22ANTICHOLINESTERASE PESTICIDES INTERACTIONS

RAMESH C. GUPTA

Murray State University, Breathitt Veterinary Center, Hopkinsville, Kentucky

DEJAN MILATOVIC

Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee

22.1 Introduction 315

22.2 Interaction between Cholinesterase Inhibitors 316

22.3 Interaction between ChE Inhibitors and non-ChEInhibitors 317

22.4 Are OPs Inhibitors or Substrates of Carboxylesterases? 318

22.5 Potentiation of Toxicity of OP Insecticides by TheirImpurities 318

22.6 Metabolism-Based Interactions 31922.6.1 Potentiation 32022.6.2 Antagonism 320

22.7 Models for Interaction Investigation 323

22.8 Conclusions 323

References 323

22.1 INTRODUCTION

We currently live in a more polluted environment than everbefore, in part because of the continual increase in the useof pesticides around the world. Among the pesticides, orga-nophosphates (OPs) and carbamates (CMs) dominatebecause of their relatively selective toxicity and reduced per-sistence in the environment compared to organochlorinepesticides. As a result of their widespread use in agriculture,horticulture, homes and gardens, human health protection,and veterinary medicine, the potential exists for exposure toa large section of the population. It is highly unlikely thathumans, animals, birds, or aquatic organisms are exposedto just a single pesticide. Instead, they are more often exposedto multiple pesticides at low doses (Castorina et al., 2003;Duggan et al., 2003; Fenske et al., 2002). Residues of thesepesticides can be found in rain, snow, fog, and air in manyparts of the world. OP and CM insecticides are often usedin combination, with the objective of achieving synergisticinteraction and to control a wide range of insects including

those that are hard to kill (resistant). In many cases, thesechemicals are also used simultaneously as ectoparasiticidesand anthelmintics in veterinary medicine. Under most cir-cumstances, exposure to a single cholinesterase (ChE) inhi-biting pesticide will be at the sub-toxic level, butsimultaneous exposure to more than one can sometimeslead to devastating health effects because of an additive orpotentiating interaction (Calabrese, 1995; Cohen, 1984;DuBois, 1961; Iyaniwura, 1990; Murphy, 1969, 1980).

Pesticide interactions can be of even greater concern tounhealthy or sick humans or animals that have less toleranceto such pesticides, partly because of inefficient metaboliccapacities. These pesticides not only have direct effects onhuman health, but may predispose humans to neurodegenera-tive diseases (such as Alzheimer’s and Parkinson’s), diabetes,and other diseases.

Many studies have investigated the toxic effects of indi-vidual OP and CM pesticides. Since the 1950s there havebeen a number of studies on the combined acute toxicity ofOP compounds following simultaneous or sequential

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

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administration (DuBois, 1961; Murphy, 1969, 1980; Murphyet al., 1959). In most studies, these compounds were adminis-tered in various combinations in laboratory animals,including dogs, to assess the resulting effect (additive, poten-tiation/synergistic, or antagonistic). From the standpoint oftoxicity, potentiation is considered to be the most seriousinteraction (Cohen, 1984). Toxic interactions were widelyrecognized when malathion toxicity was potentiated by pre-treatment with EPN (Frawley et al., 1957) and in later studieswith impurities of malathion (Umetsu et al., 1977). In a fewother investigations, interactions between OP and CM com-pounds were studied in experimental animals (Takahashiet al., 1983, 1984). In a recent publication, Padilla (2006)describes in detail the interactions and cumulative effects ofOP and CM pesticides. Toxicological studies of mixturesby nature are difficult, and understanding the risks arisingfrom simultaneous or sequential exposure to two or moreanticholinesterase (anti-ChE) pesticides or their interactionwith therapeutic drugs or other xenobiotics is the single great-est challenge to toxicologists today.

This chapter describes anti-ChE pesticide interactions inmammalian systems. The information provided here willhelp improve understanding not only of the metabolism andfate of individual pesticides, but also the extent of their nega-tive impact on the final outcome in terms of the overall tox-icity arising from exposure to a mixture of such pesticides.Understanding such interactions may allow us to developmodels for studying interactions and strategies to avoid com-binational use of certain pesticides, and to develop improvedtherapeutic interventions, because toxic interactions are ofserious concern from risk assessment as well as clinicalstandpoints.

22.2 INTERACTION BETWEENCHOLINESTERASE INHIBITORS

Organophosphorus (OP) and N-methylcarbamate (CM)insecticides are often used simultaneously to control pestsin both agricultural fields and domestic environments(Takahashi et al., 1987). Many interaction studies havedealt with two or more OPs, but interactions between OPand CM compounds in mammalian systems have rarelybeen studied. The toxicities of both classes of compoundshave been attributed to their ability to inhibit the activity ofacetylcholinesterase (AChE), which catalyzes the hydrolysisof the neurotransmitter acetylcholine (ACh) at nerve synapsesand neuromuscular junctions (NMJs). More than half a cen-tury ago, the landmark publication of Frawley and colleagues(1957) demonstrated for the first time marked potentiation inmammalian toxicity from the simultaneous administrationof two anti-ChE OP pesticides: malathion [O,O-dimethyl-S-(1,2-dicarbethoxyethyl phosphorodithioate) and EPN (O-ethyl O-p-nitrophenyl phosphonothionate]. Since then, the

phenomenon of potentiation has been confirmed in severalstudies using different pesticides and animal species.

The mechanism by which pre-treatment with EPNincreases the subsequent toxicity of malathion was investi-gated by Knaak and O’Brien (1960). In both the rat anddog, EPN resulted in a marked shift in the initial detoxifica-tion site of the malathion molecule from the carboxyl esterto the thiolophosphate bond. The percentage of administeredmalathion excreted as metabolites in urine was increased byEPN when administered in dogs but remained unchangedin rats. Malathion levels in rat tissues were increased byEPN, whereas malaoxon levels in rat blood were reduced.Potentiation appears to result from an increased persistencerather than an increased concentration of malaoxon in the tis-sues. Seume and O’Brien (1960) characterized the interactionof EPN and O,O-dimethyl S-[N-ethylcarbamoylmethyl phos-phorodithioate (CL 18706) in mice using five different ratiosand a dose-additive experimental design]. The resultsrevealed a marked toxic interaction between these two OPs,and the toxicity profile was directly related to the proportionof each in the mixture. DuBois (1961) investigated 43 pairs ofOPs for interaction and found four pairs (malathion þ EPN,malathion þ dipterex, dipterex þ guthion, and malathion þCo-Ral) to have potentiation interactions.

Cohen and Murphy (1971) investigated the mechanismsinvolved in the potentiation interaction between malathionand each of the other three OP insecticides (EPN, abate, andparathion) in mice. EPN and abate inhibited carboxylesterases(CarbEs) to the same degree in liver, lung, kidney, and plasma,but EPN potentiated malathion’s toxicity at twice the extent ofabate. However, both compounds potentiated malaoxon’s tox-icity to the same degree. This suggested that abate inhibitedthe oxidation of malathion to malaoxon. It was evident fromthis and a few other investigations that CarbE inhibitionalone is not sufficient to predict the relative capacities of var-ious compounds to potentiate the toxicity of malathion andrelated OP compounds (Lauwerys and Murphy, 1969; Polakand Cohen, 1969). In a recent study, Moser and colleagues(2005) investigated the interaction of a mixture of five OPcompounds (chlorpyrifos, diazinon, dimethoate, acephate,and malathion), and by using AChE inhibition and behavioralend points they found no more than additive interactions.Furthermore, in a recent study Timchalk and colleagues(2004) studied a pharmacokinetic-based interaction betweenchlorpyrifos and diazinon, and discovered that anythingmore than an additive interaction is unlikely at environmen-tally relevant concentrations. However, some studies haveobserved potentiation at higher doses of OPs.

McPhillips (1969) reported that pre-treatment of ratswith disulfoton resulted in increased toxic effects with acut-ely administered octamethylpyrophosphoramide (OMPA).Hagan and colleagues (1971) observed an increased sensi-tivity to acutely injected trichlorfon and physostigmine inrats pre-fed with 50 p.p.m. of either OMPA or parathion

316 ANTICHOLINESTERASE PESTICIDES INTERACTIONS

daily for 40 days. Differences were found in cholinesteraseactivities in brain, red blood cells, and plasma in OMPA-and parathion-treated rats sacrificed after 40 days of treatment.

Since the mid-1950s, much attention has focused on inves-tigating interactions between OPs. No study has ever reportedpotentiation interactions between two or more CMs. In the1980s and 1990s, a few studies investigated the interactionbetween OP and CM compounds (Gupta and Kadel, 1989;Miyaoka et al., 1984; Tsuda et al., 1984). Takahashi andcolleagues (1983, 1984) and Tsuda and co-workers (1984)demonstrated the potentiation of toxicity in mice of a CMinsecticide, 2-sec-butylphenyl N-methylcarbamate (BPMC)by P¼S type OPs (cyanophos, fenitrothion, and malathion)through metabolic blockage and not through CarbE inhi-bition. Miyaoka and colleagues (1984) reported synergismin mice treated with fenitrothion and BPMC. The acutetoxicity and plasma concentrations of BPMC were markedlyincreased when fenitrothion was given in the diet (1000p.p.m.) for 1 week. It was suggested that inhibition of first-pass metabolism in the liver was responsible for potentiationof BPMC toxicity by fenitrothion treatment (Tsuda et al.,1984). These authors further demonstrated that competitiveinhibition of BPMC metabolism by fenitrothion appears toplay a role in inhibition of BPMC detoxification, resultingin potentiation of its toxicity. It is interesting to note thatno such potentiation has been found with P¼O type OPinsecticides such as dichlorvos.

Takahashi and colleagues (1983) determined the inter-action between malathion and BPMC based on their com-bined toxicity using an acute oral toxicity (LD50) asdeterminant in rats and mice. In this investigation, theobserved LD50 of a mixture was compared with the expectedLD50, with LD50 (expected)/LD50 (observed) used as theratio of the combined effect. Of five combinations studiedin male mice, malathion þ BPMC was the only combinationto exhibit a striking synergism (ratio of 5.4). On the otherhand, no clear synergism was observed with the combinationof malathion plus any one of the other CM insecticides, suchas MTMC (3-methylphenyl N-methylcarbamate), NAC (1-naphthyl N-methylcarbamate), XMC (3,5-dimethylphenylN-methylcarbamate), or MPMC (3,4-dimethylphenyl N-methylcarbamate). When malathion or BPMC was adminis-tered alone, the toxicity in female mice or male rats was notsignificantly different from that in male mice. In femalemice, the synergism of malathion þ BPMC was similar tothat in male mice (ratio of 5.2). However, relatively lesspotent synergism was noted with the combination in malerats (ratio of 2.0). The marked synergism of malathion þBPMC could not be explained on the basis of an inhibitionof AChE by malaoxon þ BPMC, or an inhibition ofmalathion’s detoxication by BPMC. In previous studies, thesynergism of malathion toxicity by EPN or tri-o-cresyl phos-phate (TOCP) has been extensively studied and explained bythe inhibition of CarbE, which hydrolytically detoxifies

malathion and malaoxon (Cohen et al., 1972; Murphy et al.,1959). However, the marked synergism of malathion þBPMC could not be explained by the inhibition of malathion’sdetoxication by BPMC, because the activity of CarbE was notinhibited after administration of BPMC.

In an in vitro study, Hamers and colleagues (2001) found acorrelation between the concentrations of OP and CM pesti-cides in rainwater and cholinesterase inhibition. The inter-action was not more than additive. The limitation of thisinvestigation was that the end point used was cholinesteraseinhibition, but it was more sensitive than the commonlyused end point lethality.

Ito and colleagues (1995) have stated that the exposure ofagricultural workers and the general population to pesticidesis of major concern, and possible summation or synergisticeffects could occur at some levels. Some of these pesticideshave the potential for carcinogenicity. In this investigation,the acceptable daily intake (ADI) mixture of 20 pesticides(19 OPs: acephate, butamifos, chlorfenvinphos, chlorpyrifos,dichlorvos, dimethoate, edifenphos, etrimfos, fenitrothion,iprobenfos, isoxathion, malathion, methidathion, pirimi-phos-methyl, prothiophos, pyraclofos, tolclofos-methyl, tri-chlorfon, vamidothion; and one organochlorine: endosulfan)had no effect on the development of liver pre-neoplasticfoci initiated by diethylnitrosamine (DEN), although the100-times higher dose mixture demonstrated lesion-promoting potential. The authors concluded that humansare protected from carcinogenicity by current regulated pesti-cide exposure levels.

22.3 INTERACTION BETWEEN CHEINHIBITORS AND NON-CHE INHIBITORS

In a series of studies, it has been demonstrated that certaincarboxylesterase (CarbE; EC 3.1.1.1) inhibitors that do notinhibit AChE potentiate the toxicity of ChE inhibiting OPsand CMs. Pre-treatment of rats with a selective butyrylcho-linesterase (BuChE; EC 3.1.8) inhibitor tetraisopropylpyro-phosphoramide (iso-OMPA; 1 mg/kg, s.c.) 1 h prior tocarbofuran (0.5 mg/kg, s.c.), propoxur (5 mg/kg, i.p.), oraldicarb (0.1 mg/kg) potentiated the toxicity of these CMsby a factor of three (equivalent to sublethal signs producingacute toxicity observed with 1.5 mg/kg carbofuran, 5 mg/kg propoxur, and 0.4 mg/kg aldicarb). None of these CMsalone at the low dosage produced AChE inhibition or toxicsigns; however, CarbE activity in a variety of organs includ-ing brain, muscle, liver, heart, and plasma was significantlyreduced. In addition to CarbE, iso-OMPA significantly inhib-ited the activity of another serine-containing esterase,BuChE. Significant inhibition of AChE was observed afterthe combination of iso-OMPA with each one of these CMs(Gupta and Dettbarn, 1993; Gupta and Kadel, 1989,1990a, b, 1991). It was suggested that CarbEs are more

22.3 INTERACTION BETWEEN CHE INHIBITORS AND NON-CHE INHIBITORS 317

sensitive than AChE to these CMs, and inhibition ofCarbE by iso-OMPA raises the concentration of CMs avail-able to inhibit AChE, resulting in increased toxicity.Physostigmine, a CM without insecticidal activity, did notinhibit CarbE activity at the concentration that criticallyreduced AChE activity and produced toxic signs. Pre-treatment with iso-OMPA did not increase physostigmine’stoxicity, ruling out any involvement of a CarbE inhibitoryeffect in the tissues and plasma. In essence, there is nodoubt that the increase in CM insecticide toxicity resulted lar-gely from inhibition of CarbE activity, particularly in theserum and liver. This correlates well with the previouslyreported findings of OP potentiation through 2-(o-cresyl)-4H-1 :3 :2-benzodioxaphosphorin-2-oxide (CBDP), tetraiso-propylpyrophosphoramide (iso-OMPA), mipafox, or TOCPpretreatment. All these potentiating compounds bind toserine-active sites such as the CarbE, and inhibit its activityin plasma, liver, brain, muscle, and heart (Boskovic, 1979;Clement, 1984b; Dettbarn and Gupta, 1989; Grubic et al.,1988; Gupta and Kadel, 1990a, b; Gupta et al., 1985, 1986,2000; Maxwell et al., 1988). The degree of potentiationmay vary with the activity of the CarbE and susceptibilityto the given OP or CM insecticides and thus could explainthe variations between species (Maxwell, 1992; Maxwellet al., 1987; Wallace and Kemp, 1991).

In the context of OP and CM interactions, CarbEs appearto have two major features. First, they may provide partialprotection against OPs or CMs. This hypothesis was deducedfrom the findings that low level exposure to either of theseOPs or CMs caused marked inhibition of CarbE, particularlyin the serum and liver, without significantly affecting AChEactivity. It implies that CarbE has a greater affinity thanAChE for these inhibitors and thus only a reduced amountof free inhibitor concentration is left to interact with AChE.Second, in the case of pre-exposure to iso-OMPA or anyother CarbE inhibitor (Grubic et al., 1988; Gupta andKadel, 1990a, b; Maxwell et al., 1987, 1988), these non-specific binding sites are blocked. This raises the freeconcentration of the OP or CM compound, which wouldconsequently be available to inhibit AChE to a greater extent.

In essence, pre-treatment with iso-OMPA or a similarcompound (as mentioned above) is known to potentiate thetoxicity of OP or CM compounds by preventing them frombinding to non-specific esterases such as CarbE, and possiblypreventing their non-specific binding to non-enzymatic pro-teins (O’Brien, 1967). This results in a marked increase inAChE inhibition as a result of enhanced free OP or CMconcentration.

22.4 ARE OPs INHIBITORS OR SUBSTRATESOF CARBOXYLESTERASES?

Some OP insecticides that are inhibitors of AChE are alsoinhibitors as well as substrates of CarbE. Malathion is one

of the most widely used OP insecticides. It has high efficacyagainst arthropods and low toxicity to mammals (Polec et al.,1998). This feature is due to the activity of CarbEs (detoxify-ing enzymes), which hydrolyze the ester groups in thesuccinic ligand of malathion to a- and b-monoacids, whichare easily removed in the urine as metabolites (Cohen,1984; O’Brien, 1967). In insects, which lack this enzyme,malathion toxicity is much higher. Malathion is not only aninhibitor of CarbE, but also serves as a substrate for thisenzyme. CarbE in mammals is known to be an importantdetoxifying enzyme. In the 1950s and 1960s it was studiedin considerable detail with malathion as the model substrate(Knaak and O’Brien, 1960; Seume and O’Brien, 1960).The enzyme catalyzes the hydrolysis of both malathion andits active metabolite malaoxon to essentially non-toxic acidderivatives. Inhibition of CarbE by other OPs (EPN, diox-athion, trichlorfon, etc.) is therefore widely recognized asthe mechanism of potentiation of malathion toxicity (Cohenet al., 1972; Cohen and Murphy, 1974; Cook and Yip,1958; Frawley et al., 1957; Knaak and O’Brien, 1960;Murphy and DuBois, 1957; Su et al., 1971). In the rat, 60%of malathion is metabolized through the CarbE pathway(Knaak and O’Brien, 1960). It is noteworthy that the OPinsecticide phenthoate, which chemically resembles themalathion structure, also serves as a substrate for CarbEs.

22.5 POTENTIATION OF TOXICITY OF OPINSECTICIDES BY THEIR IMPURITIES

Several OP insecticides of technical grade are known to con-tain some OP impurities that potentiate the toxicity of theparent compounds. Technical malathion contains severalOP impurities that are known to inhibit the activity ofCarbEs and potentiate malathion’s toxicity in mammals(Miles et al., 1979; Umetsu et al., 1977). Aldridge and col-leagues (1979) characterized the toxicological properties ofmalathion impurities and their involvement in potentiationof OP insecticide toxicity. Malaoxon [S-1,2di(ethoxycarbo-nyl)ethyl O,O dimethyl thiophosphate] is one such impurity,and also one of the major metabolites formed during theenzymatic oxidation of malathion. Malaoxon has a greaterpotency (approximately 80-fold) for AChE inhibition thanmalathion, and also inhibits CarbE (Toia et al., 1980).Isomalathion [S-1,2-di(ethoxycarbonyl) ethyl O,S-dimethyldithiophosphate], like malaoxon, is also a technical-gradeimpurity created by thermal or photochemical isomerization(Metcalf and March, 1953). Isomalathion is highly toxic tomammals (a �1000-fold more potent anti-ChE thanmalathion). During a malaria eradication program inPakistan in 1976, from 7000 spraymen, 2800 became poi-soned and 5 died. The major cause of this poisoning hasbeen identified as isomalathion, which was present as animpurity in the malathion. Isomalathion was producedduring storage of the formulated malathion (Baker et al.,

318 ANTICHOLINESTERASE PESTICIDES INTERACTIONS

1978). Aldridge and colleagues (1979) established thequantitative correlation between isomalathion content andtoxicity in many field samples of malathion. The third mostprominent impurity in technical malathion is O,S,S-trimethylphosphorodithioate (OSS-Me). All three impurities inhibitCarbEs in rat liver and serum (Mallipudi et al., 1980;Talcott et al., 1979), which could also account for theirability to potentiate malathion toxicity.

Ryan and Fukuto (1985) have investigated the effects ofisomalathion and OSS-Me on the toxicokinetics of malathionin rats. In malathion-treated rats, malathion a- and b-mono-acids and the diacid were the predominant metabolites inthe blood. Pre-treatment of rats with isomalathion or OSS-Me followed by treatment with malathion resulted in adecrease in the total radioactive metabolites in the blood.Also, a substantial reduction in the level of malathionb-monoacid and malathion diacid was observed in theblood of impurity pre-treated rats. These results indicatethat the impurities have a stronger affinity for CarbEs, therebyinhibiting its activity. This enzyme preferentially hydrolyzesthe b-carbethoxy moiety of malathion. The major malathionmetabolites excreted in the urine of pre-treated and controlrats generally matched those present in the blood. The poten-tiation of the acute toxicity of malathion by pre-treatment withisomalathion or OSS-Me was explained by the reduction inthe rat’s capacity to degrade malathion via CarbE-catalyzedhydrolysis of the b-carbethoxy moiety. Furthermore, theseimpurities are potent inactivators of malathion CarbEs andhave also been shown to deplete cellular glutathione in rathepatocytes (Malik and Summer, 1982). For information oninteractions of the enantiomers of malaoxon and isomalathionwith AChE, readers are referred to previous publications(Berkman et al., 1993; Polec et al., 1998).

Malathion impurities O,O,S-trimethyl phosphorothioate(OOS-Me) and OSS-Me are reported to cause hemostatic dis-orders such as prolongation of blood clotting, prothrombinand thrombin time (Keadtisuke et al., 1990). Deficiency ofcoagulation factors II, V, and VII is also observed. OOS-Me and OSS-Me also cause dose-dependent increases ofb-glucuronidase in the blood, with a maximum of 15- and31-fold increases observed following treatment with60 mg/kg OOS-Me and 40 mg/kg OSS-Me, respectively.The liver endoplasmic reticulum was the source of theenzyme released into the blood. Co-treatment of OOS-Mewith 5% OOO-trimethyl phosphorothioate (OOO-Me), apotent antagonist of OOS-Me, induced delayed toxicity, pre-vented hemostatic disorders, but had no effect in reducingb-glucuronidase levels. However, pre-treatment of rats withpiperonyl butoxide reduced the amount of b-glucuronidasereleased into the blood.

As in the case of technical malathion, technical phenthoatecontains two impurities (O,O,S-trimethyl phosphorothioate,OOS-Me; O,S,S-trimethyl phosphorodithioate, OSS-Me).These impurities cause a marked inhibition of CarbE andgreatly diminish the amount of phenthoate acid formed.

OSS-Me has been found to be superior in its inhibitoryaction against rat liver CarbEs to that of OOS-Me. OSS-Mewas equipotent in inhibiting liver and plasma CarbEs, andOOS-Me was more effective in inhibiting plasma CarbEthan rat liver CarbEs (Steven et al., 1982). These authorshave demonstrated that phenthoate contains an ethoxycarbo-nyl moiety susceptible to enzymatic degradation to the corre-sponding non-toxic carboxylic acid derivative. Becausedegradation of phenthoate to the acid is most likely mediatedby a CarbE, inhibition of this enzyme by OSS-Me and OOS-Me may be the cause of their potentiation of phenthoatetoxicity.

The impurity OOS-Me present in commercial formu-lations of malathion and phenthoate has been shown topotentiate the acute toxicity of OP compounds, and causesdelayed toxicity or mortality in rats. Inhibition of tissueCarbEs, which are responsible for the hydrolytic detoxifica-tion of malathion and malaoxon, is a primary factor in thepotentiation of acute malathion toxicity. Imamura andHasegawa (1984) reported that treatment of rats with animpurity of malathion (OOS-Me) and its structural analogO,O-dimethyl S-ethyl phosphorothioate (OOS-Et) inhibitedliver microsomal malathion and phenthoate CarbEs. The inhi-bition lasted for at least 7 days following a single oral admin-istration of OOS-Me. These treatments inhibited AChE andNaþ/Kþ-dependent ATPase of erythrocyte membranes,which persisted for at least 3 days. OOS-Et was a morepotent inhibitor of all the esterases examined than OOS-Me. Pre-treatment of rats with a metabolic inducer (pheno-barbital) or a metabolic inhibitor (piperonyl butoxide) hadno influence on such inhibitory effects on liver microsomalCarbEs produced by OOS-Me or OOS-Et.

It should be mentioned that, unlike malathion or phentho-ate, during certain storage conditions for acephate or fenthiontheir impurities are formed, but they do not potentiate theirtoxicity (Toia et al., 1980; Umetsu et al., 1977).

22.6 METABOLISM-BASED INTERACTIONS

OP and CM insecticides are metabolized in many tissues, butparticularly in the liver of mammalian systems (Sams et al.,2000). OPs of the “thioate” group, such as malathion, para-thion, diazinon, and chlorpyrifos, are metabolized to “oxon”metabolites, which are much more toxic than the parent com-pounds because of their greater potency for AChE inhibition(Jokanovic, 2001; Sultatos, 2006; Tang et al., 2006). UnlikeOPs, CMs are mainly metabolized through the oxidation ofthe parent molecules to less or non-toxic products by micro-somal drug-metabolizing enzymes (Chambers et al., 1995;Kuhr and Dorough, 1976; Kulkarni and Hodgson, 1980;Tang et al., 2006). Certain drugs and xenobiotics are inducersor inhibitors of drug-metabolizing enzymes, and as a resultthey significantly modulate the toxicity (potentiation or antag-onism) of OPs or CMs.

22.6 METABOLISM-BASED INTERACTIONS 319

Phenobarbital is a model inducer of hepatic microsomalenzymes in mice. Induction of hepatic microsomal enzymesincreases the metabolism of drugs and various xenobioticsand thus decreases their half-lives in the body and their acutetoxicities. There are, however, special circumstances underwhich certain xenobiotics are activated; for example, they aremetabolized to more potent compounds by the hepatic micro-somal enzyme system (e.g., phosphorothionate insecticides).

22.6.1 Potentiation

Potentiation of OP or CM insecticides occurs primarily as aresult of blockage of their detoxification metabolism. Forexample, malathion, which contains a carboxylic ester link-age, is hydrolytically detoxified by tissue CarbEs and ismarkedly potentiated by other OP agents that inhibit theesterases (Cohen, 1984; Frawley et al., 1957; Murphy et al.,1959; Seume and O’Brien, 1960). However, inhibition ofCarbEs was not observed after BPMC administration(Takahashi et al., 1983). Alternatively, inhibition of hepaticfirst-pass metabolism of BPMC was suggested as a mechan-ism for the potentiation of BPMC toxicity, because bioavail-ability and plasma concentrations of BPMC were increasedby fenitrothion (Tsuda et al., 1984). BPMC was metabolizedby hepatic mixed-function oxidase (MFO), and the metab-olism was inhibited by fenitrothion (Takahashi et al.,1984). However, the potentiation of BPMC toxicity byfenthion treatment was only partly attributed to the increaseof plasma BPMC concentrations (Miaoka et al., 1984).The activity and amount of MFO are lowered by P¼S typeOP insecticides, which are metabolized from the P¼S typeto P¼O type through oxidative desulfuration, but not byP¼O type OP insecticides (Norman et al., 1974; Rao andAnders, 1973; Uchiyama et al., 1975; Yoshida et al., 1975,1978). Cyanophos, fenitrothion, and malathion are metab-olized via desulfuration (March et al., 1956; Miyamotoet al., 1963; Wakimura and Miyamoto, 1971).

N-methylcarbamates (BPMC, MPMC, MTMC, NAC, andXMC) are metabolized by MFO, and their major metabolitesshow no or lower anti-AChE activity than the parent chemi-cals (Cheng and Casida, 1973; Miyamoto and Fukunaga,1971; Ohkawa et al., 1974; Oonnithan and Casida, 1968).Takahashi and colleagues (1987) reported that treatmentwith fenitrothion increased plasma concentrations of otherN-methylcarbamates more than those of BPMC, althoughthe potentiation of BPMC toxicity was strongest. SKF525-A and fenitrothion treatments increased plasma BPMCconcentrations to a similar degree, but the potentiation ofBPMC toxicity by SKF 525-A was significantly less thanthat by fenitrothion. Thus, some other mechanism(s) maybe responsible for the potentiation of N-methylcarbamatetoxicities.

In a series of interaction studies, Cohen and Orzcech(1977) in mice and Satoh and Hosokawa (1998) in rats

utilized triorthotolyl phosphate (TOTP) as a selectiveCarbE inhibitor. This agent has been shown to inhibit periph-eral B-esterases, both CarbEs and pseudocholinesterases, atdoses that do not affect brain AChE activity (Cohen andMurphy, 1971). Both studies revealed that TOTP pre-treatment increased the toxicity of the local anesthetic esterprocaine and that potentiation was attributed to TOTP’s inhi-bition of liver and plasma esterases. In other experiments,Cohen (1984) demonstrated that EPN pre-treatment poten-tiated procaine toxicity by inhibition of CarbE in the liverand impairment of procaine detoxification. In similar studies,interactions between procaine and the OP insecticide Dasanit{O,O-diethyl O-[ p-(methylsulfinyl)phenyl]phosphorothioa-te}were investigated (Cohen, 1984). Dasanit treatments,which produced either minimal or no cholinergic signs,inhibited tissue CarbEs, caused elevated plasma procaineconcentrations, and potentiated procaine toxicity and lethalityin both mice and rats. Dasanit (2.5 mg/kg) pre-treatment alsopotentiated toxicity and caused lethality in rats challengedwith tricaine. It was concluded that an apparently harmlessagent became lethal by prior exposure to a dose of OP,which on its own inhibited tissue esterases but caused noovert toxicity.

22.6.2 Antagonism

Pre-treatment of experimental animals with phenobarbital(DuBois and Kinoshita, 1966; Menzer and Best, 1968) orchlorinated hydrocarbons (Triolo and Coon, 1966) reducesthe toxicity of a number of anti-ChE OP compounds. In gen-eral, reduction in the toxicities of OPs has been thought toresult from increases in the oxidation of OP compounds byhepatic microsomal enzymes.

DuBois and Kinoshita (1966) reported for the first timethat phenobarbital pre-treatment reduced the toxicity of var-ious anti-ChE OPs; however, the exact mechanism for theincreased detoxification remained unclear. Pre-treatmentwith sodium phenobarbital induces hepatic microsomalenzymes, which are responsible for the metabolic breakdownof a large number of endogenous and exogenous chemicalcompounds. Menzer and Best (1968) demonstrated that pre-treatment with phenobarbital increased the anti-ChE actionof the OP insecticide dimethoate in mice. Clement (1983)reported that phenobarbital pre-treatment decreased the tox-icity of an OP nerve agent, soman, following s.c., i.p., ori.v. administration. An increase in the amount of plasmaAChE and non-specific binding sites for soman (esterasesin liver and plasma), and not an increase in the metabolismof soman in vivo (unchanged somanase), probably accountsfor the protection afforded by phenobarbital pre-treatmentin mice. These results were in agreement with the findingsof Fonnum and Sterri (1981). Similar results can be expectedwith sarin or diisopropylphosphorofluoridate (DFP), whichbind to plasma and Red Blood Cells (RBC) aliesterase to a

320 ANTICHOLINESTERASE PESTICIDES INTERACTIONS

large extent, and only a very small portion of the administereddose is actually used to inhibit AChE. In another report,Clement (1984a) demonstrated that sodium pentobarbitalinhibited plasma and RBC AChE as well as inhibitingplasma aliesterase, thereby increasing soman toxicity. Itappears that sodium pentobarbital occupies the non-specific sites to which soman would normally be attached.Hollingworth (1969) reported that methyl iodide pre-treatment increased fenitrothion’s toxicity in mice.

OP insecticides are more toxic to weanling and newbornrats than the adult. Increased susceptibility is due in part toincomplete development of the liver microsomal enzymaticsystems, which biotransform OPs. Similarly, this decreasedmetabolic capacity alters sensitivity of the prenatal organismto the toxicity of foreign chemicals. Alary and Brodeur(1969) reported phenobarbital protected against parathiontoxicity in adult female rats. Phenobarbital stimulates thedirect degradation of parathion to diethyl phosphorothioicacid and p-nitrophenol. Detoxification of phosphorothionatecompounds, such as parathion, first involves oxidativedesulfuration to the corresponding active phosphate inhibitor,followed by hydrolysis of the phosphate. Phenobarbital pre-treatment increases liver parathion oxidase activity approxi-mately fivefold and liver paraoxonase activity 1.5-fold.Harbison (1975) reported that pre-treatment of pregnantmice with phenobarbital reduced the peak concentration ofparathion and significantly enhanced its disappearance fromfetal tissue. Passage of paraoxon into fetal tissue was pre-vented and inhibition of fetal cholinesterase blocked byphenobarbital pre-treatment, so it was concluded that thefetus was protected against lethal concentrations of paraoxonby phenobarbital treatment of the maternal mice.

Parathion must undergo oxidative desulfuration to para-oxon to inhibit AChE. In general, hydrolysis of paraoxon isthe usual pathway for detoxification. However, parathioncan undergo direct degradation to p-nitrophenol and diethyl-phosphorothioic acid in a reaction catalyzed by a NADPH-dependent microsomal enzyme system. Phenobarbitalpre-treatment of animals is known to induce the activity ofdrug-metabolizing enzymes in liver microsomes (Coonly,1967). Phenobarbital significantly increased the metabolismof parathion, involving both activation and detoxification,but the rate of formation of the end product p-nitrophenolwas significantly increased.

Knight and colleagues (1987) determined the effect ofphenobarbital pre-treatment on the in vivo metabolism of car-baryl in rats. When carbaryl was administered at a low dose(1.64 mg/kg), phenobarbital pre-treatment (75 mg/kg, i.p.for 5 days) did not quantitatively induce either the oxidativeor conjugative biotransformation of carbaryl. However, at a10-fold higher dosage of carbaryl (16.4 mg/kg), phenobarbi-tal pre-treatment enhanced only sulfate conjugation of car-baryl. In phenobarbital-treated rats, when the carbaryl dosewas increased from 1.64 mg/kg to 16.4 mg/kg an eightfold

increase in excretion of total glucuronide conjugates of car-baryl and 20-fold increase in excretion of total sulfate conju-gates of carbaryl were observed. In essence, this studydemonstrated that phenobarbital pre-treatment significantlyinfluenced the metabolism of carbaryl in rats when the car-baryl was administered at 16.4 mg/kg but not at 1.64 mg/kg. These data may be of toxicological significance inhumans ingesting drugs that are microsomal enzyme inducers,and who may inadvertently be exposed to small or largeamounts of an environmentally derived pesticide like carbaryl.

Parathion toxicity has been attributed to its metabolicproduct paraoxon, which is formed in the mammalian liverby the actions of multiple oxidative enzymes. These areinduced by barbiturates and inhibited by SKF 525-A andcimetidine. Phenobarbital increased survival by 100%,whereas cimetidine and SKF 525-A dramatically potentiatedparathion toxicity in rats. Phenobarbital increased the for-mation of p-nitrophenol, but cimetidine and SKF 525-Aproduced the opposite effect. Paraoxon and p-nitrophenolfrom parathion were decreased by cimetidine. The datastrongly suggest that parathion itself is largely responsiblefor its toxicity and the inhibition of its metabolism is harmfulrather than beneficial (Mourelle et al., 1986).

Ginsberg and colleagues (1982) described the interactionbetween the insecticide fenitrothion and the widely usedanalgesic, acetaminophen. MFOs convert acetaminophen toa reactive electrophile, which is inactivated through conju-gation with hepatic glutathione. When the amount of electro-phile is sufficient to exhaust glutathione stores, covalentbinding to cell macromolecules accelerates and hepatotoxi-city ensues. In general, agents that deplete glutathionepotentiate acetaminophen hepatotoxicity. Fenitrothion is ofrelatively low mammalian toxicity, partly due to the effectiveglutathione-dependent detoxification. Thus one might predictthat prior exposure of mice to glutathione-decreasing doses offenitrothion would increase acetaminophen hepatotoxicity.The findings of Ginsberg and colleagues (1982) revealedthat the fenitrothion antagonized acetaminophen hepatotoxi-city and lethality by diminishing the electrophile formed.Thus, in spite of reduced glutathione reserves, acetaminophenhepatotoxicity was diminished, probably as a result of feni-trothion inhibition of MFO. In other words, MFO inhibitionwas overriding the effect of glutathione depletion. So, it is dif-ficult to predict the nature of toxicologic interactions withOPs that alter both activation and inactivation pathways forspecific xenobiotics. In a similar investigation, Costa andMurphy (1984) determined the interaction between acetami-nophen and three OPs (methyl chlorpyrifos, methyl para-thion, and dichlorvos) in mice. Acetaminophen caused adose-dependent decrease of non-protein sulfhydryls(NPSH) in mouse liver. At a dose of 600 mg/kg, whichdecreased hepatic NPSH by 90%, acetaminophen did notpotentiate the effects of the OPs on esterases. On the otherhand, depletion of hepatic NPSH by diethylmaleate increased

22.6 METABOLISM-BASED INTERACTIONS 321

the toxicities of the OPs. Although the reported inhibition ofMFO activity by acetaminophen may explain the lack ofpotentiation of methyl chlorpyrifos and methyl parathion,which need to be converted to their oxygen analogs, its failurein potentiating the toxicity of dichlorvos, which does notrequire metabolic activation, suggests that other mechanismsare involved. The finding that acetaminophen decreasedNPSH only in the liver, and to a minor extent in the kidney,while diethylmaleate caused significant depletion of NPSHin several tissues, suggests that extra-hepatic glutathionemay be relevant to the detoxication of certain OPs.

Piperonyl butoxide is a classical example of chemicals thatcause inhibition of drug-metabolizing enzymes, and stronglyinhibits the conversion of parathion to paraoxon andmalathion to malaoxon. So, it is possible that the otherdrugs that are known to interfere with cytochrome P-450are able to prevent parathion- or malathion-induced toxicity.Among these substances, chloramphenicol is well known forinhibiting the biotransformation of various xenobiotics(Christensen and Skowsted, 1969; Gupta et al., 1983;Halpert et al., 1982; Sutiak et al., 1994). Hapke and col-leagues (1977) observed reduced toxicity of the methyl para-thion following chloramphenicol pre-treatment in rats as aresult of inhibited conversion of methyl parathion to methylparaoxon. The study further demonstrated that chlorampheni-col also inhibited CarbE in rat liver. In the rat, 60% of

malathion is metabolized through the CarbE pathway(Knaak and O’Brien, 1960). Chloramphenicol inhibits thecytochrome P-450-dependent oxidative desulfuration ofparathion, and thus may also inhibit the precisely analogousreaction that malathion undergoes. In a follow-up study,Gupta and colleagues (1983) demonstrated that pre-treatmentof rats with chloramphenicol completely protected ratsagainst malathion-induced signs of cholinergic toxicity.Pre-treatment with chloramphenicol also decreased theextent and duration of malathion-induced inhibition ofChE. The study concluded that the inhibition of malathiontoxicity by chloramphenicol pre-treatment is attributable toinhibition of the metabolic activation of malathion tomalaoxon by inhibition of the hepatic cytochrome P-450monooxygenase system (Fig. 22.1) in rats and mice(Adams et al., 1977; Dixon and Fouts, 1962; Halpert andNeal, 1980). Interestingly, Sutiak and colleagues (1994)reported that chloramphenicol has a protective effect againstparathion-induced pulmonary edema, which is probablyrelated to inhibition of the lung cytochrome P-450 respon-sible for parathion conversion to paraoxon. This protectiveeffect of chloramphenicol is marked in female rabbits, butthere is no inhibitory action in males. Halpert (1982) demon-strated that chloramphenicol acts as a suicide substrate of themajor phenobarbital-induced form of rat liver cytochromeP-450, by virtue of the covalent modification of the protein

Figure 22.1 Metabolism of malathion (activation and detoxification reactions). Chloramphenicol provides protection against malathiontoxicity by inhibiting cytochrome P-450 monooxygenase.

322 ANTICHOLINESTERASE PESTICIDES INTERACTIONS

rather than of the heme moiety. Adams and co-workers(1977) indicated that chloramphenicol should be adminis-tered with a great caution when given in conjunction withother drugs that are detoxified by the liver cytochromeP-450 monooxygenase system.

The toxicity of anti-ChE insecticides can be modulated byfood ingredients, metals, mycotoxins, and solvents, inaddition to therapeutic drugs (Axelrad et al., 2002; Cohen1984; Costa and Murphy, 1984; Gupta et al., 1983; Hapkeet al., 1977; Liu et al., 2007; Padilla, 2006; Pope andPadilla, 1990; Purshottam and Srivastava, 1984).

22.7 MODELS FOR INTERACTIONINVESTIGATION

Because exposure to pesticides in a mixture is more than apossibility, development of a suitable model to understandthe types of interaction was always considered necessary.Potentiation of the toxicity of some anti-ChE pesticides byothers is most interesting regarding both mechanisms andclinical aspects. Most toxic interactions occur due to inter-ference in detoxification of anti-ChE pesticides and theirbinding to target or non-target enzyme(s) or some otherproteins. So, during the last 50 years there have been attemptsto develop model(s) for understanding the interactions.Cohen and Murphy (1974) developed for the first time a sim-plified bioassay for OP detoxification and interactions. Inbrief, 0.5 nmol malaoxon or 0.15 nmol paraoxon was incu-bated with mouse brain with or without the addition of asecond, detoxifying tissue. After 25 min, ACh was added tothe flasks, and the brain ChE activities remaining were deter-mined manometrically. The difference in brain ChE activitieswas a measure of the OP detoxifying capacity of the secondtissue, and was proportional to the quantity of detoxifyingtissue added. Malaoxon detoxification was greatest inmouse liver, but lung, kidney, and plasma also possessed con-siderable activity. Hepatic subcellular fractionation studiesdemonstrated that malaoxon detoxification was greatest inthe microsomes. Administration of triorthotolyl phosphate(TOTP) and other CarbE inhibitors also inhibited malaoxondetoxification in the simplified bioassay. TOTP, an inhibitorof paraoxon binding, but not of paraoxonase, inhibited para-oxon detoxification in the simplified bioassay. TOTP (125mg/kg, 18 h) pre-treatment potentiated the anti-ChE actionof paraoxon in vivo. These authors suggested that the simpli-fied bioassay for determining detoxification of malaoxon orparaoxon in vitro may be useful in screening tests for detect-ing other synergists and antagonists of OP toxicity.

The other models for interaction may include bioassays forAChE, BuChE, CarbE, and drug-metabolizing enzymes(Cohen and Murphy, 1974; Jokanovic, 2001; Sams et al.,2000; Satoh and Hosokawa, 1998; Talcott et al., 1982;Tang et al., 2006). Of course, measurement of concentrations

of OP and CM parent compounds and their metabolites inbiological tissues and fluids is of paramount importance ininteraction studies (Jain, 2006). For information on toxico-logical evaluation of chemical mixtures, readers are referredto publications by Chaturvedi (1993) and Mauderly (1993).Recently, Padilla (2006) suggested that the study of the inter-action of anti-ChE pesticides should make use of a dose-additive experimental design with varying ratios of thecomponents to fully characterize the interaction profile.

22.8 CONCLUSIONS

From the evidence presented in this chapter, it is obvious thatexposure of humans and animals, as well as the environment,to multiple pesticides is inevitable. Under such scenarios,toxic interactions between ChE-inhibiting pesticides anddrugs or other xenobiotics are expected to occur in mamma-lian systems. Toxic or lethal potentiation by interference inthe metabolism of a pesticide by another pesticide or anyother chemical is of serious concern to health. Therefore,predicting and avoiding such interactions are the majorchallenges in this area of research.

ACKNOWLEDGMENTS

The authors’ work was supported by National Institute of Healthgrant NS057223 (D.M.).

REFERENCES

Adams HR, Isaacson EL and Masters BSS (1977) Inhibition ofhepatic microsomal enzymes by chloramphenicol. J.Pharmacol. Exp. Ther. 203:388–396.

Alary JG and Brodeur J (1969) Studies on the mechanism of pheno-barbital-induced protection against parathion in adult female rats.J. Pharmacol. Exp. Ther. 169:159–167.

Aldridge WN, Miles JW, Mount DL and Verschoyle RD (1979) Thetoxicological properties of impurities in malathion. Arch.Toxicol. 42:95–106.

Axelrad JC, Howard CV and McLean WG (2002) Interactionbetween pesticides and components of pesticide formulationsin an in vitro neurotoxicity test. Toxicology 173:259–268.

Baker EL, Zack M, Miles JW, Alderman L, Warven MW, Miller Sand Teeters WR (1978) Epidemic malathion poisoning inPakistan malaria workers. Lancet I:31–34.

Berkman CE, Quinn DA and Thompson CM (1993) Interaction ofacetylcholinesterase with the enantiomers of malaoxon and iso-malathion. Chem. Res. Toxicol. 6:724–730.

Boskovic B (1979) The influence of 2-10-cresyl-4 H-1 :3 :2-benzo-dioxa-phosphorin-2-oxide (CBDP) on organophosphate poison-ing and its therapy. Arch. Toxicol. 42:207–216.

Calabrese EJ (1995) Toxicological consequences of multiple chemi-cal interactions: A primer. Toxicology 105:121–135.

REFERENCES 323

Castorina R, Bradman A, McKone TE, Barr DB, Harnly ME andEskenazi B (2003) Cumulative organophosphate pesticideexposure and risk assessment among pregnant woman living inan agricultural community: A case study from the CHAMACOScohort. Environ. Health Perspect. 111:1640–1648.

Chambers JE, Ma T and Chambers HW (1995) Insecticide metab-olism in mammals. In: Hutson DH and Paulson GD (Eds), TheMammalian Metabolism of Agrochemicals, John Wiley andSons Ltd, Chichester, West Sussex, UK.

Chaturvedi AK (1993) Toxicological evaluation of mixtures of tenwidely used pesticides. J. Appl. Toxicol. 13:183–188.

Cheng H-M and Casida JE (1973) Metabolites and photoproducts of3-(2-butyl)phenyl N-methylcarbamate and N-benzenesulfenyl-N-methylcarbamate. J. Agric. Food Chem. 21:1037–1047.

Christensen LK and Skowsted L (1969) Inhibition of drug metab-olism by chloramphenicol. Lancet 27:1397–1399.

Clement JG (1983) Effect of pretreatment with sodium phenobarbi-tal on the toxicity of soman in mice. Biochem. Pharmacol.32:1411–1415.

Clement JG (1984a) Sodium pentobarbital alteration of the toxicityand distribution of soman (pinacolyl methylphosphonofluori-date) in mice. Biochem. Pharmacol. 33:683–685.

Clement JG (1984b) Role of aliesterase in organophosphate poison-ing. Fundam. Appl. Toxicol. 4:S96–S105.

Cohen SD (1984) Mechanism of toxicological interactions invol-ving organophosphate insecticides. Fundam. Appl. Toxicol.4:315–324.

Cohen SD and Murphy SD (1971) Carboxylesterase inhibition as anindicator of malathion potentiation in mice. J. Pharmacol. Exp.Ther. 176:733–776.

Cohen SD, Callaghan JE and Murphy SD (1972) Investigation ofmultiple mechanisms for potentiation of malaoxon’s anticholi-nesterase action by triorthotolylphosphate. Proc. Soc. Exp.Biol. Med. 141:906–910.

Cohen SD and Murphy SD (1974) A simplified bioassay for organo-phosphate detoxification and interactions. Toxicol. Appl.Pharmacol. 27:537–550.

Cohen SD and Orzech D (1977) Triorthotolyl phosphate inhibitionof carboylesterases and potentiation of procain toxicity.Biochem. Pharmacol. 26:1791–1795.

Cook JW and Yip G (1958) Malathionase II. Identity of a malathionmetabolite. J. Assoc. Offic. Agric. Chem. 41:407–411.

Coonly AH (1967) Pharmacological implications of microsomalenzyme induction. Pharmacol. Rev. 19:317–366.

Costa LG and Murphy SD (1984) Interaction between acetamino-phen and organophosphates in mice. Res. Comm. Chem.Pathol. Pharmacol. 44(3):389–400.

Dettbarn W-D and Gupta RC (1989) Role of esterases asfalse target in organophosphorus compounds toxicity. In:Aldridge WN, Hoskin FCG and Reiuer E (Eds), EsterasesHydrolyzing Organophosphorus Compounds, Ellis HorwoodLtd, Chichester, England, pp. 165–179.

Dixon RL and Fouts JR (1962) Inhibition of microsomal drug meta-bolic pathways by chloramphenicol. Biochem. Pharmacol.11:715–720.

DuBois KP (1961) Potentiation of the toxicity of organophosphoruscompounds. Adv. Pestic. Count Res. 4:117–151.

DuBois KP and Kinoshita F (1966) Stimulation of detoxificationof O-ethyl O-[(4-nitrophenyl-phenyl-phosphonothioate (EPN)]by nikethamide and phenobarbital. Proc. Soc. Exp. Biol. Med.121:59–62.

Duggan HD, Charnley G, Chen W, Chukwudebe A, Hawk R, KeigerRI, Ross J and Yarborough C. (2003) Di-alkyl phosphate biomo-nitoring data: Assessing cumulative exposure to organopho-sphate pesticides. Reg. Toxicol. Pharmacol. 37:382–395.

Fenske RA, Kedan G, Lu C, Fisker-Andersen JA and Curl CL(2002) Assessment of organophosphorus pesticide exposure inthe diets of preschool children in Washington State. J. Exp.Anal. Environ. Epidemiol. 12:21–28.

Fonnum F and Sterri SH (1981) Factors modifying the toxicity oforganophosphorus compounds including soman and sarin.Fundam. Appl. Toxicol. 1:143–147.

Frawley JP, Fuyat HN, Hagan EC, Blake JR and Fitzhugh OG(1957) Marked potentiation in mammalian toxicity from simul-taneous administration of two anticholinesterase compounds.J. Pharmacol. Exp. Ther. 121:96–106.

Ginsburg GL, Placke ME, Wyand DS and Cohen SD (1982)Protection against acetaminophen-induced hepatotoxicity byprior treatment with fenitrothion. Toxicol. Appl. Pharmacol.66:383–399.

Grubic Z, Sket D and Brzin M. (1988). Iso-OMPA-induced poten-tiation of soman toxicity in rat correlates with the inhibition ofplasma carboxylesterases. Arch. Toxicol. 62:398–399.

Gupta RC and Dettbarn W-D (1987) Iso-OMPA-induced poten-tiation of soman toxicity in rat. Arch. Toxicol. 61:58–62.

Gupta RC, Welsch F, Thornburg JE and Paul BS (1983) Effect ofchloramphenicol pretreatment on malathion-induced acute tox-icity in the rat. J. Toxicol. Environ. Health 11:897–905.

Gupta RC, Patterson GT and Dettbarn W-D (1985) Mechanisminvolved in the development of tolerance to DFP toxicity.Fundam. Appl. Toxicol. 5:517–528.

Gupta RC, Patterson GT and Dettbarn W-D (1986) Mechanisms oftoxicity and tolerance to diisopropylphosphorofluoridate at theneuromuscular junction of the rat. Toxicol. Appl. Pharmacol.84:541–550.

Gupta RC and Kadel WL (1989) Concerted role of carboxylesterasesin the potentiation of carbofuran toxicity by iso-OMPA pretereat-ment. J. Toxicol. Environ. Health 26:447–457.

Gupta RC and Kadel WL (1990a) Toxic interaction of tetraisopro-pylpyrophosphoramide and propoxur: Some insights into themechanisms. Arch. Environ. Contam. Toxicol. 19:917–920.

Gupta RC and Kadel WL (1990b) Methyl parathion acute toxicity:Prophylaxis and therapy with memantine and atropine. Arch.Int. De Pharmacod. et de Ther. 305: 208–221.

Gupta RC and Kadel WL (1991) Novel effects of memantine inantagonizing acute aldicarb toxicity: Mechanistic and appliedconsiderations. Drug Dev. Res. 24:329–341.

Gupta RC and Dettbarn W-D (1993) Role of carboxylesterases in theprevention and potentiation of N-methylcarbamate toxicity.Chem–Biol Interact. 87:295–303.

324 ANTICHOLINESTERASE PESTICIDES INTERACTIONS

Gupta RC, Goad JT, Milatovic D and Dettbarn W-D (2000)Cholinergic and noncholinergic brain biomarkers of insecticideexposure and effects. Human Exp. Toxicol. 19:297–308.

Hagan EC, Jenner PM and Jones WI (1971) Increased lethaleffects of acutely administered anticholinesterases in femalerats pre-fed with similar agents. Toxicol. Appl. Pharmacol.18:235–237.

Halpert J (1982) Further studies of the suicide inactivation ofpurified rat liver cytochrome P-450 by chloramphenicol.Mol. Pharmacol. 21:166–172.

Halpert J and Neal RA (1980) Inactivation of purified rat liver cyto-chrome P-450 by chloramphenicol. Mol. Pharmacol.17:427–431.

Halpert J, Naslund B and Betner I. (1982) Suicide inactivation of ratliver cytochrome P-450 by chloramphenicol in vivo and in vitro.Mol. Pharmacol. 23:445–452.

Hamers T, Smit MGD, Murk AJ and Koeman JH (2001) Biologicaland chemical analysis of the toxic potency of pesticides in rain-water. Chemisphere 45:609–624.

Hapke HJ, Abel J, Ghosch H, Tachampa S and Youssef S(1977) Chloramphenicol wirkungen aufenzymesysteme inner-halb des Arzneimittel Zentralbl Veterinaermed. Reiche A24:701–714.

Harbison RD (1975) Parathion-induced toxicity and phenobarbital-induced protection against parathion during prenatal develop-ment. Toxicol. Appl. Pharmacol. 32:482–493.

Hollingworth R.M. (1969). Dealkylation of organophosphate estersby mouse liver enzymes in vitro and in vivo. J. Agric. FoodChem. 17:987–996.

Imamura T and Hasegawa L (1984) Effects of an impurity onmalathion and its structural analog on rat liver carboxylesterasesand erythrocyte esterases. Pestic. Biochem. Physiol. 22:312–320.

Ito N, Hasegawa R, Imaida K, Kurata Y, Hagiwara A and Shirai T(1995) Effect of ingestion of 20 pesticides in combination atacceptable daily intake levels on rat liver carcinogenesis. FoodChem. Toxicol. 33:159–163.

Iyaniwura TT (1990) Mammalian toxicity and combined exposureto pesticides. Vet. Hum. Toxicol. 32:58–62.

Jain AV (2006) Analysis of organophosphate and carbamatepesticides and anticholinesterase therapeutic agents. In: GuptaRC (ed.), Toxicology of Organophosphate and CarbamateCompounds, Academic Press/Elsevier, Amsterdam, pp.681–701.

Jokanovic M (2001) Biotransformation of organophosphorus com-pounds. Toxicology 166:139–160.

Keadtisuke S, Dheranetra W, Nakatsugawa T and Fukuto TR (1990)Liver damage induced in rats by malathion impurities. Toxicol.Lett. 52:35–46.

Knaak J and O’Brien RD (1960) Effect of EPN on in vivo metab-olism of malathion by the rat and dog. Agric. Food Chem.8:198–203.

Knight EV, Alvares AP and Chin BH (1987) Effects of phenobarbi-tal pretreatment on the in vivo metabolism of carbaryl in rats.Bull. Environ. Contam. Toxicol. 19:815–821.

Kuhr RJ and Dorough HW (1976) In: Zweig G, (Ed.), CarbamateInsecticides: Chemistry, Biochemistry and Toxicology, CRCPress Inc., Cleveland, OH.

Kulkarni AP and Hodgson E (1980) Metabolism of insecticides bymixed function oxidase systems. Pharmacol. Ther. 8:379–475.

Lauwerys RR and Murphy SD (1969). Interaction between paraoxonand tri-o-tolyl phosphate in rats. Toxicol. Appl. Pharmacol.14:348–357.

Liu J, Gupta RC, Goad JT, Karanth S and Pope C (2007) Modulationof parathion toxicity by glucose feeding: is nitric oxide involved?Toxicol. Appl. Pharmacol. 219:106–113.

Malik JK and Summer KH (1982) Toxicity and metabolism ofmalathion and its impurities in isolated rat hepatocytes: Role ofglutathione. Toxicol. Appl. Pharmacol. 66:69–76.

Mallipudi NM, Talcott RE, Ketterman A and Fukuto TR (1980)Properties and inhibition of rat malathion carboxylesterase.J. Toxicol. Environ. Health 6:585–596.

March R, Fukuto TR, Metcalf RL and Maxon MG (1956) Fate ofP32-labeled malathion in the laying hen, white mouse andAmerican cockroach. J. Econ. Entomol. 49:185–195.

Mauderly JL (1993) Toxicological approaches to complex mixtures.Environ. Health Perspect. 101:155–165.

Maxwell DM, Brecht KM and O’Neill BL (1987) The effect of car-boxylesterase inhibition on interspecies differences in somantoxicity. Toxicol. Lett. 39:35–45.

Maxwell DM, Brecht KM, Lenz DE and O’Neill BL (1988) Theeffect of carboxylesterase inhibition on carbamate protectionagainst soman toxicity. J. Pharmacol. Exp. Ther. 246:986–991.

Maxwell DM (1992) The specificity of carboxylesterase protectionagainst the toxicity of organophosphate compounds. Toxicol.Appl. Pharmacol. 114:306–312.

McPhillips JJ (1969) Altered sensitivity to drugs following repeatedinjections of a cholinesterase inhibitor to rats. Toxicol. Appl.Pharmacol. 14:67–73.

Menzer RE and Best NH (1968) Effect of phenobarbital on the tox-icity of several organophosphorus insecticides. Toxicol. Appl.Pharmacol. 13:37–42.

Metcalf RL and March RB (1953) The isomerization of organic thio-nophosphate insecticides. J. Econ. Entomol. 46:288–294.

Miles JW, Mount DL, Staiger MA and Teeters WR (1979) S-methylisomer content of stored malathion and fenitrothion water-dispersible powders and its relationship to toxicity. J. Agric.Food Chem. 27:421–425.

Miyamoto J and Fukunaga K (1971) Metabolism of substituted phe-nylcarbamate insecticides in mammals. In: Tahori AS, (Ed.),Pesticide Terminal Residues, Butterworths, London, pp.221–229.

Miyamoto J, Sato Y, Kadota T, Fujinami A and Endo M (1963)Studies on the mode of action of organophosphorus compounds.Part I: Metabolic fate of P32 labeled sumithion and methylpar-athion in guinea pig and white rat. Agric. Biol. Chem.27:381–389.

Miyaoka T, Takahashi T, Tsuda S and Shirasu Y (1984) Potentiationof acute toxicity of 2-sec-butylphenyl N-methylcarbamate

REFERENCES 325

(BPMC) by fenitrothion in mice. Fundam. Appl. Pharmacol.4:802–807.

Moser VC, Cacey M, Hamm A, Carter JWH, Simmons JE andGennings G (2005) Neurotoxicological and statistical analysisof a mixture of five organophosphorus pesticides using a raydesign. Toxicol. Sci. 86: 101–115.

Mourelle M, Giron E and Amezcua JL (1986) Cimetidine enhancesand phenobarbital decreases parathion toxicity. J. Appl. Toxicol.6:401–404.

Murphy SD and DuBois KP (1957) Quantitative measurement ofinhibition of the enzymatic detoxification of malathion by EPN(ethyl p-nitrophenyl thiobenzene phosphate). Proc. Soc. Exp.Biol. Med. 96:813–818.

Murphy SD, Anderson RL and DuBois KP (1959) Potentiation oftoxicity of malathion by triorthotolyl phosphate. Proc. Soc.Exp. Biol. Med. 100:483–487.

Murphy SD (1969) Mechanisms of pesticide interactions in ver-tebrates. Res. Rev. 25:201–221.

Murphy SD (1980) Assessment of the potential for toxic interactionsamong environmental pollutants. In: Galli CL, Murphy SD andPaoletti R, (Eds), The Principles and Methods in ModernToxicology, Elsevier/North-Holland, Amsterdam, pp. 277–294.

Norman BJ, Poore RE and Neal RA (1974) Studies of the binding ofsulfur released in the mixed function oxidase-catalyzed metab-olism of diethyl p-nitrophenyl phosphorothionate (parathion).Biochem. Pharmacol. 23:1733–1744.

O’Brien RD (1967) Insecticides, Action and Metabolism. AcademicPress, New York, pp. 83–107.

Ohkawa H, Yoshihara R and Kohara T (1974) Metabolism ofm-tolyl N-methylcarbamate (tsumacide) in rats, houseflies andbean plants. Agric. Biol. Chem. 38:1035–1044.

Oonnithan ES and Casida JE (1968) Oxidation of methyl- anddimethylcarbamate insecticide chemicals by microsomalenzymes and anticholinesterase activity of the metabolites.J. Agric. Food Chem. 16:28–44.

Padilla S (2006) Cumulative effects of organophosphorus andcarbamate pesticides. In: Gupta RC, (Ed.), Toxicology ofOrganophosphorus and Carbamate Compounds. AcademicPress/Elsevier, Amsterdam, pp. 607–615.

Polak RL and Cohen ME (1969) The influence of triorthocresylpho-sphate on the distribution of 32P in the body of the rats after theinjection of 32P-sarin. Biochem. Pharmacol. 18:813–820.

Polec I, Cieslak L, Sledzinski B and Ksycinska H (1998) Simplesynthesis of malathion and malaoxon enantiomers and isoma-lathion diasterioisomers: toxicity–configuration relationship.Pestic. Sci. 53:165–171.

Pope C and Padilla S (1990) Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonylfluoride. J. Toxicol. Environ. Health. 31:261–273.

Purshottam T and Srivastava RK (1984) Effect of high-fat and high-protein diets on toxicity of parathion and dichlorvos. Arch.Environ. Health 39:425–430.

Rao HRG and Anders MW (1973) Inhibition of microsomal drugmetabolism by anticholinesterase insecticides. Bull. Environ.Contam. Toxicol. 9:4–9.

Ryan DL and Fukuto TR (1985) The effect of impurities on the tox-icokinetics of malathion in rats. Pestic. Biochem. Physiol.23:413–424.

Sams C, Mason HJ and Rawbone R (2000) Evidence for the acti-vation of organophosphate pesticides by cytochrome P450 3A4and 2D6 in human liver microsomes. Toxicol. Lett.116:217–221.

Satoh T and Hosokawa M (1998) The mammalian carboxyl-esterases: From molecules to functions. Ann. Rev. Pharmacol.Toxicol. 38:257–288.

Seume FW and O’Brien RD (1960) Metabolism of malathion by rattissue preparations and its modification by EPN. J. Agric. FoodChem. 8:36–41.

Steven GK, Lee T and Fukuto TR. (1982) Inhibition of rat liver andplasma carboxylesterases by impurities present in technicalphenthoate. J. Toxicol. Environ. Health 10:717–728.

Su M, Kinoshita FK, Frawley JP and DuBois KP (1971)Comparative inhibition of aliesterases and cholinesterase in ratsfed eighteen organophosphorus insecticides. Toxicol. Appl.Pharmacol. 20:241–249.

Sultatos LG (2006) Interactions of organophosphorus and carbamatecompounds with cholinesterases. In: Gupta RC, (Ed.),Toxicology of Organophosphorus and Carbamate Compounds,Academic Press/Elsevier, Amsterdam, pp. 209–218.

Sutiak V, Gustin P, Delaunois A and Ansay M. (1994) Protectiveeffect of chloramphenicol against parathion-pulmonaryoedema in rabbits: influence of sex. J. Vet. Pharmacol. Ther.17:77–79.

Takahashi H, Miyaoka T, Tsuda S and Shirasu Y (1983) Combinedtoxicity of malathion and carbamate insecticides in mice and rats.J. Pestic. Sci. 8:41–45.

Takahashi H, Miyaoka T, Tsuda S and Shirasu Y (1984)Potentiated toxicity of 2-sec-butylphenyl methylcarbamate(BPMC) by O, O-dimethyl-O-(3-methyl-4-nitrophenyl) phos-phorothioate (fenitrothione) in mice. Relationship betweenacute toxicity and metabolism of BPMC. Fundam. Appl.Toxicol. 4:718–723.

Takahashi H, Kato A, Yamashita E, Naito Y, Tsuda S and Shirasu Y(1987) Potentiation of N-methylcarbamate toxicities by organo-phosphorus insecticides in male mice. Fundam. Appl. Toxicol.8:139–146.

Talcott RE, Mallipudi NM, Umetsu N and Fukuto TR (1979)Inactivation of esterases by impurities isolated from technicalmalathion. Toxicol. Appl. Pharmacol. 49:107–112.

Talcott RE, Pond SM, Ketterman A and Becker CE (1982)Ethylesterase as indicators of liver damage. I. Studies onmalathion carboxylesterases. Toxicol. Appl. Pharmacol.65:69–74.

Tang J, Rose RL and Chambers JE (2006) Metabolism of organo-phosphorus and carbamate pesticides. In: Gupta RC (Ed.),Toxicology of Organophosphorus and Carbamate Compounds,Academic Press/Elsevier, Amsterdam, pp. 127–143.

Timchalk C, Poet TS, Hinman MN, Busby AL and Kousba AA(2004) Pharmacokinetic and pharmacodynamic interaction for

326 ANTICHOLINESTERASE PESTICIDES INTERACTIONS

a binary mixture of chlorpyrifos and diazinon in the rat. Toxicol.Appl. Pharmacol. 205:31–42.

Toia RF, March RB, Umetsu N, Mallipudi NM, Allahyari R andFukuto TR (1980) Identification and toxicological evaluationof impurities in technical malathion and fenthion. J. Agric.Food Chem. 28:599–604.

Triolo AJ and Coon JM (1966) The protective effect of aldrin againstthe toxicity of organophosphate anticholinesterase. J.Pharmacol. Exp. Ther. 154:613–623.

Tsuda S, Miyaoka T, Iwasaki M and Shirasu Y (1984)Pharmacokinetic analysis of increased toxicity of 2-sec-butyl-phenyl methylcarbamate (BPMC) by fenitrothion pretreatmentin mice. Fundam. Appl. Toxicol. 4:724–730.

Uchiyama M, Yoshida T, Homma K and Hongo T (1975) Inhibitionof hepoetic drug-metabolizing enzymes by thiophosphateinsecticides and its drug toxicological implications. Biochem.Pharmacol. 24:1221–1225.

Umetsu N, Grose F, Allahyari R, Abu-El-Haj S and Fukuto TR(1977) Effect of impurities on the mammalian toxicity oftechnical malathion and aciphate. J. Agric. Food Chem.25:946–953.

Yoshida T, Homma K, Suzuki Y and Uchiyama Y (1975) Effectof fenitrothione on hepatic microsomal components of drugmetabolizing system in mice. Chem. Pharm. Bull.23:2155–2157.

Yoshida T, Homma K, Suzuki Y and Uchiyama M (1978) Effect oforganophosphorus insecticides on hepatic microsomal cyto-chrome P-450 in mice. J. Pestic. Sci. 3:21–26.

Wakimura A and Miyamoto J (1971) Metabolism of cyanox,O,O-dimethyl O-(4-cyanophenyl)phosphorothioate in the rats.Agric. Biol. Chem. 35:410–416.

Wallace KB and Kemp JR (1991) Species specificity in the chemicalmechanisms of organophosphorus anticholinesterase activity.Chem. Res. Toxicol. 4:41–49.

REFERENCES 327