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Environmental Toxicology and Pharmacology 19 (2005) 433–446

Pharmacology and toxicology of cholinesterase inhibitors: uses and

misuses of a common mechanism of action
Carey Pope ∗ , Subramanya Karanth, Jing Liu
264 McElroy Hall, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078, USA


Cholinesterase inhibitors have been used in the treatment of human diseases, the control of insect pests, and more notoriously as chemical
warfare agents and weapons of terrorism. Most uses of cholinesterase inhibitors are based on a common mechanism of action initiated by
inhibition of acetylcholinesterase (AChE). Extensive inhibition of this enzyme leads to accumulation of the neurotransmitter acetylcholine and
enhanced stimulation of postsynaptic cholinergic receptors. This action is beneficial in cases where a reduction in cholinergic transmission
contributes to clinical symptoms, e.g., low muscle tone in the autoimmune disorder myasthenia gravis due to loss of nicotinic receptors.
Under normal conditions, however, extensive inhibition of AChE leads to excess synaptic acetylcholine levels, over-stimulation of cholinergic
receptors, alteration of postsynaptic cell function and consequent signs of cholinergic toxicity. This biochemical cascade forms the basis
for the use of anticholinesterase insecticides in pest control as well as for nerve agents in chemical warfare. Paradoxically, the short-acting
cholinesterase inhibitor pyridostigmine, an important therapeutic agent in the treatment of myasthenia gravis, was used during the Persian Gulf
War to prevent the long-term clinical consequences of possible organophosphate nerve agent exposure. As shown in the attacks in Matsumoto
and Tokyo, these same nerve agents can be effectively used to inflict urban terror. Cholinesterase inhibitors thus share a common mechanism
of pharmacological or toxicological action, ultimately modifying cholinergic signaling through disruption of acetylcholine degradation.
While the use of cholinesterase inhibitors relies on their interaction with AChE, a variety of reports indicate that a number of cholinesterase
inhibitors have additional sites of action that may have pharmacologic or toxicologic relevance. A variety of esterase and non-esterase
enzymes, neurotransmitter receptors and elements of cell signaling pathways are targeted by some anticholinesterases. In some cases, these
actions may occur at concentrations/dosages below those affecting cholinergic transmission. Studies of interactive toxicity of binary mixtures
of common organophosphorus insecticides indicate that non-cholinesterase targets may be important in cumulative toxicity. Exposure to
multiple anticholinesterases having selective effects on other macromolecules could confound the assumption of additivity in cumulative risk
assessment. Knowledge of such selective additional targets may aid, however, in the optimization of strategies for poisoning therapy and in
the further elucidation of mechanisms of toxicity for this class of compounds.
© 2005 Elsevier B.V. All rights reserved.

Keywords: Organophosphorus; Carbamates; Insecticides; Nerve agents; Mechanism; Interactive toxicity

1. History of cholinesterase inhibitors by local natives for “trial by ordeal” to determine the guilt
or innocence of an accused criminal. Basically, the accused
Knowledge of the pharmacologic and toxic properties of person was forced to consume the seeds, after which one
cholinesterase inhibitors has been available for over 100 years can assume a host of signs including breathing difficulties,
(Felter and Lloyd, 1898). The earliest records of toxic ef- uncontrolled muscle contractions and excessive salivation,
fects of cholinesterase inhibitors deal with the perennial plant lacrimation, defecation and urination were shortly noted. If
Physostigma venenosum. The beans of this plant, indigenous that person survived the acute toxic episode, he or she was
to Calabar on the coast of Nigeria in West Africa, were used pronounced innocent. If on the other hand, that individual
died, guilt was “verified”.
∗ Corresponding author. Tel.: +1 405 744 6257; fax: +1 405 744 0462. Sir Robert Christison reported on the pharmacologic and
E-mail address: (C. Pope). toxic properties of Calabar beans before the Royal Society of

1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
434 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446

Edinburgh almost 150 years ago (Christison, 1855). A pure al-

kaloid was first isolated from Calabar beans in 1864 by Jobst
and Hesse and called physostigmine (Lloyd, 1897). Fraser
first reported the ability of calabar beans to contract the pupil
in 1862 (Frazer, 1863). The further characterization of oph-
thalmic properties of physostigmine and its successful use in
the treatment of glaucoma followed in the last half of the 19th
century (Taylor, 2001). In some countries, physostigmine is
still used for this clinical condition.
A related compound neostigmine was first used in the
1930s to treat gastrointestinal or urinary bladder atony.
Shortly after, neostigmine was proven effective for treatment
of both glaucoma and myasthenia gravis. Pyridostigmine, an-
other carbamate cholinesterase inhibitor, has also been used
for decades for the treatment of myasthenia gravis and more
recently as a prophylactic agent to protect against potential
organophosphate nerve agent exposures (Gunderson et al., Fig. 1. The cholinergic synapse and cholinesterase inhibitors. Normally,
1992; Taylor, 2001). acetylcholinesterase (AChE) efficiently hydrolyzes acetylcholine released
by the presynaptic terminal. The choline released is then transported back
into the presynaptic terminal by the high affinity choline uptake (HACU)
2. Organophosphorus cholinesterase inhibitors process. Acetylcholine is formed by the synthetic enzyme choline acetyl-
transferase (ChAT) using choline and acetyl coenzyme A. Acetylcholine is
concentrated in synaptic vesicles by the vesicular acetylcholine transporter.
The first report of an organophosphorus cholinesterase in-
Upon terminal depolarization, synaptic vesicles fuse with the plasma mem-
hibitor was tetraethyl pyrophosphate (TEPP), first synthe- brane and release acetylcholine into the cleft. When sufficient cholinesterase
sized in 1854 by Philip de Clermount (Taylor, 2001). TEPP inhibitors bind to AChE to (1) block transmitter degradation, acetylcholine
was later developed in Germany during World War II as a po- (ACh) accumulates (2) in the synaptic cleft. This leads to persistent stim-
tential substitute for the insecticide nicotine (Murphy, 1980). ulation of cholinergic receptors (3) on the postsynaptic cell. The excessive
activation of these postsynaptic receptors leads to prolonged cholinergic re-
From 1938 to 1944, Schrader developed a series of fluorine-
ceptor signaling (4) and associated changes in postsynaptic cell function.
containing esters including di-isopropyl phosphorofluoridate
(DFP), sarin and soman as well as octamethylpyrophospho-
rotetramide (OMPA), parathion and paraoxon. During this
subsequently demonstrated that physostigmine, neostigmine
same period, British and American scientists were also eval-
and other carbamates including pyridostigmine were potent
uating the toxic properties of DFP and other alkyl phospho-
inhibitors of AChE. Fig. 1 shows the general mechanism of
rofluoridates. Dubois et al. (1948) reported that the toxicity
action for these drugs.
of parathion could be blocked by the antimuscarinic atropine,
Cholinergic neurons synthesize acetylcholine from
and later this same group (Dubois et al., 1949) demonstrated
choline and the cofactor acetyl coenzyme A through the
that AChE activity was markedly depressed and tissue acetyl-
action of the synthetic enzyme choline acetyltransferase. The
choline levels were significantly elevated following parathion
acetylcholine molecules are packaged into synaptic vesicles
exposure. Thus, the mechanism of toxicity for the synthetic
by the vesicular acetylcholine transporter. Upon terminal
organophosphorus toxicants was determined to be initiated
depolarization, synaptic vesicles fuse with the plasma
through the inhibition of AChE.
membrane and release acetylcholine into the synaptic cleft.
In the decade of the 1950s, the common OP insecticide
Under normal conditions, AChE rapidly and efficiently
malathion was first introduced by American Cyanamid
hydrolyzes acetylcholine. Prior to inactivation, acetylcholine
Company, Schrader produced a number of thioether
molecules interact with postsynaptic cholinergic receptors
organophosphorus toxicants including demeton, and di-
to alter cellular function, either by alterating ion flux across
alkylvinyl phosphate esters including dichlorvos were first
the postsynaptic cell membrane or through the generation
synthesized. Hundreds of organophosphorus cholinesterase
of intracellular second messengers. When cholinesterase in-
inhibitors have been synthesized to date, with 38 different OP
hibitors bind to a substantial number of (1) AChE molecules,
cholinesterase inhibitors currently being registered for use in
the efficient degradation of (2) acetylcholine is prevented
the United States as pesticides (see review by Pope, 1999).
and transmitter molecules accumulate in the synapse.
Elevated synaptic acetylcholine levels lead to persistent
3. Overview of the pharmacology of cholinesterase stimulation of (3) cholinergic receptors on postsynaptic cells
inhibitors and subsequent alteration of cholinergic receptor-mediated
(4) signaling pathways, e.g., alteration of intracellular cAMP
It was known before 1900 that atropine was an effective levels. These cellular changes lead to functional changes at
antidote to physostigmine (Felter and Lloyd, 1898). It was the tissue/organism level.
C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446 435

This cascade of cellular events elicited by AChE inhibi- preventing irreversible inactivation of those molecules by an
tion has been used therapeutically for a number of clinical organophosphate. Carbamylated enzymes recover faster, and
conditions (Taylor, 2001). As already mentioned above, the therefore as combined therapy with an anticholinergic and
efficacy of physostigmine for treatment of glaucoma was enzyme reactivator, synaptic function can be restored without
realized in the late 19th century. Physostigmine is still used requirement for de novo synthesis of new AChE molecules.
for the treatment of this ocular disorder in some countries.
Myasthenia gravis, a neuromuscular disease of autoimmune
origin, has been treated successfully with neostigmine and 4. Overview of the toxicology of cholinesterase
pyridostigmine for decades. Stimulation of the intestinal inhibitors
and urinary bladder musculature, which can often become
flaccid following surgery, is another use of cholinesterase The mechanism of toxicity for cholinesterase inhibitors is
inhibitors. A series of cholinesterase inhibitors including essentially the same as the mechanism of action for thera-
tacrine, donepezil galantamine and rivastigmine have been peutic uses. Inhibition of AChE disrupts the dynamic inter-
approved for treatment of Alzheimer’s disease. In all cases, play between acetylcholine synthesis, release and degrada-
the therapeutic strategy is to increase the persistence of tion. This leads to a net accumulation of synaptic ACh levels
synaptic acetylcholine by blockade of its degradation, such with persistent/prolonged activation of cholinergic receptors
that there is a net increase in cholinergic receptor activation. on postsynaptic cells. This relative increase in cholinergic
This overall strategy is based on a clinical condition wherein signaling leads to functional signs and symptoms of cholin-
activation of cholinergic receptors is deficient. Thus, in- ergic toxicity (Ecobichon, 2001).
creasing the residence of acetylcholine molecules within While all cholinesterase inhibitors are thought to elicit
synapses by inhibiting AChE can at least partially counteract toxicity through this general mechanism, the spectrum of
a deficiency in either the release of neurotransmitter or a cholinergic signs can be different with different inhibitors,
reduction in cholinergic receptors/signaling. the same inhibitor in different species, or the same inhibitor
Paradoxically, a cholinesterase inhibitor has been used with different routes of exposure. Generally, however, one
prophylactically to protect against the toxicity of other or more of the “classic” signs of cholinergic toxicity will
cholinesterase inhibitors. In this regard, the drug used for be evident following exposure to any inhibitor that leads to
treating myasthenia gravis, pyridostigmine, is used by the US substantial inhibition of tissue AChE activity. The biological
military to protect against potential exposures to organophos- responses to cholinesterase inhibitors are predicated upon
phorus nerve agents (Gunderson et al., 1992; Lee, 1997). the distribution and role of cholinergic neurons within
While use of one cholinesterase inhibitor to protect from the the central and peripheral nervous systems. Acetylcholine
toxicity of another at first seems contradictory, the rationale is the transmitter at pre-ganglionic terminals of both
for this therapeutic strategy resides in the differential duration parasympathetic and sympathetic nerves, postganglionic
of inhibition between the two types of inhibitors. Basically, parasympathetic terminals, neuromuscular junction of
the inhibition of AChE by pyridostigmine is much shorter striated muscle and at various synapses within the central
in duration than that following organophosphate exposure, nervous system. Thus, AChE inhibition leads to a relative
in particular if the organophosphorus inhibitor (e.g., soman) imbalance of cholinergic transmission at these sites with
is capable of rapidly aging. If some proportion of AChE elicitation of predictable functional alterations.
molecules is inhibited by pyridostigmine when exposure to One of the most recognized forms of acute toxicity
such an organophosphate occurs, those same molecules will following exposure to cholinesterase inhibitors is auto-
be protected from irreversible inactivation by the organophos- nomic dysfunction, characterized by excessive secretions at
phate. Because of the high reactivity of the organophosphorus parasympathetic end organs (Ecobichon, 2001). Excessive
inhibitors, by the time the enzymes recover from inhibition stimulation of secretory organs leads to increased secretions
by pyridostigmine, few residual organophosphate molecules at bronchial, lacrimal, salivary, sweat, intestinal and pancre-
remain in the circulation for possible enzyme re-inhibition. atic sites. Smooth muscles of the ureter also exhibit increased
Used in conjunction with standard therapies to pre- contractions leading to enhanced urination (Taylor, 2001).
vent cholinergic receptor activation (antimuscarinics) and Together, these responses leading to excessive secretions
reactivation of phosphorylated enzymes (aldoximes), pyri- at multiple sites are a characteristic of cholinergic toxicity.
dostigmine can markedly enhance survival and long-term The acronym SLUD, standing for salivation, lacrimation,
outcome from organophosphate poisoning (Xia et al., 1981; urination and defecation, is often used to refer to these types
Patocka et al., 1991). In contrast to the other pharmacolog- of autonomic signs of toxicity.
ical uses of cholinesterase inhibitors, however, the use of Loss of regulation at the neuromuscular junction leads to
pyridostigmine in this context is not based on increasing another classic sign of exposure to cholinesterase inhibitors,
synaptic acetylcholine levels by disrupting neurotransmit- involuntary movements. The residence of acetylcholine
ter breakdown. As a nerve agent antidote, the basis for released at the neuromuscular junction is generally limited
protection against nerve agent intoxication is the short- by the action of AChE such that a single nerve impulse
term carbamylation of a proportion of AChE molecules, elicits an end-plate potential and subsequent muscle action
436 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446

potential (Taylor, 2001). With extensive AChE inhibition, A working group of experts from academia, government and
acetylcholine molecules can bind multiple nicotinic recep- industry concluded that organophosphorus pesticides that
tors, prolonging the decay time of the end-plate potential work by “phosphorylating AChE and eliciting a spectrum
and disrupting the synchrony between end-plate potentials of cholinergic effects” indeed share a common mechanism
and muscle action potentials. Muscle fibrillations and of toxicity (Mileson et al., 1998). Risk assessment methods
fasciculations result, and complete muscle paralysis can be to evaluate cumulative toxicity of cholinesterase inhibitors
noted due to end-plate depolarization. have subsequently been developed, and a revised cumula-
Ocular effects can also be commonly noted following tive risk assessment of organophosphorus pesticides was re-
exposure to cholinesterase inhibitors (Rengstorff, 1994; ported in 2002 (
Moser, 1995). Miosis is a classic sign of cholinergic toxicity The approach for estimating cumulative risk of OP insecti-
resulting from constriction of the sphincter pupillae muscle cides involves the use of a toxic equivalency factor, in this
surrounding the iris. In fact, miosis is thought to be the case anticholinesterase potency relative to that of an index in-
most sensitive indicator of aerosol exposures to some of hibitor, methamidophos, to quantify relative toxicity of given
the most potent cholinesterase inhibitors, e.g., soman (Soli cholinesterase inhibitors. Potencies were based on a bench-
et al., 1980; Benschop et al., 1998). Constriction of the mark dose causing 10% cholinesterase inhibition. Further-
ciliary muscle, which blocks the accommodation reflex, also more, the cumulative risk assessment for OPs is “based on
increases outflow of aqueous humor and is the mechanism the assumption of dose additivity”.
of action for cholinesterase inhibitors for treatment of glau- It should be noted, that in the introduction to the revised
coma mentioned above. Because of the wide distribution of risk assessment, it is stated “EPA has developed a frame-
cholinergic nerves, a host of other signs and symptoms can be work for conducting cumulative risk assessments for the
associated with cholinesterase inhibitors. Cardiac effects can organophosphates (OPs) and other pesticides that have a com-
be prominent and complex (Stemler et al., 1990; Yamamoto mon mechanism of toxicity (i.e., that act in the same way in
et al., 1996; Karalliedde, 1999). Generally, bradycardia is the body)”. The concept of common mechanism for these
first observed due to negative chronotropic effects of vagal toxicants is not new. It was recognized decades ago that “. . .
muscarinic input. However, direct effects on the medullary organophosphate insecticides . . . all, in sufficient doses, in-
vasomotor center as well as stimulation of ganglionic hibit AChE in vivo and thus share a common mechanism of
nicotinic receptors can influence cardiac function. Other acute toxic action” (Murphy, 1980). A possible distinction of
common signs of intoxication of cholinesterase inhibitors importance resides in the apparent “word-splitting” between
include seizures and convulsions, hypothermia, increased common and same mechanism, however. While it is easy
excitability, dyspnea and others. Respiratory failure, through to argue that cholinesterase inhibitors share a mechanism in
a combination of excessive airway secretions, paralysis of common, one can also argue that there is ample evidence to
muscles of ventilation, and depression of the respiratory indicate they do not all “act in the same way”. We propose
control centers in the pons-medulla is generally considered that while anticholinesterases share a common mechanism,
the primary cause of death following lethal exposures. they have additional actions that can modify the toxic con-
sequences of AChE inhibition. As some non-cholinesterase
actions may occur at or below levels of exposure needed to
5. A common mechanism of toxicity for inhibit AChE, these additional sites of action may directly
cholinesterase inhibitors influence anticholinesterase potency or cholinergic signaling
and thus modify the cumulative effects of mixed exposures.
For both the pharmacological and toxicological actions
of cholinesterase inhibitors, a common mechanism of action
holds true, i.e., inhibition of AChE allows acetylcholine lev- 6. Non-cholinesterase targets of anticholinesterases
els to accumulate within the synapse, followed by enhanced
stimulation of cholinergic receptors and associated changes 6.1. Other enzymes as targets
in postsynaptic cell function. In 1996, the U.S. Congress
passed the Food Quality Protection Act, which modified ex- Other enzymes often associated with OP and carbamate
isting standards for pesticide evaluation and registration. An cholinesterase inhibitors are various esterases including
important component of that law was the requirement that neurotoxic (or neuropathy target) esterase (NTE), butyryl-
EPA consider “. . . information concerning the cumulative cholinesterase, carboxylesterases, and A-esterases, e.g.,
effects of such residues and other substances that have a com- paraoxonase or PON-1. Inhibition and subsequent “aging”
mon mechanism of toxicity, in establishing, modifying, leav- of NTE is correlated with initiation of organophosphorus-
ing in effect or revoking a tolerance for a pesticide . . .” in the induced delayed neurotoxicity (OPIDN, Johnson, 1970).
re-registration of pesticides. In essence, if two or more pesti- NTE inhibitors that are incapable of aging have no neu-
cides were considered to act through a common mechanism, ropathological capacity of their own but can block delayed
then cumulative effects of co-exposure to those pesticides neurotoxicity when given prior to OP dosing (Johnson,
would have to be considered in the evaluation of tolerances. 1980). Some of these “protective” agents were subsequently
C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446 437

shown to potentiate or promote delayed neurotoxicity when Quistad et al., 2001). Brain FAAH was inhibited by profeno-
given after a neuropathic OP (Pope and Padilla, 1990; Lotti fos and tribufos in vivo at levels of exposure lacking cholin-
et al., 1991; Lotti and Moretto, 1999). Protection afforded ergic toxicity. Anandamide modulates the release of acetyl-
by pre-exposure was correlated with NTE inhibition. choline (Gifford et al., 1999) and muscarinic receptor acti-
Potentiation of OPIDN by post-treatment does not, however, vation increases secretion of anandamide (Kim et al., 2002),
appear to involve NTE (Pope et al., 1993), but may involve thus selective inhibition of FAAH by some cholinesterase
another related esterase referred to as M200 (Moretto et al., inhibitors could potentially modulate cholinergic toxicity.
2001; Lotti, 2002). Acyl peptide hydrolase (APH) removes N-terminally
Many cholinesterase inhibitors are inactivated by direct acetylated peptides from polypeptides (Farries et al., 1991;
interaction with esterases, e.g., carboxylesterases and A- Fujino et al., 2000). DFP phosphorylates a serine residue
esterases (Clement, 1984; Pond et al., 1995; Maxwell and on APH (Scaloni et al., 1992). Methyl chlorpyrifos oxon,
Brecht, 2001; Chemnitius et al., 1983; Walker and Mackness, dichlorvos and DFP are potent inhibitors of APH (IC50 val-
1987; Li et al., 2000). While carboxylesterase or A-esterase ues from 18 to 199 nM), with in vitro potencies 6–10 times
detoxification can affect the toxicity of individual inhibitors, those for inhibiting AChE (Richards et al., 2000).
cumulative toxicity of combined exposures may also be in- Chlorpyrifos oxon (50 ␮M) activated extracellular signal-
fluenced by these detoxifying enzymes in a complex manner regulated kinases (ERK) two to three-fold in Chinese hamster
(Frawley et al., 1957; Murphy et al., 1959; Karanth et al., ovary cells (Bomser and Casida, 2000). Increased ERK ac-
2001). tivity may be the result of inhibition of diacylglycerol lipase
Some anticholinesterases can bind to and inhibit a num- activity (Bomser et al., 2002). Such actions could affect sig-
ber of other enzymes. O,O -Diisopropylphosphorofluoridate naling pathways of a variety of growth factors and mitogens.
(DFP) is a well-known protease inhibitor used in protein iso-
lation procedures (Scopes, 1982). Chymotrypsin is inhibited 6.2. Cholinergic receptors as macromolecular targets of
by a number of OP anticholinesterases (Hamilton et al., 1975; cholinesterase inhibitors
Johnson and Clothier, 1980). Various liver proteases have
been reported sensitive to a number of anticholinesterases It has been known for decades that some cholinesterase
(Mantle et al., 1997; Saleem et al., 1998). inhibitors directly interact with cholinergic receptors
Evaluation of 20 OP inhibitors on the activity of AChE, (Frederickson, 1958). DFP, phospholine and paraoxon
NTE, trypsin, and an enzyme involved in mitogen-induced acted as reversible antagonists on nicotinic receptors in
activation of T lymphocytes indicated a range of differ- Electrophorus cells (Bartles and Nachmansohn, 1969).
ential selectivity towards inhibition of the four enzymes Azinphos-methyl, dichlorvos, dicrotophos, monocrotophos,
(Pruett et al., 1994). Profenofos, tribufos and phenyl sali- echothiophate and VX all bound to neuromuscular nicotinic
genin cyclic phosphonate inhibited thrombin, trypsin and receptors of the Torpedo electric organ (Eldefrawi and
elastase, but at higher concentrations than needed to inhibit Eldefrawi, 1983; Bakry et al., 1988).
AChE (Quistad and Casida, 2000). Under some conditions, Some carbamate cholinesterase inhibitors interact
soman and parathion inhibit choline acetyltransferase (ChAT; directly with nicotinic receptors (Seifert and Eldefrawi,
Murumatsu and Kuriyama, 1976; Thompson and Thomas, 1974). Physostigmine potentiated acetylcholine-induced
1985). Sivam et al. (1984) reported, however, that neither ion flux at low concentrations (1–10 ␮M) but blocked
DFP, tabun, sarin or soman had any effect on brain regional acetylcholine actions at higher concentrations (Zwart et al.,
ChAT activity. 2000). Physostigmine binds to nicotinic receptors at a site
Kynurenine formamidase, an enzyme involved in distinct from the agonist binding site (Albuquerque et al.,
the metabolism of l-tryptophan, is inhibited by some 1985). Pyridostigmine and neostigmine also bound to an
organophosphorus and carbamate cholinesterase inhibitors allosteric site on the nicotinic receptor, but at higher concen-
(Seifert and Casida, 1978; Henderson and Kitos, 1982; Seifert trations than noted with physostigmine (Akaike et al., 1984;
and Pewnim, 1992; Pewnim and Seifert, 1993). Different Albuquerque et al., 1984). It was also noted that physostig-
metabolites of kynurenine have been reported to be neuro- mine could activate nicotinic receptors even following prior
protective (kynurenic acid, Stone, 2000) and neurotoxic (3- receptor desensitization to agonist (Kuhlmann et al., 1991).
hydroxykynurenine and quinolinic acid, Okuda et al., 1998; [3 H]Phencyclidine (PCP) binding to the nicotinic channel
Vasquez et al., 2000). Pirimiphos-ethyl (20 mg/kg) and di- can be used to evaluate both activation and agonist-induced
azinon (10 mg/kg) almost completely inhibited (>90%) liver desensitization (Eldefrawi et al., 1980). Several OP and
kynurenine formamidase in mice (Seifert and Pewnim, 1992; carbamate anticholinesterases modify PCP (or [3 H]thienyl-
Pewnim and Seifert, 1993). cyclohexylpiperidine [TCP], an analog of phencyclidine)
Fatty acid amide hydrolase (FAAH) is involved in the binding to nicotinic receptors (Mansour et al., 1987;
metabolism of the endocannabinoid, anandamide and is Eldefrawi et al., 1988; Katz et al., 1997).
inhibited by a number of cholinesterase inhibitors (Quistad et A number of studies have evaluated effects of anti-
al., 2001). Chlorpyrifos oxon was a potent inhibitor of FAAH cholinesterases on muscarinic receptor binding. Dichlor-
activity in mouse brain and liver (IC50 values = 40–56 nM, vos, paraoxon and tetraethylpyrophosphate (TEPP) inhibited
438 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446

binding to the non-selective muscarinic antagonist quinu- at increasing neurotransmitter release, while soman had no
clidinyl benzilate (QNB) at low concentrations (5–50 nM, effect (Rocha et al., 1996b). The effects of VX on GABA re-
Volpe et al., 1985). Paraoxon blocked QNB binding to lease were blocked by atropine (Rocha et al., 1999). Sarin
M2 and M3 subtypes of receptors at extremely low levels (0.1–3 nM) blocked the stimulated GABA release in hip-
(as low as 10−15 M, Katz and Marquis, 1989). Echothio- pocampal slices independent of AChE inhibition (Chebabo
phate, paraoxon, VX, soman and tabun all blocked [3 H]cis- et al., 1999).
dioxolane (CD) binding in vitro in membranes from rat
heart at sub-micromolar concentrations (Silveira et al., 1990).
Paraoxon inhibited CD binding in rat striatal cells (Jett 7. Other actions of cholinesterase inhibitors
et al., 1991). Paraoxon and malaoxon blocked CD bind-
ing in rat hippocampus and cortex in vitro (Ward et al., The above studies suggest that both muscarinic and
1993). Chlorpyrifos oxon inhibited CD binding in rat striatum nicotinic receptor subtypes may be sensitive to direct
(IC50 = 22 nM; Huff et al., 1994). Paraoxon, malaoxon and binding by a number of anticholinesterases. Other types
chlorpyrifos oxon inhibited CD binding in a concentration- of neurotransmitter receptors are also sensitive to some
dependent manner at sub-micromolar concentrations (po- cholinesterase inhibitors. Cyanofenphos, leptophos, salithion
tencies: chlorpyrifos oxon > paraoxon > malaoxon; Ward and and tri-ortho-cresyl phosphate (TOCP) blocked cardiac
Mundy, 1996). [3 H]norepinephrine binding in vitro with potencies similar to
CD preferentially labels the M2 receptor subtype (Huff that of propranolol (El-Sebae et al., 1981). DFP, dichlorvos,
and Abou-Donia, 1994). Chlorpyrifos oxon diethylphos- cyanophos and mipafox inhibited 3-((+)-2-carboxypiperazin-
phorylated the rat cardiac M2 muscarinic receptor in vitro 4-yl)-[1,2-3 H]propyl-1-phosphonic acid ([3 H]CPP) binding
(Bomser and Casida, 2001). Chlorpyrifos oxon, paraoxon to the NMDA receptor in rat brain (Johnson and Michaelis,
and methyl paraoxon displaced [3 H]oxotremorine-M bind- 1992). In contrast, binding to other NMDA ligands including
ing to rat cardiac membrane muscarinic receptors (Howard [3 H]AMPA, [3 H]glycine and [3 H]TCP was not affected by
and Pope, 2002), with chlorpyrifos oxon being most potent any of the cholinesterase inhibitors. Some OP inhibitors
(IC50 = 7 nM). Presynaptic muscarinic autoreceptors in the are relatively potent inhibitors of GABAA receptor binding
mammalian brain are primarily the M2 subtype and can in- to t-[35 S]butyl-bicyclophophorothionate in membranes
hibit acetylcholine release (Weiler, 1989; Feuerstein et al., from Torpedo californica (Gant et al., 1987). Chlorpyrifos
1992; Kitaichi et al., 1999). Selective actions at these recep- oxon and methyl chlorpyrifos oxon were potent inhibitors
tors could modulate regulation of acetylcholine release and (IC50 = 14 and 64 nM, respectively) of in vitro binding
play a role in selective toxicity (Liu et al., 2002). to a cannabinoid CB1 receptor subtype ligand ([3 H]CP
Paraoxon (<1 ␮M) enhanced GABA and glutamate release 55,490) in mouse brain membranes whereas other inhibitors
in primary neurons (Rocha et al., 1996a). Acetylcholine had (paraoxon, diazoxon and dichlorvos) were markedly less
no effect in this system, suggesting that paraoxon did not act potent (IC50 = 1.2–4.2 ␮M, Quistad et al., 2002). Tribufos
through AChE inhibition. VX was more potent than paraoxon exposure (50 mg/kg) inhibited [3 H]CP 55,490 binding to

Table 1
Additional targets for anticholinesterases and known or possible consequences of their interaction
Target Known or possible effects
Acetylcholinesterase Cholinergic toxicity, neurodevelopmental deficits
Acyl peptide hydrolase Changes in peptide/neuropeptide turnover
Adenylyl cyclase Altered transduction for a variety of extracellular signals
Butyrylcholinesterase Altered cholinergic neurotransmission, neurodevelopmental deficits
Cannabinoid receptors Changes in neuromodulation
Carboxylesterase Modulation of toxicity with combined exposures
Cardiac adrenergic receptors Modulation of heart function, arrythmia
Choline acetyltransferase Alteration of choline utilization, acetylcholine synthesis
Choline transport Alteration of acetylcholine synthesis
Cholinergic autoreceptors Modulation of acetylcholine release
Diacylglycerol kinase Altered growth factor/mitogen signaling
Fatty acid amide hydrolase Changes in neuroactive amide (e.g., endocannabinoid) metabolism
Kynurenine formamidase Modulation of l-tryptophan metabolism, production of NAD+
M-200 Exacerbation of delayed neurotoxicity
Muscarinic receptors Variety of central/peripheral neurochemical changes
Neurotoxic esterase Delayed neurotoxicity
Na+ ,K+ -ATPase alpha 1 Arrythmias
Nicotinic receptors Variety of central/peripheral neurochemical changes
NMDA receptors Excitotoxicity, altered neuromodulation
Proteases Alteration of protein turnover, modulation of clotting
C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446 439

cannabinoid receptors in mouse brain in an apparently of cholinesterase inhibition elicited differential signs of
irreversible manner. Together, these findings suggest that a cholinergic toxicity (Chaudhuri et al., 1993; Pope et al.,
number of non-cholinergic neurotransmitter receptors may 1995; Liu and Pope, 1998). In essence, parathion elicited
be targeted by anticholinesterases. more extensive signs of toxicity in the presence of similar
As noted above, changes in cardiac function often changes in cholinesterase activity. We hypothesized that
occur following exposure to cholinesterase inhibitors. non-cholinesterase actions must be responsible for the dif-
Cardiotoxicity from exposure to some cholinesterase ferential toxicity of parathion and chlorpyrifos. Muscarinic
inhibitors may occur independent of AChE inhibition. autoreceptor function, important in the feedback inhibition of
Dimethoate caused cardiac failure in guinea pigs, apparently acetylcholine release (Weiler, 1989; Feuerstein et al., 1992),
unrelated to AChE inhibition (Marosi et al., 1985). VX was affected in an OP-selective manner in vivo (Pope et al.,
elicited delayed after-depolarizations in guinea pig ventricle 1995; Liu and Pope, 1998). Furthermore, paraoxon and chlor-
while carbachol and physostigmine had no effect under pyrifos oxon had qualitatively different actions on muscarinic
similar conditions (Corbier and Robineau, 1989). Inhibition autoreceptor function in vitro (Liu et al., 2002). These selec-
of the cardiac specific alpha-1 Na+ /K+ -ATPase isoform tive non-cholinesterase actions were proposed to contribute
may contribute to VX-induced cardiotoxicity (Robineau to the differential toxicity noted with these two insecticides
et al., 1991). Oral dosing with diazinon or fenthion led with dosages causing similar changes in AChE activity.
to cholinergic toxicity and extensive AChE inhibition, but
intravenous exposure to these same inhibitors elicited tonic
convulsions and opisthotonus resistant to atropine and only 9. Can non-cholinesterase targets modify cumulative
slight AChE inhibition (Takahashi et al., 1991). A number cholinergic toxicity?
of reports suggest that some cholinesterase inhibitors can
influence neurite extension and neurodevelopment (Dupree The interactive toxicity of cholinesterase inhibitors has
and Bigbee, 1994; Campbell et al., 1997; Dam et al., been studied for almost half a century. A number of ear-
1998; Sternfeld et al., 1998; Bigbee et al., 1999; Das and lier studies noted marked potentiation of the toxicity of
Barone, 1999). A recent study suggests that chlorpyrifos the organophosphorus insecticide malathion following pre-
and chlorpyrifos oxon potently increase the phosphorylation exposure to other inhibitors. Frawley et al. (1957) reported
of the transcription factor Ca2+ /cAMP response element that the toxicity of malathion was increased approximately
binding protein (CREB), potentially influencing a number of 10-fold in rats and 50-fold in dogs by pre-treatment with
neurodevelopmental pathways (Schuh et al., 2002). Table 1 the OP insecticide, EPN. Murphy et al. (1959) reported that
lists a number of additional targets for anticholinesterases tri-ortho-cresyl phosphate could increase malathion toxicity
and their possible consequences. 88–134-fold. Ramakrishna and Ramachandran (1978) also
reported that fenitrothion and parathion could markedly in-
crease the toxicity of malathion.
8. Can non-cholinesterase targets modify cholinergic Malathion is a succinic acid derivative containing a
toxicity? carboxyester linkage. Carboxylester, distributed in various
tissues, efficiently inactivate malathion. Prior blockade of
The review above provides ample evidence for the po- carboxylesterases was shown to be partially responsible for
tential of some cholinesterase inhibitors to interact in a toxicokinetic potentiation of malathion toxicity by various
selective manner with non-cholinesterase targets. Some of agents (Cohen et al., 1972). Thus, non-cholinesterase
these non-cholinesterase actions may modulate cholinergic actions of some cholinesterase inhibitors have been known
or non-cholinergic systems. We hypothesized that some non- for decades to modify cumulative toxicity with multiple
cholinesterase actions can influence cumulative toxicity from cholinesterase inhibitor exposures. Table 2 lists all the
mixed cholinesterase inhibitor exposures. As noted before, anticholinesterases discussed herein along with their uses.
qualitative differences in the expression of OPIDN have
been reported based on the sequence of administration of 9.1. Interactive toxicity of parathion and chlorpyrifos
two esterase inhibitors (Pope and Padilla, 1990; Lotti et al.,
1991). We used this same strategy, i.e., exposure to two We hypothesized that the differential toxicity noted
compounds but in differential sequence of administration, with “equitoxic” dosages of parathion and chlorpyrifos
to probe for potential non-cholinesterase actions of the com- (Chaudhuri et al., 1993; Pope et al., 1995; Liu and Pope,
mon organophosphorus insecticides chlorpyrifos, parathion 1998) was due to non-cholinesterase actions of one or both
or methyl parathion. insecticides, and that such additional actions would influence
Chlorpyrifos and parathion are structural analogs, cumulative toxicity with combined exposures. As noted
derivatives of diethyl thiophosphoric acid. They are both above, chlorpyrifos oxon and paraoxon have differential
activated in vivo to oxons (chlorpyrifos oxon and paraoxon), direct effects on muscarinic autoreceptors (Liu et al., 2002).
potent inhibitors of AChE (Sultatos et al., 1985). Dosages of Chlorpyrifos oxon is also a much better substrate for the
chlorpyrifos or parathion causing similar rates and degrees A-esterase mediated detoxification pathways. Either of these
440 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446

Table 2
Anticholinesterases listed and their uses
Common/chemical/trade name Practical or experimental use
Carbamate inhibitors
Physostigmine (antilirium) Therapeutic (glaucoma)
Neostigmine (prostigmin) Therapeutic (glaucoma, myasthenia gravis)
Pyridostigmine (mestinon) Therapeutic (myasthenia gravis), prophylactic (nerve agent)
Rivastigmine (exelon) Therapeutic (Alzheimer’s)
Organophosphorus inhibitors
Azinphos-methyl (guthion) Insecticide
Chlorpyrifos (dursban) Insecticide
Chlorpyrifos-methyl oxon Metabolite of chlorpyrifos methyl
Chlorpyrifos oxon Metabolite of chlorpyrifos
Cyanofenphos Insecticide
Cyanophos (cyanox) Insecticide
Demeton (systox) Insecticide, acaracide
Diazinon (spectracide) Insecticide
Diazoxon Metabolite of diazinon
Dichlorvos (DDVP) Insecticide, nematocide, fumigant
Dicrotophos (bidrin) Insecticide, acaracide
DFP (diisopropylphosphorofluoridate) Experimental (serine hydroxyl phosphorylation), therapeutic (glaucoma)
Dimethoate (cygon) Insecticide, acarcide
Echothiophate (phospholine) Therapeutic (glaucoma)
EPN (phenyl, O-ethyl, p-nitrophenyl thiophosphonate) Insecticide, acaracide
Fenitrothion (sumithion) Insecticide
Fenthion (baytex) Insecticide
Iso-OMPA (tetraisopropylpyrophosphoramide) Experimental (selective butyrylcholinesterase inhibitor)
Leptophos (phosvel) Insecticide
Malaoxon Metabolite of malathion
Malathion (sumitox) Insecticide, acaracide
Paraoxon Metabolite of parathion
Parathion Insecticide
Parathion-methyl Insecticide
Phenyl saligenin cyclic phosphonate Experimental (selective inducer of OPIDN)
Pirimiphos-ethyl (primicid) Insecticide
Profenofos (curacron) Insecticide
Methamidophos (monitor) Insecticide
Mipafox Experimental (selective NTE inhibitor)
Monocrotophos (azodrin) Insecticide
Salithion Insecticide
Sarin Nerve agent
Soman Nerve agent
Tabun Nerve agent
Tetraethylpyrophosphate First synthetic anticholinesterase
Tribufos (DEF) Herbicide, defoliant
Tri-ortho-cresyl phosphate (TOCP) Neurotoxic isomer of plasticizer, flame retardant
VX (O-ethyl, S-[2-(diisopropylamino)ethyl]methylphosphonothiolate) Nerve agent

non-cholinesterase sites of action/loss could influence cumu- signs of toxicity (SLUD signs, involuntary movements)
lative toxicity with mixed exposures including chlorpyrifos. compared to animals given the same dosages of both
We therefore probed the relevance of possible non- toxicants but with parathion first (Karanth et al., 2001).
cholinesterase actions of chlorpyrifos and parathion by eval- Table 3 shows cumulative lethality in these studies. Rats
uating differential toxicity following sequential exposures. exposed to chlorpyrifos prior to parathion exhibited more
Adult rats were challenged with equitoxic dosages (0.5, extensive lethality than in rats given the identical dosages
0.75 or 1 × LD1 , where LD1 is the estimated dosage required of both toxicants but with their sequence of administration
to elicit 1% lethality) of both parathion (4 mg/kg, p.o.) and reversed. Fig. 2 shows cholinesterase inhibition in frontal
chlorpyrifos (80 mg/kg, p.o.). In some rats, parathion was cortex and diaphragm at 4, 8 or 24 h after the first exposure.
given 4 h prior to chlorpyrifos, while in others the same As shown in this figure, brain and diaphragm
dosages were used but in reverse sequence of administration. cholinesterase inhibition was more extensive at each
Autonomic (SLUD) signs and lethality were recorded for 4 time-point in the animals given chlorpyrifos first. The
days. With all binary combinations of exposures, rats given results suggested that toxicokinetic factors contribute to
chlorpyrifos first exhibited significantly greater functional sequence-dependent differences in toxicity under these
C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446 441

Table 3 calcium was added, and a 14-fold increase in the absence of

Interactive toxicity of chlorpyrifos and parathion calcium (IC50 : no liver = 3.5 nM; liver + calcium = 280 nM;
Dosages (×LD1 )a First exposure 4-Day lethality liver + EGTA, 48 nM). With paraoxon, there was only about
Chlorpyrifos 2/8 a threefold increase in IC50 with added liver, and calcium had
Parathion 0/8 no additional effect (IC50 : no liver, 16.5 nM; liver + calcium,
Chlorpyrifos 3/8 57 nM; liver + EGTA, 56 nM). These results suggested that
Parathion 0/8 both carboxylesterase and A-esterase-mediated detoxifica-
Chlorpyrifos 7/8 tion of chlorpyrifos oxon appeared more robust compared to
1.0 paraoxon. When rats were treated with one insecticide and
Parathion 2/8
a The respective LD1 dosages of chlorpyrifos and parathion were 80 and the ex vivo detoxification of the alternate oxon measured,
4 mg/kg, p.o., respectively. Rats (n = 8 per treatment group) were given one parathion exposure had relatively little effect on hepatic
OP insecticide (first exposure) by gavage in peanut oil (1 ml/kg) 4 h prior detoxification of chlorpyrifos oxon when calcium was
to the second OP and cumulative lethality was recorded for 4 days (from included (IC50 = 225 nM) but carboxylesterase-mediated
Karanth et al., 2001).
detoxification was markedly reduced (IC50 in the absence of
calcium = 7.6 nM). In contrast, when rats were treated with
conditions. It is known that both paraoxon and chlorpyrifos
chlorpyrifos and ex vivo detoxification of paraoxon was eval-
oxon are detoxified by carboxylesterases. In contrast, several
uated, no detoxification capacity was evident (IC50 = 16 nM,
studies suggest that A-esterase mediated detoxification may
with or without calcium). We concluded from these studies
be important in the metabolism of chlorpyrifos but have
that sequence-dependent differences in response to chlor-
little relevant role in the metabolism of parathion. Thus,
pyrifos and parathion were at least partially due to selective
we proposed that prior blockade of carboxylesterase by
differences in the hepatic detoxification of the oxons and
chlorpyrifos would have a more dominant effect on the
the relative importance of blocking carboxylesterases. Other
subsequent metabolism of parathion than the effects of
factors could be important, however. For example, chlor-
parathion pre-exposure on chlorpyrifos metabolism.
pyrifos has been shown to block cytochrome P450-mediated
metabolism of the carbamate anticholinesterase carbaryl in
9.2. Does differential detoxification contribute to
vitro (Tang et al., 2002). Thus, differential alteration of phos-
sequence-dependent toxicity of chlorpyrifos and
phorothioate bioactivation could also potentially contribute
to exposure sequence-dependent differences in response.
To evaluate the role of differential detoxification in
sequence-dependent toxicity, we pretreated rats with one 9.3. Interactive toxicity of chlorpyrifos and methyl
insecticide and then evaluated the ex vivo hepatic detoxifica- parathion
tion of the alternate oxon using an indirect inhibition assay
(Karanth et al., 2001) similar to that reported by Chambers Carboxylesterases are thought to play a lesser role in the
et al. (1990) and Chanda et al. (1997). Briefly, the rat was detoxification of methyl parathion than with either parathion
treated with either parathion or chlorpyrifos, and the liver or chlorpyrifos (Chambers and Carr, 1993). We therefore
was removed 4 h later to evaluate oxon detoxification. proposed that if prior blockade of carboxylesterase-mediated
Using livers from un-treated rats, there was an approxi- detoxification of paraoxon by chlorpyrifos contributed to
mate 80-fold increase in the IC50 of chlorpyrifos oxon when the sequence-dependent toxicity, lesser sequence-dependent

Fig. 2. Interactive toxicity of chlorpyrifos and parathion in adult male rats. Rats were given one OP insecticide (either chlorpyrifos or parathion) 4 h prior to
challenge with “equitoxic” dosage of the other (0.5 × LD1 ; 40 mg/kg CPF, 2 mg/kg PS). Cortical and diaphragm cholinesterase activity was measured by the
radiometric method of Johnson and Russell (1975) using [3 H]acetylcholine as the substrate (1 mM). Cholinesterase activity was significantly lower in rats
pre-exposed to chlorpyrifos at all time-points evaluated.
442 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446

Table 4 or methyl parathion. In contrast, pharmacodynamic factors

Interactive toxicity of chlorpyrifos and methyl parathion may also contribute, e.g., differential effects on acetylcholine
Dosages (×LD1 )a First exposure 4-Day lethality release (Liu et al., 2002). Regardless of the mechanism(s)
Chlorpyrifos 1/8 leading to differences in toxic response with sequential ex-
Methyl parathion 0/8 posures, these studies using a limited number of compounds
Chlorpyrifos 8/8 suggest a departure from additivity with mixed exposures.
Methyl parathion 0/8 While the dosages used were relatively high, studies with
a The respective LD dosages of chlorpyrifos and methyl parathion were
1 lower dosages (0.5 × LD1 ) also suggested non-additive
80 and 4 mg/kg, p.o., respectively. Rats (n = 8 per treatment group) were interactions when based on cholinesterase inhibition. While
given one OP insecticide (First exposure) by gavage in peanut oil (1 ml/kg) cholinesterase inhibiting pesticides all share a common
4 h prior to the second OP and cumulative lethality was recorded for 4 days
mechanism of toxicity, additional non-cholinesterase targets
(from Karanth et al., 2004).
of some anticholinesterases may selectively alter cumulative
toxicity. Knowledge of such non-cholinesterase actions will
differences in toxicity would be noted with combined improve the overall estimate of cumulative toxicity when
chlorpyrifos and methyl parathion dosing (Karanth et al., exposures to selected anticholinesterases are anticipated.
2004). Rats were given sequential dosing as above (either 0.5
or 1 × the LD1 , i.e., 4 mg/kg methyl parathion and 80 mg/kg,
chlorpyrifos, p.o.), with 4 h separating the two treatments
as before. Functional toxicity and cumulative lethality were
recorded. Table 4 shows cumulative lethality with interactive 10. Conclusions
As shown in this table, lethality was much more extensive The therapeutic and toxic actions of cholinesterase in-
in rats given chlorpyrifos before methyl parathion than hibitors are based on inhibition of the enzyme AChE, with
in those rats given identical dosages of both but with the consequent accumulation of synaptic acetylcholine levels and
sequence reversed. Functional signs of cholinergic toxicity enhanced stimulation of cholinergic receptors in the cen-
were also markedly greater in those rats given chlorpyrifos tral and/or peripheral nervous systems. In clinical conditions
prior to methyl parathion (data not shown). Fig. 3 shows exhibiting reduced activation of cholinergic receptors, e.g.,
cholinesterase inhibition in cortex and diaphragm with myasthenia gravis, such effects can be therapeutically ad-
interactive chlorpyrifos and methyl parathion exposures. vantageous. In contrast, under normal conditions the over-
As noted in the interactive parathion–chlorpyrifos studies, stimulation of cholinergic receptors leads to an imbalance in
cholinesterase inhibition was also more extensive in rats neurotransmission and signs of cholinergic toxicity. While
pre-exposed to chlorpyrifos and then given methyl parathion this basic common mechanism is employed in both thera-
compared to animals given the reverse sequence of ad- peutic and toxic uses, considerable information suggests that
ministration. Thus, this study suggested that other factors some organophosphorus and carbamate anticholinesterases
besides differential detoxification by carboxylesterases may also bind to other macromolecular targets that can poten-
be important in the sequence-dependent toxicity of OP in- tially modify the consequences of AChE inhibition. Such
secticides. For example, parathion and methyl parathion pre- non-cholinesterase actions may influence the cholinesterase
exposure may inhibit the activation of chlorpyrifos to its oxon inhibitor’s own toxicity as well as cumulative toxicity of
more than chlorpyrifos can inhibit the activation of parathion mixed exposures.

Fig. 3. Interactive toxicity of chlorpyrifos and methyl parathion in adult male rats. Rats were given one OP insecticide (either chlorpyrifos or parathion) 4 h
prior to challenge with “equitoxic” dosage of the other (0.5 × LD1 ; 40 mg/kg CPF, 2 mg/kg MPS). Cortical and diaphragm cholinesterase activity was measured
by the radiometric method of Johnson and Russell (1975) using [3 H]acetylcholine as the substrate (1 mM). Cholinesterase activity was significantly lower in
rats pre-exposed to chlorpyrifos at all time-points evaluated.
C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446 443

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