BACKGROUND
the use of repellents against biting arthropods was probably developed a thousand years ago  <cit> ; however, a real breakthrough occurred in  <dig> with the discovery of the synthetic repellent n,n-diethyl-3-methylbenzamide , 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  <cit> .
despite the widespread and increased interest in the use of deet in public health programmes  <cit> , 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  <cit>  suggested that deet may block electrophysiological responses of olfactory sensory neurons to attractive odours in anopheles gambiae giles  and drosophila melanogaster meigen .
by contrast, syed and leal  <cit>  have recently reported that mosquitoes detect deet by means of olfaction, a physiological mechanism that directly initiates avoidance behaviour .
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  <cit> .
in the same context, if deet is considered to have a relatively good toxicological profile  <cit> , 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  <cit> .
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   <cit> .
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  in both insect and mammal neuronal preparations, and have investigated the consequences of its interactions with carbamate insecticides on the cholinergic system.
RESULTS
insecticidal effect of deet on insects
to elucidate repellent toxicity in insects, we first assessed the sensitivity of the dengue vector aedes aegypti l.  to deet -treated filter papers using world health organization  bioassays  <cit> .
figure  <dig> shows that deet caused dose-dependent mortality at doses ranging from  <dig> to  <dig> μg/cm <dig>  <cit> .
this range corresponds to the lower range of doses usually applied to human skin for personal protection.
topical applications of deet on the mosquito culex pipiens quinquefasciatus say  resulted in an ld <dig>  and an ld <dig> of deet for adult females of  <dig>  ±  <dig>   and  <dig>  ±  <dig>  ng of active ingredient/mg of mosquito .
for comparison, the ld <dig> and ld <dig> of propoxur  phenol methylcarbamate) were  <dig>  ±  <dig>  and  <dig>  ±  <dig>  ng a.i./mg female, respectively, indicating that the amount of deet required to kill mosquitoes was about  <dig> times higher than that for propoxur, an acetylcholinesterase  inhibitor.
however, the slopes of the regression lines for mosquito mortality for deet  and propoxur  did not differ significantly.
this indicated a similar heterogeneity of response by the mosquitoes with respect to the toxic effect of the two molecules.
neurophysiological effects of deet on insect and mammalian neuronal preparations
based on these observations, we investigated the neurophysiological effects of deet on the cercal-afferent giant-interneuron synapses in the terminal abdominal ganglion of the cockroach periplaneta americana l. , known to present many functional analogies with other insect systems  <cit> .
the single-fibre oil-gap method  <cit>  is a well-adapted electrophysiological technique for the cockroach cns, which allows investigation of the effects of such compounds at the synaptic level.
deet dissolved in physiological saline was applied at two concentrations  on the synaptic preparation by superfusion into the experimental chamber.
bath application of deet produced a biphasic effect on excitatory post synaptic potential  amplitudes.
as illustrated in figure 2a , deet  produced an increase in epsp amplitude within the first  <dig> min .
this effect was also observed at  <dig>  μm deet .
after  <dig> min, a time-dependent decrease in epsp amplitude was observed compared with controls, which was more pronounced with the higher concentration .
this typical biphasic effect, previously observed with anticholinesterase compounds such as carbamates  <cit> , reflected changes in synaptic transmission activity.
indeed, treatment with carbamates can cause an increase in acetylcholine  concentration that is sufficient to activate negative feedback acting through presynaptic muscarinic receptors, which thereby decrease subsequent release of ach  <cit> .
as the deet-induced biphasic effect on epsp amplitude was very similar to that reported with classical anticholinesterase compounds, it is possible that deet might cause an elevation of ach concentration into the synaptic cleft via an inhibition of ache.
to test this hypothesis, we conducted additional experiments in the presence of atropine, known to block muscarinic receptors in insect synaptic transmission  <cit> .
as illustrated in figure 2a , pre-treatment with atropine  for  <dig> min counteracted the epsp depression previously observed with deet, producing only a time-dependent increase in epsp amplitude .
these data confirm the participation of pre-synaptic muscarinic receptors in the modulation of ach release in the synapses after bath application of deet  <cit> .
it is also interesting to note that application of deet on synaptic preparations, pre-treated with  <dig> μm atropine, increased both composite  and unitary epsp amplitudes , which result from the spontaneous activity of presynaptic cercal mechanoreceptors.
all these findings clearly indicate that ach is not efficiently hydrolyzed by ache in the presence of deet.
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  <dig> μm deet prolonged by about threefold the decay time constant of synaptic potentials on endplate regions of the muscle fibre .
this prolongation of the time course of synaptic potentials, which is known to occur after ache inhibition  <cit>  or in the absence of ache expression  <cit> , 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  compared with cockroach synaptic preparations .
characterization of cholinesterase inhibition by deet
to ascertain the inhibition of cholinesterases by deet, we analysed, in vitro, the effect of deet on the activity of purified ache from d. melanogaster  and both acetyl and butyrylcholinesterases  from human .
as illustrated in figures 3a, b, and 3c, incubation of each enzyme with the substrate and deet  resulted in a strong reduction of enzyme activity.
this indicates that deet is capable of inhibiting the hydrolysis of acetylthiocholine  and butyrylthiocholine  by aches.
as preincubation of the enzyme with deet in the absence of substrate did not change the extent of inhibition, and as dilution of the inhibited enzyme restored enzyme activity, deet can be considered as a reversible inhibitor of cholinesterases.
deet has also the capacity to diminish the rate of ache carbamoylation by propoxur , indicating that both molecules act as competitive inhibitors for the enzyme.
the kinetics of substrate hydrolysis by cholinesterases are complex.
the substrate first binds to a peripheral site, located at the entrance of the active site gorge, and then slides down to the catalytic site, buried  <dig> Å inside the protein .
simultaneous kinetic analyses of inhibition of substrate hydrolysis and carbamoylation allowed us to estimate the binding constants of deet for the two substrate binding sites of cholinesterases.
binding of deet at the peripheral site was estimated to be  <dig>  ±  <dig> ,  <dig>  ±  <dig>  and  <dig>  ±  <dig>  mm for dmache, huache and hubche, respectively .
binding of deet at the catalytic site located at the bottom of the active site gorge was not necessary to describe inhibition of dmache and was estimated as  <dig>  and  <dig>  mm for huache and hubche, respectively .
thus, deet would enter into the active site gorge of huache and hubche, but not that of dmache, resulting in a stronger inhibition of human enzymes.
this hypothesis was consistent with structural data showing the active site gorge of dmache to be about 50% narrower than the active site of huache  <cit> .
to determine whether the accommodation and binding of deet was possible within the active site of vertebrate ache, it was docked as a tetrahedral adduct on the catalytic serine of human ache crystal structure .
minimal adaptation of the side chains of adjacent residues in the active side of huache suggests the accommodation of deet in a catalytic site is possible .
atch = acetylthiocholine; btch = butyrylthiocholine; dmache = acetylcholinesterase from d. melanogaster; huache = acetylcholinesterase from human; hubche = butyrylcholinesterase from human.
interactions between deet and cholinesterase compounds
having established that deet binds to the active site of cholinesterases and then hinders the entrance of substrates, we investigated its potential interaction with carbamate insecticides.
the effects of topical applications of a range of deet doses combined with a range of propoxur doses applied to c. quinquefasciatus were not in agreement with a model based on the hypothesis of an additive effect for the two compounds .
several models of interactions between the two chemicals were subsequently tested.
the best fit took into account a synergistic interaction involving the effect of deet on the insecticidal effect of propoxur .
further electrophysiological experiments were conducted on p. americana preparations to assess deet and propoxur interactions at the synaptic level .
after pre-treatment of atropine and when applied alone, both propoxur  and deet  significantly increased epsp amplitude compared with the control .
in the presence of atropine, however, subsequent application of deet + propoxur  did not cause a greater effect on post-synaptic potentials than that caused by deet alone at the same concentrations.
propoxur+d <dig> was almost equal to d1; the difference between them was not significant .
similarly, there was no difference between p+d <dig> and d <dig> .
this indicates that propoxur and deet acted similarly on the same target site in the insect cholinergic system.
however, a different trend was noted on mouse isolated phrenic hemidiaphragm muscles.
indeed, when neostigmine  was perfused in the continuous presence of  <dig> μm deet, the decay time constant of synaptic responses was about two fold more prolonged than with deet alone .
recordings of full  size endplate potentials  in response to a single or paired nerve stimuli, either in the presence of  <dig> μm deet or in the presence of deet plus  <dig> μm neostigmine, showed a marked prolongation of the decay phase of the epps in the presence of deet and neostigmine .
these results indicated that deet  had an inhibitory action on ache in mouse hemidiaphragm endplates that was not maximal at the concentration used;  did not prevent subsequent action of neostigmine on endplate ache; and  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 <cit> ;
CONCLUSIONS
in vivo toxic interactions between deet and propoxur, pirimiphos-methyl, or pyridostigmine bromide  for cockroaches and mosquitoes have been reported previously  <cit> .
in adult hens, abou-donia et al.
<cit>  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  <cit> .
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 use of repellents against biting arthropods was probably developed a thousand years ago; however, a real breakthrough occurred in with the discovery of the synthetic repellent n,n-diethyl-3-methylbenzamide, 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, 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 and drosophila melanogaster meigen. by contrast, syed and leal have recently reported that mosquitoes detect deet by means of olfaction, a physiological mechanism that directly initiates avoidance behaviour. 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. 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. 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. 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 in both insect and mammal neuronal preparations, and have investigated the consequences of its interactions with carbamate insecticides on the cholinergic system.  insecticidal effect of deet on insects to elucidate repellent toxicity in insects, we first assessed the sensitivity of the dengue vector aedes aegypti l. to deet -treated filter papers using world health organization bioassays. figure shows that deet caused dose-dependent mortality at doses ranging from to μg/cm. this range corresponds to the lower range of doses usually applied to human skin for personal protection. topical applications of deet on the mosquito culex pipiens quinquefasciatus say resulted in an ld and an ld of deet for adult females of ± and ± ng of active ingredient/mg of mosquito. for comparison, the ld and ld of propoxur phenol methylcarbamate) were ± and ± ng a.i./mg female, respectively, indicating that the amount of deet required to kill mosquitoes was about times higher than that for propoxur, an acetylcholinesterase inhibitor. however, the slopes of the regression lines for mosquito mortality for deet and propoxur did not differ significantly. this indicated a similar heterogeneity of response by the mosquitoes with respect to the toxic effect of the two molecules. neurophysiological effects of deet on insect and mammalian neuronal preparations based on these observations, we investigated the neurophysiological effects of deet on the cercal-afferent giant-interneuron synapses in the terminal abdominal ganglion of the cockroach periplaneta americana l., known to present many functional analogies with other insect systems. the single-fibre oil-gap method is a well-adapted electrophysiological technique for the cockroach cns, which allows investigation of the effects of such compounds at the synaptic level. deet dissolved in physiological saline was applied at two concentrations on the synaptic preparation by superfusion into the experimental chamber. bath application of deet produced a biphasic effect on excitatory post synaptic potential amplitudes. as illustrated in figure 2a, deet produced an increase in epsp amplitude within the first min. this effect was also observed at μm deet. after min, a time-dependent decrease in epsp amplitude was observed compared with controls, which was more pronounced with the higher concentration. this typical biphasic effect, previously observed with anticholinesterase compounds such as carbamates, reflected changes in synaptic transmission activity. indeed, treatment with carbamates can cause an increase in acetylcholine concentration that is sufficient to activate negative feedback acting through presynaptic muscarinic receptors, which thereby decrease subsequent release of ach. as the deet-induced biphasic effect on epsp amplitude was very similar to that reported with classical anticholinesterase compounds, it is possible that deet might cause an elevation of ach concentration into the synaptic cleft via an inhibition of ache. to test this hypothesis, we conducted additional experiments in the presence of atropine, known to block muscarinic receptors in insect synaptic transmission. as illustrated in figure 2a, pre-treatment with atropine for min counteracted the epsp depression previously observed with deet, producing only a time-dependent increase in epsp amplitude. these data confirm the participation of pre-synaptic muscarinic receptors in the modulation of ach release in the synapses after bath application of deet. it is also interesting to note that application of deet on synaptic preparations, pre-treated with μm atropine, increased both composite and unitary epsp amplitudes, which result from the spontaneous activity of presynaptic cercal mechanoreceptors. all these findings clearly indicate that ach is not efficiently hydrolyzed by ache in the presence of deet. 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 μm deet prolonged by about threefold the decay time constant of synaptic potentials on endplate regions of the muscle fibre. this prolongation of the time course of synaptic potentials, which is known to occur after ache inhibition 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 compared with cockroach synaptic preparations. characterization of cholinesterase inhibition by deet to ascertain the inhibition of cholinesterases by deet, we analysed, in vitro, the effect of deet on the activity of purified ache from d. melanogaster and both acetyl and butyrylcholinesterases from human. as illustrated in figures 3a, b, and 3c, incubation of each enzyme with the substrate and deet resulted in a strong reduction of enzyme activity. this indicates that deet is capable of inhibiting the hydrolysis of acetylthiocholine and butyrylthiocholine by aches. as preincubation of the enzyme with deet in the absence of substrate did not change the extent of inhibition, and as dilution of the inhibited enzyme restored enzyme activity, deet can be considered as a reversible inhibitor of cholinesterases. deet has also the capacity to diminish the rate of ache carbamoylation by propoxur, indicating that both molecules act as competitive inhibitors for the enzyme. the kinetics of substrate hydrolysis by cholinesterases are complex. the substrate first binds to a peripheral site, located at the entrance of the active site gorge, and then slides down to the catalytic site, buried Å inside the protein. simultaneous kinetic analyses of inhibition of substrate hydrolysis and carbamoylation allowed us to estimate the binding constants of deet for the two substrate binding sites of cholinesterases. binding of deet at the peripheral site was estimated to be ±, ± and ± mm for dmache, huache and hubche, respectively. binding of deet at the catalytic site located at the bottom of the active site gorge was not necessary to describe inhibition of dmache and was estimated as and mm for huache and hubche, respectively. thus, deet would enter into the active site gorge of huache and hubche, but not that of dmache, resulting in a stronger inhibition of human enzymes. this hypothesis was consistent with structural data showing the active site gorge of dmache to be about 50% narrower than the active site of huache. to determine whether the accommodation and binding of deet was possible within the active site of vertebrate ache, it was docked as a tetrahedral adduct on the catalytic serine of human ache crystal structure. minimal adaptation of the side chains of adjacent residues in the active side of huache suggests the accommodation of deet in a catalytic site is possible. atch = acetylthiocholine; btch = butyrylthiocholine; dmache = acetylcholinesterase from d. melanogaster; huache = acetylcholinesterase from human; hubche = butyrylcholinesterase from human. interactions between deet and cholinesterase compounds having established that deet binds to the active site of cholinesterases and then hinders the entrance of substrates, we investigated its potential interaction with carbamate insecticides. the effects of topical applications of a range of deet doses combined with a range of propoxur doses applied to c. quinquefasciatus were not in agreement with a model based on the hypothesis of an additive effect for the two compounds. several models of interactions between the two chemicals were subsequently tested. the best fit took into account a synergistic interaction involving the effect of deet on the insecticidal effect of propoxur. further electrophysiological experiments were conducted on p. americana preparations to assess deet and propoxur interactions at the synaptic level. after pre-treatment of atropine and when applied alone, both propoxur and deet significantly increased epsp amplitude compared with the control. in the presence of atropine, however, subsequent application of deet + propoxur did not cause a greater effect on post-synaptic potentials than that caused by deet alone at the same concentrations. propoxur+d was almost equal to d1; the difference between them was not significant. similarly, there was no difference between p+d and d. this indicates that propoxur and deet acted similarly on the same target site in the insect cholinergic system. however, a different trend was noted on mouse isolated phrenic hemidiaphragm muscles. indeed, when neostigmine was perfused in the continuous presence of μm deet, the decay time constant of synaptic responses was about two fold more prolonged than with deet alone. recordings of full size endplate potentials in response to a single or paired nerve stimuli, either in the presence of μm deet or in the presence of deet plus μm neostigmine, showed a marked prolongation of the decay phase of the epps in the presence of deet and neostigmine. these results indicated that deet had an inhibitory action on ache in mouse hemidiaphragm endplates that was not maximal at the concentration used; did not prevent subsequent action of neostigmine on endplate ache; and 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 for cockroaches and mosquitoes have been reported previously. 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. 
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. here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system. 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.
 n,n-diethyl-3-methylbenzamide remains the gold standard for insect repellents. about million people use it every year and over billion doses have been applied over the past 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. 