Maria Stankiewicz

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

BackgroundN,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.ResultsBy 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.ConclusionThese 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 Putative Bioactive Surface of Insect-selective Scorpion Excitatory Neurotoxins

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.

Scorpion α‐like toxins, toxic to both mammals and insects, differentially interact with receptor site 3 on voltage‐gated sodium channels in mammals and insects

α‐Like toxins, a unique group designated among the scorpion α‐toxin class that inhibit sodium channel inactivation, are highly toxic to mice but do not compete for α‐toxin binding to receptor site 3 on rat brain sodium channels. We analysed the sequence of a new α‐like toxin, which was also highly active on insects, and studied its action and binding on both mammalian and insect sodium channels. Action of the α‐like toxin on isolated cockroach axon is similar to that of an α‐toxin, and the radioactive toxin binds with a high affinity to insect sodium channels. Other sodium channel neurotoxins interact competitively or allosterically with the insect α‐like toxin receptor site, similarly to α‐toxins, suggesting that the α‐like toxin receptor site is closely related to receptor site 3. Conversely, on rat brain sodium channels, specific binding of 125I‐α‐like toxin could not be detected, although at high concentration it inhibits sodium current inactivation on rat brain sodium channels. The difficulty in measuring binding to rat brain channels may be attributed to low‐affinity binding due to the acidic properties of the α‐like toxins that also impair the interaction with receptor site 3. The results suggest that α‐like toxins bind to a distinct receptor site on sodium channels that is differentially related to receptor site 3 on mammalian and insect sodium channels.

Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.

Refined electrophysiological analysis suggests that a depressant toxin is a sodium channel opener rather than a blocker.

The effects of a recombinant depressant insect toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltage-clamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na+ channels, but may be attributed to modified Na+ channels which open at more negative potential, activate slowly and do not inactivate normally.

Effects of a centipede venom fraction on insect nervous system, a native Xenopus oocyte receptor and on an expressed Drosophila muscarinic receptor.

Centipede venoms are complex protein mixtures; very few is known about their pharmacological actions. Application of a Scolopendra sp. venom fraction (SC1) on the cockroach giant axon induced an increase in the leak current correlated with a decrease in the membrane resistance, suggesting the presence in SC1 of components opening non-specific pores in the axonal membrane. On a cockroach central cholinergic synapse, microinjection of SC1 induced a small transient depolarization of the postsynaptic membrane, followed by a slow stable depolarization and a drastic decrease in the evoked subthreshold excitatory postsynaptic potential amplitude. A pretreatment of the ganglion with atropine or scopolamine reduced the amplitude of the SC1-induced depolarizing wave, suggesting a possible cholinergic muscarinic target. On control Xenopus oocytes, SC1 induced an inward oscillatory Ca2(+)-dependent Cl- current mediated through the activation of native lysophosphatidic acid receptors (LPAr). Indeed, pretreatment of oocytes with 1 microM N-palmitoyl-tyrosine phosphoric acid, a selective competitive antagonist of LPAr, decreased responses to SC1 by 70%. Application of SC1 to oocytes expressing a cloned Drosophila muscarinic receptor (Dml) induced a biphasic response comprising: (1) a large fast Cl- current that was abolished by pretreatment with atropine and scopolamine and (2) a slow and small oscillating Cl- current corresponding to the response observed in control oocytes. These observations confirm the presence of muscarinic agonists in SCI and reveal their direct action on an insect muscarinic receptor subtype homologous to mammalian M1-M3 receptors.

Biophysical properties of scorpion α‐toxin‐sensitive background sodium channel contributing to the pacemaker activity in insect neurosecretory cells (DUM neurons)

A scorpion α‐toxin‐sensitive background sodium channel was characterized in short‐term cultured adult cockroach dorsal unpaired median (DUM) neurons using the cell‐attached patch‐clamp configuration. Under control conditions, spontaneous sodium currents were recorded at different steady‐state holding potentials, including the range of normal resting membrane potential. At −50 mV, the sodium current was observed as unclustered, single openings. For potentials more negative than −70 mV, investigated patches contained large unitary current steps appearing generally in bursts. These background channels were blocked by tetrodotoxin (TTX, 100 nm), and replacing sodium with TMA‐Cl led to a complete loss of channel activity. The current–voltage relationship has a slope conductance of 36 pS. At −50 mV, the mean open time constant was 0.22 ± 0.05 ms (n = 5). The curve of the open probability versus holding potentials was bell‐shaped, with its maximum (0.008 ± 0.004; n = 5) at –50 mV. LqhαIT (10–8m) altered the background channel activity in a time‐dependent manner. At −50 mV, the channel activity appeared in bursts. The linear current–voltage relationship of the LqhαIT‐modified sodium current determined for the first three well‐resolved open states gave three conductance levels: 34, 69 and 104 pS, and reversed at the same extrapolated reversal potential (+52 mV). LqhαIT increased the open probability but did not affect either the bell‐shaped voltage dependence or the open time constant. Mammal toxin AaHII induced very similar effects on background sodium channels but at a concentration 100 × higher than LqhαIT. At 10–7m, LqhαIT produced longer silence periods interrupted by bursts of increased channel activity. Whole‐cell experiments suggested that background sodium channels can provide the depolarizing drive for DUM neurons essential to maintain beating pacemaker activity, and revealed that 10–7 m LqhαIT transformed a beating pacemaker activity into a rhythmic bursting.

Sequence and electrophysiological characterization of two insect‐selective excitatory toxins from the venom of the Chinese scorpion Buthus martensi

The two insecticidal peptides Bm32‐VI and Bm33‐I, isolated from the venom of the Chinese scorpion Buthus martensi induce paralytical symptoms typical of insect contractive toxins. They show, respectively, 74% and 77% homology with AaIT from Androctonus australis, comparable insecticidal activity and no vertebrate toxicity. Under voltage‐clamp conditions, both toxins induced (1) an increased fast Na+ current, (2) a shift in voltage dependence of Na+ current activation, (3) the occurrence of a delayed current, and (4) a slow development of a holding current. Increased Na+ conductance at negative potential values is responsible for axonal hyperexcitability and the contractive paralysis of insect prey.

Bot IT2, a toxin paralytic to insects from the Buthus occitanus tunetanus venom modifying the activity of insect sodium channels

The effects of insect toxin Bot IT2, purified from the venom of the scorpion Buthus occitanus tunetanus, were investigated on the isolated giant axon and on isolated dorsal unpaired median (DUM) neurone of the cockroach Periplaneta americana under current- and voltage-clamp conditions, using the double-oil-gap technique and the patch-clamp technique, respectively. In both preparations, Bot IT2, induces a limited depolarization together with the development of a repetitive activity in axon and an increase of spontaneous discharge frequency in DUM neurone. After artificial hyperpolarization to the normal resting level, plateau potentials can be evoked in both preparations. Under voltage-clamp conditions, Bot IT2 induces a similar effect in axon and DUM neurone by acting specifically on sodium channels. The peak sodium current is decreased and simultaneously a new current with very slow activation-deactivation kinetics is developed. Voltage dependence of this slow current is not very different from that of the control. The inactivation of the fast component is incomplete because it is masked by the development of the slow component. These results suggest that Bot IT2 modifies the kinetics of insect sodium channel activation, and the transformation of normal fast channels into slow ones is discussed.

Nervous System of Periplaneta americana Cockroach as a Model in Toxinological Studies: A Short Historical and Actual View

Nervous system of Periplaneta americana cockroach is used in a wide range of pharmacological studies, including electrophysiological techniques. This paper presents its role as a preparation in the development of toxinological studies in the following electrophysiological methods: double-oil-gap technique on isolated giant axon, patch-clamp on DUM (dorsal unpaired median) neurons, microelectrode technique in situ conditions on axon in connective and DUM neurons in ganglion, and single-fiber oil-gap technique on last abdominal ganglion synapse. At the end the application of cockroach synaptosomal preparation is mentioned.