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OCR for page 146
5
Acety~cho'i nesterase
inhibitors: Case Study of
Mixtures of Contaminants with
Similar Biologic Effects
Acetylcholine, a neurotransmitter normally present in many parts of the
nervous system, is hydrolyzed by the enzyme acetylcholinesterase. Chemicals
that inhibit the action of acetylcholinesterase at doses or concentrations sub-
stantially lower than those required for other kinds of biologic effects are
pharmacologically classified as anticholinesterases. Anticholinesterases ap-
pear to mimic the stimulation of cholinergic nerves or receptors in the central
and peripheral nervous systems.
This chapter discusses the toxicity and interactions of the two groups of
chemicals most often associated with anticholinesterase activity the organic
triesters of phosphoric (P-O) or phosphorothioic (P=S) acid (i.e., organ-
ophosphorus compounds) and several carbamates (esters of carbamic acid).
Chemicals in both groups are widely used as insecticides. However, not all
organophosphorus triesters or all carbamates are insecticidal, nor can all of
them be classified as anticholinesterases. It is important to keep that in mind
to avoid overgeneralizing when discussing these chemicals (either as phar-
macologic classes or as chemical classes) with respect to their toxic actions
and the regulatory decisions concerning them.
Although organophosphorus compounds and carbamates are often used as
insecticides, the anticholinesterases have other applications as well. The drug
physostigmine, obtained from the calabar bean, is an aromatic carbamate
ester that was first used therapeutically in 1877 in the treatment of glaucoma
and still has some use for this purpose. Other related carbamates (such as
neostigmine and edrophonium) and a few organophosphorus esters (such as
diisopropyl phosphorofluoridate, octamethyl pyrophosphoramide, and echo-
thiophate) have been used clinically to stimulate the smooth muscles of the
146
OCR for page 147
Acetylcholinesterase Inhibitors 147
ileum or the urinary bladder in paralytic ileus and atony of the urinary bladder,
to decrease intraocular tension in glaucoma, and to overcome the muscular
weakness and rapid fatigability of skeletal muscle in myasthenia gravis.
Except for physostigmine and neostigmine, however, the clinical uses of
anticholinesterases are very limited.
Large stocks of organic triesters of phosphoric acid that are potent anti-
cholinesterases have been stored for potential use as chemical warfare agents.
In fact, the use of the organophosphorus compounds in agriculture, as well
as clinical medicine, was an outgrowth of the chemical-warfare research
during World War II. The agents could become environmental contaminants
in areas where they have been tested in military field operations or as a result
of leakage from disposal sites.
In summary, the anticholinesterases are primarily in two chemical classes:
the organic triesters of phosphoric or phosphorothioic acid and the carba-
mates. The chemicals have been developed for use as chemical-warfare
agents, as insecticides, and in clinical medicine; their most probable source
as surface-water and groundwater contaminants is insecticides.
TOXICITY
The known toxic effects of the anticholinesterases are predominantly the
acute effects elicited by single doses (Murphy, 19861. Both the organo-
phosphorus and carbamate classes of anticholinesterases contain compounds
whose acute lethal dosages range from a few milligrams per kilogram to
greater than a gram per kilogram (Murphy, 19861. The manifestation of
cumulative toxic action is generally the same as that of the action produced
by a large single dose. The effects usually appear in several organs, because
acetylcholine accumulates at the synapses of cholinergic nerves when ace-
tylcholinesterase is inhibited and has muscarinic, nicotinic, and central ner-
vous system actions. Some organophosphorus compounds or carbamates have
other toxic actions, such as carcinogenicity or teratogenicity, that are not
associated with the anticholinesterase action. The chronic effects of the com-
pounds are generally compound-specific and cannot be defined as charac-
teristic of the class. A possible exception is delayed peripheral neuropathy,
known as organophosphorus-compound-induced delayed neurotoxicity (OP-
IDN), which reflects a primary axonal degeneration caused by some of the
organophosphorus triesters. Many, but not all, organophosphorus triesters
that produce OPIDN are also strong inhibitors of acetylcholinesterase. The
inhibition of acetylcholinesterase appears to be unrelated to the mechanism
of production of OPIDN. In fact, in many cases the capacity of a chemical
to produce delayed OPIDN has been discovered only when doses greater
than the dose that would be lethal owing to anticholinesterase or cholinergic
action could be administered to test animals (usually fully grown hens). Such
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148 DRINKING WATER AND HEALTH
testing is possible with the use of atropine, which protects the muscarinic
receptors from accumulating acetylcholine.
The signs and symptoms of acute poisoning by the anticholinesterases
usually reflect the actions of acetylcholine at muscarinic receptors in smooth
muscle, the heart, and exocrine glands. They include tightness in the chest,
wheezing, and increases in bronchial secretion, salivation, lacrimation,
sweating, and gastrointestinal tone and peristalsis, with the consequent de-
velopment of nausea, vomiting, abdominal cramps, and diarrhea. There can
be slowing of the heart (which can progress to heart block), infrequent and
involuntary urination, and constriction of the pupils.
The signs of poisoning by anticholinesterases that are associated with
stimulation of nicotinic receptors include contractions of skeletal muscle,
leading first to scattered and then generalized fasciculations and finally to
muscular weakness and ultimately paralysis. The skeletal muscles include
the muscles of respiration, and their paralysis is often the immediate cause
of death. Nicotinic actions also include those at autonomic ganglia; in severe
intoxication, the effects at synapses in the autonomic ganglia can mask the
more usual muscarinic effects.
Accumulation of acetylcholine in the central nervous system can be re-
sponsible for the tension, anxiety, restlessness, insomnia, headaches, emo-
tional instability and neurosis, excessive dreaming and nightmares, apathy,
confusion, and forgetfulness reported by persons poisoned with anticholin-
esterases. Generally, if a person survives an episode of acute poisoning,
recovery is complete. However, chronic sequelae involving the central ner-
vous system (such as forgetfulness, dreaming, and electroencephalographic
changes) have been reported to persist for a long time.
All the signs and symptoms described above can result from a single dose
of an anticholinesterase agent that passes the blood-brain barrier, gains access
to cells in the central nervous system, and acts at synapses of peripheral
nerves. Smaller doses can be tolerated without these signs, but frequent
repetition of the smaller doses can lead eventually to their onset when the
accumulated inhibition of acetylcholinesterase allows acetylcholine to reach
an excessive concentration. Results of most studies in experimental laboratory
animals, as well as clinical observations and research, have indicated that
inhibition of acetylcholinesterase must be substantial (e.g., 50% before signs
typical of acute poisoning become manifest. However, there is some reason
to suspect that subtle and unrecognized effects in the central nervous system
can occur with smaller degrees of inhibition (Roney et al., 19861.
. . . . . . . ... .
Mechanisms
The actual manifestation of acetylcholinesterase-related poisoning is me-
diated by the accumulation of the endogenous neurotransmitter acetylcholine,
which affects receptors in effecter organs and the brain.
OCR for page 149
Acetylcholinesterase Inhibitors 149
The organophosphorus triesters phosphorylate the chemically active sites
of the enzyme acetylcholinesterase, and the carbamate insecticides carba-
mylate the same sites. In a sense, they both act as alternative substrates,
with the normal substrate being acetylcholine. The acetylated site dissociates
very rapidly, the carbamylated site dissociates slowly, and the phosphorylated
site dissociates even more slowly. Hence, cholinesterase inhibition by the
phosphate compounds generally lasts longer than that by the carbamate com-
pounds. With the carbamate compounds, spontaneous reversal of cholines-
terase inhibition occurs when the excessive inhibitor has been metabolized
or otherwise removed generally within a few minutes to a few hours. The
phospho~ylated acetylcholinesterase of the organophosphorus compounds tends
to be much more stable, and spontaneous dephosphorylation and regeneration
of the uninhibited enzyme can take many hours to several days. On exposure
to organophosphorus compounds, a portion of the inhibited enzyme is never
spontaneously dephosphorylated; hence, some inhibition of cholinesterase
can last for weeks, or until synthesis of new enzyme fully restores normal
activity. This implies that the recovery mechanism is more complex than
simple first-order kinetics.
Knowledge of the primary biochemical lesion associated with poisoning
by the two classes of compounds has resulted in a convenient means for
following the course of poisoning measurement of the cholinesterase ac-
tivity in erythrocytes or plasma. Inhibition of acetylcholinesterase activity in
erythrocytes is thought to reflect the course of inhibition and reversal of
inhibition in nerve tissue. A problem in the assay of carbamate compounds
with cholinesterase inhibition is that rapid spontaneous reversal of inhibition
can occur in vitro after blood has been drawn. It can also occur in vivo.
Thus, if a cholinesterase assay of blood from a severely poisoned person is
not conducted very promptly, it might fail to confirm carbamate poisoning.
The slower reversibility of the inhibition caused by the organophosphorus
compounds lessens this diagnostic problem.
There is substantial evidence from studies in laboratory animals, as well
as some indication from studies in humans, that repeated exposures to sub-
acute doses of organophosphorus compounds or persistent exposures to car-
bamate anticholinesterases can cause a fob of tolerance to these compounds
or a refractoriness of direct-acting cholinergic agonists. The phenomenon
appears to be due to a reduction in the responsiveness of the cholinergic
receptor system-a reduction in the density of cholinergic receptors- and
it has been demonstrated for both muscarinic and nicotinic cholinergic re-
ceptors (Bombinski and DuBois, 1958; Brodeur and DuBois, 1964; Costa
et al., 1982a,b; Schwab et al., 19811. Induction of such tolerance appears
to require a prolonged inhibition of acetylcholinesterase (which results in a
prolonged increase in acetylcholine at the receptor site) and thus has generally
been reported for only the more persistent anticholinesterase compounds. If
mixtures of anticholinesterases act to prolong inhibition, it is conceivable
OCR for page 150
150 DRINKING WATER AND HEALTH
that early additivity or even synergism might give way to tolerance or apparent
antagonism with prolonged exposures. If the manifestation of cholinergic
effects were used as the end point, the tolerance might be interpreted as
apparent antagonism; but if acetylcholinesterase inhibition were the end point,
antagonism would not be apparent.
The preponderance of reported evidence indicates that all whole-organism
signs and symptoms caused by anticholinesterases are preceded or accom-
panied by a significant inhibition of acetylcholinesterase. However, there are
reports that some behavioral changes have persisted long after cholinesterase
activity has returned to normal. Furthermore, some (sparse) experimental
data (Roney et al., 1986) indicate that tests of subtle learned behaviors in
laboratory animals can be altered with very little reduction in blood cholin
esterase.
Some organophosphorus and carbamate compounds are not strong inhib-
itors of acetylcholinesterases and are not properly classed with the anticho-
linesterases. Those chemicals have their own cholinesterase-independent toxic
action and cannot be grouped with the anticholinesterases for regulatory
purposes. Examples are the triaryl phosphates, including the classic peripheral
neurotoxic compound tri-o-cresylphosphate, the dithiocarbamate fungicides,
and some of the carbamate herbicides.
Metabolism-Toxicity Relationships
ORGANOPHOSPHORUS COMPOUNDS
The broad class of organophosphorus anticholinesterases includes several
types of compounds. Some are esters of phosphoric acid, (RO)3 P=O,
some are esters of phosphorothioic acid, (RO)3 P=S, and a few are phos-
phonates and phosphoroamidates. The phosphoric acid triesters (P=0 com-
pounds) are active insecticides and are generally direct inhibitors of
acetylcholinesterase. That is, when added to a solution of purified or partially
purified acetylcholinesterase or to a crude homogenate or extract of tissue
containing acetylcholinesterase, they exhibit potent in vitro anticholinesterase
action, often at concentrations of 1o-~0-lO-7 M. When the derivatives of
phosphorothioic acid (P=S compounds) are added to a cholinesterase-con-
taining preparation, they are not strong direct inhibitors of cholinesterase.
They are, however, active in vivo at relatively low doses, and tissues taken
from animals poisoned with low doses of these compounds have greatly
reduced concentrations of acetylcholinesterase.
It is now well established that the P=S compounds must be activated by
other enzymes in the body to a form that is highly reactive with the acetyl-
cholinesteraseactive center. The most common case with the I' S derivatives
is conversion to the P=0 (phosphate) form of the triester, which is a direct
OCR for page 151
HO S
Wp-
/ \
RO OAr
~ Vl
HO O
Acetylcholinesterase Inhibitors 151
RO S ,,, RO S
If
/ \
RO OAr
Parent insecticide
RO O
Sp'
/ \
RO OH
I Vl1
RO - O
`` ,' ~ IV `` p ,' V ~ `- ,'
RO OAr
IX
Aliesterase
inhibition
Consequence uncertain
RO OAr
Oxygen analog
or
\ V111
RO OH
Acetylcholinesterase (ChE)
inhibition
Acetylcholine
accumulation
t
Poisoning
FIGURE 5-1 General scheme of metabolism and mechanism of toxic action of dialkyl aryl phos-
phorothioates. From Murphy, 1980, with permission.
inhibitor of acetylcholinesterase (I in Figure 5-11. Hence, factors that alter
the rate of metabolism of these indirect inhibitors to their directly inhibiting
forms can alter the toxicity of the compounds.
Furthermore, many of the P=0 compounds can be attacked directly by
hydrolases, sometimes called A-esterases, that split the (RO)2P~OAr
bond (V in Figure 5-1) and result in products that do not inhibit acetylcho-
linesterase. The P=S compounds are generally resistant to those hydrolases
and are hydrolyzed only after they are converted to their oxygen analogue
(I and V in Figure 5-11. It is now well established that most of the P=S
compounds can be oxidatively cleaved to a phosphorothioic diester and an
aryl hydroxy group (III in Figure 5-1~. The oxidative cleavage step and the
hydrolytic step (V) are detoxifying, and the products of these pathways do
not inhibit acetylcholinesterase. To complicate the matter, the enzyme that
oxidatively cleaves (III in Figure 5-1 ) and oxidatively desulfurates (I in Figure
5-1) phosphorothioates is either the same enzyme or two very closely related
enzymes. They are classed as mixed-function oxidases, which are well known
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152 DRINKING WATER AND HEALTH
to be induced and inhibited by many other compounds (Murphy, 19861. In
addition to the oxidative activation and the oxidative and hydrolytic inacti-
vation of the phosphorothioate compounds, some of the compounds are also
detoxified via glutathione alkyltransferases that remove an alkyl group from
the phosphate and render it inactive as an anticholinesterase (II and IV in
Figure 5-11. In a few cases, organophosphorus compounds have also been
demonstrated to be dealkylated by oxidative enzymes. That is also a detox-
ifying step and is probably another form of a mixed-function oxidase.
In summary, oxidative metabolic pathways can either activate or detoxify
the phosphorothioate insecticides. Such factors as inducers or inhibitors of
mixed-function oxidases and competition by other compounds for the reactive
sites in the mixed-function oxidases can alter the quantity of the active direct
inhibitor of acetylcholinesterase at critical sites in nerve tissue (VIII in Figure
5-1) that will be present with any given dose at any given time. In addition,
a different set of enzymes, the soluble glutathione alkyltransferases, might
also detoxify some of the compounds. One further means of detoxification
is the reaction of the organophosphorus compounds with other noncritical
enzymes (IX in Figure 5-1) that can serve as a sink to divert the active
phosphates from critical sites and spare acetylcholinesterase.
The complex multiple pathway of metabolism renders it extremely difficult
to predict the possibility or quality of toxic interactions among mixtures of
the organophosphorus compounds. In addition, conditions that might predict
results with one compound or homologues of a compound might not apply
for other, equally closely related compounds. For example, it has been dem-
onstrated that inhibition of mixed-function oxidases by piperonyl butoxide
or SKF 525A in mice moderately increases the toxicity of ethyl parathion,
but protects strongly against the toxicity of methyl parathion (Levine and
Murphy, 1977a,b). The reason appears to be that the alternate pathway for
detoxification through glutathione transferase is effective for methyl para-
thion, but not for ethyl parathion.
A few of the organophosphorus compounds have chemical groups that can
be attacked by other enzymes. Notable among them are chemicals that have
carboxyl ester or carboxy amide linkages. The ester linkages can be attacked
by widely distributed carboxyl esterase or carboxy amidase in tissues. The
action of those hydrolases generally leads to a loss of anticholinesterase action
by the phosphate or carbamate that contains the groups. Hence, compounds
(including many insecticidal and noninsecticidal organophosphorus com-
pounds) that inhibit carboxyl esterases can increase the toxicity of other
organophosphorus compounds whose anticholinesterase activity depends on
an intact carboxyl ester or carboxy amide linkage (DuBois, 1969; Murphy,
19691. The best-known synergism of this kind is with malathion (Casida et
al., 1963; Cohen and Murphy, 1971; Frawley et al., 1957; Murphy et al.,
1959), which is usually considered a relatively safe insecticide. Many lab
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Acetylcholinesterase Inhibitors 153
oratory studies and a field accident (Baker et al., 1978) have demonstrated
that malathion becomes much more toxic under conditions in which carboxyl
esterases are inhibited.
Finally, it has been demonstrated that some organophosphorus triesters
that are not always potent inhibitors of acetylcholinesterase can compete with
other anticholinesterases for a noncritical group of enzymes, sometimes re-
ferred to as aliesterases (IX in Figure 5-11; these include nonspecific carboxyl
esterases. The competition can block a sink of noncritical binding sites that
normally act to spare acetylcholinesterase (the critical binding site) from
being inhibited by the organophosphates. Synergism among some organo-
phosphorus compounds might depend on such action (Fleisher et al., 1963;
Lauwerys and Murphy, 1969; Murphy et al., 1976; Polak and Cohen, 19694.
CARBAMATES
The carbamate insecticides also have multiple pathways of metabolism,
which are also predominantly oxidative and hydrolytic. Hydrolysis of the
carbamate ester invariably reduces its anticholinesterase activity, but oxi-
dative reactions that occur on the ring or alkyl portions of the carbamate
insecticides can increase or decrease anticholinesterase activity. For example,
in the case of the carbamate insecticide propoxur, hydrolysis of the carbamate
ester linkage reduces the anticholinesterase potency by a factor of 100. With
the carbamate ester intact, oxidative removal of the isopropoxy group reduces
toxicity by a factor of only about 5-6, hydroxylation of the N-methyl group
reduces toxicity by a factor of only 4, and hydroxylation of the aromatic
ring without other changes actually increases the anticholinesterase potency
by a factor of 3 (Oonnithan and Casida~ 19681. There is much less information
on the interactions that can occur between carbamates or between carbamates
and phosphate anticholinesterases than there is on interactions between the
phosphate anticholinesterases. However, a recent report (Takahashi et al.~
1987) indicated that the toxicity of N-methyl carbamate compounds in mice
can be increased by organophosphorus insecticides. The extent of synergism
varied widely, from a factor of 2 to a factor of 1S, depending on the organ-
ophosphorus compounds tested. The precise mechanism of the synergism is
not entirely clear from the results of the study.
I NTERACTIONS
Reported Data
The conceptual model that is usually applied to pesticides is the dose-
additive model, and in the remainder of this chapter when the words syn
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154 DRINKING WATER AND HEALTH
ergism, antagonism and interaction are used they imply departures from
dose additivity.
Over 30 years ago, Frawley and coworkers (1957) reported a marked
synergism of two organophosphorus insecticides, ethyl p-nitrophenyl thion-
obenzenephosphonate (EPN) and malathion. For several years thereafter, the
Food and Drug Administration required that all safety-evaluations on all
anticholinesterase insecticides for which food-residue tolerances were estab-
lished include tests of the toxicity of combinations. Most of the tests con-
ducted in response to the regulation were acute-toxicity tests that used
simultaneous administration of two chemicals (binary mixtures).
DuBois (1961) reported on studies in which various combinations of 13
organophosphorus insecticides were tested for acute toxicity in rats. Toxicities
of 21 pairs showed dose additivity, of 18 pairs were less than additive, and
of 4 pairs were synergistic. Administration of half the ~D50 of each of the
two compounds in each pair, which should have led to 505to mortality was
followed by 100% mortality in 4 pairs. Three of the 4 pairs included mal-
athion. A few more pairs involving newer compounds have since been shown
to be synergistic in acute-toxicity tests. However, combinations of several
organophosphorus insecticides that were incorporated into experimental diets
at residue-tolerance limits did not show greater than additive toxicity in
chronic feeding studies. The regulation requiring tests of anticholinesterase
insecticides for synergism was lifted a few years after it was instituted when
investigators failed to demonstrate synergism at the residue-tolerance limits.
The likely mechanisms of synergism among organophosphorus insecticides
have been reviewed by DuBois (1969) and Murphy (19691. Inhibition of
detoxification by tissue carboxyl esterases and amidases and competition for
nonvital binding sites that normally act as a buffer system to spare the vital
acetylcholinesterase appeared to be the two major mechanisms!involved in
the synergism among or~anophosphorus compounds. One of the insecticides
most often observed to be synergistic with other or~anophosphorus insecti-
cides was malathion. Malathion, normally a relatively safe compound is
detoxified by carboxyl esterases that are inhibited by other organophosphorus
insecticides (Murphy et al.. 19591.
Clear evidence of the synergistic action of organophosphorus compounds
in humans did not emerge until an incident among spraymen in a mosquito
control program in Pakistan in the late 1970s resulted in several deaths and
thousands of clinical poisonings. The incident was attributed to an increase
in the toxicity of malathion due to interaction with other or~anophosphorus
compounds that were strong inhibitors of carboxyl esterases and that con-
stituted impurities in the malathion (Baker et al.. 19781.
The other proposed mechanism of synergism is competition for noncritical
binding sites. It has been suggested that the carboxyl esterases might represent
one type of noncritical binding site. Compounds that are more potent inhib
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Acetylcholinesterase Inhibitors 155
itors of carboxyl esterases than of cholinesterase might be the most likely to
interact synergistically. The anticholinesterase action appears to be increased
by a factor of 3-4 (i.e., the dose for an equitoxic effect is reduced to one-
third or one-fourth) in acute doses of combinations of this type (DuBois,
1961; McCollister et al., 1959; Murphy, 1969, 19761.
Although the possibility of interactions among anticholinesterase com-
pounds has been less studied, Takahashi et al. (1987) recently demonstrated
increases in the toxicity of five N-methyl carbamates by simultaneous treat-
ments or pretreatments with one-twentieth of the ~D50 of some organophos-
phorus compounds. The toxicity of 2-sec-butylphenyl N-methyl carbamate
(BPMC) increased by a factor of approximately 15 at the most sensitive time
tested. Because the phosphorothioate (P=S) type of organophosphorus in-
secticides had a synergistic effect on BPMC and the direct-acting organo-
phosphorus insecticide dichlorvos (100 type) did not? the investigators
suggested that inhibition of mixed-function oxidases, which occurs only with
the P=S type, is a probable mechanism of this synergism. However, several
additional tests of that hypothesis suggested that some other mechanism could
also be operative for organophosphate synergism with N-methyl carbamates
(Takahashi et al., 19871.
DEGREES OF INTERACTION
The greatest departure from dose additivity reported among anticholin-
esterase insecticides appears to be an increase in malathion toxicity by a
factor of about 100 achieved with acute doses and rather unrealistic routes
of exposure (Murphy et al., 19591. The first reported example of substantial
synergism among anticholinesterase compounds involved binary mixtures of
malathion and EPN (Frawley et al., 19571. Both chemicals are anticholin-
esterase organophosphorothioate insecticides. Tests of the acute toxicity of
an equitoxic mixture of the two in rats revealed about a lO-fold increase in
toxic mortality. DuBois (1961) reviewed similar but less extensive acute-
toxicity tests on dogs that suggested approximately a 50-fold increase. In
addition, early feeding studies with EPN at 3 ppm and malathion at 8 ppm
in the diet (these were the legal tolerance limits for these chemicals in fruits
and vegetables) resulted in increased toxicity~ as indicated by erythrocyte
cholinesterase inhibition.
As noted earlier, DuBois (1961) tested a lar~e number of binary mixtures
of organophosphorus insecticides. He applied the principle of dose additiv-
ity that is, that two compounds with the same mode of action. parallel
dose-mortality curves, and similar time and mechanism of action exhibit dose
additivity (not synergism) if the simultaneous administration of half the LDS,,
of each results in 50% mortality. Four pairs (malathion and EPN. malathion
and dipterex, malathion and Co-Ral, and dipterex and Guthion) resulted in
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156 DRINKING WATER AND HEALTH
synergism by the dose-additivity definition, as indicated by 100% mortality.
Further acute-toxicity tests in rats with a range of doses of equitoxic mixtures
of the same pairs resulted in a measure of the degree of synergism when the
ratios of the expected ~D50 of the mixture (if additive) to the observed ~D50
were calculated. The ratios (degree of synergism) ranged from 1.5:1 (for
dipterex and Guthion) to 2.4:1 (for malathion and Co-Ral). The comparison
technique probably can be extended to combinations of three or more chem-
icals, although this does not yet appear to have been done in a published
paper.
Using the dose-additive model, McCollister et al. (1959) reported acute
toxicity of 50-50 mixtures of the organophosphorus insecticide O,O-di-
methyl-0-~2,4,5-trichlorophenyl) phosphorothioate (Ronnel) with each of 10
other organophosphorus insecticides and calculated the ratios of expected (if
additive) to observed ~D50. Tests of Ronnel with each of six chemicals yielded
ratios greater than 1.0:1 (1.3: 1 with Systox. 1.4:1 with phosdrin~ 1.7:1 with
Guthion? 1.8:1 with parathion, 2.1:1 with malathion and 3.2:1 with EPN);
tests with four other pairs yielded ratios of 1.0:1 or less. Of the compounds
cited above only malathion contains carboxyl ester moieties. which are
vulnerable to attack by carboxyl esterases which in turn are known to be
sensitive to inhibition by several organophosphorus compounds (DuBois,
1969; Murphy, 19691.
A few other published studies have revealed a slight to moderate (less
than 10 times) degree of synergism of acute toxicity of oraanochosohorus
insecticides given simultaneously as binary mixtures to laboratory animals.
One criterion that appears to apply to most of the cases of reported synergism
is that at least one of the compounds has a higher potency as a carboxyl
esterase inhibitor than as an anticholinesterase. DuBois ( 1961 ) suggested that
the residue-tolerance limits for such compounds should be based on the
dosages that inhibit their detoxification enzymes rather than on the less
sensitive acetylcholinesterase inhibition. That suggestion has not to our
knowledge been adopted as a regulatory rule. From the standpoint of pro-
tectin~ against synergism among or~anophosphorus compounds standards
for individual compounds based on this detoxification principle might not
include any special considerations or extra safety factors (other than the
assumption of dose additivity) required for drinking water standards for
~_
mixtures containing this class of compounds.
If one considers a case of minimally detectable synergism demonstrated
in laboratory animals with a binary mixture of or~anophosphorus insecti-
cides i.e., feeding the maximal acceptable dietary-tolerance limits of mal-
athion at 8 ppm and EPN at 3 ppm (Frawley et al.. 1957) one can draw
some conclusions regarding the relationship of doses carrying some risk to
the dose that might be obtained from drinking water. Assuming ingestion of
1 keg of food all of which contains maximal food-tolerance limits a human
OCR for page 157
Acetylcholinesterase Inhibitors 157
would ingest 8 mg of malathion and 3 mg of EPN. There are few data
available regarding measured concentrations of those compounds in ground-
water (or drinking water), but data from California (NRC, 1986) indicate
that the highest concentration of malathion observed in groundwater is 23
parts per billion (ppb). If an adult consumed 2 liters of this water, the total
dose of malathion would be only 0.046 mg-slightly more than 0.5~o of the
lowest reported daily amount of malathion for a detectable synergistic effect
in chronic dietary feeding tests.
The committee could find no groundwater or drinking water concentration
data on EPN. However, for several compounds with similar uses-e.g.,
parathion, diazinon, and Delnav-the maximal groundwater concentrations
reported for California were 4, 9, and 25 ppb, respectively, i.e., no more
than 0.050 mg per adult ingesting 2 liters/day, or about 1% or less of the
reported minimal dosage of EPN that produced a detectable synergistic effect
in animal feeding studies. In fact, Moeller and Rider (1960) tested human
response to the dosages that might be obtained at the food-tolerance limits
and reported that 3 mg of EPN and 8 mg of malathion in the daily diet of
healthy men for 6 weeks led to no observation of depression of plasma or
erythrocyte cholinesterase. On the basis of that most-studied example of joint
action by organophosphorus insecticides, it appears that no excess risk of
cholinesterase inhibition in healthy men is likely if intake of EPN and mal-
athion does not exceed the maximum that could result from legal food res-
idues.
No similar data base is available for other combinations of anticholinest-
erase organophosphorus or carbamate compounds. There are apparently no
reports of tests on interactions that include the carbamate insecticide aldicarb,
which has been found in many groundwater samples. In California, maximal
concentrations of 47 ppb in groundwater would be equivalent to a 0.094-mg
dose in 2 liters of water ingested by adults. Contamination at 47 ppb is more
than 4 times EPA's health advisory not to exceed 10 ~g/liter (10 ppb) for a
10-kg child. If aldicarb acts in synergy with other anticholinesterases, as
Takahashi et al. (1987) have reported for some other carbamates, the risk
of occurrence of adverse interactions could be substantially increased.
A related approach based on the dose-additive model is to use the concept
of toxic equivalence (Berlin and Barnes, 1987; Eadon et al., 19861. A possible
toxic-equivalence scheme for regulation could be used for a mixture of al-
dicarb and its transformation products aldicarb sulfoxide and aldicarb sulfone.
All those compounds are cholinesterase inhibitors, and their potencies relative
to that of aldicarb (as measured by 1/NOAEL) can be e-xpressed (Table 5-
11. Aldicarb has a lifetime health advisory guideline of 10 ~g/liter (EPA?
1987), and the toxic equivalents of aldicarb can be compared with this value.
A liter of water containing a mixture of aldicarb at 2 ~g/liter, aldicarb
sulfoxide at 5 ~g/liter, and aldicarb sulfone at 30 ~g/liter could be expressed
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158 DRINKING WATER AND HEALTH
TABLE 5-1 Relative Potencies and Toxic-Equivalent Concentrations of
Aldicarb and Its Transformation Products
Toxic-Equivatent
NOAEL. Relative Concentration. Concentration.
Compound mg/kg Potency ~.~/liter /liter
Aldicarb 0.125 1 2 ~
Aldicarb sulfoxide 0.125 1 5 5
Aldicarb sulfone 0.6 0.9 30 6
in toxic equivalents of aldicarb on the basis of relative potencies. The toxic-
equivalent concentrations for the individual compounds are obtained by mul-
tiplying the concentration by the relative potency. For example, for aldicarb
sulfone, the concentration of toxic equivalence is
(30 log of aldicarb sulfone/liter) (0.2) = 6 log of aldicarb/liter.
It should also be noted that the toxic equivalence of the mixture is 13 log/
liter the sum of the concentrations in the last column which can be com-
pared with the health advisory guideline of 10 /liter REPAY 19871. This
approach assumes. as explained in an earlier chapter, that these compounds
do not act synergistically.
CONCLUSIONS
The known mechanisms of anticholinesterase synergism depend on inter-
ference with or competition for metabolic mechanisms of detoxification of
the anticholinesterases or their precursors. Therefore one might predict that
synergism will occur only when the dosage exceeds the theshold where
metabolism becomes a rate-limiting factor in toxicity. Of course, that dosage
becomes smaller as critical pathways of detoxification are inhibited by other
compounds.
Without specific knowledge of the mechanism of synergism and without
quantitative data on response to a range of doses of interactive chemicals, it
is not possible to determine at precisely what concentrations interaction oc-
curs. From acute-toxicity studies? it appears likely, at least for the compounds
discussed in this chapters that there are dosages below which interactions do
not occur and that these can be predicted from data on individual compounds.
With regard to the interaction resulting from the existence of cholinesterase
inhibition as a common action, one would anticipate that at most this inter-
action would result in additive activity. In fact, DuBois (1961) reported that
the oxygen analo~gues of EPN and malathion are strictly additive with respect
to their anticholinesterase action in vitro and. in contrast. synergistic in vivo.
If the compounds compete for the same active catalytic sites on the acetyl
OCR for page 159
Acetylcholinesterase Inhibitors 159
cholinesterase molecules, chemicals that are intrinsically less effective as
inhibitors might sometimes occupy these sites at the expense of intrinsically
more active inhibitors. When that happens, the combined action will be
manifested as antagonistic, according to the principles put forward by Veld-
stra (19561.
RESEARCH RECOMMENDATIONS FOR M IXTURES OF
ANTICHOLI N ESTERASES
· Whether interactions with active inhibitors at a primary biochemical
target (i.e., acetylcholinesterase-active center) produce other than additive
responses on exposure to multiple chemicals is not known and should be the
subject of research. The additivity of multiple compounds at low doses or
concentrations should be tested. The resulting knowledge would help to
validate the usefulness of a summation or hazard-index approach to rec-
ommending quality standards.
· The role of inhibition of carboxyl esterases or other noncritical (silent)
receptors in the loss of anticholinesterases, whether or not they involve
carboxyl ester linkage, should be investigated further.
· The mechanisms of interaction of carbamate and organophosphorous
insecticides recently reported by Takahashi et al. ( 1987) need better definition
to determine whether new concepts or methods for testing interaction potential
can be developed.
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160 DRINKING WATER AND HEALTH
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Representative terms from entire chapter:
drinking water
