimetric determination of acetylcholinester­ ase activity. Biochem. Pharmacol. 7, 88–95. Engelhart, E., and Loewi, O. (1930). Fermentative azetylcholinspaltung im blut und ihre hemmung. Naunyn-Schiedeberg’s Arch. Exp. Pathol. Pharmakol. 150, 1–13. Faerman, C., Ripoll, D., Bon, S., Le Feuvre, Y., Morel, N., Massoulie, J., Sussman, J. L., and Silman, I. (1996). Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function. FEBS Lett. 386, 65–71. Francis, P. T., Palmer, A. M., Snape, M., and Wilcock, G. K. (1999). The cholinergic hypothesis of Alzheimer’s disease, a review of progress. J. Neurol. Neurosurg. Psychiatr. 66, 137–147. Furlong, C. E., Li, W.-F., Richtei, R. J., Shih, D. M., Lusis, A. J., Alleva, E., and Costa, L. G. (2000). Genetic and temporal determinants of pes­ ticide sensitivity, role of paraoxonase (PON1). Neurotoxicology 21(1/2), 91–100. Gage, J. C. (1967). The significance of blood cholinesterase activity measurements. Residue Rev. 18, 159–173.

1475

Gallo, M. A., and Lawryk, N. J. (1991). Organic phosphorus pesticides. In “Handbook of Pesticide Toxicology” (W. J. Hayes Jr. and E. R. Laws Jr., eds.), pp. 917–1123. Academic Press, New York. Gamson, R. M., Robinson, D. W., and Goodman, A. (1973). Test for anticholinesterase materials in water. Environ. Sci. Technol. 7, 1137–1140. Geller, A. M., Abdel-Rahman, A. A., Peiffer, R. L., Abou-Donia, M. B., and Boyes, W. K. (1998). The organophosphate pesticide chlorpyrifos affects form deprivation myopia. Investigative Ophthalmol. Visual Sci. 39, 1290–1294. Gnatt, A., Lowenstein, Y., Yaron, A., Schwarz, M., and Soreq, H. (1994). Site-directed mutagenesis of active site residues reveals plasticity of human butyrylcholinesterase in substrate and inhibitor interactions. J. Neurochem. 62, 749–755. Groff, W. A., Kaminskis, A., and Ellin, R. I. (1976). Interconversion of cholinesterase enzyme activity units by the manual pH method and recommended automated method. Clin. Toxicol. 9, 353–358. Gupta, P. K., Roy, J. K., and Prasad, M. (1999). DNA chips, microarrays and genomics. Curr. Sci. 77, 875–884. Haley, R. W., Billecke, S., and LaDu, B. N. (1999). Association of low PON1 Type Q (Type A) arylesterase activity with neurologic symptom com­ plexes in Gulf War veterans. Toxicol. Appl. Pharmacol. 157, 227–233. Hamm, J. T., Wilson, B. W., and Hinton, D. E. (1998). Organophosphateinduced acetylcholinesterase inhibition and embryonic retinal cell necrosis in vivo in the teleost (Oryzias latipes). Neurotoxicology 19, 853–869. Hansen, M. E., and Wilson, B. W. (1999). Oxime reactivation of RBC acetylcholinesterases for biomonitoring. Arch. Environ. Contam. Toxicol. 37, 283–289. Harlin, K. S., and Ross, P. F. (1990). Enzymatic-spectrophotometric method for determination of cholinesterase activity in whole blood, collaborative study. J. Assoc. Off. Anal. Chem. 73, 616–619. Henn, B. C., McMaster, S., and Padilla, S. (2006). Measuring cholines­ terase activity in human saliva. J. Toxicol. Environ. Health A 69(19), 1805–1818. Hestrin, S. (1949). The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine, and its analytical application. J. Biol. Chem. 180, 249–261. Hille, T., Mueller, W., Asmussen, B., Levy, A., and Meshulam, Y. (1995). Pharmaceutical formulation for prophylaxis or preliminary treatment against poisoning by organophosphorus cholinesterase inhibitors. Ger. Offen., 6. Hoffman, R. S., Henry, G. C., Wax, P. M., Weisman, R. S., Howland, M. A., and Goldfrank, L. R. (1992). Decreased plasma cholinesterase activity enhances cocaine toxicity in mice. J. Pharmacol. Exp. Ther. 263, 698–702. Hoffmann, W. E., Kramer, J., Main, A. R., and Torres, J. L. (1989). Clinical enzymology. In “The Clinical Chemistry of Laboratory Animals” (W. F. Loeb and F. W. Quimby, eds.), pp. 217–278. Pergamon, New York. Holmstedt, B. (1957). A modification of the thiocholine method for the determination of cholinesterase: I. Biochemical evaluation of selec­ tive inhibitors. Acta Physiol. Scand. 40, 322–330. Holmstedt, B. (1963). Structure–activity relationships of the organo­ phosphorus anticholinesterase agents. In “Cholinesterases and Anticholinesterase Agents: Handbuch der Experimentellen Pharmakologie” (G. B. Koelle, ed.), Vol. 15, pp. 428–485. SpringerVerlag, Berlin. Humiston, C. G., and Wright, G. W. (1967). An automated method for the determination of cholinesterase activity. Toxicol. Appl. Pharmacol. 10, 467–480.

1476

Husain, K. (1994). Phenylvalerate and choline ester hydrolases in the platelets of human, hen, rat and mouse. Hum. Exp. Toxicol. 13, 157–159. Jaga, K., and Dharmani, C. (2006). Ocular toxicity from pesticide expo­ sure: A recent review. Environ. Health Prev. Med. 11(3), 102–107. Johnson, C. D., and Russell, R. L. (1975). Rapid simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal. Biochem. 64(1), 229–238. Kasprzak, H., and Salpeter, M. M. (1985). Recovery of acetylcholinester­ ase at intact neuromuscular junctions after in vivo inactivation with diisopropylfluorophosphate. J. Neurosci. 5, 951–955. Katz, E. J., Cortes, V. I., Eldefrawi, M. E., and Eldefrawi, A. T. (1997). Chlorpyrifos, parathion, and their oxons bind to and desensitize a nicotinic acetylcholine receptor; Relevance to their toxicities. Toxicol. Appl. Pharmacol. 146, 227–236. Keifer, M., Rivas, F., Moon, J. D., and Checkoway, H. (1996). Symptoms and cholinesterase activity among rural residents living near cotton fields in Nicaragua. Occup. Environ. Med. 53, 726–729. Koelle, G. B. (1963). “Cholinesterases and Anticholinesterase Agents: Handbuch der Experimentellen Pharmakologie.” Springer-Verlag, Berlin. Krasowski, M. D., McGehee, D. S., and Moss, J. (1997). Natural inhibi­ tors of cholinesterases; Implications for adverse drug reactions. Can. J. Anaesth. 44(5 Pt 1), 525–534. Krejci, E. (1998). The building of acetylcholinesterase collagen-tailed forms. In “Structure and Function of Cholinesterases and Related Proteins” (B. P. Doctor et al, eds.), pp. 57–63. Plenum, New York. Kurt, T. L. (1998). Epidemiological association in U.S. veterans between Gulf War illness and exposures to anticholinesterases. Toxicol. Lett. 102/103, 523–526. La Du, B. N., Aviram, M., Billecke, S., Navab, M., Primo-Parmo, S., Sorenson, R. C., and Standiford, T. J. (1999). On the physiological role(s) of the paraoxonases. Chem. Biol. Interact. 119/120, 379–388. Layer, P. G., Keller, M., Mack, A., Willbold, E., and Robitzki, A. (1998). Nonenzymatic roles of cholinesterases in avian neurogenesis. In “Structure and Function of Cholinesterases and Related Proteins” (B. P. Doctor et al, eds.), pp. 569–576. Plenum, New York. Leonard, J. P., and Salpeter, M. M. (1979). Agonist-induced myopathy at the neuromuscular junction is mediated by calcium. J. Cell Biol. 82, 811–819. Li, B., Stribley, J. A., Ticu, A., Xie, W., Schopfer, L. M., Hammond, P., Brimijoin, S., Hinrichs, S. H., and Lockridge, O. (2000). Abundant tissue butyrylcholinesterase and its possible function in the acetyl­ cholinesterase knockout mouse. J. Neurochem. 75(3), 1320–1331. Li, W. F., Furlong, C. E., and Costa, L. G. (1995). Paraoxonase protects against chlorpyrifos toxicity in mice. Toxicol. Lett. 76, 219–226. Loewi, O., and Navratil, E. (1926). Uber humorale Ubertragbarkeit der Herzenwirkung X. Uber das Schicksal des Vagusstoffes. Arch. Ges. Physiol. 214, 678. London, L., Thompson, M. L., Sacks, S., Fuller, B., Bachman, O. M., and Myers, J. E. (1995). Repeatability and validity of a field kit for estima­ tion of cholinesterase in whole blood. Occup. Environ. Med. 52, 57–64. Loof, I. (1992). Experience with the Ellman method: Proposal for a modi­ fication and an alternative method (PAP). In “U.S. Environmental Protection Agency (EPA) Workshop on Cholinesterase Methodologies,” pp. 119–139. Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC. Magnotti, R. A., and Eberly, J. P. (1996). Test-mate ChE: New simpli­ fied cholinesterase assay system. Med. Def. Biosci. Rev. Proc. 3, 1192–1201.

Hayes’ Handbook of Pesticide Toxicology

Magnotti, R. A. Jr., Dowling, K., Eberly, J. P., and McConnell, R. S. (1988). Field measurement of plasma and erythrocyte cholinester­ ases. Clin. Chim. Acta 176(3), 315–332. Marchot, P., Bourne, Y., Prowse, C. N., Bougis, P. E., and Taylor, P. (1998). Inhibition of mouse acetylcholinesterase by fasciculin, crys­ tal structure of the complex and mutagenesis of fasciculin. Toxicon 36, 1613–1622. Massoulie, J., Pezzementi, L., Bon, S., Krejci, E., and Vallette, F. M. (1993). Molecular and cellular biology of cholinesterases. Prog. Neurobiol. 41, 31–91. Massoulie, J., Anselmet, A., Bon, S., Krejci, E., Legay, C., Morel, N., and Simon, S. (1999). The polymorphism of acetylcholinesterase posttranslational processing, quaternary associations and localization. Chem. Biol. Interac. 119/120, 29–42. McGehee, D. S., Krasowski, M. D., Fung, D. L., Wilson, B., Gronert, G. A., and Moss, J. (2000). Cholinesterase inhibition by potato glyco­ alkaloids slows mivacurium metabolism. Anesthesiology 93, 510–519. Meshul, C. K. (1989). Calcium channel blocker reverses anticholinester­ ase-induced myopathy. Brain Res. 497, 142–168. Metcalf, R. L. (1951). The colorimetric microestimation of human blood cholinesterases and its application to poisoning by organic phosphate insecticides. J. Econ. Entomol. 44, 883–890. Michel, H. O. (1949). An electrometric method for the determination of red blood cell and plasma cholinesterase activity. J. Lab. Clin. Med. 34, 1564–1568. Mikalsen, A., Andersen, R. A., and Alexander, J. (1986). Use of etho­ propazine and BW 284C51 as selective inhibitors for cholinesterases from various species. Comp. Biochem. Physiol. C Comp. Pharmacol. 83, 447–449. Mileson, B. E., Chambers, J. E., Chen, W. L., Dettbarn, W., Ehrich, M., Eldefrawi, A. T., Gaylor, D. W., Hamernik, K., Hodgson, E., Karczmar, A. G., Padilla, S., Pope, C. N., Richardson, R. J., Saunders, D. R., Sheets, L. P., Sultatos, L. G., and Wallace, K. B. (1998). Common mechanism of toxicity: A case study of organo­ phosphorus pesticides. Toxicol. Sci. 41, 8–20. Mortinsen, S. R., Brimijoin, S., Hooper, M. J., and Padilla, S. (1998). Comparison of the in vitro sensitivity of rat acetylcholinesterase to chlorpyrifosoxon. What do tissue IC50 values represent? Toxicol. Appl. Pharmacol. 148, 46–49. Mosca, A., Onelli, E., Rosti, E., Paleari, R., Luzzana, M., and Imbimbo, B. P. (1995). A patient-side technique for real-time measurement of acetylcholinesterase activity during monitoring of eptastigmine treat­ ment. Ther. Drug Monitor 17, 230–238. Myers, D. K. (1953). Studies on cholinesterase. Species variation in the speci­ ficity patterns of the pseudo-cholinesterase. Biochem. J. 55, 67–69. Nabb, D. P., and Whitfield, F. (1967). Determination of cholinesterases by an automated pH stat method. Arch. Environ. Health 15, 147–154. Nachmansohn, D., and Wilson, I. B. (1951). The enzymic hydrolysis and synthesis of acetylcholine. Adv. Enzymol. 12, 259–339. Nagata, K., Huang, C. S., Song, J. H., and Narahashi, T. (1997). Direct actions of anticholinesterases on the neuronal nicotinic acetylcholine receptor channels. Brain Res. 769, 211–218. National Academy of Sciences. (1999). “Review of the U.S. Army’s Health Risk Assessments for Oral Exposure to Six Chemical-Warfare Agents” (R. Snyder, ed.). National Academy Press, Washington, DC. Nigg, H. N., and Knaak, J. B. (2000). Blood cholinesterase as human biomarkers of organophosphorus pesticide exposure. Rev. Environ. Contamin. Toxicol. 163, 29–111. Nigg, H. N., Ramos, L. E., Graham, E. M., Sterling, J., Brown, S., and Cornell, J. A. (1996). Inhibition of human plasma and serum

Chapter | 68  Cholinesterases

butyrylcholinesterase (EC 3.1.1.8) by alpha-chaconine and alpha solanine. Fundam. Appl. Pharmacol. 33, 272–281. Nordberg, A., and Svensson, A. L. (1998). Cholinesterase inhibitors in the treatment of Alzheimer’s disease: A comparison of tolerability and pharmacology. Drug Saf. 19, 465–480. Nostrandt, A. C., Duncan, J. A., and Padilla, S. (1993). A modified spectrophotometric method appropriate for measuring cholinester­ ase activity in tissue from carbaryl-treated animals. Fundam. Appl. Toxicol. 21, 196–203. O’Brien, R. D. (1967). “Insecticides, Actions and Metabolism”, p. 83. Academic Press, New York. Okabe, H., Sagesaka, K., Nakajima, N., and Noma, A. (1977). New enzy­ matic assay of cholinesterase activity. Clin. Chim. Acta 80, 87–94. Oliveira, G. H., Henderson, J. D., and Wilson, B. W. (2002). Cholinesterase measurements with an automated kit. Am. J. Ind. Med Suppl. 2, 49–53. Orwell, G. (1945). “Animal Farm; A Fairy Story”. Seeker & Warburg, London. Patocka, J. (1998). Huperzine A – An interesting anticholinesterase compound from the Chinese herbal medicine. Acta Med. (Hradec Kralove) 41(4), 155–157. Paulus, J.-M., Maigne, J., and Keyhani, E. (1981). Mouse megakaryocytes secrete acetylcholinesterase. Blood 58, 1100–1106. Pezzementi, L., Sutherland, D., Sanders, M., Soong, W., Milner, D., McClellan, J. S., Sapp, M., Coblentz, W. B., Rulewicz, G., and Merritt, S. (1998). Structure and function of cholinesterases from agnathans and cephalochordates: Implications for the evolution of cholinesterases. In “Structure and Function of Cholinesterases and Related Proteins” (B. P. Doctor et al, eds.), pp. 105–110. Plenum, New York. Polhuijs, M., Langenberg, J. P., and Benschop, H. P. (1997). New method for retrospective detection of exposure to organophosphorus anticho­ linesterases: Application to alleged sarin victims of Japanese terror­ ists. Toxicol. Appl. Pharmacol. 146, 156–161. Polhuijs, M., Langenberg, J. P., Noort, D., Hulst, A. G., Benschop, H. P., and De Jong, L. P. A. (1999). Retrospective detection of expo­ sure to organophosphates: Analyses in blood of human beings and rhesus monkeys. NATO Science Series 1. In “NBC Risks: Current Capabilities and Future Perspectives for Protection”, Vol. 25, pp. 513–521. Kluwer, Dordrecht, The Netherlands. Pope, C. N. (1999). Organophosphorus pesticides: Do they all have the same mechanism of toxicity? J. Toxicol. Environ. Health. B Crit. Rev. 2, 161–181. Potter, W. T., Garry, V. F., Kelly, J. T., Tarone, R., Griffith, J., and Nelson, R. L. (1993). Radiometric assay of red cell and plasma cholinesterase in pesticide appliers from Minnesota. Toxicol. Appl. Pharmacol. 119, 150–155. Prall, Y. G., Gambhir, K. K., and Ampy, F. R. (1998). Acetylcholinesterase, an enzymatic marker of human red blood cell aging. Life Sci. 63, 177–184. Raveh, L., Cohen, G., Grauer, E., Alkali, D., Chapman, S., Kapon, Y., Sahar, R., Kadar, T., and Shapira, S. (1996). Efficacy of prophylac­ tic treatments against sarin poisoning in rats. Med. Def. Biosci. Rev. Proc. 1, 539–547. Reiner, E., Rudolf, V. S., Doctor, B. P., Furlong, C. E., Johnson, M. K., Lotti, M., Silman, I., and Taylor, P. (1999). Esterases reacting with organophosphorus compounds. Chem. Biol. Interact. 119/120, 1–619. Rider, J. A., Hodges, J. L., Swader, J., and Wiggins, A. D. (1957). Plasma and red cell cholinesterase in 800 “healthy” blood donors. J. Lab. Clin. Med. 50, 376–383.

1477

Robitzki, A., Mack, A., Hoppe, U., Chatonnet, A., and Layer, P. G. (1997). Regulation of cholinesterase gene expression affects neuronal differentiation as revealed by transfection studies on reaggregating embryonic chicken retinal cells. Eur. J. Neurosci. 9, 2394–2405. Rocha, E. S., Swanson, K. L., Aracava, Y., Goolsby, J. E., Maelicke, A., and Albuquerque, E. X. (1996). Paraoxon, cholinesteraseindependent stimulation of transmitter release and selective block of ligand-gated ion channels in cultured hippocampal neurons. J. Pharmacol. Exp. Ther. 278, 1175–1187. Rodgers, K., and Xiong, S. (1997). Effect of administration of malathion for 14 days on macrophage function and mast cell degranulation. Fundam. Appl. Toxicol. 37, 95–99. Rodgers, K. E., and Ellefson, D. D. (1990). Modulation of respiratory burst activity and mitogenic response of human peripheral blood mononuclear cells and murine splenocytes and peritoneal cells by malathion. Fundam. Appl. Toxicol. 14, 309–317. Rogers, K. R., Cao, C. J., Valdes, J. J., Eldefrawi, A. T., and Eldefrawi, M. E. (1991). Acetylcholinesterase fiber-optic biosensor for detection of anticholinesterases. Fundam. Appl. Toxicol. 16, 810–820. Romano, J. A. Jr., Lukey, B. J., and Salem, H. (eds) (2008). “Chemical Warfare Agents,” 2nd ed. CRC Press, Boca Raton, FL. Rosenberry, T. L., Mallender, W. D., Thomas, P. J., and Szegletes, T. (1998). Substrate binding to the peripheral site occurs on the catalytic pathway of acetylcholinesterase and leads to substrate inhibition. In “Structure and Function of Cholinesterases and Related Proteins” (B. P. Doctor et al, eds.), pp. 189–196. Plenum, New York. Ryhanen, R., Narhi, M., Puhakainen, E., Hanninen, O., and KontturiNarhi, V. (1983). Pseudocholinesterase activity and its origin in human oral fluid. J. Dental Res. 62, 20–23. Sanz, P., Rodriguez-Vicente, M. C., Diaz, D., Repetto, J., and Repetto, M. (1991). Red blood cell and total acetylcholinesterase and plasma pseudocholinesterase in humans: Observed variances. Clin. Toxicol. 29, 81–90. Senanayake, N., and Karalliedde, L. (1987). Neurotoxic effects of organo­ phosphorus insecticides. An intermediate syndrome. N. Engl. J. Med. 316, 761–763. Sharma, K. V., and Bigbee, J. W. (1998). Acetylcholinesterase antibody treatment results in neurite detachment and reduced outgrowth from cultured neurons: Further evidence for a cell adhesive role for neuro­ nal acetylcholinesterase. J. Neurosci. Res. 53, 454–464. Shih, D. M., Gu, L., Xia, Y.-R., Navab, M., Li, W.-F., Hama, S., Castellani, L. W., Furlong, C. E., Costa, L. G., Fogelman, A. M., and Lusis, A. J. (1998). Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature (London) 394, 284–287. Sidell, F. R., and Kamiskis, A. (1975). Temporal intrapersonal physiologi­ cal variability of cholinesterase activity in human plasma and eryth­ rocytes. Clin. Chem. 21, 1961–1963. Sigma Diagnostics (1989). “Cholinesterase (PTC) Procedure No. 422”. Sigma Diagnostics, St. Louis. Silk, E., King, J., and Whittaker, M. (1979). Assay of cholinesterase in clinical chemistry. Ann. Clin. Biochem. 16, 57–75. Silman, I., and Sussman, J. L. (1998). Structural and functional studies on acetylcholinesterase: A perspective. In “Structure and Function of Cholinesterases and Related Proteins” (B. P. Doctor et al., eds.), pp. 25–33. Plenum, New York. Silman, I., and Sussman, J. L. (2008). Acetylcholinesterase: How is struc­ ture related to function? Chem. Biol. Interact. 175(1-3), 3–10. Silver, A., (1974). “The Biology of Cholinesterases: Frontiers of Biology”. North-Holland, Amsterdam.

1478

Sket, D., Dettbarn, W.-D., Clinton, M. E., Misulis, K. E., Sketelj, J., Cucke, D., and Brizin, M. (1991). Prevention of diisopropylphospho­ rofluoridate-induced myopathy by botulinum toxin type A blockage of quantal release of acetylcholine. Acta Neuropathol. 82, 134–142. Spencer, P. S., Schaumburg, H. H., and Ludolph, A. C. (2000). “Experimental and Clinical Neurotoxicology”, 2nd ed. Oxford University Press, New York. Sperling, L. E., Steinert, G., Boutter, J., Landgraf, D., Hescheler, J., Pollet, D., and Layer, P. G. (2008). Characterisation of cholinesterase expression during murine embryonic stem cell differentiation. Chem. Biol. Interact. 175(1-3), 156–160. Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991). Atomic structure of acetylcholinesterase from Torpedo californica: A prototypic acetylcholine-binding protein. Science 253, 872–879. Talesa, V., Grauso, M., Arpagaus, M., Giovannini, E., Romani, R., and Rosi, G. (1999). Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens, a new member of the cholinesterase family resistant to diisopropyl fluorophosphate. J. Neurochem. 72(3), 1250–1258. Taylor, P. (1994). The cholinesterases: From genes to proteins. Annu. Rev. Pharmacol. Toxicol. 34, 281–320. Taylor, P. (1996). Cholinesterase agents. In “Goodman and Gilman’s the Pharmacological Basis of Therapeutics” (J. G. Hardman et al, eds.), 9th ed., pp. 161–176. McGraw-Hill, New York. Taylor, P. (1999). Esterases reacting with organophosphorus compounds. Chem. Biol. Interact. 119/120, 1–620. Thomsen, T., Kewitz, H., and Pleul, O. (1988). Estimation of cholinester­ ase activity (EC 3.1.17; 3.1.1.8) in undiluted plasma and erythrocytes as a tool for measuring in vivo effects of reversible inhibitors. J. Clin. Chem. Clin. Biochem. 26, 469–475. Thomsen, T., Kewitz, H., and Pleul, O. (1989). A suitable method to monitor inhibition of cholinesterase activities in tissues as induced by reversible enzyme inhibitors. Enzyme 42, 219–224. Thyssen, J., and Machemer, L. (1999). Imidacloprid: toxicology and metabolism. In “Nicotinoid Insecticides and the Nicotinic Acetylcholine Receptor” (I. Yamamoto and J. E. Casida, eds.), pp. 213–222. Springer, Tokyo. Traina, M. E., and Serpietri, L. A. (1984). Changes in the levels and forms of rat plasma cholinesterases during chronic diisopropylphosphofluo­ ridate intoxication. Biochem. Pharmacol. 33, 645–653. Tuovinen, K., Kaliste-Korhonen, E., Raushel, F. M., and Hanninen, O. (1999). Success of pyridostigmine, physostigmine, eptastigmine and phosphotriesterase treatments in acute sarin intoxication. Toxicology 134, 169–178. U.S. Environmental Protection Agency (EPA) (1992). “Workshop on Cholinesterase Methodologies”. Office of Pesticide Programs, U.S. EPA, Washington, DC. van den Beukel, I., van Kleef, R. G., and Oortgissen, M. (1998). Differential effects of physostigmine and organophosphates on nico­ tinic receptors in neuronal cells of different species. Neurotoxicology 19, 777–787. Willig, S., Hunter, D. L., Dass, P. D., and Padilla, S. (1996). Validation of the use of 6,6-dithiodinicotinic acid as a chromogen in the Ellman method for cholinesterase determinations. Vet. Hum. Toxicol. 38, 249–253. Wills, J. H. (1972). The measurement and significance of changes in the cholinesterase activities of erythrocytes and plasma in man and ani­ mals. CRC Crit. Rev. Toxicol. 1, 153–199. Wilson, B. W. (1999). Cholinesterases. In “Clinical Chemistry of Laboratory Animals” (E. Quimby and W. Loeb, eds.), 2nd ed., pp. 430–440. Taylor & Francis, Philadelphia.

Hayes’ Handbook of Pesticide Toxicology

Wilson, I. B., and Ginsburg, S. (1955). A powerful reactivator of alkyl phos­ phate-inhibited acetylcholinesterase. Biochim. Biophys. Acta 18, 168–170. Wilson, B. W., and Henderson, J. D. (1992). Blood esterase determinations as markers of exposure. Rev. Environ. Contam. Toxicol. 128, 55–69. Wilson, B. W., and Henderson, J. D. (2007). Determination of cholines­ terase in blood and tissue. Curr. Protocols Toxicol.(Suppl. 34), 1–16. Wilson, B. W., Jaeger, B., and Baetcke, K. (eds) (1992a). “Proceedings of the U.S. EPA Workshop on Cholinesterase Methodologies, Arlington, VA, Dec. 4–5, 1991”. Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC. Wilson, B. W., Hooper, M. J., Hansen, M. E., and Nieberg, P. S. (1992b). Reactivation of organophosphate inhibited AChE with oximes. In “Organophosphates: Chemistry, Fate, and Effects” (J. E. Chambers and P. E. Levi, eds.), pp. 107–137. Academic Press, New York. Wilson, B. W., Padilla, S., and Henderson, J. D. (1996). Factors in stan­ dardizing automated cholinesterase assays. J. Toxicol. Environ. Health 48, 187–195. Wilson, B. W., Sanborn, J. R., O’Malley, M. A., Henderson, J. D., and Billitti, J. R. (1997). Monitoring the pesticide-exposed worker. Occup. Med. 12, 347–363. Wilson, B. W., McCurdy, S. A., Henderson, J. D., McCarthy, S. A., and Billitti, J. E. (1998). Cholinesterases and agriculture, humans, laboratory animals and wildlife. In “Structure and Function of Cholinesterases and Related Proteins” (B. P. Doctor et al, eds.), pp. 539–546. Plenum, New York. Wilson, B. W., Henderson, J. D., Bosworth, D. H., and Oliveira, G. H. (2000). “Standardization of cholinesterase measurements for moni­ toring human exposures”. Book of Abstracts. 219th ACS National Meeting, San Francisco, CA, March 26–30. Wilson, B. W., Henderson, J. D., Furman, J. L., Zeller, B. E., and Michaelson, D. (2009). Blood cholinesterases from Washington State orchard workers. Bull. Environ. Contam. Toxicol. 83(1), 59–61. Witter, R. F. (1963). Measurement of blood cholinesterase. Arch. Environ. Health, 6, 537–563. Wolfe, A. D., Rush, R. S., Doctor, B. P., Koplovitz, I., and Jones, D. (1987). Acetylcholinesterase prophylaxis against organophosphate toxicity. Fundam. Appl. Toxicol. 9, 266–270. Wright, C. I., and Sabine, J. C. (1948). Cholinesterases of human eryth­ rocyte and plasma and their inhibition by antimalarial drugs. J. Pharmacol. 93, 230–239. Wyttenbach, C. R., and Thompson, S. C. (1985). The effects of the organophosphate insecticide malathion on very young chick embryos, malformations detected by histological examination. Am. J. Anat. 174, 187–202. Xie, W., Stribley, J. A., Chatonnet, A., Wilder, P. J., Rizzino, A., McComb, R. D., Taylor, P., Hinrichs, S. H., and Lockridge, O. (2000). Postnatal developmental delay and supersensitivity to organo­ phosphate in gene-targeted mice lacking acetylcholinesterase. J. Pharmacol. Exp. Ther. 293(3), 896–902. Yamalik, N., Ozer, N., Caglayan, F., and Caglayan, G. (1990). Determination of pseudocholinesterase activity in the gingival crevic­ ular fluid, saliva, and serum from patients with juvenile periodontitis and rapidly progressive periodontitis. J. Dental Res. 69, 87–89. Yeary, R. A., Eaton, J., Gilmore, E., North, B., and Singell, J. (1993). A multiyear study of blood cholinesterase activity in urban pesticide applicators. J. Toxicol. Environ. Health 39, 11–25. Yuknavage, K. L., Fenske, R. A., Kalman, D. A., Keifer, M. C., and Furlong, C. E. (1997). Simulated dermal contamination with capillary samples and field cholinesterase biomonitoring. J. Toxicol. Environ. Health 51, 35–55. Zajicek, J. (1957). Studies on the histogenesis of blood platelets and megakaryocytes. Acta Physiol. Scand. 40(Suppl. 138), 1–32.