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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

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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.

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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

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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

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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

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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

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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. REFERENCES Baker, E. L., Jr., M. Warren, M. Backs R. D. Dobbin, J. W. Miles. S. Miller. L. Alderman. and W. R. Teeters. 1978. Epidemic malathion poisoning in Pakistan malaria workers. Lancet 1(8054):31-34. Bellin, J. S., and D. G. Barnes. 1987. Interim Procedures for Estimating Risk Associated with Exposures to Mixtures of Chlorinated Dibenzo-p-dioxins and Dibenzofuran (CDDs and CDF). U.S. EnvironmentalProtection Agency Report No. EPA/625/3-87/012. Washington D.C.: Risk Assessment Forum, U.S. Environmental Protection Agency. 27 pp. + appen- dixes. Bombinski, T. J., and K. P. DuBois. 1958. Toxicity and mechanism of action of DiSyston. A.M.A. Arch. Ind. Health 17: 192- 199. Brodeur, J., and K. P. DuBois. 1964. Studies on the mechanism of acquired tolerance by rats to 0,0 diethyl S-[2-(ethylthio)ethyl] phosphorodithioate (Di-Syston). Arch. Int. Pharma- codyn. 149:560-570. Casida, J. E., R. L. Baron, M. Eto, and J. L. Engel. 1963. Potentiation and neurotoxicity induced by certain organophosphates. Biochem. Pharmacol. 12:73-83. Cohen, S. D., and S. D. Murphy. 1971. Malathion potentiation and inhibition of hydrolysis of various carboxylic esters by triorthotolyl phosphate (TOTP) in mice. Biochem. Pharmacol. 20:575-587. Costa, L. G., B. W. Schwab, and S. D. Murphy. 1982a. Differential alterations of cholinergic

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160 DRINKING WATER AND HEALTH muscarinic receptors during chronic and acute tolerance to organophosphorus Biochem. Pharmacol. 31 :3407-3413. . . . Insecticides. Costa, L. G.? B. W. Schwab, and S. D. Murphy. 1982b. Tolerance to anticholinesterase compounds in mammals. Toxicology 25:79-97. DuBois, K. P. 1961. Potentiation of the toxicity of organophosphorus compounds. Adv. Pest Control Res. 4: 117-151. DuBois, K. P. 1969. Combined effects of pesticides. Can. Med Assoc. J. 100:173-179. Eadon, G., L. Kaminsky, J. Silkworth, K. Aldous, D. Hilker, P. O'Keefe, R. Smith, J. Gierthy, J. Hawley, N. Kim, and A. DeCaprio. 1986. Calculation of 2,3,7,8-TCDD equiv- alent concentrations of complex environmental contaminant mixtures. Environ. Health Per- spect. 70:221-227. EPA (U.S. Environmental Protection Agency). 1987. Aldicarb (Sulfoxide and Sulfone). Health Advisory (Draft). Washington, D.C.: Office of Drinking Water, U.S. Environmental Pro- tection Agency. 16 pp. Fleisher, J. H., L. W. Harris, C. Prudhomme, and J. Bursel. 1963. Effects of ethyl p- nitrophenyl thionobenzene phosphonate (EPN) on the toxicity of isopropyl methyl phos- phonofluoridate (GB). J. Pharmacol. Exp. Ther. 139:390-396. Frawley, J. P., H. N. Fuyat, E. C. Hagan, J. R. Blake, and O. G. Fitzhugh. 1957. Marked potentiation in mammalian toxicity from simultaneous administration of two anticholinest- erase compounds. J. Pharmacol. Exp. Ther. 121:96-106. Lauwerys, R. R., and S. D. Murphy. 1969. Interaction between paraoxon and tri-o-tolyl phosphate in rats. Toxicol. Appl. Pharmacol. 14:348-357. Levine. B. S., and S. D. Murphy. 1977a. Esterase inhibition and reactivation in relation to piperonyl butoxide-phosphorothionate interactions. Toxicol. Appl. Pharmacol. 40:379-391. Levine, B. S., and S. D. Murphy. 1977b. Effect of piperonyl butoxide on the metabolism of dimethyl and diethyl phosphorothionate insecticides. Toxicol. Appl. Pharmacol. 40:393- 406. McCollister, D. D., F. Oyen, and V. K. Rowe. 1959. Toxicological studies of O O-dimethyl- 0-(2,4,5-trichlorophenyl) phosphorothionate (Ronnel) in laboratory animals. J. A=ric. Food Chem. 7:689. Moeller, H.C., and J. A. Rider. 1960. Cholinesterase depression by EPN and Malathion. Pharmacologist 2:84. Murphy, S. D. 1969. Mechanisms ofpesticideinteractionsin vertebrates. Residue Rev. 25:201- 222. Murphy. S. D. 1980. Assessment of the potential for toxic interactions among environmental pollutants. Pp. 277-294 in The Principles and Methods in Modern Toxicology C. L. Galli. S. D. Murphy, and R. Paoletti, eds. Amsterdam: Elsevier/North Holland. Murphy. S. D. 1986. Toxic effects of pesticides. Pp. 519-581 in Casarett and Doull's Tox- icology: The Basic Science of Poisons 3rd Ed. J. Doull, C. S. Klassen and M. O. Amdur? eds. New York: MacMillan. Murphy, S. D.. R. L. Anderson~ and K. P. DuBois. 1959. Potentiation of the toxicity of malathion by triorthotolyl phosphate. Proc. Soc. Exp. Biol. Med. 100:483-487. Murphy S. D. K. L. Cheever. A. Y. K. Chow and M. Brewster. 1976. Or~anophosphate insecticide potentiation by carboxylesterase inhibitors. Proc. Eur. Soc. Tox. XVII~ Esc. Med. Int. Cong. 376:292-300. NRC (National Research Council). 1986. Pesticides and Groundwater Quality: Issues and Problems in Four States. Written by Patrick W. Holden. Washington~ D.C.: National Acad- emy Press. 124 pp. Oonnithan, E. S. and J. E. Casida. 1968. Oxidation of methyl and methyl carbonate insecticide

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