Chemico-Biological Interactions 195 (2012) 189–198

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Chemico-Biological Interactions

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Differential sensitivity of plasma carboxylesterase-null mice to parathion,chlorpyrifos and chlorpyrifos oxon, but not to diazinon, dichlorvos,diisopropylfluorophosphate, cresyl saligenin phosphate, cyclosarin thiocholine,tabun thiocholine, and carbofuran

Ellen G. Duysen a, John R. Cashman b, Lawrence M. Schopfer a, Florian Nachon c, Patrick Masson a,c,Oksana Lockridge a,⇑a Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198-5950, USAb Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121-2804, USAc Toxicology Dept., Institut de Recherche Biomédicale des Armées, 38702 La Tronche, France

a r t i c l e i n f o

Article history:Received 20 November 2011Received in revised form 16 December 2011Accepted 16 December 2011Available online 24 December 2011

Keywords:OrganochlorineOrganophosphatesPesticidesCarboxylesterase knockout mouse

0009-2797/$ - see front matter � 2011 Elsevier Irelandoi:10.1016/j.cbi.2011.12.006

Abbreviations: AChE, acetylcholinesterase; BChE,cresylbenzodioxaphosphorin; chlorpyrifos or CF, O,Opyridyl phosphorothioate; chlorpyrifos oxon or CPO,-2-pyridyl phosphate; diazinon, O,O-diethyl-O-(2-iso4-yl)phosphorothioate; cyclosarin thiocholine, 2-[(dphorylthio]-N,N,N-trimethylethanaminium iodide S(p2,2-dichlorovinyl phosphate; DFP, O,O-diisopropyl flcarboxylesterase; ES1�/�, homozygous carboxylesterheterozygous carboxylesterase knockout mouse; ES1National Center for Biotechnology Information; OP, oparaoxon, O,O-diethyl O-p-nitrophenyl phosphate;nitrophenyl phosphorothioate; sc, subcutaneously; taylamino)(ethoxy)phosphorylthio]-N,N,N-trimethyleth⇑ Corresponding author. Tel.: +1 402 559 6032; fax

E-mail addresses: ellenduysen@hotmail.com (E.G.(J.R. Cashman), lmschopf@unmc.edu (L.M. Scho(F. Nachon), pmasson@unmc.edu (P. Masson), olockri

a b s t r a c t

Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase,butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the lat-ter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition ofacetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinester-ase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has noknown effect on an animal’s well being. Paraoxonase hydrolyzes organophosphorus compounds and isnot inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency onresponse to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygousplasma carboxylesterase deficient ES1�/�mice and wild-type littermates were observed for toxic signs andchanges in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasmaacetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found thatwild-type mice were protected from the toxicity of 12.5 mg/kg parathion applied subcutaneously. How-ever, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophos-phate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpectedresult was the finding that transdermal application of chlorpyrifos at 100 mg/kg and chlorpyrifos oxonat 14 mg/kg was lethal to wild-type but not to ES1�/� mice, showing that with this organochlorine, thepresence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterasein mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is toolow to protect from low toxicity compounds that require high doses to inhibit acetylcholinesterase.

� 2011 Elsevier Ireland Ltd. All rights reserved.

d Ltd. All rights reserved.

butyrylcholinesterase; CBDP,-diethyl O-3,5,6-trichloro-2-O,O-diethyl O-3,5,6-trichloro

propyl-6-methyl-pyrimidine-imethylamino)(ethoxy)phos-); dichlorvos, O,O-dimethyl

uorophosphate; ES1, plasmaase knockout mouse; ES1+/�,

+/+, wild-type mouse; NCBI,rganophosphorus compound;parathion, O,O-diethyl O-p-bun thiocholine, 2-[(dimeth-

anaminium iodide S(p).: +1 402 559 4651.Duysen), jcashman@hbri.orgpfer), fnachon@nachon.net

d@unmc.edu (O. Lockridge).

1. Introduction

The acute toxicity of organophosphorus poisons is due to inhibi-tion of acetylcholinesterase. Carboxylesterase acts as a bioscavengerby stoichiometrically binding and inactivating organophosphoruspoisons (OP), thereby reducing the number of OP molecules avail-able for inhibiting AChE [1]. Studies in rats have concluded that plas-ma carboxylesterase plays a major role in protecting rats from thetoxicity of parathion, paraoxon, chlorpyrifos, soman, sarin, and ta-bun, but not dichlorvos, diisopropylfluorophosphate, and VX [1–4].

Carboxylesterase in mouse plasma is a 70 kDa glycoproteinproduct of the ES1 gene on mouse chromosome 8, where it isone of 16 homologous carboxylesterase genes and pseudogenes.Humans have no carboxylesterase in plasma, whereas laboratory

190 E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198

animals including mice, rats, guinea pigs, and rabbits have abun-dant carboxylesterase in plasma [5]. The presence of carboxylester-ase in the plasma of mice makes them poor models for studies oforganophosphorus intoxication in humans.

ES1 carboxylesterase in plasma is distinct from the carboxyles-terases in liver and intestine, which are transcribed from the CES1and CES2 gene clusters on chromosome 8 and not from the ES1gene. Humans and all mammals have CES1 and CES2 carboxylester-ases in liver, intestine, and other organs [6]. Carboxylesterase in ratplasma, but not in liver, intestine, and other organs is inhibited bytoxic doses of soman, suggesting that the carboxylesterase in plas-ma is the most important OP detoxifying carboxylesterase [7,8].

In a previous report we described the plasma carboxylesteraseknockout mouse (ES1�/�), created as a small animal model forstudies that mirror human response to organophosphorus agents[9]. These mice have no detectable carboxylesterase activity inplasma, but have normal carboxylesterase activity in liver, intes-tine, and other organs. Our goal in the present report was to com-pare the response of ES1�/� and ES1+/+ wild-type mice to variousorganophosphorus toxicants and one carbamate pesticide.

2. Materials and methods

2.1. Materials

The following were from Chem Service Inc. (West Chester, PA):chlorpyrifos (PS-674); chlorpyrifos oxon (Met-674B); trichloropy-ridinol (Met-674A); parathion (PS-95); paraoxon (PS610); anddichlorvos (PS-89).

The following were from Sigma–Aldrich (St. Louis, MO): carbo-furan (# 426008); DFP (# D0879); Cremophor EL (C5135); andPolyethylene glycol average Mn 400 (#202398).

Diazinon was from Ciba-Geigy Corp. (Switzerland). CBDP was agift from Dr. Wolf-Dietrich Dettbarn (Vanderbilt University, Nash-ville, TN) and Dr. David Lenz (US Army Institute of Chemical De-fense, Aberdeen Proving Gd, MD). It was custom synthesized byStarks Associates, Buffalo, NY and was 99.5% pure. The nerve agentmodel compounds, cyclosarin thiocholine S(p) and tabun thiocho-line S(p) were custom synthesized in the laboratory of Dr. John R.Cashman (Human BioMolecular Research Institute, San Diego,CA) as described [10]. The stereoisomers had the S(p) configura-tion, which is more reactive with acetylcholinesterase than theR(p) configuration [10].

2.2. ES1 knockout mouse

Animal work was conducted in accordance with the Guide forthe Care and Use of Laboratory Animals as adopted by the US Na-tional Institutes of Health. Formal approval to conduct animalexperiments was obtained from the Institutional Animal Care andUse Committee (IACUC) of the University of Nebraska Medical Cen-ter. C57BL/6 mice completely deficient in plasma carboxylesteraseactivity were created by homologous recombination of the ES1 geneon mouse chromosome 8 [9]. The ES1 gene was inactivated bydeleting exon 5 and introducing a frame shift for amino acids trans-lated from exons 6 to 13, thus deleting the catalytic triad residuesSer, Glu, His. The ES1�/�mice are healthy and fertile. They have nor-mal acetylcholinesterase, butyrylcholinesterase, and paraoxonase-1 activity in plasma and organs. ES1+/� mice were bred to createES1+/+, ES1+/� and ES1�/� mice. This breeding protocol allowedsiblings to be utilized in experiments, reducing the possibility of ef-fects from genetic drift when comparing the response of wild-typeand knockout mice to intoxication.

The ES1 knockout mouse in strain C57BL/6 is available from TheJackson Laboratory Repository (Bar Harbor, ME) http://

jaxmice.jax.org/query where it is listed as JAX Stock No. 014096C57BL/6-Ces1ctm1.1Loc/J. An alternative name for the ES1 gene isCes1c. The Jackson Laboratory provides heterozygous animals thatmust be bred to produce animals completely deficient in plasmacarboxylesterase.

2.3. Determination of ES1 phenotype

Pups were phenotyped by assaying plasma carboxylesteraseactivity of blood collected from the saphenous vein. Carboxylester-ase activity was measured with alpha-naphthyl acetate [4,11] inthe presence of eserine to inhibit AChE and BChE and in the pres-ence of EDTA to inhibit paraoxonase-1. Measured activity was 9–13 lmol/ml in ES1+/+, 4–7 lmol/ml in ES1+/� and 0.5–0.7 lmol/ml in ES1�/� plasma of adult mice. The residual activity in ES1�/�

plasma was due to hydrolysis of alpha-naphthyl acetate by albu-min [9,12].

2.4. Challenge with organophosphorus compounds and a carbamatepesticide

Adult male ES1+/+ and ES1�/� mice (n = 3–6/genotype) werechallenged with organophosphorus compounds or a carbamatepesticide to determine differences in toxicity and in effects on plas-ma BChE, AChE and carboxylesterase activities. Dose finding exper-iments were conducted in ES1+/+ mice to find non-lethal doses thatwould produce toxic signs or significantly decrease esterase activ-ity. Challenge compounds were delivered subcutaneously (sc) withthe exception of chlorpyrifos and chlorpyrifos oxon, which weredelivered transdermally. Challenge compounds, doses, and deliv-ery solvents were as follows: chlorpyrifos (CF) 100 mg/kg inacetone; chlorpyrifos oxon (CPO) 14 mg/kg in acetone; trichloropy-ridinol 100 mg/kg in acetone; parathion 15 mg/kg in ethanol;paraoxon 0.2 mg/kg in 50% ethanol; carbofuran 1.0 mg/kg in 5%dimethylsulfoxide; CBDP 20 mg/kg in polyethylene glycol 400;DFP 4 mg/kg in 2% ethanol/saline solution; diazinon 50 mg/kg in7% Cremophor EL/10% ethanol/saline solution; dichlorvos 7.5 mg/kg in saline; cyclosarin thiocholine 0.05 mg/kg in ethanol; tabunthiocholine 6.0 mg/kg in ethanol. Toxicant solutions were freshlyprepared immediately before use.

2.5. Functional observational battery

Mice were observed for the behavioral toxic signs described byMcDaniel and Moser [14] including posture, involuntary motormovements, tremors, seizures, convulsions, palpebral closure,reactivity to being handled, lacrimation, salivation, piloerection,gait, mobility, arousal, stereotypy, straub tail, vocalization, rightingreflex, and temperature. Mice challenged with a single sublethaldose displayed many of these toxic signs, but none had convulsionsor stereotypical behavior.

2.6. Temperature

Axial body temperature was measured with a digital thermom-eter, Thermalert model TH-5, attached to a surface MicroprobeMT-D, type T thermocouple (Physitemp Instruments, Clifton, NJ).Temperature was recorded prior to challenge, at 5 min intervalsfor the first hour post-challenge, hourly through 8 h, and finallyat 24, 48, and 96 h post dosing.

2.7. Determination of carboxylesterase, AChE, and BChE activity

Blood was collected prior to challenge and at various times postdosing via the saphenous vein (50 ll) into heparinized collectiontubes. Plasma carboxylesterase, AChE, and BChE activities were

E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198 191

determined at each time point. Paraoxonase-1 activity was notmeasured because paraoxonase is not inhibited by organophos-phorus compounds. Mice have a significant amount of solubleAChE in plasma [15], whereas humans have negligible AChE inplasma, but have membrane bound AChE on red blood cells whereAChE constitutes the Yt blood group antigen [16].

All three enzyme activities were measured at 25 �C in a 2-mlreaction volume using a Gilford spectrophotometer interfaced witha MacLab data recorder (ADInstruments Pty Ltd., Castle Hill,Australia).

Carboxylesterase activity was determined by monitoring thehydrolysis rate of alpha-naphthyl acetate [4,11]. Three microlitersof plasma were added to 1.9 ml of 0.1 M potassium phosphate buf-fer pH 7.0 in the presence of 0.01 mM eserine (to inhibit AChE andBChE) and 1.3 mM EDTA (to inhibit paraoxonase-1). The sampleswere incubated at 25 �C for 15 min to allow complete inhibitionof AChE, BChE, and paraoxonase. After incubation, 100 ll of 0.1 Malpha-naphthyl acetate in ethanol was added to the cuvette andthe rate of hydrolysis was followed at 321 nm. The extinction coef-ficient for the reaction product was 2220 M�1 cm�1.

AChE and BChE activities were assayed at 412 nm using anextinction coefficient of 13,600 M�1 cm�1 [17]. AChE activity wasassayed by adding 3 ll of plasma to 2 ml of 0.1 M potassium phos-phate pH 7.0, containing 1 mM acetylthiocholine and 0.5 mMdithiobisnitrobenzoic acid, at 25 �C in the presence of 0.01 mM eth-opropazine to inhibit BChE. BChE activity was assayed by adding3 ll of plasma to 2.0 ml of 0.1 M potassium phosphate pH 7.0, con-taining 1 mM butyrylthiocholine and 0.5 mM dithiobisnitrobenzo-ic acid at 25 �C.

A unit of AChE, BChE, or carboxylesterase activity was defined asone micromole of substrate hydrolyzed per minute. The concentra-tions of AChE, BChE, and carboxylesterase in mouse plasma are0.2 mg/L, 2.6 mg/L, and 80 mg/L, respectively [5]. The subunitmolecular weights are 70 kDa for AChE, 85 kDa for BChE, and70 kDa for carboxylesterase. These molecular weights are heavierthan the protein molecular weights calculated for the primary ami-no acid sequences because they include the mass of N-linked gly-cans. Database codes for these mouse enzymes are: AChE(GenBank ID gi:49845; UniProt ID P21836), BChE (GenBank IDgi:191580; UniProt ID M99492), and ES1 carboxylesterase (GenBankID gi:247269929; UniProt ID P23953; previously gi:6679689, whichis 97% identical to the updated version gi:247269929).

2.8. Statistics

Comparison of means was by paired samples t-test with 95%confidence interval (±std dev). Analysis was conducted using SPSSsoftware (IBM Corporation Chicago, IL).

Fig. 1. Structures of chemicals used for challenge trials. The Chemical AbstractsService (CAS) registry number is given for each compound.

3. Results

3.1. Plasma carboxylesterase protects against parathion challenge

3.1.1. ParathionParathion is a phosphorothioate pesticide (Fig. 1). Its toxicity is

mediated through its oxon metabolite, paraoxon, which is gener-ated by cytochrome P450 enzymes in the liver [18,19]. ES1�/�

and ES1+/+ mice (n = 3 per genotype) were treated sc with12.5 mg/kg parathion. Inhibition of plasma AChE, BChE, and ES1carboxylesterase activities began to be apparent 0.5 h after treat-ment. Inhibition levels increased with time reaching maximumlevels at 6–24 h (Fig. 2A–C). The lag between exposure to parathionand inhibition of plasma esterases reflects the time to induce thecytochrome P450 enzymes that activate parathion to paraoxon.At 6 h post dosing, plasma AChE was significantly more inhibited

in ES1�/� than in ES1+/+ mice (73.1 ± 4.6% versus 56 ± 2.3%) (Table1). At the same time, BChE was equally inhibited in both genotypes(71–76%) and ES1 in wild-type mice was inhibited 85%. One ES1�/�

mouse was dead at 24 h post dosing. Surface body temperature ofES1�/� mice dropped from 38 to 33 �C at 3 h, and to 30 �C at 6 h(Fig. 2D). Behavioral toxic signs observed in ES1�/� mice at 6 hpost-dosing included muscle fasciculation, flattened posture, hea-vy mucus covering the eyes, palpebral closure, decreased handlingreaction, ataxic gait, reduced mobility, decreased arousal andpiloerection. Surface body temperature of ES1+/+ mice dropped

Fig. 2. Parathion challenge. (A) plasma AChE activity; (B) plasma BChE activity; (C)plasma carboxylesterase activity; (D) surface body temperature. Mice werechallenged with 12.5 mg/kg parathion sc (n = 3 per genotype). Points are shown±std dev. Wild-type mice, ES1+/+, are represented by white bars and white squares.ES1�/� mice are represented by black bars and black squares. ES1�/� mice hadbackground plasma carboxylesterase activity of 0.6 lmol/ml due to hydrolysis ofalpha-naphthyl acetate by albumin [12]. In the body temperature panel, measure-ments marked with an ‘‘a’’ are significantly different from those marked with a ‘‘b’’based on a two tailed t-test p 6 0.05.

192 E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198

2 �C at 6 h, but mice displayed no behavioral toxic signs. By 48 hpost dosing, body temperatures had recovered to baseline in bothgenotypes and all mice were free of behavioral toxic signs. Theseresults indicate that plasma carboxylesterase plays a role in reduc-ing the toxicity of parathion.

Table 1Average percent inhibition of BChE, AChE and carboxylesterase ES1 activity in plasma of Etreatment. (±std dev).

Compound Dose (mg/kg) Time post-dose (hours) ES1 genotype B

Carbofuran 1.0 0.5 �/� 2Carbofuran 1.0 0.5 +/+ 2CBDP 20.0 0.5 �/� 9CBDP 20.0 0.5 +/+ 9Chlorpyrifos 100.0 6.0 �/� 7Chlorpyrifos 100.0 6.0 +/+ 7Chlorpyrifos oxon 14.0 0.5 �/� 9Chlorpyrifos oxon 14.0 0.5 +/+ 9DFP 4.0 0.5 �/� 5DFP 4.0 0.5 +/+ 5Diazinon 50.0 2.0 �/� 8Diazinon 50.0 2.0 +/+ 8Dichlorvos 7.5 0.5 �/� 2Dichlorvos 7.5 0.5 +/+ 2Paraoxon 0.2 0.5 �/� 6Paraoxon 0.2 0.5 +/+ 7Parathion 12.5 6.0 �/� 7Parathion 12.5 6.0 +/+ 7Cyclosarin thiocholine 0.05 0.5 �/� 6Cyclosarin thiocholine 0.05 0.5 +/+ 4Tabun thiocholine 6.0 0.5 �/� 9Tabun thiocholine 6.0 0.5 +/+ 9Soman coumarin(e) 3.0 4.0 �/� 6Soman coumarin 3.0 4.0 +/+ 6Soman coumarin 3.0 48.0 �/� 8Soman coumarin 3.0 48.0 +/+ 1

ND, no detectable activity in ES1�/� plasma. n = 3–6 per genotype.(a) is significantly different than (b) (p = 0.02); by paired samples t-test.(c) is significantly different than (d) (p < 0.01) by paired samples t-test.(e) Soman coumarin studies are from [9].

3.1.2. ParaoxonParaoxon is the active metabolite of parathion (Fig. 1). Mice

(n = 3 per genotype) were challenged sc with 0.2 mg/kg paraoxon.There was no lag time between administration of paraoxon and theappearance of behavioral toxic signs. AChE and BChE inhibition at0.5 h post dosing (Fig. 3A and B) was equivalent between the geno-types (61–71% for BChE and 57–61% for AChE). There was 91% inhi-bition of ES1 in the wild-type mice (Fig. 3C). Behavioral toxic signsand changes in body temperature through 4 h were similar forboth the ES1+/+ and ES1�/� mice. Behavioral toxic signs were thesame as in the parathion treated animals. Body temperaturedropped from 38 to 28 �C at 1 h post-dosing (Fig. 3D). Both AChEand BChE activities recovered to baseline by 24 h in both genotypeswhile carboxylesterase ES1 activity in the wild-type mice was 60%of baseline by this time point. By 24 h body temperature had re-turned to normal and all behavioral signs of toxicity were gonein both genotypes. We conclude that although mouse plasma carb-oxylesterase protected against parathion toxicity it played no sig-nificant role in protection against the active metabolite paraoxon.

3.2. Wild-type mice die, but ES1 knockouts survive challenge withchlorpyrifos and chlorpyrifos oxon

3.2.1. Chlorpyrifos (CF)Chlorpyrifos is a phosphorothioate, organochlorine pesticide

(Fig. 1). Chlorpyrifos is metabolically activated by oxidative desulf-uration to the oxon. The oxon, but not the parent compound, istoxic. Mice (n = 6 per genotype) were treated by transdermal appli-cation with 100 mg/kg CF. No toxic signs were evident until 6 hpost dosing when wild-type ES1+/+ mice, but not ES1�/� mice,started to display decreased surface body temperature (Fig. 4D)and decreased motor activity. At 6 h post dosing, AChE activitywas inhibited 70% in both genotypes, BChE activity was inhibited75–77% in both genotypes, and there was complete inhibition of

S1�/� and +/+ mice after challenge with carbamate and OP at 0.5, 2, 4, 6, or 48 h after

ChE % inhibition AChE % inhibition ES1 % inhibition Behavioral toxic signs

8.9 ± 2.1 52.4 ± 4.1 ND Yes5.8 ± 2.9 52.0 ± 5.6 94.2 ± 1.4 Yes8.1 ± 0.7 39.7 ± 2.8 ND No7.8 ± 0.2 32.2 ± 5.4 99.2 ± 0.6 No5.4 ± 3.3 70.1 ± 15.1 ND Yes6.8 ± 4.8 70.4 ± 6.6 99.2 ± 0.8 Yes1.2 ± 1.5 58.8 ± 1.2 ND Yes1.5 ± 0.4 62.8 ± 9.9 100 ± 0.0 Yes5.2 ± 2.7 59.9 ± 4.8 ND Yes5.3 ± 1.8 71.4 ± 8.6 84.3 ± 2.3 Yes1.7 ± 1.1 93.5 ± 0.5 ND Yes2.0 ± 3.1 88.2 ± 2.6 100 ± 0.0 Yes5.5 ± 3.6 53.7 ± 4.8(a) ND Yes0.4 ± 1.8 33.4 ± 3.9(b) 79.4 ± 2.7 Yes1.0 ± 5.5 61.4 ± 2.2 ND Yes0.5 ± 1.8 57.5 ± 1.4 90.9 ± 2.3 Yes6.0 ± 4.4 73.1 ± 4.6(c) ND Yes1.5 ± 3.9 56.0 ± 2.3(d) 85.5 ± 3.4 No1.2 ± 0.8 67.5 ± 1.7(c) ND Yes9.9 ± 2.8 32.2 ± 4.6(d) 8.6 ± 3.4 Yes7.1 ± 2.3 78.0 ± 17.8 ND Yes7.5 ± 1.7 83.3 ± 11.6 24.1 ± 0.9 Yes3.3 ± 11.5 97.1 ± 1.9 ND Yes3.0 ± 11.2 83.6 ± 15.4 98.6 ± 1.1 Yes9.1 72.5 ND Yes0.3 ± 12.1 0 18.0 ± 3.0 Yes

Fig. 3. Paraoxon challenge. (A) plasma AChE activity; (B) plasma BChE activity; (C)plasma carboxylesterase activity; (D) body temperature. Mice were challenged with0.2 mg/kg paraoxon sc (n = 3 per genotype). Points are shown ±std dev. Wild-typemice, ES1+/+, are represented by white bars and white squares. ES1�/� mice arerepresented by black bars and black squares.

Fig. 4. Chlorpyrifos challenge. (A) plasma AChE activity; (B) plasma BChE activity;(C) plasma carboxylesterase activity; (D) surface body temperature. Mice werechallenged with 100 mg/kg chlorpyrifos (CF) transdermally (n = 6 per genotype).Points are shown ±std dev. Wild-type mice, ES1+/+, are represented by white barsand white squares. ES1�/� mice are represented by black bars and black squares. At24 h, 4 of the 6 ES1+/+ mice were moribund. At 48 h all wild-type mice (ES1+/+) weremoribund or dead, whereas ES1�/� mice had no signs of toxicity. In the bodytemperature panel, measurements marked with an ‘‘a’’ are significantly differentfrom those marked with a ‘‘b’’ based on a two tailed t-test p 6 0.05.

E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198 193

ES1 carboxylesterase activity in the wild-type mice (Table 1). At24 h post-dosing, the ES1�/� mice had no behavioral toxic signs,but 4 of the 6 ES1+/+ wild-type mice were moribund with behav-ioral signs of cholinergic toxicity including mucus covered eyes,flattened posture, piloerection, and whole body tremor. Therewas also a drop in body temperature from 38 to 32 �C in theES1+/+ mice (Fig. 4D). At 48 h post-dosing, all wild-type mice weremoribund or dead, whereas the ES1�/� mice displayed no signs oftoxicity. These observations are precisely opposite to expectation.

Additional observations include the following. At 24 h post-dos-ing with chlorpyrifos, plasma AChE activity in ES1�/�mice had par-tially recovered to 53% inhibition (Fig. 4A). In contrast, plasmaAChE activity in wild-type mice had not recovered, with the inhi-bition level remaining at 70%. At 24 h post-dosing, BChE activityin both genotypes (Fig. 4B) was more inhibited than at 6 h (BChEwas 92% inhibited in ES1�/�mice and 99% inhibited in ES1+/+ mice).At 24 h, carboxylesterase activity in wild-type plasma was still 99%inhibited (Fig. 4C). Surface body temperatures of CF-treated micewere normal up to 6 h. At 24 and 48 h, there was a significant dropin the body temperature of ES1+/+ mice correlating to their mori-bund status (Fig. 4D). Baseline AChE activity was recovered inthe ES1�/� mice by 9 days while BChE activity had not recoveredby 25 days post-dosing. We conclude that plasma carboxylesterasehas an effect opposite to that expected. Rather than protectingfrom chlorpyrifos toxicity, plasma carboxylesterase increased thetoxicity of chlorpyrifos. This observation in mice differs from thereported protective effect of carboxylesterase in chlorpyrifos trea-ted rats [1,2].

3.2.2. Chlorpyrifos oxon (CPO)Chlorpyrifos oxon (Fig. 1) is produced by oxidative desulfuration

of chlorpyrifos through the action of cytochrome P450 enzymes[20]. The oxon is very toxic. Mice (n = 6 per genotype) were treatedtransdermally with 14 mg/kg CPO. At 0.5 h post dosing, plasmaAChE activity was inhibited 59–63% in both genotypes, plasma BChEwas inhibited 91% in both genotypes and plasma ES1 carboxylester-ase activity was completely inhibited in the wild-type ES1+/+ mice(Fig. 5A–C). Between 2 and 6 h post-dosing, mice of both

genotypes had flattened posture, abnormal gait, decreased responseto handling, and a drop in body temperature. At 24 h, ES1+/+ micewere either moribund (n = 4) or dead (n = 2) while the ES1�/� miceappeared to have recovered. At 24 h, the body temperature ofES1�/� mice was back to normal (Fig. 5D) and they were free ofbehavioral toxic signs. At 24 h, plasma AChE activity in ES1�/�micehad partially recovered to an average inhibition of 53% compared to63% inhibition in the moribund ES1+/+ mice. At 24 h, BChE activitywas inhibited 84% in the ES1�/� mice and 89% in the moribundES1+/+ mice. Baseline AChE activity was recovered in the ES1�/�miceby 6 days while BChE activity was recovered by 15 days post-dosing.Carboxylesterase activity was still completely inhibited in ES1+/+

mice at 24 h and could not be measured at later times because allES1+/+ mice were dead. We conclude that chlorpyrifos oxon, like itsparent compound chlorpyrifos, is more toxic to wild-type mice thanto mice deficient in plasma carboxylesterase.

3.2.3. Trichloropyridinol3,5,6-Trichloro-2-pyridinol is released from both chlorpyrifos

and chlorpyrifos oxon by hydrolysis of the ester bond (Fig. 1).Wild-type mice would be expected to produce more of thismetabolite than ES1�/� mice, because wild-type mice have morecarboxylesterase. The possibility was tested that the higher levelsof trichloropyridinol in wild-type mice might explain their greatersusceptibility to poisoning by chlorpyrifos and chlorpyrifos oxoncompared to ES1�/� mice. Mice (n = 3 per genotype) were treatedtransdermally with 100 mg/kg trichloropyridinol dissolved in ace-tone. The dose and route of exposure were identical to those forchlorpyrifos. Mice showed no toxic signs at any time up to 48 h.Hanley et al. have also shown that trichloropyridinol is nontoxic[21]. It was concluded that poisoning by trichloropyridinol didnot explain why wild-type mice, but not carboxylesterase defi-cient mice, died after treatment with chlorpyrifos or chlorpyrifosoxon.

Fig. 5. Chlorpyrifos oxon challenge. (A) Plasma AChE activity; (B) plasma BChEactivity; (C) plasma carboxylesterase activity; (D) body temperature. Mice werechallenged with 14 mg/kg chlorpyrifos oxon (CPO) transdermally (n = 6 pergenotype). Points are shown ±std dev. Wild-type mice, ES1+/+, are represented bywhite bars and white squares. ES1�/� mice are represented by black bars and blacksquares. At 24 h the wild-type mice (ES1+/+) were either moribund (n = 4) or dead(n = 2) while the ES1�/� mice had no toxic signs. In the body temperature panel,measurements marked with an ‘‘a’’ are significantly different from those markedwith a ‘‘b’’ based on a two tailed t-test p 6 0.05.

194 E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198

3.3. Response of wild-type and ES1�/� mice to carbofuran, CBDP, DFP,diazinon, dichlorvos, cyclosarin thiocholine, and tabun thiocholinesuggests a minor protective role of plasma carboxylesterase

3.3.1. CarbofuranCarbofuran is a carbamate pesticide (Fig. 1) that inhibits rat

carboxylesterase at doses that do not significantly inhibit AChE[22], suggesting that carboxylesterase could protect against acutetoxicity from carbofuran. To test the contribution of plasma carb-oxylesterase to protection against carbofuran toxicity we comparedthe effect of carbofuran on wild-type and plasma carboxylesteraseknockout mice. ES1+/+ and ES1�/� mice (n = 3 per genotype) weretreated sc with 1 mg/kg carbofuran. Carbofuran-inhibited cholines-terases spontaneously reactivate with a half-life of about 2 h [23].Therefore plasma samples were analyzed for activity immediatelyafter collection to limit the degree of enzyme reactivation. AChEin both genotypes was inhibited to a greater extent (52%) thanwas BChE (26–29%) (Table 1). Plasma carboxylesterase activitywas 94% inhibited in wild-type mice, confirming that carboxylest-erase is more sensitive than AChE to inhibition by carbofuran.Behavioral signs of toxicity were similar for both genotypes includ-ing straub tail, ataxic gait, muscle fasciculations, clonic tremor, flat-tened posture, bugged eyes, salivation, lacrimation, arched back,and decreased arousal. The surface body temperature of ES1�/�

mice dropped to 30–31 �C by 0.5 h and remained at that low tem-perature for 1 h before beginning to rise (Fig. 6A). The body temper-ature of ES1+/+ mice dropped to 31.5 �C for only 15 min. After 2 h,both genotypes had completely recovered normal body tempera-ture and showed no behavioral signs of toxicity. By 24 h postdosing, the activity of plasma AChE, BChE and ES1 had returnedto normal (data not shown). We conclude that plasma carboxylest-erase has only a minor role in protection from carbofuran toxicity.

3.3.2. CBDPCBDP is a cyclic organophosphorus compound (Fig. 1) that is

used to inhibit rodent carboxylesterase in studies designed to

equalize species responses to nerve agents [24]. CBDP is the activemetabolite of tri-o-cresyl phosphate, a chemical implicated inaerotoxic syndrome [25]. The rate constant for inhibition of humanBChE by CBDP is 100-fold faster than for inhibition of AChE [25]. Itwas of interest to determine the contribution of plasma carboxy-lesterase to protection from CBDP toxicity. Mice (n = 3 per geno-type) were treated sc with 20 mg/kg CBDP. At 30 min postdosing, AChE was inhibited 32% in wild-type and 40% in ES1�/�

plasma, while BChE and ES1 carboxylesterase activity were inhib-ited 98–99%, respectively (Table 1). The nearly complete loss ofplasma BChE and ES1 activity was not accompanied by behavioralsigns of toxicity. The only notable sign of intoxication was a 1 de-gree increase in surface body temperature in both genotypes(Fig. 6B). Body temperature returned to normal by 24 h post dos-ing. Although AChE activity was 20% less inhibited in wild-typethan in ES1�/� plasma, the levels were not significantly different.We conclude that plasma carboxylesterase may afford only minorprotection against AChE inhibition by CBDP.

3.3.3. Diisopropylfluorophosphate (DFP)DFP is a research chemical (Fig. 1) that is sometimes used as a

surrogate for the nerve agent sarin. At one time DFP was used forthe treatment of glaucoma [26]. Mice (n = 3 per genotype) werechallenged sc with 4.0 mg/kg DFP. AChE and BChE plasma activitieswere not significantly different between the genotypes at 30 minpost-dosing (60–70% inhibited AChE and 55% inhibited BChE; Table1). ES1 carboxylesterase activity was inhibited 84% in wild-typemice at 30 min post dosing. ES1�/� mice had significantly lowersurface body temperatures than wild-type mice, from 1 to 3 h postdosing with a recovery of body temperature by 4 h post-dosing(Fig. 6C). Both genotypes displayed similar behavioral toxic signsincluding mild muscle fasciculation, decreased handling response,decreased arousal and hunched posture through 4 h post-dosing.We conclude that plasma carboxylesterase has a minor protectiverole in mice against DFP toxicity.

3.3.4. DiazinonDiazinon is an OP pesticide (Fig. 1) whose toxicity is mediated

through its metabolite diazoxon. The rate constant for inhibitionof human BChE by diazoxon is 100-fold faster than for inhibitionof AChE [27]. Mice (n = 3 per genotype) were challenged sc with50 mg/kg diazinon. At 2 h post-dosing, plasma AChE was inhibited88% in wild-type and 94% in ES1�/� mice, BChE was inhibited 82%in both genotypes, and ES1 was inhibited 100% in wild-type (Table1). At 2–4 h, all mice developed mild behavioral toxic signs includ-ing mucus covered eyes, palpebral closure, hunched posture, re-duced handling reaction and reduced arousal. No behavioral toxicsigns were observed at 24 h post dosing. AChE activity had recov-ered to baseline levels in all animals by 48 h while BChE activitywas still inhibited 40% in both genotypes at 11 days post dosing(data not shown). Body temperatures in both genotypes dropped2 �C at 4 h post-dosing but returned to normal by 24 h (Fig. 6D).We conclude that mouse plasma carboxylesterase does not havea significant role in protection from the toxicity of diazinon.

3.3.5. DichlorvosDichlorvos is an active OP pesticide (Fig. 1) that does not require

activation to become a cholinesterase inhibitor. Dichlorvos is inac-tivated by paraoxonase-1 (‘‘arylesterase’’ or ‘‘A-esterase’’ in theolder literature) and is considered relatively nontoxic [28,29].Carboxylesterases have been reported to be unimportant in detox-ication of dichlorvos in mice [29]. Mice (n = 3 per genotype) werechallenged sc with 7.5 mg/kg dichlorvos. ES1�/� mice had signifi-cantly greater inhibition of plasma AChE at 30 min post dosing(54% inhibition) compared to ES1+/+ mice (33% inhibition) whileBChE inhibition was equivalent between the genotypes. ES1

Fig. 6. Average surface body temperatures of mice after challenge with a single sublethal dose of a variety of toxicants. Points are shown ±std dev. Panel A, carbofuran (1 mg/kg); panel B, CBDP (20 mg/kg); panel C, DFP (4 mg/kg); panel D, diazinon (50 mg/kg); panel E, dichlorvos (7.5 mg/kg); panel F, cyclosarin thiocholine (0.05 mg/kg); and panelG, tabun thiocholine (6 mg/kg). The organophosphorus compounds were applied subcutaneously (n = 3 per genotype). Wild-type mice, ES1+/+, are represented by whitecircles. ES1�/� mice are represented by black squares. Measurements marked with an ‘‘a’’ are significantly different from those marked with a ‘‘b’’ based on a two tailed t-testp 6 0.05.

E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198 195

carboxylesterase was inhibited 80% in wild-type mice (Table 1).Behavioral toxic signs were similar for both genotypes. These signswere severe including muscle fasciculation, ataxic gait, splayedhindlimbs, straub tail, decreased arousal, mobility and handlingreaction. AChE and BChE activities had recovered by 48 h post dos-ing in both genotypes. Decreases in surface body temperature post-dosing were equivalent between the genotypes, dropping to27.5 �C at 8 h and returning to normal by 24 h (Fig. 6E). We con-clude that plasma carboxylesterase in mice plays a minor role inprotection from dichlorvos toxicity.

3.3.6. Cyclosarin thiocholineCyclosarin thiocholine is a nerve agent model compound that dif-

fers from authentic cyclosarin by the presence of a thiocholine groupin place of a fluoride ion (Fig. 1). Thiocholine is a poorer leavinggroup than fluoride, making the model compound less reactiveand less toxic than authentic cyclosarin [10]. The thiocholinenerve agent model compounds are quaternary amines and posi-tively charged but nevertheless appear to get into the brain (unpub-lished observations). Because carboxylesterase is resistant toinhibition by positively charged inhibitors [30] it was expected that

196 E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198

carboxylesterase would have no significant role in protection fromthe toxicity of this model compound. Mice (n = 3 per genotype) werechallenged sc with 0.05 mg/kg cyclosarin thiocholine. Behavioraltoxic signs were similar between the genotypes including hunchedposture, palpebral closure, decreased handling reaction and arousal,decreased body tone, lacrimation, salivation, reddened paws andnose, ataxic gait, and straub tail. Plasma AChE was significantly moreinhibited at 30 min post dosing in the ES1�/� mice (68%) than inES1+/+ mice (32%) (p < 0.02) while 9% of the plasma carboxylester-ase was inhibited in the wild-type mice at this time point (Table1). The concentration of carboxylesterase in plasma is about 400-fold greater than the concentration of AChE, suggesting that carb-oxylesterase scavenged more of the cyclosarin thiocholine thanwas able to react with AChE, even though the carboxylesterase inhi-bition level was only 9%. By 1 h post dosing, BChE was inhibitedabout 70% in both genotypes, whereas AChE activity had returnedto normal. BChE activity returned to normal after 5 days. ES1 carb-oxylesterase inhibition remained constant at 9%, through 24 h. Sur-face body temperature in both genotypes dropped to an average of30 �C at 1 h post-dosing and returned to normal by 24 h (Fig. 6F).We conclude that plasma carboxylesterase has a minor protectiverole against the toxicity of cyclosarin thiocholine.

3.3.7. Tabun thiocholineTabun thiocholine is a nerve agent model compound (Fig. 1)

that differs from authentic tabun by the presence of a thiocholinegroup in place of cyanide. Thiocholine is a poorer leaving groupthan cyanide, making tabun thiocholine less reactive and less toxic[10]. Mice (n = 3 per genotype) were challenged sc with 6.0 mg/kgtabun thiocholine. Plasma AChE inhibition at 30 min post dosingwas about 80% for both genotypes. Almost complete inhibition ofBChE was found for both genotypes. Plasma carboxylesterase wasinhibited 24% in the wild-type mice (Table 1). At 24% inhibition,carboxylesterase scavenged about 100 times more of the OP thanwas scavenged by AChE, and about 10 times more than was scav-enged by BChE. Behavioral toxic signs and surface body tempera-ture (Fig. 6G) were similar between the genotypes. Behavioraltoxic signs included hunched flattened posture, myoclonic jerks,palpebral closure, decreased handling reactivity, ataxic gait, de-creased mobility and arousal, straub tail, vocalization when held,and decreased body temperature through 4 h. At 24 h mice hadno behavioral toxic signs; their body temperature and plasmaAChE activity had returned to normal, though BChE activity wasstill inhibited 85%. In conclusion, plasma carboxylesterase has aminor role in protection against the toxicity of tabun thiocholine.

4. Discussion

4.1. Carboxylesterase as a bioscavenger of OP

In agreement with Karanth and Pope (2001) we found that plas-ma carboxylesterase contributed to protection from the toxicity ofparathion [1]. However, plasma carboxylesterase had only a minorrole in protection from the toxicity for most of the compoundstested. An explanation for the latter finding takes into account therelative amounts of plasma carboxylesterase and toxicant in a25 g mouse. The total amount of ES1 carboxylesterase in thewild-type mouse is estimated to be a minimum of 2 nmol. The OPcompounds used in our study were administered in amountsslightly less than lethal. Table 1 shows the doses of OP. Conversionof mg/kg into nmol/kg (based on a 25 g mouse) indicates that OPamounts between 5 nmol (for cyclosarin-thiocholine) and4100 nmol (for diazinon) were administered per mouse. Aftercyclosarin-thiocholine, the next lowest doses were for paraoxon(18 nmol) and DFP (540 nmol). It is clear that stoichiometric scav-

enging by carboxylesterase could provide significant protectionagainst only cyclosarin-thiocholine and paraoxon. Cyclosarin-thi-ocholine is cationic and therefore does not react well with carboxy-lesterase [30]. Nevertheless, plasma AChE was less inhibited inwild-type than in ES1�/�mice, suggesting some protection by plas-ma carboxylesterase. In rats the toxicity from paraoxon was barelyenhanced by depletion of carboxylesterase through reaction withCBDP [4], making the absence of a difference between wild-typeand ES1�/� mice for paraoxon in our study within experimental er-ror of the result with rats.

The dose of parathion that we used was about 60-fold higherthan the dose of paraoxon, yet plasma carboxylesterase protectedmice from parathion toxicity. This observation can be partly ex-plained by the fact that parathion is itself not toxic, and that itmust be metabolically activated to the toxic oxon. The lag time ob-served between administration of parathion and appearance oftoxic signs suggests that induction of cytochrome P450 desulfura-tion enzymes is required before activation can occur. Therefore,activation would be expected to take time. Consequently, the toxicoxon would be slowly produced and slowly released into the circu-lation [31] where it could be scavenged by carboxylesterase. Wehave no explanation for the observation that AChE activity is re-stored to normal 72 h after parathion at a time when BChE activityis still inhibited. If BChE were more sensitive than AChE to inhibi-tion by paraoxon, or if paraoxon-inhibited BChE aged more rapidlythan paraoxon-inhibited AChE, then BChE should have remainedinhibited at 24 h in Fig. 3B. However, both AChE and BChE activitiesreturned to normal in 24 h, suggesting that there are no significantdifferences in the rates of inhibition and aging.

We agree with Maxwell that plasma carboxylesterase has no ef-fect on the toxicity of DFP [4]. Maxwell used CBDP to deplete carb-oxylesterase in rats, whereas our study used mice geneticallyengineered to have no carboxylesterase in plasma. The lack of pro-tection from DFP toxicity is explained as follows. DFP reacts poorlywith AChE (2.9 � 104 M�1 min�1) [30] and therefore causes toxic-ity only at high doses, i.e. doses that are much higher than the con-centration of carboxylesterase in the subject. Even though DFPreacts readily with carboxylesterase (3.5 � 106 M�1 min�1) [30],the carboxylesterase is used up before it can absorb a significantfraction of DFP, thereby affording no protection.

The time post-dosing when body temperature dropped corre-lated well with behavioral toxic signs and with reduced plasma AChEactivity. However, the level of inhibition did not predict body tem-perature. For example at 6 h the parathion-treated ES1�/� micehad 73% of their plasma AChE activity inhibited and a drop in bodytemperature from 38 to 30 �C. In contrast the diazinon-treatedES1�/� mice had 93% of their plasma AChE activity inhibited at 2 h,and a drop in body temperature of 2 �C. Hypothermia lasting up to24 h is a characteristic response of rodents to anticholinesterases[32].

4.2. Unexpected result: ES1+/+ mice are more sensitive than ES1�/�

mice to chlorpyrifos and chlorpyrifos oxon

ES1+/+ mice became severely intoxicated by 6 h after treatmentwith chlorpyrifos or chlorpyrifos oxon and were moribund or deadby 24–48 h. In contrast, ES1�/� mice showed the same behavioralsigns of intoxication as the wild-type at 6 h, but recovered fromthe toxic effects by 24–48 h. This observation is contrary to theexpectation that plasma carboxylesterase protects from the toxicityof OP. It was a surprise to find that mice with no plasma carboxylest-erase, and therefore with reduced OP scavenging capacity, fared bet-ter than wild-type mice with the higher OP scavenging capacityafforded by the presence of carboxylesterase.

CPO and CF were applied transdermally, whereas all other tox-icants were applied subcutaneously. The route of administration

E.G. Duysen et al. / Chemico-Biological Interactions 195 (2012) 189–198 197

does not explain why wild-type mice are more sensitive to chlor-pyrifos and chlorpyrifos oxon than carboxylesterase deficient micebecause the route of administration was the same for wild-typeand ES1�/� mice.

A possible explanation for this unexpected result is based on lit-erature reports that organochlorines including chlorpyrifos inducethe expression of cytochrome P450 isomers [33–35] and that cyto-chrome P450 isomers are capable of both dearylation (inactivation)and oxidative desulfuration (activation) of phosphorothioates. Thedearylation step detoxifies chlorpyrifos by removing trichloropy-ridinol, whereas oxidative desulfuration activates the phosphoro-thioate by replacing the phosphoryl thiol with oxygen [36]. Somecytochrome P450 isomers are more proficient at dearylation thanat oxidative desulfuration [37,38]. Chlorpyrifos induces the expres-sion of isomers that are more active at dearylation (inactivation)than at desulfuration (activation) [37,39]. The levels of inducedcytochrome P450 correlate with the amount of chlorpyrifos in cir-culation, so that higher levels of the detoxifying isomer are ex-pected in response to higher levels of chlorpyrifos [33].

We hypothesize that in ES1�/� mice, which lack plasma carb-oxylesterase, higher levels of chlorpyrifos reached the liver micro-somes, causing more cytochrome P450 to be induced. We furtherhypothesize that more of the detoxifying isomers than of the acti-vating isomers were induced. Consequently, CF was more quicklyinactivated in the ES1�/� mice than in the ES1+/+ mice. Supportfor this hypothesis comes from a study in rats which found thattreatment with chlorpyrifos induced 4-fold more dearylation thandesulfuration, whereas treatment with parathion induced 4 to 8-fold more desulfuration than dearylation [39]. This hypothesis isexpected to apply only to a limited range of chlorpyrifos or chlor-pyrifos oxon doses. We found that higher doses of either com-pound were lethal to animals of both genotypes, while lowerdoses caused no differences in toxic effects.

This hypothesis does not apply to chlorpyrifos oxon because theoxon is not a major substrate for any P450 isomer. Furthermore,the oxon does not need to be metabolized to be toxic, so the like-lihood of induction of differential P450 gene expression is muchsmaller for the oxon than for chlorpyrifos.

Not all phosphorothioates showed this paradoxical effect. Para-thion and diazinon caused greater toxicity in the ES1�/� mice. Themost obvious difference between these OP and chlorpyrifos (orchlorpyrifos oxon) is the organochlorine nature of the latter. Thissuggests that the protective CYP orthologs are induced specificallyby organochlorines and not by phosphorothioates in general. Insupport of this suggestion, a study in rats found that treatmentwith chlorpyrifos induced 4-fold more dearylation than desulfura-tion, whereas treatment with parathion induced 4- to 8-fold moredesulfuration than dearylation [39].

Induction of new, detoxifying cytochrome P450 enzymes takestime. A time-dependent induction of protective cytochrome P450is consistent with the observation that both wild-type and ES�/�

mice displayed the same toxic signs over the first 6 h, but thatthe ES�/� mice had a better long term outcome. Dearylation inac-tivates chlorpyrifos by removing the trichloropyridinol group, buttrichloropyridinol by itself is nontoxic [21] (and the results fromthis paper).

4.3. AChE activity returns to normal within 48 h

We observed return of plasma AChE activity to baseline levelswithin 48 h after treatment with a variety of OP. Gupta et al. foundthat AChE activity in brain and muscle recovers by 48–72 h aftertreatment of rats with soman [8]. It is generally accepted that AChEis irreversibly inhibited by OP, although limited spontaneous reac-tivation has been reported. Examples of spontaneous reactivationinclude (1) an in vitro study of human erythrocyte dimethylphos-

phoryl-AChE which spontaneously regained 75% active enzymewithin 2 h [40]; and (2) a study on homogenates of mouse braininhibited with dichlorvos which showed that AChE regained 50%of its original activity in 2 h [41]. In contrast homogenates ofmouse brain inhibited with paraoxon did not regain AChE activity[41]. In neither of these examples did reactivation restore fullactivity. It follows that the return to normal plasma AChE activitycannot be due simply to reactivation, but most likely involvesthe synthesis of new molecules of AChE as well. This would beespecially true for the regain of AChE activity in our in vivo mousestudies with paraoxon where no reactivation would be predictedbased on the example above. Synthesis of new AChE after treat-ment of an organism with OP is supported by gene expressionstudies that have shown that mRNA for AChE in brain increaseswhen mice are treated with sarin [42]. New synthesis is also sup-ported by the observation that AChE levels can rise above baselineafter treatment with OP. For example, cultured chick myoblaststreated briefly with paraoxon had 2-fold more AChE activity com-pared to control cells [43]. Treatment of mice with a variety of OPincreased plasma AChE activity 2.5-fold, a result attributed toinduction of AChE synthesis [44].

5. Conclusion

Plasma carboxylesterase had only a minor role in protectionfrom sublethal doses of the majority of toxicants tested. This resultagrees with the finding of Maxwell [4] that plasma carboxylester-ase is protective only against OP that are effective AChE inhibitorsat low doses, notably the OP nerve agents. Less reactive OP, such asthose used in the present report, must be administered in highdoses to achieve AChE inhibition and toxicity. High doses far ex-ceed the binding capacity of endogenous plasma carboxylesterase,thus explaining the lack of protection in mice against less potentOP. Thus, the wild-type mouse is as good a model as the ES1 knock-out mouse for studies with OP pesticides. We predict that theES1�/� mice will be more sensitive than ES1+/+ mice to highlyreactive nerve agents such as soman and sarin.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

Part of the work was funded by the National Institutes of HealthCounterACT Program through the National Institute of Neurologi-cal Disorders and Stroke (Award # U01 NS058038) to J.R.C. andthe Direction Générale de l’Armement (Contract 08ca501) to F.N.

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