Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet
© Corbel et al; licensee BioMed Central Ltd. 2009
Received: 13 March 2009
Accepted: 5 August 2009
Published: 5 August 2009
N,N-Diethyl-3-methylbenzamide (deet) remains the gold standard for insect repellents. About 200 million people use it every year and over 8 billion doses have been applied over the past 50 years. Despite the widespread and increased interest in the use of deet in public health programmes, controversies remain concerning both the identification of its target sites at the olfactory system and its mechanism of toxicity in insects, mammals and humans. Here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system.
By using toxicological, biochemical and electrophysiological techniques, we show that deet is not simply a behaviour-modifying chemical but that it also inhibits cholinesterase activity, in both insect and mammalian neuronal preparations. Deet is commonly used in combination with insecticides and we show that deet has the capacity to strengthen the toxicity of carbamates, a class of insecticides known to block acetylcholinesterase.
These findings question the safety of deet, particularly in combination with other chemicals, and they highlight the importance of a multidisciplinary approach to the development of safer insect repellents for use in public health.
The use of repellents against biting arthropods was probably developed a thousand years ago ; however, a real breakthrough occurred in 1953 with the discovery of the synthetic repellent N,N-Diethyl-3-methylbenzamide (deet), which became the most commonly used active ingredient of topically applied insect repellent due to its efficacy against a broad spectrum of medically important pests, including mosquitoes . Despite the widespread and increased interest in the use of deet in public health programmes [3–5], controversies remain concerning both the identification of its target sites at the molecular level and its exact mechanism of action in insects. Ditzen and colleagues  suggested that deet may block electrophysiological responses of olfactory sensory neurons to attractive odours in Anopheles gambiae Giles (Diptera:Culicidae) and Drosophila melanogaster Meigen (Diptera:Drosophilidae). By contrast, Syed and Leal  have recently reported that mosquitoes detect deet by means of olfaction, a physiological mechanism that directly initiates avoidance behaviour (i.e., deet does not cause a loss of attractive chemical signal).
Although the debate concerning the 'olfactory' mode of action of deet is still a topical question, other laboratory bioassays and field experiments have revealed that deet also exerts a deterrent effect in insects and has insecticidal properties [8–10]. In the same context, if deet is considered to have a relatively good toxicological profile , other authors have shown that excessive doses of deet could be toxic to humans and could cause severe seizures and lethality when combined with other active ingredients, such as pesticides [12–14]. It has been reported previously that symptoms related to deet poisoning in invertebrates, mammals and humans reflect an apparent action on the central nervous system (CNS) [15–18]. Based on these findings, we have investigated further the potential mechanisms of deet toxicity. For the first time, we have identified a molecular target site for deet (i.e., cholinesterases) in both insect and mammal neuronal preparations, and have investigated the consequences of its interactions with carbamate insecticides on the cholinergic system.
Results and discussion
Insecticidal effect of deeton insects
Neurophysiological effects of deet on insect and mammalian neuronal preparations
Based on these unexpected results and because AChE is an ubiquitous enzyme in both insect and mammalian nervous systems, additional electrophysiological studies were performed on isolated mouse phrenic hemidiaphragm muscles. We showed that 500 μM deet prolonged by about threefold the decay time constant of synaptic potentials on endplate regions of the muscle fibre (Figure 2d). This prolongation of the time course of synaptic potentials, which is known to occur after AChE inhibition [25, 26] or in the absence of AChE expression , was shown to be due to the lack of ACh hydrolysis, allowing ACh to persist in the synaptic cleft and to activate endplate nicotinic ACh receptors repeatedly. Considering our data, higher concentrations of deet were, however, required to prolong the decay time constant of synaptic events on mammalian neuromuscular preparations (500 μM) compared with cockroach synaptic preparations (1 μM).
Characterization of cholinesterase inhibition by deet
Characteristic kinetic constants for the hydrolysis of ATCh and BTCh by DmAChE, HuAChE and HuBChE
k 3 [s-1]
13471 ± 2171
1082 ± 44
K ip [mM]
5.43 ± 1.0
11.3 ± 0.5
0.0181 ± 0.0054
0.0019 ± 0.0008
9.23 ± 6.58
12.3 ± 5.4
k 2 [s-1]
14375 ± 2182
420 ± 20
1.64 ± 0.36
1.32 ± 0.07
0.11 ± 0.03
39.6 ± 13.5
K s = K p *K L [mM]
K ss = K p *K LL [mM]
K ip [mM]
1.02 ± 0.03
8.39 ± 6.97
0.37 ± 0.05
0.083 ± 0.078
1.54 ± 0.45
0.56 ± -0.41
29 ± 5.4
1.49 ± 0.99
0.19 ± 0.18
0.52 ± 0.09
K is = K ip *K iL [mM]
K iss = K ip *K iLL [mM]
Interactions between deet and cholinesterase compounds
However, a different trend was noted on mouse isolated phrenic hemidiaphragm muscles. Indeed, when neostigmine (3 μM) was perfused in the continuous presence of 500 μM deet, the decay time constant of synaptic responses was about two fold more prolonged than with deet alone (Figure 4d). Recordings of full (maximum) size endplate potentials (EPPs) in response to a single or paired nerve stimuli, either in the presence of 500 μM deet or in the presence of deet plus 3 μM neostigmine, showed a marked prolongation of the decay phase of the EPPs in the presence of deet and neostigmine (Figure 4e). These results indicated that deet (i) had an inhibitory action on AChE in mouse hemidiaphragm endplates that was not maximal at the concentration used; (ii) did not prevent subsequent action of neostigmine on endplate AChE; and (iii) was less active, on an equimolar basis, than neostigmine in mouse hemidiaphragm junctions. We have shown that deet causes an equal or even greater in vitro inhibitory effect on purified human enzyme than on insect AChE, and therefore speculate that deet may be a less potent inhibitor of the asymmetric forms of AChEs, which are anchored to the basal lamina of the mouse skeletal neuromuscular junction;
In vivo toxic interactions between deet and propoxur, pirimiphos-methyl, or pyridostigmine bromide (PB) for cockroaches and mosquitoes have been reported previously [8, 30, 31]. In adult hens, Abou-Donia et al.  demonstrated that co-exposure to sub-neurotoxic doses of PB, deet and chlorpyrifos resulted in increased toxicity characterized by neurological dysfunction and neuropathological lesions. In the central cholinergic system of rats, application of physiologically relevant doses of pyridostigmine and deet, in combination, led to neurobehavioural deficits and region-specific alterations in AChE and nicotinic receptors . More investigations are urgently needed to confirm or dismiss the potential neurotoxicity to humans arising from the combined use of deet with different cholinesterase inhibitors.
The standard insecticide susceptible strains 'S-Lab' of C. quinquefasciatus and 'Bora' of A. aegypti were used in bioassays. These two strains have been colonized for many years at IRD-LIN in Montpellier and are free of any detectable insecticide resistance mechanisms.
Treated filter papers bioassay
Mortality resulting from tarsal contact with treated filter paper was measured using WHO test kits  against adult females of A. aegypti. Four batches of 25 non-blood-fed females, two to five days old, were introduced into WHO bioassay holding tubes for a period of 60 min. They were then transferred to exposure tubes, which were held vertically for 60 min under subdued light. Mortality was recorded 24 h after exposure. Each solution was tested four times and each test was replicated three times with different cohorts of insects to take into account inter-batch variability.
Adult male cockroaches P. americana were taken from our laboratory stock colonies which are maintained under standard conditions (29°C photo-cycle 12 h light/12 h dark). Cockroaches were pinned dorsal side up in a dissection dish and dorsal cuticles were removed to allow access to the ventral nerve cord. The terminal abdominal ganglion (TAG) with the nerve cord were carefully dissected and placed in normal cockroach saline containing (in mM): NaCl 208, KCl 3.1, CaCl2 10, sucrose 26, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10; pH was adjusted to 7.2 with NaOH. The synaptic preparation was composed of a cercus, the corresponding cercal nerve XI, the de-sheathed TAG (containing the studied synapse) and the abdominal part of the nerve cord. Electrophysiological recordings of synaptic events were obtained using the single-fibre oil-gap method . With this technique it is possible to record unitary excitatory postsynaptic potentials (uEPSP) resulting from the activity of pre-synaptic cercal mechanoreceptors and composite EPSP. These potentials were triggered in response to short electrical pre-synaptic stimulation applied at a frequency of 0.1 Hz to the ipsilateral cercal nerve XI and are the main subject of observations to study synaptic transmission. During experiments, the resting potential was continuously monitored on a pen chart recorder. The uEPSPs and EPSPs were recorded using a Hameg oscilloscope and stored on a PC computer with Hameg software. Experiments were conducted at room temperature (20°C). Data were expressed as a mean ± s.e.m. when quantified. Electrophysiological data were analysed for statistical significance using a one-way Analysis of Variance (ANOVA) followed by a post-hoc Tukey test. Differences among data were judged to be significant when P < 0.05. Data analysis was performed using STATISTICA (StatSoft, Cracow, Poland).
In all electrophysiological experiments, deet and propoxur were prepared in dimethylsulfoxide (DMSO, stock solution 10 mM) and absolute ethanol (stock solution 10 mM), respectively. Final dilutions in physiological saline contained at most 0.1% DMSO and absolute ethanol. These concentrations of solvents had no effect on synaptic transmission. All compounds used were purchased from Sigma Chemicals (L'isle d'Abeau Chesnes, France) and propoxur was bought from Bayer AG (Leverkusen, Germany).
All experiments on mice were performed in accordance with French and European Community guidelines for laboratory animal handling . Adult male Swiss-Webster mice (20 to 25 g body weight) purchased from IFFA CREDO (Saint Germain sur l'Arbresle, France) were anaesthetized with Isoflurane (AErrane®, Baxter S.A., Lessines, Belgium) inhalation, and euthanized by dislocation of the cervical vertebrae followed by immediate exsanguination. The left mouse hemidiaphragm with its associated phrenic nerve was dissected out from the animal and mounted in a silicone-lined organ bath (4 ml volume). Isolated preparations were perfused with standard Krebs-Ringer solution of the following composition: 154.0 mM NaCl, 5.0 mM KCl, 2 mM CaCl2, 1.0 mM MgCl2, 5.0 mM HEPES, and 11.0 mM glucose. The solution gassed with pure O2 had a pH of 7.4.
Electrophysiological recordings on isolated phrenic hemidiaphragm muscles were performed using conventional techniques . Briefly, membrane and synaptic potentials were recorded from endplate regions, at room temperature (22°C), with intracellular microelectrodes filled with 3 M KCl (8–12 MΩ resistance), or with extracellular microelectrodes (filled with Krebs-Ringer solution, 1–3 MΩ resistance) and an Axoclamp-2A system (Axon Instruments, Foster City, CA, USA). Electrical signals after amplification were displayed on a digital oscilloscope, collected and digitized at a sampling rate of 25 kHz with the aid of a PC computer and a Digidata 1322A unit (Axon Instruments). Computerized data acquisition and analysis was performed with the program WinWCP (V3.8), provided by Dr John Dempster (University of Strathclyde, Strathclyde, Scotland). The motor nerve of isolated neuromuscular preparations was stimulated via a suction microelectrode, adapted to the diameter of the nerve, with square wave pulses of 0.1 ms duration, generated by a S-44 stimulator (Grass Instruments, AstroMed, W. Warwick, RI, USA), and supramaximal intensity (typically 3–8 V). Studies on EPPs were performed in standard physiological solution containing 1.6 μM μ-conotoxin Conus Geographus (GIIIB) (Alomone Labs, Jerusalem, Israel) to block voltage-dependent sodium channels of skeletal muscle fibres . The amplitudes of full-sized EPPs and MEPPs recorded on junctions treated with μ-conotoxin GIIIB were normalized to a membrane potential of -75 mV. MEPPs and EPPs were analysed individually for amplitude and time course. For each condition studied, four to six individual experiments were performed and the results were averaged to give the presented mean ± s.e.m. The statistical significance of differences between controls and test values was assessed with Student's t-Test (two-tailed), or the Kolmogorov-Smirnov two-sample test. Differences were considered significant if P < 0.05.
DmAChE was produced in the baculovirus system and purified as previously described . The native human AChE and BChE used for kinetic studies were from Sigma Chemical Co. (St Louis, MO, USA). Hydrolysis of ATCh was measured spectrophotometrically at 412 nm by the Ellman method  at 25°C, in 25 mM phosphate buffer, pH 7. Substrate concentrations were 4 μM-200 mM, with a minimum of five repetitions per concentration. Activity was followed for 1 min after addition of the enzyme to the mixture and spontaneous hydrolysis of the substrate was subtracted. Rates of carbamoylation were estimated by incubation of AChEs with various concentrations of propoxur for different periods of time. The remaining activity was measured for 30 sec following 10-fold dilutions in Ellman reaction medium supplemented with 1 mM acetylthiocholine. Data were analysed using the model and equation of Stojan and colleagues  for ATCh hydrolysis inhibition and using the model of pseudo first order irreversible inhibition for carbamoylation rate. Fits were performed simultaneously on both equations by multiple non-linear regressions using the program GOSA .
Molecular docking of deet into AChE
The accommodation and binding of deet inside the active site of HuAChE was made by building a 3D structure of deet using MOLDEN, a processing program of molecular and electronic structure, and then optimized quantum mechanically in vacuo by Gaussian 03, an electronic structure program. For the calculation we used 6–31 g* basis set at the Hartree-Fock level. For molecular mechanics, energy and dynamic calculations we assigned atomic types for the deet molecule already existing in the CHARMM distribution C27n1 topology file. Charges were calculated by Mullikan's approximation and the missing parameters were searched until a satisfactory fit of the model to the ab initio energy potentials and geometry was obtained.
In the next step we manually docked the deet in the active site above the catalytic serine (S203) of the HuAChE molecule: the appropriate three atoms of deet were superimposed on the corresponding atoms of substrate analogue molecule situated in the active site of torpedo AChE (PDB code 2C5F) with the carbonyl oxygen pointing into the oxyanion hole. The structure was then fully relaxed without moving any of the protein atoms. Finally, our 3D model of HuAChE and docked deet was subjected to two successive 50-step QMMM refinements, assigning the deet molecule, catalytic serine (S203) and histidine (H447) quantum mechanically (49 QM atoms and two link atoms), while the rest of protein and water molecules (193 of them) were treated mechanically. During QMMM relaxation of the complex between HuAChE and the deet molecule, the latter was accommodated in a tetrahedral adduct conformation.
We thank J Bonnet for his technical assistance and Dr P Agnew, Prof J Hemingway, Prof IJ Russell and Dr G Gibson for critical reviews of the manuscript. This study was financially supported by the Agence Nationale pour la Recherche (ANR, programme REAC SEST06 030 01).
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