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Chemico-Biological Interactions 190 (2011) 79–83

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

n vitro kinetic interactions of DEET, pyridostigmine and organophosphorusesticides with human cholinesterases

imo Wille, Horst Thiermann, Franz Worek ∗

undeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany

r t i c l e i n f o

rticle history:eceived 11 February 2011eceived in revised form 17 February 2011ccepted 18 February 2011vailable online 25 February 2011

eywords:uman cholinesteraseEET

a b s t r a c t

The simultaneous use of the repellent DEET, pyridostigmine, and organophosphorus pesticides has beenassumed as a potential cause for the Gulf War Illness and combinations have been tested in differentanimal models. However, human in vitro data on interactions of DEET with other compounds are scarceand provoked the present in vitro study scrutinizing the interactions of DEET, pyridostigmine and pesti-cides with human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE). DEET showed to bea weak and reversible inhibitor of hAChE and hBChE. The IC50 of DEET was calculated to be 21.7 mMDEET for hAChE and 3.2 mM DEET for hBChE. The determination of the inhibition kinetics of pyri-dostigmine, malaoxon and chlorpyrifos oxon with hAChE in the presence of 5 mM DEET resulted in amoderate reduction of the inhibition rate constant k . The decarbamoylation velocity of pyridostigmine-

yridostigmine

alaoxonhlorpyrifos oxon

n vitro kinetics

i

inhibited hAChE was not affected by DEET. In conclusion, the in vitro investigation of interactions betweenhuman cholinesterases, DEET, pyridostigmine, malaoxon and chlorpyrifos oxon showed a weak inhibi-tion of hAChE and hBChE by DEET. The inhibitory potency of the tested cholinesterase inhibitors wasnot enhanced by DEET and it did not affect the regeneration velocity of pyridostigmine-inhibited AChE.Hence, this in vitro study does not give any evidence of a synergistic effect of the tested compounds on

human cholinesterases.

. Introduction

The interaction of various chemicals such as pyridostigmine, thensect repellent N,N-diethyl-m-toluamide (DEET) and organophos-horus pesticides has been extensively discussed as a potentialause for the Gulf War Illness and thus been tested in animal models1–6].

The reversible acetylcholinesterase (AChE) inhibitor pyridostig-ine (Fig. 1) is a quaternary dimethyl carbamate that was originally

nvented for the treatment of myasthenia gravis and approved byhe FDA in 1955. In addition, it is used by different countries asre-treatment in case of anticipated use of chemical warfare nervegents, primarily soman and tabun [7,8]. The daily dosage of pyri-ostigmine is 90 mg if used as a pre-treatment [7] and up to 500 mgor treatment of myasthenia gravis.

Military pest control during the Gulf War included sur-ace spraying and environmental fogging with chlorpyrifos and

alathion, two of the most commonly used organophosphorusesticides for domestic, agricultural and industrial purposes [9].oth are pre-poisons that require a metabolic activation to becomehe potent phosphorylating agents chlorpyrifos oxon and malaoxon

∗ Corresponding author. Tel.: +49 89 3168 2930; fax: +49 89 3168 2333.E-mail address: FranzWorek@Bundeswehr.org (F. Worek).

009-2797/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2011.02.022

© 2011 Elsevier Ireland Ltd. All rights reserved.

(Fig. 1). However, the activation of chlorpyrifos into its oxon-formis extremely variable and depends on the individual enzyme con-stitution [10].

The insect repellent DEET (Fig. 1) was invented and patented forthe American military in 1946 by the US government and registeredfor the general population in 1957 [11]. Clinical studies have provenDEET as most effective repellent for insects and arthropods [12].Due to its safety profile the WHO highly recommends the use ofDEET-containing repellents and after a re-appraisal of DEET the USEnvironmental Protection Agency decided that an application ofDEET according to the instruction guidelines causes no health risk[13].

Several retrospective studies on human exposure to DEET havebeen conducted on base of reports to the American Associationof Poison Control Centers [11,14]. The analysis revealed predom-inantly mild dermal symptoms in a study from 1993 to 1997 with20,764 requests to the Poison Control Centers registered and mod-erate or worse effects in 4% [14]. In the period from 1995 to 2001296 moderate and major severity cases were detected – a low num-ber if one accounts for the billions of applications during this time

[11,15]. Nevertheless some case reports with fatal outcome weredescribed for children using products with a high content of DEET[16–18] or in intentional oral ingestion [19].

The pharmacodynamic mechanism is known for the carbamatepyridostigmine and organophosphorus pesticides but remains

80 T. Wille et al. / Chemico-Biological In

DEET Chlorpyrifos oxon

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nclear for DEET. Some publications favour the olfactory system asarget [20,21] while a recent publication postulated an inhibition ofhe crucial enzyme acetylcholinesterase (AChE) as molecular tar-et of DEET especially when it interacts with carbamates [15]. Suchn interaction could be relevant in a military scenario since differ-nt armed forces still provide DEET-based repellents for personalrotection against biting insects and continue to consider pyri-ostigmine pre-treatment as part of their medical defence strategygainst poisoning by nerve agents, especially soman [2].

Of note, the repellent DEET is often used in combination withesticides against insects [9]. This is of special interest as there areonflicting reports in the literature on the ability of DEET to alterhe pharmacokinetics of pesticides and pyridostigmine. Althougheveral animal studies were published [2,3,22–24] human in vitroata are scarce. In order to provide more insight into potential

nteractions between DEET, pyridostigmine and organophospho-us pesticides the present study was initiated using human AChEnd butyrylcholinesterase (BChE) as in vitro test system [25].

. Materials and methods

.1. Chemicals

Acetylthiocholine iodide (ATCh), S-butyrylthiocholine iodideBTCh), 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB, Ellman’seagent), N,N-diethyl-m-toluamide (DEET), pyridostigmine bro-ide and human butyrylcholinesterase (hBChE) were obtained

rom Sigma–Aldrich (Taufkirchen, Germany). Chlorpyrifos oxon97%) and malaoxon (97%) were purchased from Dr. EhrenstorferAugsburg, Germany) and all other chemicals were from MerckDarmstadt, Germany).

.2. Preparation of hemoglobin-free erythrocyte ghosts

Hemoglobin-free erythrocyte ghosts were prepared accordingo Dodge et al. [26] with minor modifications [27] as source ofuman erythrocyte acetylcholinesterase (hAChE). Aliquots of therythrocyte ghosts were stored at −80 ◦C. Prior to use, aliquots

ere homogenized on ice with a Sonoplus HD 2070 ultrasonicomogenator (Bandelin electronic, Berlin, Germany), three times

or 5 s with 30 s intervals, to achieve a homogeneous matrix for theinetic studies.

teractions 190 (2011) 79–83

2.3. Inhibition of isolated human AChE and BChE by DEET

The enzyme activities were measured spectrophotometrically(Cary 3Bio, Varian, Darmstadt) at 412 nm with a modified Ellmanassay [25,28,29]. The assay mixture (3165 �l) contained 0.3 mMDTNB (100 �l) as a chromogen in 0.1 M phosphate buffer (3000 �l,pH 7.4). To the pre-warmed cuvette (37 ◦C), 10 �l enzyme (hBChEor hAChE) and 5 �l DEET in isopropanol (<0.15% final isopropanolconcentration) were added to achieve final DEET concentrations of1 �M–5 mM. The mixture was then incubated for further 2 min at37 ◦C and finally 50 �l ATCh (0.45 mM) or BTCh (1 mM) was addedas substrate. The activity of the cholinesterases was measuredfor 1 min and analyzed by linear regression analysis. To scruti-nize a potential time dependence of cholinesterase inhibition byDEET additional tests were performed with 1, 10 and 20 min pre-incubation before adding ATCh or BTCh.

2.4. Reversibility of cholinesterase inhibition by DEET

To investigate the reversibility of cholinesterase inhibition byDEET, hAChE and hBChE (100 �l) were incubated with DEET (5 mM)or isopropanol for 5 min. Then 10 �l of the sample was added toa cuvette filled with pre-warmed phosphate buffer (0.1 M), DTNB(0.3 mM) and ATCh (0.45 mM) or BTCh (1 mM) to dilute the DEETconcentration by a factor of 316 and to measure enzyme activities.

2.5. Determination of IC50

The IC50 was calculated from semi-logarithmic plots of the DEETconcentration versus the hAChE or hBChE activity.

2.6. Interaction of pyridostigmine, malaoxon and chlorpyrifosoxon with DEET

The second-order inhibition rate constants (ki) were determinedin the presence of substrate according to Forsberg and Puu [30] withslight modifications [31]. In brief, 10 �l hAChE and 5 �l DEET (5 mMfinal concentration) or isopropanol were added to a cuvette withphosphate buffer and DTNB (0.3 mM). Then 5 �l pyridostigmine,malaoxon (both 3–10 �M final concentration, 8 different concen-trations) or chlorpyrifos oxon (40–110 nM final concentration, 8different concentrations) were added. The reaction was startedwith ATCh (0.45 mM) and the absorbance change was continuouslymonitored for up to 30 min. The recorded curves were analyzed bynon-linear regression analysis and used for the further determina-tion of ki.

2.7. Spontaneous decarbamoylation

200 �l hAChE and 2 �l pyridostigmine (5 �M final concentra-tion) were incubated for 30 min at 37 ◦C and were then diluted500-fold in phosphate buffer containing 0.2% pig gelatine [32] tostabilize hAChE activity during the prolonged incubation at 37 ◦C.Then, DEET (1 or 5 mM) or isopropanol was added. 3000 �l sam-ples were taken at 0, 15, 30, 45, 60, 90, 120, 150 and 180 minand transferred into cuvettes that had previously been filled with100 �l DTNB (10 mM) to achieve a final concentration of 0.3 mMchromogen. hAChE activities were measured after addition of ATCh(0.45 mM) and referred to native controls.

2.8. Data analysis

The analysis of the data and the calculation of the kinetic con-stants by linear and non-linear regression analysis were performedwith GraphPad Prism 5.0 (GraphPad, San Diego, USA). All experi-ments were carried out at a minimum of n = 4 and data are given

T. Wille et al. / Chemico-Biological Interactions 190 (2011) 79–83 81

Fit*

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Fig. 3. Effect of 1 mM or 5 mM DEET on the decarbamoylation of pyridostigmine-inhibited hAChE. hAChE was inhibited with pyridostigmine (5 �M) for 30 min at

◦

and reached 82% and 50% of control activity with 1 and 5 mM DEET,respectively, and was different compared to control.

ig. 2. Effect of DEET on the activity of hAChE and hBChE. hAChE and hBChE werencubated with various DEET concentrations (1 �M–5 mM) at 37 ◦C for 2 min andhe residual enzyme activity was measured. Data are shown as means ± SD (n = 4).Significantly different from control (p < 0.05).

s means ± SD. Data obtained from two groups were compared byeans of Student t test and comparisons among multiple groupsere performed using an analysis of variance (ANOVA) with Bon-

erroni post hoc comparisons. A p-value of 0.05 was consideredignificant.

. Results

.1. Inhibition of hAChE and hBChE by DEET

Incubation of hAChE with different DEET concentrations1 �M–5 mM) did not affect hAChE activity up to 100 �M DEET andesulted in a maximum inhibition of 25% at 5 mM DEET (Fig. 2).EET caused a more pronounced effect with hBChE resulting in aaximum inhibition of 57% at 5 mM (Fig. 2).The incubation of hAChE and hBChE with 5 mM DEET for

–20 min did not result in significantly different enzyme activitiesTable 1) which indicates the absence of a time-dependent DEETffect.

.2. Reversibility of DEET-inhibited ChE

The reversibility of DEET inhibition of hAChE and hBChE wasested by incubating the cholinesterase with 5 mM DEET followedy extensive dilution (316-fold). In treated samples, the hAChE149.9 ± 1.5 mA/min) and hBChE (199.5 ± 3.1 mA/min) activitiesere almost identical to hAChE (148.6 ± 0.8 mA/min) and hBChE

198.4 ± 1.9 mA/min) activities of corresponding isopropanol con-rols indicating a reversible mode of inhibition with a completeecovery of the enzyme activities.

.3. Calculation of IC50

DEET concentrations were used up to 5 mM without achievingcomplete inhibition of cholinesterase activities suggesting a low

able 1ffect of DEET incubation time on the activity of hAChE and hBChE. hAChE and hBChEctivities were measured after 1, 10 and 20 min incubation with 5 mM DEET at 37 ◦C.ata (% of control) are shown as means ± SD (n = 5).

Enzyme Pre-incubation time (min)

1 10 20

hBChE 43.6 ± 1.0 46.2 ± 1.1 46.2 ± 2.6hAChE 67.7 ± 5.1 67.7 ± 1.8 66.8 ± 1.6

37 C. After 500-fold dilution in phosphate buffer with 0.2% gelatine DEET was addedto achieve final concentrations of 1 or 5 mM. Samples were taken at several timeintervals and hAChE activity was measured. Data are shown as means ± SD (n = 4).

inhibitory potency (Fig. 2). These data were used to estimate theIC50 which was calculated to be 21.7 mM DEET (R2 = 0.98) for hAChEand 3.2 mM DEET (R2 = 0.99) for hBChE.

3.4. Interaction of pyridostigmine, malaoxon and chlorpyrifosoxon with DEET

The determination of the inhibition constants of pyridostigmine,malaoxon and chlorpyrifos oxon with or without DEET (5 mM)revealed only a moderate effect of DEET. The inhibition constantswere lowered by a factor of 1.22 for chlorpyrifos oxon, 1.37 forpyridostigmine and 1.47 for malaoxon (Table 2).

3.5. Spontaneous decarbamoylation

The decarbamoylation kinetics of pyridostigmine-inhibitedhAChE was determined in extensively diluted samples in theabsence and presence of DEET (1 or 5 mM) for up to 3 h (Fig. 3).The decarbamoylation half life was almost identical for control(26.0 ± 0.8 min), 1 mM DEET (26.1 ± 0.5 min) and 5 mM DEET sam-ples (26.6 ± 1.3 min). However, the maximum hAChE activity at theend of the observation period depended on the DEET concentration

Table 2Inhibition rate constants (ki) of pyridostigmine, malaoxon and chlorpyrifos oxonwith hAChE in the presence of DEET. Inhibition rate constants ki were determinedunder pseudo first-order conditions with pyridostigmine, malaoxon and chlorpyri-fos oxon with or without DEET (5 mM) in the presence of 0.45 mM ATCh at pH 7.4and 37 ◦C, the impact of substrate on the inhibition by pyridostigmine and pesticideswas corrected according to Forsberg and Puu [30]. Data are shown as means ± SD(n = 4).

ki (M−1 min−1)

Control +DEET (5 mM) Ratio

Pyridostigmine 5.29 ± 0.29 × 105 3.85 ± 0.34 × 105* 1.37Malaoxon 5.77 ± 0.45 × 105 3.93 ± 0.59 × 105* 1.47Chlorpyrifos oxon 11.43 ± 1.39 × 106 9.39 ± 0.97 × 106 1.22

* Significantly different from control (p < 0.05).

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. Discussion

The results of the present study demonstrate a low inhibitoryotency of DEET with human AChE and BChE which confirms previ-us results [15]. In fact, a significant inhibition of hAChE and hBChEctivity was reached only with DEET concentrations exceedingmM and 100 �M, respectively. The DEET effect was not time-ependent and completely reversible which indicated that DEEToes not bind covalently to the active site but prevents access of theubstrate either by attachment to the peripheral site or by enteringhe active site gorge [15].

Up to now, DEET is widely used as an effective insect repellentnd it is considered to cause no health risk if applied in due form33]. Only few cases of fatal outcome were reported after inad-quate use of DEET preparations in children and intentional oralngestion [16,19,34]. Hereby, notably high blood DEET concentra-ions up to 1.25 mM were determined. In view of the results ofhe present study the described deleterious toxic effects of DEETannot be attributed to inhibition of hAChE. This is in line withhaney et al. [2] who could not find an inhibition of peripheral ChEith 1 mM DEET in a rat model. In addition, a human volunteer

tudy with dermal application of DEET showed a rapid eliminationf the compound indicating that hardly detectable DEET plasmaoncentrations occur in case of appropriate use of DEET containingepellents [35].

The simultaneous use of DEET, pyridostigmine and organophos-horus pesticides in a military environment was assumed toe a potential trigger for the development of the so-calledulf War Illness [36] and numerous studies were undertaken

o investigate clinical effects and pathophysiologic mechanisms.bou-Donia and co-workers exposed hens to pyridostigmine, DEETnd chlorpyrifos, either alone or in combination and found anncreased neurotoxicity of combinations [1]. However, the indi-idual compounds produced already relevant clinical effects, i.e.he animals were exposed to agent doses which were alreadyoxic. In contrast, a human volunteer study with relevant expo-ure levels of DEET, pyridostigmine and permethrin did notesult in any short-term physical or neurocognitive impairment22].

The results of the present in vitro study with human AChE andChE support the view that DEET does not aggravate the inhibitionf the enzymes by pyridostigmine or pesticides. Hereby, it has toe kept in mind that an effect of DEET on the inhibition kinetics ofhlorpyrifos oxon, malaoxon and pyridostigmine as well as on theecarbamoylation of pyridostigmine-inhibited AChE could only bechieved at an extremely high DEET concentration.

In contrast to Corbel and co-workers [15] who concluded anncreased toxicity of the carbamates propoxur in the presence ofEET our results indicate a DEET-induced reduction of the inhi-ition of hAChE by pyridostigmine and pesticides which maye attributed to a competitive action of DEET and the testedholinesterase inhibitors.

The safe use of pyridostigmine as a pre-treatment against nervegent exposure is dependent on its pharmacokinetics and interac-ion with AChE, i.e. carbamoylation and decarbamoylation kinetics37]. Hence, it was tempting to determine the effect of DEET onhe decarbamoylation of hAChE. Up to 5 mM DEET did not affecthe decarbamoylation velocity of pyridostigmine-inhibited hAChEhich indicates that DEET does not impair the regeneration of AChE

ctivity after treatment with this carbamate.In conclusion, the in vitro investigation of interaction between

uman cholinesterases, DEET, pyridostigmine, malaoxon andhlorpyrifos oxon showed a weak inhibition of hAChE and hBChE byEET. The inhibitory potency of the tested cholinesterase inhibitorsas not enhanced by DEET and it did not affect the regeneration

elocity of pyridostigmine-inhibited AChE. Hence, this in vitro study

[

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teractions 190 (2011) 79–83

does not give any evidence of a synergistic effect of the testedcompounds on human cholinesterase.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

The study was funded by the German Ministry of Defence. How-ever, the design, performance, data interpretation and manuscriptwriting was under the complete control of the authors and hasnever been influenced.

The authors are grateful to T. Hannig for expert technical assis-tance.

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