J BIOCHEM MOLECULAR TOXICOLOGYVolume 26, Number 1, 2012

Pyridostigmine Bromide Protection againstAcetylcholinesterase Inhibition by PesticidesJohn D. Henderson,1 Gabriela Glucksman,1 Bryan Leong,1 Andras Tigyi,1

Anna Ankirskaia,1 Imteaz Siddique,1 Helen Lam,1 Ed DePeters,1

and Barry W. Wilson1,2

1Animal Science Department, University of California, Davis, CA 95616, USA; E-mail: bwwilson@ucdavis.edu2Environmental Toxicology Department, University of California, Davis, CA 95616, USA

Received 19 May 2011; revised 15 July 2011; accepted 31 July 2011

ABSTRACT: Pyridostigmine bromide (PB) has beenused to protect soldiers from the toxic effects ofsoman, a chemical warfare agent. Recent researchshows that pyridostigmine bromide protects a sig-nificant percentage of acetylcholinesterase in isolatedhuman intercostal muscle. Findings presented hereindicate that red blood cell acetylcholinesterase issimilarly protected by pyridostigmine bromide fromthe action of diisopropyl fluorophosphate and severalorganophosphate pesticides including chlorpyrifos-oxon, diazinon-oxon, and paraoxon, but not malaoxon,using the bovine red blood cell as a subject. Thesefindings suggest that pretreatment with PB may pro-tect growers, farmworkers, first responders, and thepublic, in general, from the effects of selected pes-ticides. C© 2011 Wiley Periodicals, Inc. J Biochem MolToxicol 26:31–34, 2012; View this article online atwileyonlinelibrary.com. DOI 10:1002/jbt.20410

KEYWORDS: Pyridostigmine Bromide; Acetylcholines-terase; Organophosphate; Pesticides

INTRODUCTION

Muscle contraction and nerve transmission dueto acetylcholine (ACh) is regulated in part by thepresence of the enzyme acetylcholinesterase (AChE;E.C. 3.1.1.7) at synapses and motor endplates [1].Organophosphate (OP) ester pesticides and chemicalwarfare agents inhibit AChE disrupting these processesby increasing the concentration and resident time of

Correspondence to: Barry Wilson.Contract Grant Sponsor: U.S. Department of Agriculture West-

ern Regional Project W-1045.Contract Grant Number: 7504-RR.Contract Grant Sponsor: University of California, Department

of Animal Science research allocation (ANSFBWW).c© 2011 Wiley Periodicals, Inc.

the ACh molecules, prolonging their pharmacologicaleffects [2].

The presence of the carbamate pyridostigmine bro-mide (PB) has been shown to protect a significant frac-tion of the AChE from OP inhibition by the nerve gassoman, a potent AChE inhibitor [1,3,4]. The mechanismis thought to be a lessening of the effect of the moretightly bound OP by the presence of the more readilyhydrolysable carbamate [1].

The U.S. military has been using 30 mg 3× dailydoses of PB as a pretreatment to protect troops fromOP warfare agents since the first Gulf War [1]. Lessattention has been given to the possible use of PB inthe civilian sector as a protectant from anti-AChE pes-ticides. This study used bovine red blood cell (RBC)AChE to investigate whether PB will also protect AChEagainst the activated (oxon) forms of several widelyused pesticides. Human and bovine RBC AChEs havesimilar biochemical properties, and cow blood lacksserum butyrylcholinesterase, eliminating possible in-terference by this enzyme in the study [5].

MATERIALS AND METHODS

Materials

Activated OP pesticides, paraoxon (PO), diazinon-oxon (DZO), chlorpyrifos-oxon (CPO), and malaoxon(MXO) were obtained from Chem Services (WestChester, PA). PB, diisopropyl fluorophosphate (DFP),and other reagents were obtained from Sigma-Aldrich(St Louis, MO). Chemical structures are shown inFigure 1.

Blood Collection

RBCs were obtained from fresh bovine blood usingthe U.C. Davis dairy herd. The blood was collected in

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32 HENDERSON ET AL. Volume 26, Number 1, 2011

FIGURE 1. Chemical structures.

heparinized vacutainers, pooled as necessary, and re-frigerated for up to 2 days. Collection was carried outunder a protocol approved by the U.C. Davis Institu-tional Animal Care and Use Committee.

Blood Treatment

OP-oxon pesticide stocks were prepared in 10%ethanol in phosphate-buffered saline. 270 μL aliquots ofpooled whole blood were pretreated with 30 μL salineor 100 μM PB (final concentration 10 μM PB) for 20 minat room temperature (RT; 23.5 ± 1.0◦C). The suspen-sion was centrifuged for 10 min at 1000 × g, 4◦C. The

RBCs were resuspended with 1000 μL of (a) saline, orsaline with: (b) 10 μM PB, (c) OP, or (d) 10 μM PB + OP,and incubated for 10 min at RT. OP concentrations arelisted in Table 1. Treatments contained 1%–2% ethanol.Treated RBCs were centrifuged and resuspended with500 μL of saline. The RBCs were washed twice by recen-trifugation and resuspended in 500 μL saline to removeresidual PB or OP, and incubated at RT for up to 9 h.Samples were removed at various times throughouttreatment, and frozen at −80◦C until assayed. Frozensamples were thawed, diluted 1/10, and solubilizedwith 0.5% Triton X-100, 0.1 M sodium phosphate pH 8buffer for AChE determinations.

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Volume 26, Number 1, 2011 PROTECTING AChE FROM PESTICIDES 33

TABLE 1. Acetylcholinesterase Activities in Treated Bovine Red Blood Cells

Treatment N Salinea PBb OPb PB + OPb P Protects

DFP 10μM 4 0.776 ± 0.064 35.2 ± 4.1 6.5 ± 5.5 37.0 ± 6.8 <0.01 +CPO 200μM 4 0.719 ± 0.112 50.8 ± 15.3 2.0 ± 1.8 23.2 ± 8.4 <0.01 +DZO 200μM 3 0.627 ± 0.215 42.1 ± 3.1 1.0 ± 2.0 23.4 ± 10.6 <0.05 +PO 10μM 6 0.646 ± 0.238 42.3 ± 7.6 2.2 ± 4.5 11.8 ± 3.8 <0.01 +MXO 1 mM 5 0.827 ± 0.133 45.8 ± 7.2 5.7 ± 3.3 7.5 ± 4.1 >0.10 –

Cells are pretreated with saline or pyridostigmine bromide (PB).Organophosphate (OP) treatments: diisopropyl fluorophosphate (DFP); chlorpyrifos-oxon (CPO); diazinon-oxon (DZO); paraoxon (PO); malaoxon (MXO).N: number of replicated experiments.P : Difference between OP and PB + OP groups; Paired t-test, 1-tailed.Activities determined in prewash samples.a Activity of control in μmol/min/mL.b Activity expressed as a percentage of saline control activity.

AChE Determination

AChE activity (μmol acetylthiocholine hy-drolyzed/min/mL) was determined in triplicateaccording to the colorimetric method of Ellman et al.[6], modified [7] for use with an automated 96-wellmicroplate reader (PowerWavex, Bio-Tek Instruments,Inc., Winooski, Vt). Separate blanks included wellslacking substrate and those lacking sample.

Statistical Analysis

AChE activities were tested for statistical signifi-cance using a one-tailed Student’s t-test [8].

RESULTS

The AChE activities of RBCs treated with PB wereless inhibited by DFP, PO, DZO, and CPO than thosetreated with saline (Table 1), demonstrating the protec-tive effect of PB [3,9].

PB initially protected approximately 25% of theAChE of the cells with CPO; the activity recovered to40% after washing (Figure 2). Interestingly, no such pro-tection occurred with MXO (Table 1 and Figure 3).

PB-treated RBCs recovered AChE activity over a9-h incubation period (Figure 2), as did those treatedwith PB + DFP (Figure 4). There was no further recov-ery of PB + OP treated RBCs with the pesticides tested:CPO, DZO, PO, and MXO. Indeed, OP pesticide treatedcells protected with PB lost activity after 9 h of incuba-tion. Microscopic examination indicated that the cellsdid not break up, maintaining their integrity for at least24 h.

DISCUSSION

Recent work from U.C. Davis [3] showed that pre-treatment with PB protected approximately 20% of the

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FIGURE 2. Pyridostigmine bromide (PB) pretreatment protected25% of red blood cell acetylcholinesterase (AChE) from chlorpyrifos-oxon (CPO) inhibition. Activity is mean ± S.D., n = 3.

AChE of human intercostal muscle fibers from inhibi-tion by the warfare agent soman and partially protectedthe electrophysiological responses associated with OPintoxication. Although a “no effect” level for AChEinhibition by OPs has not been quantitatively estab-lished, it is likely that protection of even a fractionof the AChE at motor end plates may be sufficientto prevent the worst effects of OP exposures. Thus,pretreatment with PB may be useful in protecting pes-ticide users when methods such as protective equip-ment are either unsuitable or unavailable. Unfortu-nately, PB did not protect AChE against all OP pesti-cides. It did not affect the inhibition of AChE by MXO,but it did affect the inhibition of AChE by DZO, CPO,and PO.

PB-treated cells did not always recover from AChEinhibition. Gordon et al. [10] reported recovery of AChEin PB-treated lysed human blood after soman ex vivoexposure. But Maselli et al. [3] did not find recovery ofAChE activity in human muscle treated with PB andsoman. Here, RBCs treated with PB and OP pesticides

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34 HENDERSON ET AL. Volume 26, Number 1, 2011

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FIGURE 3. Pyridostigmine bromide (PB) pretreatment did notprotect red blood cell acetylcholinesterase (AChE) from malaoxon(MXO) inhibition. Activity is mean ± S.D., n = 3.

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FIGURE 4. Pyridostigmine bromide (PB) pretreatment protected30% of red blood cell acetylcholinesterase (AChE) from diisopropylfluorophosphate (DFP) inhibition. There was recovery in PB + DFPto 88% of control activity over a 9-h period. Activity is mean ± S.D.,n = 3.

had no recovery of AChE activity, though RBCs treatedwith PB and DFP did. Further work will be necessaryto understand the complexities of carbamate and OPester interactions with such a highly structured proteinas AChE.

Recently, the direction of much chemical counter-terrorism research has focused on agents like somanrather than on the plethora of other available OP andcarbamate AChE inhibitors [1]. This pilot study demon-strates that PB, approved for military applications, maybe important for farmworker and public safety, espe-cially in light of the relative ease of access of registeredpesticides to potential terrorists [11].

ACKNOWLEDGMENTS

We thank Kevin Bauer, Katie Cassinerio, ArielleHines, and Kara Ortega for their assistance in obtain-ing cow blood. This work has been presented by un-dergraduate researchers Gabriela Glucksman, BryanLeong, Andras Tigyi, Anna Ankirskaia, Imteaz Sid-dique, and Helen Lam at the 21st and 22nd Annual U.C.Davis Undergraduate Research, Scholarship & CreativeActivities Conferences sponsored by the Office of theProvost and by the Office of the Vice Chancellor forStudent Affairs.

REFERENCES

1. Wilson B. Cholinesterases. In: Krieger R, editor.Hayes handbook of pesticide toxicology, 3rd edi-tion. London: Elsevier; 2010. Ch 68. pp 1457–1478.

2. Brunton LL, Lazo JS, Parker KL, editors. Goodman &Gilman’s The pharmacological basis of therapeutics, 11thEdition. New York: McGraw-Hill;2006. 2021 p.

3. Maselli RA, Henderson JD, Ng J, Follette D,Graves G, Wilson BW. Protection of human mus-cle acetylcholinesterase from soman by pyri-dostigmine bromide. Muscle Nerve 2011;43:591–595.

4. Tuovinen K, Kaliste-Korhonen E, Raushel FM, HanninenO. Success of pyridostigmine, physostigmine, eptastig-mine and phosphotriesterase treatments in acute sarinintoxication. Toxicology 1999;134:169–178.

5. Arrieta D, Ramirez A, DePeters E, Bosworth D,Wilson BW. Bovine red blood cell ghost cholinesteraseas a monitoring standard. Bull Environ Contam Toxicol2003;71:447–452.

6. Ellman GL, Courtney KD, Andres VJ, Featherstone R.A new and rapid colorimetric determination of acetyl-cholinesterase activity. Biochem Pharmacol 1961;7:88–95.

7. Wilson BW, Henderson JD. Determination ofcholinesterase in blood and tissue. Curr ProtocolsToxicol 2007;34(12.13):1–16.

8. Sokal RR, Rohlf FJ. Biometry: the principles and practiceof statistics in biological research, 3rd edition. New York:W. H. Freeman; 1995,887 p.

9. Herkert NM, Eckert S, Eyer P, Bumm R, Weber G, Thier-mann H, Worek F. Identical kinetics of human ery-throcyte and muscle acetylcholinesterase with respectto carbamate pre-treatment, residual activity upon so-man challenge and spontaneous reactivation after with-drawal of the inhibitors. Toxicology 2008;246(2–3):188–192.

10. Gordon RK, Haigh JR, Gregory E, Garcia GE, Feaster SR,Riel MA, Lenz DE, Aisen PS, Doctor BP. Oral adminis-tration of pyridostigmine bromide and huperzine A pro-tects human whole blood cholinesterases from ex vivo ex-posure to soman. Chem-Biol Interact 2005;157–158:239–246.

11. Wilson BW, Gunderson P. Biological and chemical ter-rorism and the agricultural health and safety committee.J Agromed 2005;10(2):5–6.

J Biochem Molecular Toxicology DOI 10:1002/jbt