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1457Hayes’ Handbook of Pesticide ToxicologyCopyright © Elsevier Inc. All rights reserved2010
Cholinesterases
Barry W. WilsonUniversity of California, Davis, California
Chapter 68
68.1 IntroductIon
Cholinesterases (ChEs) are specialized carboxylic ester hydrolases that break down esters of choline. Two of special concern to the pesticide toxicologist are acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) and butyrylcholinesterase (BuChE; acylcholine acylhydrolase, EC 3.1.1.8), also known as nonspecific cholinesterase or pseudocholinesterase. The preferred substrate for AChE is acetylcholine (ACh). Nonspecific cholinesterases prefer butyrylcholine and/or propionylcholine, depending on the species (Silver, 1974). This chapter discusses these enzymes, their importance in understanding the toxicity of organophosphate ester (OP) and carbamate (CB) pesticides, and their application to risk assessment (Taylor, 1999).
ChEs are classed among the Besterases, 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 Besterases include the carboxylesterases (CarbE, EC 3.1.1.1.), one of which is neuropathy target esterase (NTE), the enzyme associated with organophosphateinduced delayed neuropathy (OPIDN) discussed in other chapters. The Aesterases 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.
Today, techniques such as sitedirected mutagenesis, knockout mutants, and highresolution xray crystallography 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 structure 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 fullcolor molecular structures displayed 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 warfare 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 agricultural 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 invertebrates has made antiChE agents popular and effective pesticides. Molecular forms of ChE similar to those in vertebrates 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 vertebratelike AChE form has been reported in Paramecium, a ciliated protozoan (Corrado et al., 1999, 2005).
Hayes’ Handbook of Pesticide Toxicology1458
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 erythrocytes [red blood cells (RBCs)] of mammals, megakaryocytes, 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 relatively 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 specificities 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 antiChE agents.
BuChEs and AChEs are also found together at synapses, 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 antiChE agents. A number of plant toxins have antiChE 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
Loewi, 1930). Other examples of naturally occurring antiChE 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 stimulus for modern studies of natural antiChE 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 transmission. 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 successful 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 disease in Japan). These studies, reviewed by Dementi (1994) and Jaga and Dharmani (2006), have stimulated studies by the U.S. Environmental Protection Agency (EPA) (Atkinson et al., 1994; Boyes et al., 1994), although without 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.
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
aManometric assay at N 3. Mean human AChE was 2180 l CO2/ 30 min/mg nitrogen. ACh substrate.bN 2.Adapted from Zajicek (1957).
1459chapter | 68 Cholinesterases
68.3 substrate preferences and selectIve InhIbItors
AChEs prefer ACh as a substrate. Substrate preferences and activities of BuChEs vary with the species. For example, 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 acetylmethylcholine. AChEs are selectively inhibited by several agents, including the CB BW284c51 (1,5bis(4allyldimethylammoniumphenyl)pentan3one
dibromide) (Austin and Berry, 1953; Holmstedt, 1957; Silver, 1974) and ARA 1327 (Augustinsson et al., 1978). BuChEs are preferentially inhibited by isoOMPA (tetra isopropylpyrophosphoramide; 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 starting points for testing are 0.1–0.01 mM.
68.4 MultIple Molecular forMs and lIfe hIstory
ChEs are polymorphic proteins, occurring in multiple molecular 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
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.aManometric assay. lCO2/0.1 ml plasma/30 min. N 3 or more animals.Source: Augustinsson (1959b).
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. aManometric assay. lCO2/0.1 ml plasma/30 min. N 3 or more animals.Source: Augustinsson (1959a).
Hayes’ Handbook of Pesticide Toxicology1460
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 synthesized as catalytic globular monomers (G1) that oligomerize 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. Collagentailed 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 sitedirected mutagenesis (Doctor et al., 1998).
AChE and BuChE forms are each coded by single genes. The gene coding sequence for the collagentailed forms contains a Cterminal extension of 40 amino acids, the Tpeptide, that interacts by a short prolinerich attachment domain with the collagen. A single collagen gene (ColQ) is associated
s
ss
ss
s
s s
Hs
s s
s s
s
ss ss
ss
s ss s
s
s s ss ss
Hs
Hs
s
sss
s
s
Hs
HsH
sHs
s
ssss
s
AI2
A 4
G1
G2A8
G4
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 represent collagenlike tails. Disulfide bridge locations from studies of eel electroplax AChE, assumed to reflect ChEs from many sources (after Brimijoin, 1992).
Plasma membrane
Extracellular fluids
Membrane boundExtracellular matrix
NucleusAChEmRNA
RER
Synthesis,sequesteringglycosylation,assembly ofglobular forms,degradation
Processing,assembly ofasymmetricforms
Transport
Secretion,membraneincorporation andturnover
Binding in basal lamina
20–30%
Golgi
Degradation70–80%
Vesicles
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 intracellular proteases after ribosomal translation and before transit of the Golgi. In the Golgi, secretory forms (black triangles) are sequestered from membranebound forms (open triangles), collagenlike 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 polyanionrich, fibrous matrix of the extracellular synaptic basal lamina (after Brimijoin, 1992).
1461chapter | 68 Cholinesterases
with both the AChE and the BuChE collagentailed forms. The globular forms are synthesized by an alternative splice variant, the Hexon, that is expressed instead of the Texon [see Krejci (1998) for a model and Massoulie et al. (1999) for further information].
The threedimensional 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 choline 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 speculation; 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 excellent 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 determining 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
E AX EAX EA X E A
← → → →kk K K
1
1 2 3
where E is the enzyme, AX is the substrate (ACh) or inhibitor, EAX is the reversible enzyme complex, X is Ch
449
341
447
334
295
297
286124 74
86286
124
72
74
86
282
449
341
334
447
295
297 203
282
283
72
fIgure 68.3 Threedimensional 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 3D 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 Toxicology1462
(choline), and ks are reaction rate constants (Rosenberry et al., 1998; Taylor, 1996).
The kinetics of ACh hydrolysis is a complicated multistep 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, producing acetic acid and regenerating the enzyme (E A). A similar reaction scheme applies to BuChEs (Taylor, 1996). The realtime kinetics of the enzyme reactions has been described by one biochemist (Quinn, personal communication) 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 molecules of ACh per molecule of enzyme per minute, a turnover time of 150 s (Taylor, 1996). The upshot of this rapid rate of hydrolysis is that k1 becomes the ratelimiting step for hydrolysis of ACh and its analog ATCh. Perhaps this
represents the movement of substrate to the active center. The deacylation step (k3) is considered rate limiting to carbamoylating and phosphorylating agents.
A major distinction between AChEs and BuChEs is the inhibition of AChE activity with increasing substrate concentration [S]. A plot of activity versus [S] for ACh, ATCh, and, in mammals, acetylmethylcholine, 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 substrate may be due to interactions at the peripheral site. Rosenberry et al. (1998) present evidence that excess substrate 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.
JDiisopropyl
phosphoryl enzyme
MOLECULE carbon oxygen nitrogen hydrogen phosphorus fluorine
KAged
phosphoryl enzyme
LReactivation of
phosphoryl enzyme
lDiisopropyl-
fluorophosphate
EEdrophonium
AEnzyme-substrate
complex
BTetrahedralintermediate
CAcetyl enzyme
DHydrolysis ofacyl enzyme
FNeostigmine
GHydrolysis of dimethyl
carbamoyl enzyme
HDimethyl carbamoyl
enzyme
Trp 86
Ser 203
His 447
Glu 334
Trp 86
Ser 203His 447
Glu 334
Trp 86
Ser 203His 447
Glu 334
Trp 86
Gly 122Gly 121
Gly 122Gly 121
Ser 203
His 447
Glu 334
Trp 86
Ser 203His 447
Glu 334
Trp 86
Ser 203His 447
Glu 334
Trp 86
Gly 122Gly 121
Ser 203
His 447
Glu 334
Trp 86
Ser 203
His 447
Glu 334
Trp 86
Ser 203His 447
Glu 334
Trp 86
2PAM
Gly 122Gly 121
Ser 203
His 447
Glu 334
Trp 86
Ser 203
His 447
Glu 334
Trp 86
Ser 203His 447
Glu 334
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 (2PAM) to regenerate active enzyme (after Taylor, 1996).
1463chapter | 68 Cholinesterases
68.6 toxIcItIes of antIcholInesterases
The knowledge of the threedimensional structure of the ChEs has led to a better understanding of the mechanisms 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 serine 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 longer if tertiary alkyl groups are involved. Loss of one of the alkyl groups, a phenomenon known as aging, further stabilizes 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 enzymeinhibitor 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 chlorpyrifosoxon with tissues from 4dayold and adult rats (Table 68.4). The IC50s from young and adult brains were similar; the IC50s from the other tissues were not, differing 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
0.1 1
[Acetylthiocholine] (mM)
ChE
act
ivity
(µm
ol/m
in/m
l)
10
pH 7.2pH 8.0
6
5
4
3
2
1
0
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).
17,18
14
11
9
6, 7, 8
31
32
202321
1916
15
22
2524
260.1
1 2 4 6
Pl50
8 10 12
0.2
0.5
1
2
5
10
20
50
100
200
500
1,000
1
2
3
5
10
1213
4
LD50
(m
g/kg
)
fIgure 68.6 Toxicity in vivo of directly acting OPs versus their inhibition of AChE in vitro. (1) Dipterex. (2) O,Odiethyl4chlorophenylphosphate. (3) O,Odiethylbisdimethyl pyrophosphorodiamide (sym). (4) TIPP. (5) O,Odiethylphosphostigmine. (6) Isodemeton sulfoxide. (7) Isodemeton. (8) Isodemeton sulfone. (9) DFP. (10) Diethylamidoethoxyphosphoryl cyanide. (11) O,OdimethylO,Odiisopropyl pyrophosphate (asy). (12) Diethyl; amidomethoxyphosphoryl cyanide. (13) Tetramethyl pyrophosphate. (14) O,Odiethyl phosphorocyanidate. (15) O,OdimethylO,Odiethyl pyrophosphate (asym). (16) Soman. (17) TEPP. (18) Oisopropylethylphosphonefluoridate. (19) Tabun. (20) Amiton. (21) Diethylamidoisopropoxyphosphoryl cyanide. (22) O,OdiethylS(2diethylaminoethyl)phosphorothioate. (23) Sarin. (24) O,Odiethyl-S(2triethylammoniumethyl)thiophosphate iodide. (25) Echothiophate. (26) Methylfluorophosphorylcholine iodide. (27) Methylfluorophosphorylmethylcholine iodide. (28) Oethylmethylphosphorylthiocholine iodide. (29) Methylfluorophosphorylhomocholine 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 Toxicology1464
BuChE inhibitor isoOMPA. Aesterase destruction, carboxyesterase binding, and sequestration of the lipophilic OP were considered possible factors accounting for the differences between the in vitro and in vivo findings.
One example of a carboxyesterase is serum paraoxonase (PON1), an Aesterase associated with highdensity 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 demonstration that mice exposed to chlorpyrifos were protected against cholinesterase inhibition and toxicity by administration of purified PON1 (Li et al., 1995). Shih et al. (1998) demonstrated that knockout PON1deficient mice were more sensitive to chlorpyrifos and chlorpyrifosoxon 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 purified AChE can protect mice and other animals from exposure to OPs (Doctor et al., 1991).
Many physiological actions of antiChEs are those expected from an excess of ACh caused by the inhibition of its catalysis. Specific symptoms depend on the chemicals 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 salivation (Spencer et al., 2000; Taylor, 1996). Overstimulation at skeletal nicotinic neuromuscular junctions (motor endplates) causes muscle fasciculation (disorganized twitching) and, at higher doses, muscle paralysis. Increased ACh at cholinergic junctions of the sympathetic and parasympathetic autonomic ganglia affects the eye, bladder, heart, and salivary glands. Finally, antiChEs 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.
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 example, 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 evidence 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).
AChinduced 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 transient muscle damage in humans has been termed intermediate 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 evidence for other modes of action of these agents. AntiChE pesticides have been shown to directly affect pre and postsynaptic events (Pope, 1999). Electrophysiological studies suggest that choline may act as a regulator of nicotinic receptors 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 chlorpyrifos) 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 antiChE treatments intensifies the toxic effect of cocaine (Hoffman et al., 1992).
Table 68.4 Tissue IC50 Values for 4-Day-Old and Adult Mice after Chlorpyrifos-Oxon
Tissue Neonate (nM; mean SE)
Adult (nM; mean SE)
Brain 9.6 0.1 10 0.2
Liver 96 3 530 50
Plasma 18 1.5 330 28
Purified 3 nM for all tissues
Source: Mortinsen et al. (1998).
Table 68.5 Necrosis of Rat Muscle after DFP and Botulinum Toxina
Treatment EDL SOL
Saline 0 0
DFP 85.5 28
Btx 0 0
DFP Btx 0 1.59
Btx, botulinum toxin; DFP, diisopropyl fluorophosphate; EDL, extensor digitorum longus; SOL, soleus muscle.aNecroses/1000 fibers. DFP injected 48 h after Btx; sampled 24 h later – 1.5 mg/kg sc.Source: Sket et al. (1991).
1465chapter | 68 Cholinesterases
Genetic variation between individuals can play an important role in the toxicity of antiChEs. One example is humans with inherited low levels of plasma BuChEs. Although usually 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 serious 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 radiometric 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 hydrolysis of acetylcholine used a Warburg manometer to measure 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 development 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 periodic estimation of blood cholinesterase levels may provide 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 reddish purple complex read at 515 nm. Metcalf (1951) adapted the method for drops of blood obtained with a springloaded 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
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 aqueous phase, quenching its scintillation. The hydrolyzed radioactive acetate moves into the organic phase and is counted. The degree of substrate hydrolysis is determined by comparison 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 encountered with “reversible” carbamate chemicals are minimized, many tubes may be measured at once, the sample size is small, and readouts may be computerized. However, radioactive assays involve a high initial investment and costly disposal 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, 14Clabeled 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 determines 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 inexpensive. However, they are limited by their relative insensitivity; they often have larger sample requirements and lower outputs than radiometric methods. Several micropH methods 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
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).
Hayes’ Handbook of Pesticide Toxicology1466
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 investigators … 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 abnormally low value and has probably been exposed to a ChE inhibitor.” Such “redalert 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, replacing 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 automated 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 sulfhydrylsensitive 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 96well 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 sensitivity 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 concentration and low pH of the Boehringer–Mannheim kit introduce 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 substrate for human AChE is 1 or 2 mM and the optimum pH for the assay is 8.0, whereas the kit uses a substrate concentration 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 chromogen that absorbs in the nearultraviolet at 340 nm, to avoid the interference of Hb at 410 nm, thus divorcing the wavelength of the assay from the Soret band of Hb (Christenson et al., 1994; Loof, 1992; Willig et al., 1996). However, instruments reading in the nearultraviolet are often more costly than those that register in the visual range. The stability of modern instruments should be sufficient to overcome the increased noise level of the assay at 410 nm, providing activity levels are sufficiently high. The substrate 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 68.7 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
apH/h/0.02 ml RBCs or plasma, 25°C, mean SD.Source: Rider et al. (1957).
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).
1467chapter | 68 Cholinesterases
kit substrate, as a compromise substrate for both enzymes. Under the conditions of their assay, it was no better a substrate 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 excellent 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 specific inhibitors such as isoOMPA 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 conditions 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) indicate that, with other species, the relative activities of AChE and BuChEs, and even the properties of BuChEs, may differ from those of human enzymes. Lack of consideration for this may result in possible discrepancies and misinformation. 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 roundrobin 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 cholinesterase is low or absent. Another problem arises when studies are undertaken with species that lack RBC AChE; the literature 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 registrationgeared 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 platelet 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 thiocholinebased assays is the presence of a transient nonlinear “thiol oxidase” reaction with the color reagent
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
aATCh 0.8 mM (RBC); 7 mM (plasma); Autotechnicon analyzer.Adapted from Humiston and Wright (1967, Table 4).
Table 68.9 Relative Substrate Specificity of Plasma ChEsa
Species Propionyl Butyryl Benzoyl
Butyryl favoring
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
Propionyl favoring
Hamster 153 128 24
Rat 211 119 17
Chicken 147 83 6
Mouse 139 75 11
aNormalized to plasma ChE hydrolysis of ACh 100%.Adapted from Hoffmann et al. (1989, Table 11.4) and Myers (1953).
Hayes’ Handbook of Pesticide Toxicology1468
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 appropriate 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 regulatory 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 indeterminate error will occur when the Boehringer–Mannheim kit assays are applied to rats without preincubating the samples. 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 sensitivity of the assay requires dilution of the enzyme samples, but the act of dilution itself promotes rehydrolysis of the
enzyme. Several investigators have suggested ways to minimize the problem, including Thomsen et al. (1988) for the radiometric assays and Nostrandt et al. (1993) for Ellmantype thiocholinebased 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 contrary, as discussed elsewhere in this handbook, ChE inhibitions by methylOPs such as azinphosmethyl (Guthion) have relatively rapid rates of spontaneous reactivation at room temperature; in this case, the halflife of recovery of activity for azinphosmethyl 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 interpret the results of otherwise excellently designed and executed studies such as that of Yeary et al. (1993). Although the instructions that accompany the commercial ChE monitoring 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 samples 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 conditions 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 activities obtained from one experiment to another (Carakostas
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
aEighteen 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
Cat Male/femaleb 30 5450
aManometric, lCO2/30 min/mgN; N 3.bN 2.Adapted from Zajicek (1957).
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).aTransient activity, first 5–10 min, Ellman method.
1469chapter | 68 Cholinesterases
and Landis, 1991; Wilson et al., 1992a). Perhaps this is what has led to the idea that thiocholinebased 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 autoanalyzer. 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 methods 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 saponin hemolyzed 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 Sshaped substrate–concentration curve with butyrylthiocholine. They established that 0.01 mM eserine 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 laboratory, 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 activity 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 approximately 12 University of California, Davis, volunteers (Figures 68.7 and 68.8).
One problem to circumvent is the possible contamination 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 lowtemperature (75°C) freezer temperatures for more than 250 days. Its latest use is by a clinical
laboratory (Wilson et al., 2009) conducting courtordered monitoring of blood of orchard workers in the state of Washington.
68.9 fIeld kIts
Welldesigned, reliable field kits for cholinesterase determinations would be valuable for monitoring the health of those who apply pesticides and those who work in agricultural workplaces subject to pesticide spraying. Enzymeimpregnated 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 TestMate kit (Magnotti and Eberly, 1996; Magnotti et al., 1988). It uses a solidstate device and the Ellman method to measure ChE activity in a drop of whole
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
aBuChE 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
Enzyme Mean SEa n
AChE
Male 4.99 0.14 72
Female 5.18 0.18 71
Pooled 5.08 0.11 143
BuChE
Male 2.26 0.04 101
Female 2.46 0.09 71
Pooled 2.34 0.05 172
aIU/ml whole blood; Ellman assay.Modified from Bellino et al. (1978).
Hayes’ Handbook of Pesticide Toxicology1470
blood obtained by a fingerstick. Several models have been marketed. The TestMate 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 recommended 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 TestMate models have attempted to circumvent this by using higher wavelengths, sacrificing sensitivity of the chromogen for a lower noise level (Wilson and Henderson, 1992). A second problem with the device is that the TestMate does not display the raw absorbance values of the reaction. Instead, it displays values normalized to those expected at 25°C, employing a temperature sensor and a builtin algorithm that has been criticized (Amaya et al., 1996; London et al., 1995; Oliveira et al., 2002; Wilson et al., 1998).
1 3 5 7 9 11
AChE activity (nmol/min/mg Hb)
13 15 17 19 21 23 25 27 29 310
1
2
3
4
5
6
Num
ber
of s
ubje
cts
fIgure 68.7 Fingerstick AChE activities of University of California, Davis, volunteers. Whole blood hemolyzed with Triton X100 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).
1 3 5 7 9 11 13AChE activity (nmol/min/mg Hb)
15 17 19 21 23 25 27 29 31
200
180
160
140
120
100
80
60
40
20
0
Num
ber
of s
ubje
cts
fIgure 68.8 Fingerstick AChE activities of migrant family center residents. Bloodfilled 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).
1471chapter | 68 Cholinesterases
68.10 regulatory Matters: are che InhIbItIons adverse effects?
Soon after the introduction of OP pesticides, the experiences 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 cholinesterase 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 choosing 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 antiChEs tend to be steep, this “slippery slope” provides a realistic statistically significant 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 concerning the short and longterm health of individuals, populations, 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 putative exposed group and an unexposed group, or compared to an accepted “normal” range, is often accepted as indicating that a potentially hazardous exposure to an antiChE chemical has occurred. AntiChE chemicals are not usually longlasting within the body, and blood ChE activity can be expected to recover relatively rapidly from inhibition by an OP or CB. In the human, RBCs (and their AChE activities) are replaced at approximately 0.9% per day (a 120day 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, restoring enzyme activity, provided an important treatment for OP poisonings. It also opened the door to using reactivation 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 performed on a routine basis by clinical laboratories known to the author. Benschop and colleagues treated OPinhibited serum ChE with potassium fluoride at pH 4 to chemically
detect the presence of the inhibited OPChE. The fluoride ions reactivated the inhibited enzyme, converting the OP moiety into the corresponding phosphofluoridate and permitting 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 multibilliondollar, worldwide pesticide industry, they would still be of great interest to physiologists, cell biologists, and biochemists. ChEs have important roles in regulating neural transmission, and they are targets for pharmacological interventions 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 noeffect 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 agricultural workplace by monitoring those who could be exposed to dangerous levels of antiChE chemicals (i.e., mixer loaders and applicators). A fourth role is to decide whether poisoning episodes have involved ChEinhibiting agents. One factor in favor of using ChEs for such clinical and regulatory ends is the ease with which they may be rapidly and inexpensively assayed compared to analyzing for the cholinergic 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 experimental animals after short and longterm 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 noobservableadverseeffect 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 perform batteries of behavioral tests under controlled laboratory
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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 toxicity 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 animalderived 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 corrected 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 antiChE inhibitors rather than to insist they be considered quantitative indicators of physiological effects, thus supporting their use as early warning signs and as important weightofevidence 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 antiChE 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 AChinduced transient nicotinic inward currents in mouse N1E115 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 contrast, 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
at which these electrophysiological effects occur strongly suggest they should be taken into account when considering the effects of ChEinhibiting 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 (2PAM). Doses depend on the extent of exposure and species. Atropine binds to the muscarinic ACh receptor (mAChR), reducing 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 OPinhibited AChEs [Wilson and Ginsburg (1955) reviewed in Wilson et al. (1992b)]. 2PAM 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 transfer 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 warfare 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 Pretreatment Sets (NAPS),” contains 30mg tablets of pyridostigmine bromide to be taken orally three times a day. A Swedish autoinjector contains HI6 (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 permanent phosphorylation by soman and other OPs (Tuovinen et al., 1999). Three 30mg doses of pyridostigmine bromide per day were given to the Allied Forces during the first Gulf War under an experimentaldrug 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 symptoms termed the Gulf War syndrome (AbouDonia et al., 1996a,b; Kurt, 1998). An alternative approach to OP poisoning 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 phosphotriesterases to destroy them (Tuovinen et al., 1999; Wolfe et al., 1987).
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Experimental evidence of nerve damage (in this case to chickens) was reported by AbouDonia and coworkers after combined treatments of pyridostigmine bromide, DEET (an insect repellent), chlorpyrifos (AbouDonia et al., 1996a), or permethrin (AbouDonia 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 antiChE 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 laboratory, the clinic, and the environment. Nevertheless, problems 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 difficulties encountered when using ChE activities for regulatory 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 healthbased 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, garden, 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 healthbased 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, government regulators, and toxicologists specializing in agricultural 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 characterization 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.” ChEinhibiting chemicals played a leading role in this venture when a panel of distinguished toxicologists asserted that inhibition of AChE was a “common mechanism of toxicity” for OPs. They
concluded that “OP pesticides act by a common mechanism 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 pharmacologically simplistic, but regulatory valuable, view will win the day. Pope (1999), one of the authors of the “common 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 pesticides. 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 mid20th 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 anticholinergic 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 foreseeable 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 studies 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 pharmaceuticals, enzyme action, development of the nervous system, and many other basic features of living systems will benefit from studies of ChEs for many years to come. Also, dismayingly, the simplicity of synthesizing and deploying antiChEs as weapons of war and terror is likely to be as tempting to governments and to terrorists of the 21st century as it was to some in the 20th century. Today’s scientists leave a legacy of knowledge for those who come after,
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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.
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