Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Insecticidal effect of deet on mosquitoes. a) Mortality rate among A. aegypti exposed for 1 h to paper impregnated with deet in World Health Organisation bioassays. Doses applied on paper were compared with standard skin applications of commercially available formulations containing deet (lower and upper deet formulation concentrations 5 to 100% were taken from the review of Xue et al. [20]). Deet formulation concentrations (%) were converted to doses (μg/cm²) based on 5 ml being the average volume required to cover a human arm [4]. The figure showed that the doses used on skin (400 μg/cm² to 8000 μg/cm²) were equivalent or greater than doses showing insecticidal properties in the case of close contact (LD₅₀ = 830 ± 30 μg/cm², s.e.m; LD₉₅ = 1,180 ± 50 μg/cm²).

Figure 2

Effects of deet on insect and mammalian neuronal preparations. a) The Histogram illustrates the excitatory postsynaptic potential (EPSP) amplitudes (% of control) versus time (min) after exposure to 1 μM of deet in absence (blue bars) and in the presence of atropine (red bars) in P. americana central nervous system. Application of deet without atropine (blue bars) induced a biphasic effect on EPSP amplitudes. Within the first 3 min, application of deet induced a significant increase of EPSP amplitude which reflected an elevation of acetylcholine (ACh) concentration in the synaptic cleft (see text for details). After 3 min, a significant EPSP depression was observed, suggesting a regulation of ACh concentration in the synaptic cleft through an activation of presynaptic muscarinic receptors. Pre-treatment for 10 min with atropine 1 μM (red bars) clearly reversed the EPSP depression observed with deet 1 μM, confirming the participation of the muscarinic receptors in the negative feedback of ACh release following deet exposure. Data are means ± S.E.M. b) and c) Typical examples of cockroach composite (b) and unitary (c) EPSP following deet application. Experiments were done in the presence of atropine (1 μm) to prevent an action of presynaptic muscarinic receptors. Note the increase of unitary EPSP frequency and amplitude (c) following deet application (0.5 μM) in the synapses. d) Effect of deet on the time course of full size endplate potentials (EPPs) recorded in a mouse hemidiaphragm preparation bathed with a standard Krebs-Ringer solution supplemented with 1.6 μM μ-conotoxin GIIIB to selectively block sodium channels in muscle fibres. The EPPs recorded under control conditions (trace 1), and after the addition of 500 μM deet to the medium (trace 2); note the prolongation of the decay phase of EPPs in the presence of deet, with little change in the amplitude and time to peak; the mean decay-time constant were 11.1 ± 0.7 and 3.8 ± 0.08 ms for deet-treated and controls, respectively (n = 6, P < 0.001).

Figure 3

Effects of deet on cholinesterase enzymatic activities. a) and b) Inhibition of D. melanogaster (a) and Human (b) acetylcholinesterases (AChEs) by deet. Note the dose-dependant decrease of ATCh hydrolysis by AChE following deet application. [ATCh]: Acetylthiocholine concentration in micromole per liter; v/[Et] specific activity in s-1. c) Inhibition of human (Hu) butyrylcholinesterase by deet. As previously observed with ATCh, deet is also capable of strongly decreasing the BTCh hydrolysis by human BChE. [BTCh]: butyrylthiocholine concentration in micromole per liter; v/[Et] specific activity in s-1. d and e) Dose-dependant effect of deet on Drosophila (d) and Human (e) AChE carbamoylation rates by propoxur (carbamate). The curves clearly show the strong reduction of the second order rate constant (ki) for the carbamoylation of HuAChE by propoxur in presence of deet. At high concentration (10 mM), protection of AChE by deet is total. f) Accommodation and binding of deet inside the active site of Human AChE. The picture was created by VMD, a Visual Molecular Dynamics program. After QMMM relaxation of the complex between HuAChE and deet molecule, the latter was accommodated in a tetrahedral adduct conformation. Minimal adaptation of the side chains of adjacent residues in the active side of HuAChE suggests that the accommodation of deet in a position favourable for enzymatic hydrolysis is possible.

Figure 4

Interactions between deet and anti-cholinesterasic compounds. a) and b) Toxic interactions between deet (μg/mg mosquito) and propoxur (μg/mg mosquito) for C. quinquefasciatus by topical application. The model including a synergistic interaction (b) between the two molecules provided a better description of the data than a model based on simply additive effects (a); see equations where ED is the effective dose, D the dose and ic the interaction coefficient. The interaction coefficient (ic = 3.07 ± 0.98) was significantly greater than 0, indicating that deet synergised propoxur toxicity in insects. c) Effects of propoxur (P) and deet (D), alone and in combination (P+D), on cockroach synaptic activity. All synaptic preparations were pretreated (10 min) with atropine (1 μM). NS not significant (P > 0.05). d) Effect of deet and neostigmine on the time course of full size EPPS recorded in mouse hemidiaphragm preparations. Mean values (± s.e.m, n = 6) of the half-decay time of EPPs (ms) under control conditions (2.8 ± 0.05 ms, blue column), 500 μM deet (6.1 ± 0.36 ms, red column) and in the continuous presence of deet and 3 μM neostigmine (10.5 ± 0.55 ms, yellow column). * denotes a significant difference from controls (P < 0.001). e) Examples of full size endplate potentials (EPPs) in response to a single or paired stimulus in the presence of 500 μM deet and in the presence of deet (upper part) and 3 μM neostigmine (lower part). * denote significant difference from control P < 0.001.