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

Abstract

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

1382-6689/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.etap.2004.12.048
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
This cascade of cellular events elicited by AChE inhibi-
tion has been used therapeutically for a number of clinical
conditions (Taylor, 2001). As already mentioned above, the
efficacy of physostigmine for treatment of glaucoma was
realized in the late 19th century. Physostigmine is still used
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
pyridostigmine for decades. Stimulation of the intestinal
and urinary bladder musculature, which can often become
flaccid following surgery, is another use of cholinesterase
inhibitors. A series of cholinesterase inhibitors including
tacrine, donepezil galantamine and rivastigmine have been
approved for treatment of Alzheimer’s disease. In all cases,
the therapeutic strategy is to increase the persistence of
synaptic acetylcholine by blockade of its degradation, such
that there is a net increase in cholinergic receptor activation.
This overall strategy is based on a clinical condition wherein
activation of cholinergic receptors is deficient. Thus, in-
creasing the residence of acetylcholine molecules within
synapses by inhibiting AChE can at least partially counteract
a deficiency in either the release of neurotransmitter or a
reduction in cholinergic receptors/signaling.
Paradoxically, a cholinesterase inhibitor has been used
prophylactically to protect against the toxicity of other
cholinesterase inhibitors. In this regard, the drug used for
treating myasthenia gravis, pyridostigmine, is used by the US
military to protect against potential exposures to organophos-
phorus nerve agents (Gunderson et al., 1992; Lee, 1997).
While use of one cholinesterase inhibitor to protect from the
toxicity of another at first seems contradictory, the rationale
for this therapeutic strategy resides in the differential duration
of inhibition between the two types of inhibitors. Basically,
the inhibition of AChE by pyridostigmine is much shorter
in duration than that following organophosphate exposure,
in particular if the organophosphorus inhibitor (e.g., soman)
is capable of rapidly aging. If some proportion of AChE
molecules is inhibited by pyridostigmine when exposure to
such an organophosphate occurs, those same molecules will
be protected from irreversible inactivation by the organophos-
phate. Because of the high reactivity of the organophosphorus
inhibitors, by the time the enzymes recover from inhibition
by pyridostigmine, few residual organophosphate molecules
remain in the circulation for possible enzyme re-inhibition.
Used in conjunction with standard therapies to pre-
vent cholinergic receptor activation (antimuscarinics) and
reactivation of phosphorylated enzymes (aldoximes), pyri-
dostigmine can markedly enhance survival and long-term
outcome from organophosphate poisoning (Xia et al., 1981;
Patocka et al., 1991). In contrast to the other pharmacolog-
ical uses of cholinesterase inhibitors, however, the use of
pyridostigmine in this context is not based on increasing
synaptic acetylcholine levels by disrupting neurotransmit-
ter breakdown. As a nerve agent antidote, the basis for
protection against nerve agent intoxication is the short-
term carbamylation of a proportion of AChE molecules,
435
preventing irreversible inactivation of those molecules by an
organophosphate. Carbamylated enzymes recover faster, and
therefore as combined therapy with an anticholinergic and
enzyme reactivator, synaptic function can be restored without
requirement for de novo synthesis of new AChE molecules.
4. Overview of the toxicology of cholinesterase
inhibitors
The mechanism of toxicity for cholinesterase inhibitors is
essentially the same as the mechanism of action for thera-
peutic uses. Inhibition of AChE disrupts the dynamic inter-
play between acetylcholine synthesis, release and degrada-
tion. This leads to a net accumulation of synaptic ACh levels
with persistent/prolonged activation of cholinergic receptors
on postsynaptic cells. This relative increase in cholinergic
signaling leads to functional signs and symptoms of cholin-
ergic toxicity (Ecobichon, 2001).
While all cholinesterase inhibitors are thought to elicit
toxicity through this general mechanism, the spectrum of
cholinergic signs can be different with different inhibitors,
the same inhibitor in different species, or the same inhibitor
with different routes of exposure. Generally, however, one
or more of the “classic” signs of cholinergic toxicity will
be evident following exposure to any inhibitor that leads to
substantial inhibition of tissue AChE activity. The biological
responses to cholinesterase inhibitors are predicated upon
the distribution and role of cholinergic neurons within
the central and peripheral nervous systems. Acetylcholine
is the transmitter at pre-ganglionic terminals of both
parasympathetic and sympathetic nerves, postganglionic
parasympathetic terminals, neuromuscular junction of
striated muscle and at various synapses within the central
nervous system. Thus, AChE inhibition leads to a relative
imbalance of cholinergic transmission at these sites with
elicitation of predictable functional alterations.
One of the most recognized forms of acute toxicity
following exposure to cholinesterase inhibitors is auto-
nomic dysfunction, characterized by excessive secretions at
parasympathetic end organs (Ecobichon, 2001). Excessive
stimulation of secretory organs leads to increased secretions
at bronchial, lacrimal, salivary, sweat, intestinal and pancre-
atic sites. Smooth muscles of the ureter also exhibit increased
contractions leading to enhanced urination (Taylor, 2001).
Together, these responses leading to excessive secretions
at multiple sites are a characteristic of cholinergic toxicity.
The acronym SLUD, standing for salivation, lacrimation,
urination and defecation, is often used to refer to these types
of autonomic signs of toxicity.
Loss of regulation at the neuromuscular junction leads to
another classic sign of exposure to cholinesterase inhibitors,
involuntary movements. The residence of acetylcholine
released at the neuromuscular junction is generally limited
by the action of AChE such that a single nerve impulse
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,
acetylcholine molecules can bind multiple nicotinic recep-
tors, prolonging the decay time of the end-plate potential
and disrupting the synchrony between end-plate potentials
and muscle action potentials. Muscle fibrillations and
fasciculations result, and complete muscle paralysis can be
noted due to end-plate depolarization.
Ocular effects can also be commonly noted following
exposure to cholinesterase inhibitors (Rengstorff, 1994;
Moser, 1995). Miosis is a classic sign of cholinergic toxicity
resulting from constriction of the sphincter pupillae muscle
surrounding the iris. In fact, miosis is thought to be the
most sensitive indicator of aerosol exposures to some of
the most potent cholinesterase inhibitors, e.g., soman (Soli
et al., 1980; Benschop et al., 1998). Constriction of the
ciliary muscle, which blocks the accommodation reflex, also
increases outflow of aqueous humor and is the mechanism
of action for cholinesterase inhibitors for treatment of glau-
coma mentioned above. Because of the wide distribution of
cholinergic nerves, a host of other signs and symptoms can be
associated with cholinesterase inhibitors. Cardiac effects can
be prominent and complex (Stemler et al., 1990; Yamamoto
et al., 1996; Karalliedde, 1999). Generally, bradycardia is
first observed due to negative chronotropic effects of vagal
muscarinic input. However, direct effects on the medullary
vasomotor center as well as stimulation of ganglionic
nicotinic receptors can influence cardiac function. Other
common signs of intoxication of cholinesterase inhibitors
include seizures and convulsions, hypothermia, increased
excitability, dyspnea and others. Respiratory failure, through
a combination of excessive airway secretions, paralysis of
muscles of ventilation, and depression of the respiratory
control centers in the pons-medulla is generally considered
the primary cause of death following lethal exposures.
5. A common mechanism of toxicity for
cholinesterase inhibitors
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-
els to accumulate within the synapse, followed by enhanced
stimulation of cholinergic receptors and associated changes
in postsynaptic cell function. In 1996, the U.S. Congress
passed the Food Quality Protection Act, which modified ex-
isting standards for pesticide evaluation and registration. An
important component of that law was the requirement that
EPA consider “. . . information concerning the cumulative
effects of such residues and other substances that have a com-
mon mechanism of toxicity, in establishing, modifying, leav-
ing in effect or revoking a tolerance for a pesticide . . .” in the
re-registration of pesticides. In essence, if two or more pesti-
cides were considered to act through a common mechanism,
then cumulative effects of co-exposure to those pesticides
would have to be considered in the evaluation of tolerances.
A working group of experts from academia, government and
industry concluded that organophosphorus pesticides that
work by “phosphorylating AChE and eliciting a spectrum
of cholinergic effects” indeed share a common mechanism
of toxicity (Mileson et al., 1998). Risk assessment methods
to evaluate cumulative toxicity of cholinesterase inhibitors
have subsequently been developed, and a revised cumula-
tive risk assessment of organophosphorus pesticides was re-
ported in 2002 (http://www.epa.gov/pesticides/cumulative/).
The approach for estimating cumulative risk of OP insecti-
cides involves the use of a toxic equivalency factor, in this
case anticholinesterase potency relative to that of an index in-
hibitor, methamidophos, to quantify relative toxicity of given
cholinesterase inhibitors. Potencies were based on a bench-
mark dose causing 10% cholinesterase inhibition. Further-
more, the cumulative risk assessment for OPs is “based on
the assumption of dose additivity”.
It should be noted, that in the introduction to the revised
risk assessment, it is stated “EPA has developed a frame-
work for conducting cumulative risk assessments for the
organophosphates (OPs) and other pesticides that have a com-
mon mechanism of toxicity (i.e., that act in the same way in
the body)”. The concept of common mechanism for these
toxicants is not new. It was recognized decades ago that “. . .
organophosphate insecticides . . . all, in sufficient doses, in-
hibit AChE in vivo and thus share a common mechanism of
acute toxic action” (Murphy, 1980). A possible distinction of
importance resides in the apparent “word-splitting” between
common and same mechanism, however. While it is easy
to argue that cholinesterase inhibitors share a mechanism in
common, one can also argue that there is ample evidence to
indicate they do not all “act in the same way”. We propose
that while anticholinesterases share a common mechanism,
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
inhibit AChE, these additional sites of action may directly
influence anticholinesterase potency or cholinergic signaling
and thus modify the cumulative effects of mixed exposures.
6. Non-cholinesterase targets of anticholinesterases
6.1. Other enzymes as targets
Other enzymes often associated with OP and carbamate
cholinesterase inhibitors are various esterases including
neurotoxic (or neuropathy target) esterase (NTE), butyryl-
cholinesterase, carboxylesterases, and A-esterases, e.g.,
paraoxonase or PON-1. Inhibition and subsequent “aging”
of NTE is correlated with initiation of organophosphorus-
induced delayed neurotoxicity (OPIDN, Johnson, 1970).
NTE inhibitors that are incapable of aging have no neu-
ropathological capacity of their own but can block delayed
neurotoxicity when given prior to OP dosing (Johnson,
1980). Some of these “protective” agents were subsequently
 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446
shown to potentiate or promote delayed neurotoxicity when
given after a neuropathic OP (Pope and Padilla, 1990; Lotti
et al., 1991; Lotti and Moretto, 1999). Protection afforded
by pre-exposure was correlated with NTE inhibition.
Potentiation of OPIDN by post-treatment does not, however,
appear to involve NTE (Pope et al., 1993), but may involve
another related esterase referred to as M200 (Moretto et al.,
2001; Lotti, 2002).
Many cholinesterase inhibitors are inactivated by direct
interaction with esterases, e.g., carboxylesterases and A-
esterases (Clement, 1984; Pond et al., 1995; Maxwell and
Brecht, 2001; Chemnitius et al., 1983; Walker and Mackness,
1987; Li et al., 2000). While carboxylesterase or A-esterase
detoxification can affect the toxicity of individual inhibitors,
cumulative toxicity of combined exposures may also be in-
fluenced by these detoxifying enzymes in a complex manner
(Frawley et al., 1957; Murphy et al., 1959; Karanth et al.,
2001).
Some anticholinesterases can bind to and inhibit a num-
ber of other enzymes. O,O -Diisopropylphosphorofluoridate
(DFP) is a well-known protease inhibitor used in protein iso-
lation procedures (Scopes, 1982). Chymotrypsin is inhibited
by a number of OP anticholinesterases (Hamilton et al., 1975;
Johnson and Clothier, 1980). Various liver proteases have
been reported sensitive to a number of anticholinesterases
(Mantle et al., 1997; Saleem et al., 1998).
Evaluation of 20 OP inhibitors on the activity of AChE,
NTE, trypsin, and an enzyme involved in mitogen-induced
activation of T lymphocytes indicated a range of differ-
ential selectivity towards inhibition of the four enzymes
(Pruett et al., 1994). Profenofos, tribufos and phenyl sali-
genin cyclic phosphonate inhibited thrombin, trypsin and
elastase, but at higher concentrations than needed to inhibit
AChE (Quistad and Casida, 2000). Under some conditions,
soman and parathion inhibit choline acetyltransferase (ChAT;
Murumatsu and Kuriyama, 1976; Thompson and Thomas,
1985). Sivam et al. (1984) reported, however, that neither
DFP, tabun, sarin or soman had any effect on brain regional
ChAT activity.
Kynurenine formamidase, an enzyme involved in
the metabolism of l-tryptophan, is inhibited by some
organophosphorus and carbamate cholinesterase inhibitors
(Seifert and Casida, 1978; Henderson and Kitos, 1982; Seifert
and Pewnim, 1992; Pewnim and Seifert, 1993). Different
metabolites of kynurenine have been reported to be neuro-
protective (kynurenic acid, Stone, 2000) and neurotoxic (3-
hydroxykynurenine and quinolinic acid, Okuda et al., 1998;
Vasquez et al., 2000). Pirimiphos-ethyl (20 mg/kg) and di-
azinon (10 mg/kg) almost completely inhibited (>90%) liver
kynurenine formamidase in mice (Seifert and Pewnim, 1992;
Pewnim and Seifert, 1993).
Fatty acid amide hydrolase (FAAH) is involved in the
metabolism of the endocannabinoid, anandamide and is
inhibited by a number of cholinesterase inhibitors (Quistad et
al., 2001). Chlorpyrifos oxon was a potent inhibitor of FAAH
activity in mouse brain and liver (IC50 values = 40–56 nM,
437
Quistad et al., 2001). Brain FAAH was inhibited by profeno-
fos and tribufos in vivo at levels of exposure lacking cholin-
ergic toxicity. Anandamide modulates the release of acetyl-
choline (Gifford et al., 1999) and muscarinic receptor acti-
vation increases secretion of anandamide (Kim et al., 2002),
thus selective inhibition of FAAH by some cholinesterase
inhibitors could potentially modulate cholinergic toxicity.
Acyl peptide hydrolase (APH) removes N-terminally
acetylated peptides from polypeptides (Farries et al., 1991;
Fujino et al., 2000). DFP phosphorylates a serine residue
on APH (Scaloni et al., 1992). Methyl chlorpyrifos oxon,
dichlorvos and DFP are potent inhibitors of APH (IC50 val-
ues from 18 to 199 nM), with in vitro potencies 6–10 times
those for inhibiting AChE (Richards et al., 2000).
Chlorpyrifos oxon (50 ␮M) activated extracellular signal-
regulated kinases (ERK) two to three-fold in Chinese hamster
ovary cells (Bomser and Casida, 2000). Increased ERK ac-
tivity may be the result of inhibition of diacylglycerol lipase
activity (Bomser et al., 2002). Such actions could affect sig-
naling pathways of a variety of growth factors and mitogens.
6.2. Cholinergic receptors as macromolecular targets of
cholinesterase inhibitors
It has been known for decades that some cholinesterase
inhibitors directly interact with cholinergic receptors
(Frederickson, 1958). DFP, phospholine and paraoxon
acted as reversible antagonists on nicotinic receptors in
Electrophorus cells (Bartles and Nachmansohn, 1969).
Azinphos-methyl, dichlorvos, dicrotophos, monocrotophos,
echothiophate and VX all bound to neuromuscular nicotinic
receptors of the Torpedo electric organ (Eldefrawi and
Eldefrawi, 1983; Bakry et al., 1988).
Some carbamate cholinesterase inhibitors interact
directly with nicotinic receptors (Seifert and Eldefrawi,
1974). Physostigmine potentiated acetylcholine-induced
ion flux at low concentrations (1–10 ␮M) but blocked
acetylcholine actions at higher concentrations (Zwart et al.,
2000). Physostigmine binds to nicotinic receptors at a site
distinct from the agonist binding site (Albuquerque et al.,
1985). Pyridostigmine and neostigmine also bound to an
allosteric site on the nicotinic receptor, but at higher concen-
trations than noted with physostigmine (Akaike et al., 1984;
Albuquerque et al., 1984). It was also noted that physostig-
mine could activate nicotinic receptors even following prior
receptor desensitization to agonist (Kuhlmann et al., 1991).
[3 H]Phencyclidine (PCP) binding to the nicotinic channel
can be used to evaluate both activation and agonist-induced
desensitization (Eldefrawi et al., 1980). Several OP and
carbamate anticholinesterases modify PCP (or [3 H]thienyl-
cyclohexylpiperidine [TCP], an analog of phencyclidine)
binding to nicotinic receptors (Mansour et al., 1987;
Eldefrawi et al., 1988; Katz et al., 1997).
A number of studies have evaluated effects of anti-
cholinesterases on muscarinic receptor binding. Dichlor-
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-
clidinyl benzilate (QNB) at low concentrations (5–50 nM,
Volpe et al., 1985). Paraoxon blocked QNB binding to
M2 and M3 subtypes of receptors at extremely low levels
(as low as 10−15 M, Katz and Marquis, 1989). Echothio-
phate, paraoxon, VX, soman and tabun all blocked [3 H]cis-
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
et al., 1991). Paraoxon and malaoxon blocked CD bind-
ing in rat hippocampus and cortex in vitro (Ward et al.,
1993). Chlorpyrifos oxon inhibited CD binding in rat striatum
(IC50 = 22 nM; Huff et al., 1994). Paraoxon, malaoxon and
chlorpyrifos oxon inhibited CD binding in a concentration-
dependent manner at sub-micromolar concentrations (po-
tencies: chlorpyrifos oxon > paraoxon > malaoxon; Ward and
Mundy, 1996).
CD preferentially labels the M2 receptor subtype (Huff
and Abou-Donia, 1994). Chlorpyrifos oxon diethylphos-
phorylated the rat cardiac M2 muscarinic receptor in vitro
(Bomser and Casida, 2001). Chlorpyrifos oxon, paraoxon
and methyl paraoxon displaced [3 H]oxotremorine-M bind-
ing to rat cardiac membrane muscarinic receptors (Howard
and Pope, 2002), with chlorpyrifos oxon being most potent
(IC50 = 7 nM). Presynaptic muscarinic autoreceptors in the
mammalian brain are primarily the M2 subtype and can in-
hibit acetylcholine release (Weiler, 1989; Feuerstein et al.,
1992; Kitaichi et al., 1999). Selective actions at these recep-
tors could modulate regulation of acetylcholine release and
play a role in selective toxicity (Liu et al., 2002).
Paraoxon (<1 ␮M) enhanced GABA and glutamate release
in primary neurons (Rocha et al., 1996a). Acetylcholine had
no effect in this system, suggesting that paraoxon did not act
through AChE inhibition. VX was more potent than paraoxon
Table 1
Additional targets for anticholinesterases and known or possible consequences of their interaction
Target
Acetylcholinesterase
Acyl peptide hydrolase
Adenylyl cyclase
Butyrylcholinesterase
Cannabinoid receptors
Carboxylesterase
Cardiac adrenergic receptors
Choline acetyltransferase
Choline transport
Cholinergic autoreceptors
Diacylglycerol kinase
Fatty acid amide hydrolase
Kynurenine formamidase
M-200
Muscarinic receptors
Neurotoxic esterase
Na+ ,K+ -ATPase alpha 1
Nicotinic receptors
NMDA receptors
Proteases
at increasing neurotransmitter release, while soman had no
effect (Rocha et al., 1996b). The effects of VX on GABA re-
lease were blocked by atropine (Rocha et al., 1999). Sarin
(0.1–3 nM) blocked the stimulated GABA release in hip-
pocampal slices independent of AChE inhibition (Chebabo
et al., 1999).
7. Other actions of cholinesterase inhibitors
The above studies suggest that both muscarinic and
nicotinic receptor subtypes may be sensitive to direct
binding by a number of anticholinesterases. Other types
of neurotransmitter receptors are also sensitive to some
cholinesterase inhibitors. Cyanofenphos, leptophos, salithion
and tri-ortho-cresyl phosphate (TOCP) blocked cardiac
[3 H]norepinephrine binding in vitro with potencies similar to
that of propranolol (El-Sebae et al., 1981). DFP, dichlorvos,
cyanophos and mipafox inhibited 3-((+)-2-carboxypiperazin-
4-yl)-[1,2-3 H]propyl-1-phosphonic acid ([3 H]CPP) binding
to the NMDA receptor in rat brain (Johnson and Michaelis,
1992). In contrast, binding to other NMDA ligands including
[3 H]AMPA, [3 H]glycine and [3 H]TCP was not affected by
any of the cholinesterase inhibitors. Some OP inhibitors
are relatively potent inhibitors of GABAA receptor binding
to t-[35 S]butyl-bicyclophophorothionate in membranes
from Torpedo californica (Gant et al., 1987). Chlorpyrifos
oxon and methyl chlorpyrifos oxon were potent inhibitors
(IC50 = 14 and 64 nM, respectively) of in vitro binding
to a cannabinoid CB1 receptor subtype ligand ([3 H]CP
55,490) in mouse brain membranes whereas other inhibitors
(paraoxon, diazoxon and dichlorvos) were markedly less
potent (IC50 = 1.2–4.2 ␮M, Quistad et al., 2002). Tribufos
exposure (50 mg/kg) inhibited [3 H]CP 55,490 binding to
Known or possible effects
Cholinergic toxicity, neurodevelopmental deficits
Changes in peptide/neuropeptide turnover
Altered transduction for a variety of extracellular signals
Altered cholinergic neurotransmission, neurodevelopmental deficits
Changes in neuromodulation
Modulation of toxicity with combined exposures
Modulation of heart function, arrythmia
Alteration of choline utilization, acetylcholine synthesis
Alteration of acetylcholine synthesis
Modulation of acetylcholine release
Altered growth factor/mitogen signaling
Changes in neuroactive amide (e.g., endocannabinoid) metabolism
Modulation of l-tryptophan metabolism, production of NAD+
Exacerbation of delayed neurotoxicity
Variety of central/peripheral neurochemical changes
Delayed neurotoxicity
Arrythmias
Variety of central/peripheral neurochemical changes
Excitotoxicity, altered neuromodulation
Alteration of protein turnover, modulation of clotting
 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446
cannabinoid receptors in mouse brain in an apparently
irreversible manner. Together, these findings suggest that a
number of non-cholinergic neurotransmitter receptors may
be targeted by anticholinesterases.
As noted above, changes in cardiac function often
occur following exposure to cholinesterase inhibitors.
Cardiotoxicity from exposure to some cholinesterase
inhibitors may occur independent of AChE inhibition.
Dimethoate caused cardiac failure in guinea pigs, apparently
unrelated to AChE inhibition (Marosi et al., 1985). VX
elicited delayed after-depolarizations in guinea pig ventricle
while carbachol and physostigmine had no effect under
similar conditions (Corbier and Robineau, 1989). Inhibition
of the cardiac specific alpha-1 Na+ /K+ -ATPase isoform
may contribute to VX-induced cardiotoxicity (Robineau
et al., 1991). Oral dosing with diazinon or fenthion led
to cholinergic toxicity and extensive AChE inhibition, but
intravenous exposure to these same inhibitors elicited tonic
convulsions and opisthotonus resistant to atropine and only
slight AChE inhibition (Takahashi et al., 1991). A number
of reports suggest that some cholinesterase inhibitors can
influence neurite extension and neurodevelopment (Dupree
and Bigbee, 1994; Campbell et al., 1997; Dam et al.,
1998; Sternfeld et al., 1998; Bigbee et al., 1999; Das and
Barone, 1999). A recent study suggests that chlorpyrifos
and chlorpyrifos oxon potently increase the phosphorylation
of the transcription factor Ca2+ /cAMP response element
binding protein (CREB), potentially influencing a number of
neurodevelopmental pathways (Schuh et al., 2002). Table 1
lists a number of additional targets for anticholinesterases
and their possible consequences.
8. Can non-cholinesterase targets modify cholinergic
toxicity?
The review above provides ample evidence for the po-
tential of some cholinesterase inhibitors to interact in a
selective manner with non-cholinesterase targets. Some of
these non-cholinesterase actions may modulate cholinergic
or non-cholinergic systems. We hypothesized that some non-
cholinesterase actions can influence cumulative toxicity from
mixed cholinesterase inhibitor exposures. As noted before,
qualitative differences in the expression of OPIDN have
been reported based on the sequence of administration of
two esterase inhibitors (Pope and Padilla, 1990; Lotti et al.,
1991). We used this same strategy, i.e., exposure to two
compounds but in differential sequence of administration,
to probe for potential non-cholinesterase actions of the com-
mon organophosphorus insecticides chlorpyrifos, parathion
or methyl parathion.
Chlorpyrifos and parathion are structural analogs,
derivatives of diethyl thiophosphoric acid. They are both
activated in vivo to oxons (chlorpyrifos oxon and paraoxon),
potent inhibitors of AChE (Sultatos et al., 1985). Dosages of
chlorpyrifos or parathion causing similar rates and degrees
439
of cholinesterase inhibition elicited differential signs of
cholinergic toxicity (Chaudhuri et al., 1993; Pope et al.,
1995; Liu and Pope, 1998). In essence, parathion elicited
more extensive signs of toxicity in the presence of similar
changes in cholinesterase activity. We hypothesized that
non-cholinesterase actions must be responsible for the dif-
ferential toxicity of parathion and chlorpyrifos. Muscarinic
autoreceptor function, important in the feedback inhibition of
acetylcholine release (Weiler, 1989; Feuerstein et al., 1992),
was affected in an OP-selective manner in vivo (Pope et al.,
1995; Liu and Pope, 1998). Furthermore, paraoxon and chlor-
pyrifos oxon had qualitatively different actions on muscarinic
autoreceptor function in vitro (Liu et al., 2002). These selec-
tive non-cholinesterase actions were proposed to contribute
to the differential toxicity noted with these two insecticides
with dosages causing similar changes in AChE activity.
9. Can non-cholinesterase targets modify cumulative
cholinergic toxicity?
The interactive toxicity of cholinesterase inhibitors has
been studied for almost half a century. A number of ear-
lier studies noted marked potentiation of the toxicity of
the organophosphorus insecticide malathion following pre-
exposure to other inhibitors. Frawley et al. (1957) reported
that the toxicity of malathion was increased approximately
10-fold in rats and 50-fold in dogs by pre-treatment with
the OP insecticide, EPN. Murphy et al. (1959) reported that
tri-ortho-cresyl phosphate could increase malathion toxicity
88–134-fold. Ramakrishna and Ramachandran (1978) also
reported that fenitrothion and parathion could markedly in-
crease the toxicity of malathion.
Malathion is a succinic acid derivative containing a
carboxyester linkage. Carboxylester, distributed in various
tissues, efficiently inactivate malathion. Prior blockade of
carboxylesterases was shown to be partially responsible for
toxicokinetic potentiation of malathion toxicity by various
agents (Cohen et al., 1972). Thus, non-cholinesterase
actions of some cholinesterase inhibitors have been known
for decades to modify cumulative toxicity with multiple
cholinesterase inhibitor exposures. Table 2 lists all the
anticholinesterases discussed herein along with their uses.
9.1. Interactive toxicity of parathion and chlorpyrifos
We hypothesized that the differential toxicity noted
with “equitoxic” dosages of parathion and chlorpyrifos
(Chaudhuri et al., 1993; Pope et al., 1995; Liu and Pope,
1998) was due to non-cholinesterase actions of one or both
insecticides, and that such additional actions would influence
cumulative toxicity with combined exposures. As noted
above, chlorpyrifos oxon and paraoxon have differential
direct effects on muscarinic autoreceptors (Liu et al., 2002).
Chlorpyrifos oxon is also a much better substrate for the
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-
lative toxicity with mixed exposures including chlorpyrifos.
We therefore probed the relevance of possible non-
cholinesterase actions of chlorpyrifos and parathion by eval-
uating differential toxicity following sequential exposures.
Adult rats were challenged with equitoxic dosages (0.5,
0.75 or 1 × LD1 , where LD1 is the estimated dosage required
to elicit 1% lethality) of both parathion (4 mg/kg, p.o.) and
chlorpyrifos (80 mg/kg, p.o.). In some rats, parathion was
given 4 h prior to chlorpyrifos, while in others the same
dosages were used but in reverse sequence of administration.
Autonomic (SLUD) signs and lethality were recorded for 4
days. With all binary combinations of exposures, rats given
chlorpyrifos first exhibited significantly greater functional
signs of toxicity (SLUD signs, involuntary movements)
compared to animals given the same dosages of both
toxicants but with parathion first (Karanth et al., 2001).
Table 3 shows cumulative lethality in these studies. Rats
exposed to chlorpyrifos prior to parathion exhibited more
extensive lethality than in rats given the identical dosages
of both toxicants but with their sequence of administration
reversed. Fig. 2 shows cholinesterase inhibition in frontal
cortex and diaphragm at 4, 8 or 24 h after the first exposure.
As shown in this figure, brain and diaphragm
cholinesterase inhibition was more extensive at each
time-point in the animals given chlorpyrifos first. The
results suggested that toxicokinetic factors contribute to
sequence-dependent differences in toxicity under these
 C. Pope et al. / Environmental Toxicology and Pharmacology 19 (2005) 433–446
Table 3
Interactive toxicity of chlorpyrifos and parathion
Dosages (×LD1 )a First exposure 4-Day lethality
Chlorpyrifos 2/8
0.5
Parathion 0/8
Chlorpyrifos 3/8
0.75
Parathion 0/8
Chlorpyrifos 7/8
1.0
Parathion 2/8
a The respective LD1 dosages of chlorpyrifos and parathion were 80 and
4 mg/kg, p.o., respectively. Rats (n = 8 per treatment group) were given one
OP insecticide (first exposure) by gavage in peanut oil (1 ml/kg) 4 h prior
to the second OP and cumulative lethality was recorded for 4 days (from
Karanth et al., 2001).
conditions. It is known that both paraoxon and chlorpyrifos
oxon are detoxified by carboxylesterases. In contrast, several
studies suggest that A-esterase mediated detoxification may
be important in the metabolism of chlorpyrifos but have
little relevant role in the metabolism of parathion. Thus,
we proposed that prior blockade of carboxylesterase by
chlorpyrifos would have a more dominant effect on the
subsequent metabolism of parathion than the effects of
parathion pre-exposure on chlorpyrifos metabolism.
9.2. Does differential detoxification contribute to
sequence-dependent toxicity of chlorpyrifos and
parathion?
To evaluate the role of differential detoxification in
sequence-dependent toxicity, we pretreated rats with one
insecticide and then evaluated the ex vivo hepatic detoxifica-
tion of the alternate oxon using an indirect inhibition assay
(Karanth et al., 2001) similar to that reported by Chambers
et al. (1990) and Chanda et al. (1997). Briefly, the rat was
treated with either parathion or chlorpyrifos, and the liver
was removed 4 h later to evaluate oxon detoxification.
Using livers from un-treated rats, there was an approxi-
mate 80-fold increase in the IC50 of chlorpyrifos oxon when
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.
441
calcium was added, and a 14-fold increase in the absence of
calcium (IC50 : no liver = 3.5 nM; liver + calcium = 280 nM;
liver + EGTA, 48 nM). With paraoxon, there was only about
a threefold increase in IC50 with added liver, and calcium had
no additional effect (IC50 : no liver, 16.5 nM; liver + calcium,
57 nM; liver + EGTA, 56 nM). These results suggested that
both carboxylesterase and A-esterase-mediated detoxifica-
tion of chlorpyrifos oxon appeared more robust compared to
paraoxon. When rats were treated with one insecticide and
the ex vivo detoxification of the alternate oxon measured,
parathion exposure had relatively little effect on hepatic
detoxification of chlorpyrifos oxon when calcium was
included (IC50 = 225 nM) but carboxylesterase-mediated
detoxification was markedly reduced (IC50 in the absence of
calcium = 7.6 nM). In contrast, when rats were treated with
chlorpyrifos and ex vivo detoxification of paraoxon was eval-
uated, no detoxification capacity was evident (IC50 = 16 nM,
with or without calcium). We concluded from these studies
that sequence-dependent differences in response to chlor-
pyrifos and parathion were at least partially due to selective
differences in the hepatic detoxification of the oxons and
the relative importance of blocking carboxylesterases. Other
factors could be important, however. For example, chlor-
pyrifos has been shown to block cytochrome P450-mediated
metabolism of the carbamate anticholinesterase carbaryl in
vitro (Tang et al., 2002). Thus, differential alteration of phos-
phorothioate bioactivation could also potentially contribute
to exposure sequence-dependent differences in response.
9.3. Interactive toxicity of chlorpyrifos and methyl
parathion
Carboxylesterases are thought to play a lesser role in the
detoxification of methyl parathion than with either parathion
or chlorpyrifos (Chambers and Carr, 1993). We therefore
proposed that if prior blockade of carboxylesterase-mediated
detoxification of paraoxon by chlorpyrifos contributed to
the sequence-dependent toxicity, lesser sequence-dependent
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
Dosages (×LD1 )a First exposure 4-Day lethality
Chlorpyrifos 1/8
0.5
Methyl parathion 0/8
Chlorpyrifos 8/8
1.0
Methyl parathion 0/8
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
given one OP insecticide (First exposure) by gavage in peanut oil (1 ml/kg)
4 h prior to the second OP and cumulative lethality was recorded for 4 days
(from Karanth et al., 2004).
differences in toxicity would be noted with combined
chlorpyrifos and methyl parathion dosing (Karanth et al.,
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
exposures.
As shown in this table, lethality was much more extensive
in rats given chlorpyrifos before methyl parathion than
in those rats given identical dosages of both but with the
sequence reversed. Functional signs of cholinergic toxicity
were also markedly greater in those rats given chlorpyrifos
prior to methyl parathion (data not shown). Fig. 3 shows
cholinesterase inhibition in cortex and diaphragm with
interactive chlorpyrifos and methyl parathion exposures.
As noted in the interactive parathion–chlorpyrifos studies,
cholinesterase inhibition was also more extensive in rats
pre-exposed to chlorpyrifos and then given methyl parathion
compared to animals given the reverse sequence of ad-
ministration. Thus, this study suggested that other factors
besides differential detoxification by carboxylesterases may
be important in the sequence-dependent toxicity of OP in-
secticides. For example, parathion and methyl parathion pre-
exposure may inhibit the activation of chlorpyrifos to its oxon
more than chlorpyrifos can inhibit the activation of parathion
may also contribute, e.g., differential effects on acetylcholine
release (Liu et al., 2002). Regardless of the mechanism(s)
leading to differences in toxic response with sequential ex-
posures, these studies using a limited number of compounds
suggest a departure from additivity with mixed exposures.
While the dosages used were relatively high, studies with
interactions when based on cholinesterase inhibition. While
cholinesterase inhibiting pesticides all share a common
mechanism of toxicity, additional non-cholinesterase targets
of some anticholinesterases may selectively alter cumulative
toxicity. Knowledge of such non-cholinesterase actions will
improve the overall estimate of cumulative toxicity when
exposures to selected anticholinesterases are anticipated.
10. Conclusions
The therapeutic and toxic actions of cholinesterase in-
hibitors are based on inhibition of the enzyme AChE, with
consequent accumulation of synaptic acetylcholine levels and
enhanced stimulation of cholinergic receptors in the cen-
tral and/or peripheral nervous systems. In clinical conditions
exhibiting reduced activation of cholinergic receptors, e.g.,
myasthenia gravis, such effects can be therapeutically ad-
vantageous. In contrast, under normal conditions the over-
stimulation of cholinergic receptors leads to an imbalance in
neurotransmission and signs of cholinergic toxicity. While
this basic common mechanism is employed in both thera-
peutic and toxic uses, considerable information suggests that
some organophosphorus and carbamate anticholinesterases
also bind to other macromolecular targets that can poten-
tially modify the consequences of AChE inhibition. Such
non-cholinesterase actions may influence the cholinesterase
inhibitor’s own toxicity as well as cumulative toxicity of
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
Acknowledgements
This research was supported by STAR grant R825811
from the United States Environmental Protection Agency,
R01 ES009119 from the National Institute of Environmental
Health Sciences, the University of Louisiana Board of Re-
gents, the Oklahoma State University Board of Regents, and
a conference grant from the United States Army.
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