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1Institut de Recherche sur la Biologie de l'Insecte, UMR Centre National de la Recherche Scientifique 6035, Université de Tours, F-37200 Tours; 2Laboratoire de Neurophysiologie, Unité Propre de Recherche de l'Enseignement Supérieur Equipe d'Accueil 2647, Université d'Angers, Unité de Formation et de Recherche Sciences, F-49045 Angers Cedex 01; 3Unité Mixte de Recherche Physiologie Moléculaire des Semences, F-49045 Angers Cedex 01; 4Laboratoire de Préconditionnement et remodelage du myocarde, Université d'Angers, Unité de Formation et de Recherche Sciences médicales, F-49045 Angers Cedex 01, France; and 5Laboratory of Biophysics, N. Copernicus University, 87–100 Torun, Poland
Submitted 6 December 2002; accepted in final form 2 February 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Some of plants can produce, in response to insect attacks,
volatile secondary compounds, also known as chemical defensives, which alter
insect metabolism and nervous system activity
(Rauscher 1992). By contrast,
these substances are ineffective against specialist insects
(Brattsen 1992), in which the
consumption of these compounds induces the induction of detoxification
enzymes, such as P450 enzymes, glutathione S-transferase enzymatic
system, and 
-glucosidases
(Feyereisen 1999;
Lindroth 1988;
Sogorb and Vilanova 2002)
Among plants producing the volatile secondary compounds, Allium plant
species, and particularly the leek Allium porrum, can release in the
atmosphere, when they are damaged, sulfur volatile compounds such as
thiosulfinates, which can lead to the formation of disulfides [e.g., dimethyl
disulfide (DMDS), tested in this study]
(Auger et al. 1989). Because
they are lethal (via a hypothetical neurotoxic activity) for many insect
species, nonspecialists of these plants
(Dugravot et al. 2002) they
could be used in plant protection and particularly in the seed storage systems
as fumigant (Arthur 1996;
Dugravot et al. 2002). Up to
date, the most commonly used fumigant, methyl bromide (CH3Br),
acting in the gaseous state, diffuses through and into large quantities of
seeds. It is particularly effective on insect eggs and adults. However,
despite its high toxicity to pests, many disadvantages are attributed to
CH3Br, including for instance a reduction of seed germination and a
major contribution to a depletion of ozone in the upper atmosphere
(Watson 1992). Elimination of
all uses and production of CH3Br is expected by 2005 in
industrialized countries and 2015 in developing countries
(Bell 2000;
Taylor 1996). Consequently,
sulfur compounds such as DMDS could represent a new alternative way to fight
insect predators while avoiding the use of environmentally aggressive
chemicals. However, to date, the precise insecticidal neurotoxic mode of
action of DMDS still remains unknown. Consequently, the susceptibility to DMDS
of two insect species developing in seed storage system, the Coleoptera
Bruchidae Callosobruchus maculatus and its parasitoid hymenoptera
Dinarmus basalis, were analyzed in the present study. We also
evaluated the susceptibility to DMDS of 1) the coackroach
Periplaneta americana, known to cause many inconveniences in urban
entomology, and 2) adult mice, to examine if DMDS could induce
toxicity in mammals. Finally, to characterize the insecticide target site of
DMDS at cellular and molecular levels, electrophysiological and
pharmacological experiments were carried out on the CNS of Periplaneta
americana, which is known to be a suitable model for better understanding
the effects of insecticides (Buckingham et
al. 1997; Lapied et al.
2001; Pelhate et al.
1990). Different preparations including isolated giant axon,
synaptic transmission, and short-term cultured cockroach neurosecretory cells
identified as dorsal unpaired median (DUM) neurons were used to investigate
this neurotoxic action. These experiments, associated with complementary
approaches such as oximetry and spectrophotometry adapted to mitochondria
purified from the pea Pisum sativum seeds, have allowed
demonstration, for the first time, that DMDS exerts an uncommon complex mode
of action through mitochondrial dysfunction, like the complex IV inhibitor
that induces an activation of neuronal ATP-sensitive potassium channels, never
identified, until now, in insect neuronal preparations. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The Coleoptera Bruchidae C. maculatus and its parasitoid
hymenoptera D. basalis originated from West Africa were mass reared
under the following conditions: 33–23°C, 12-h light/12-h dark, 70%
relative humidity, and synchronous photo- and thermoperiods as described by
(Ouedraogo et al. 1996). The
adults emerging from the seeds were placed in petri dishes and were fed with
10% sucrose solution, renewed every 2 days before the experiments were carried
out. Adult male cockroaches P. americana were obtained from our
laboratory stock colony and maintained at 29°C with a photoperiod of
12-hour light/12-hour dark.
Susceptibility of adult C. maculatus, D. basalis, and P. americana to DMDS
To analyze the toxicity of DMDS, 20 pairs of 2-day-old adults of C.
maculatus and D. basalis and 10 adult male P. americana
were placed for 24 h in 3-l hermetically sealed glass jars containing variable
concentrations of this volatile compound (0.33, 0.66, 1, 1.33, and 1.66
µl/l). At the end of the exposure, adults still alive were removed from the
jar and isolated in petri dishes for 24 h (C. maculatus and D.
basalis) or for48 h (P. americana). During this isolation
period, some individuals ambulated weakly became moribund and died. The other
individuals progressively recovered their mobility. The mortality rates were
determined at the end of this isolation period. Under each experimental
condition, three replicates were made. A dose-response curve was established
for each species. The lethal concentration causing 50% of mortality
(LC50) was determined by Probit analysis
(Finney 1971). Maximum
likelihood program software was employed for analysis of the dose-mortality
response. ANOVA and 
2 tests were used for the comparison of
the data.
Susceptibility of adult mice to DMDS
Male Swiss mice weighing 35 ± 5 g were used for this study. All procedures were performed in accordance with the regulations of the French ministry of Agriculture for the care and use of laboratory animals. The determination of the LC50 of the mice population exposed to DMDS was performed as followed. Each increasing concentration of DMDS, between 1 and 10 µl/l air, was tested on a 30-mouse group subdivided into 6 subgroups of 5 mice. A 30-mouse control group was exposed to confined air. After a 24-h exposure, the number of dead mice was noted, and the results were expressed as percentages in relation to the different concentrations used. During exposure to DMDS, each mice subgroup was placed in a 40-l experimental chamber during 24 h. At first, mice were placed in a small plastic transparent box with water and food. Then, each box perforated on two sides was heightened in an experimental chamber to inhibit all CO2 effects. In fact, CO2 and H2O were respectively captured with potassium hydroxide and calcium dichloride. The temperature was controlled with a thermometer and maintained to 21 ± 1°C. Just before airtight closing the chamber, DMDS was quickly applied, on an absorbent paper (Whatman paper) placed above a glass pot, for a swift and complete spraying of product.
Electrophysiology
SYNAPTIC TRANSMISSION—SINGLE FIBER OIL-GAP RECORDINGS. All
experiments were performed on adult male cockroaches (P. americana).
Briefly, the single fiber oil-gap technique
(Hue and Callec 1990) was used
to record composite excitatory postsynaptic potentials (cEPSPs) in response to
electrical presynaptic stimulation applied at a frequency of 0.1 Hz to the
ipsilateral cercal nerve. Direct activation of cholinergic postsynaptic
receptors located on dendritic membranes of the isolated giant interneuron
(GI) was achieved by means of ionophoretic micro-injection of carbamylcholine
(CCh) within the neuropile of the terminal abdominal ganglion (TAG). To assess
the physiological properties of the axonal membrane, action potentials were
evoked in the intraganglionic part of the TAG by passing suprathreshold square
current pulses using a Wheastone bridge circuit. During the experiments,
resting potential and unitary EPSPs were continuously monitored on a pen chart
recorder. The cEPSPs were recorded as the average of three sweeps. The
desheathed TAG was superfused with a saline of the following composition (in
mM): 208 NaCl, 3.1 KCl, 10 CaCl2, 2 NaHCO3, and 26
sucrose, pH 7.4. DMDS (100 µM) was bath-applied directly onto the TAG
during periods of 30–60 min. Quantitative effects of DMDS were expressed
as mean ± SE (n = 3).
ISOLATED DORSAL UNPAIRED MEDIAN NEURONS—WHOLE CELL PATCH-CLAMP
RECORDINGS. Experiments were performed on dorsal unpaired median (DUM)
neuron cell bodies isolated from the midline of the TAG of the nerve cord of
adult male cockroaches, P. americana, as previously described
(Lapied et al. 1989). DUM
neuron cell bodies used were maintained at 29°C for 24 h before
electrophysiological experiments were carried out. The whole cell patch-clamp
recording configuration was used to record ionic currents (voltage-clamp mode)
and action potentials (current-clamp mode). Signals were recorded using an
Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Patch pipettes
were pulled from borosilicate glass capillary tubes (Clark Electromedical
Instruments, Harvard Apparatus, Edenbridge, UK) with a PP-83 electrode puller
(Narishige, Japan) and had resistances of 0.9–1.3 M
when filled
with the pipette solution (see composition below). The liquid junction
potential between bath and internal pipette solution was compensated before
the formation of a gigaohm seal (>3 G).
For current-clamp experiments, depolarizing current pulses were elicited at
0.5 Hz with a programmable stimulator (SMP 310, Biologic, Claix, France)
Evoked action potentials and membrane potential were displayed and stored on
the hard disk of an IBM pentium 100 computer with the pClamp software control
(pClamp version 6.03, Axon Instruments). The computer was connected to a
125-kHz labmaster DMA data acquisition system (TL-1–125 interface, Axon
Instruments). The bathing solution contained (in mM) 200 NaCl, 3.1 KCl, 5
CaCl2, 4 MgCl2, 10 HEPES buffer, and the pH was adjusted
to 7.4 with NaOH. The recording electrode was filled with (in mM) 150
K-aspartate, 10 KF, 10 NaCl, 1 MgCl2, 3 ATP-Mg, 0.5
CaCl2, 10 EGTA, and 10 HEPES buffer, pH 7.4. For voltage-clamp
studies of the inward sodium current, step voltage pulses were generated by
the computer using pClamp software. Cells were clamped at a holding potential
of –90 mV, and test pulses of 30 ms in duration were applied at 0.3 Hz.
The procedure used to record the DUM neuron inward sodium currents is
described elsewhere (Lapied et al.
1990,
2001).
In all electrophysiological studies, DMDS stock solution (100 mM) was prepared in dimethylsulphoxide (DMSO). Final dilution contained at most 0.1% DMSO. These concentrations of solvent were found to be without effect on both axonal and neuronal electrophysiological properties. All compounds were purchased from Sigma Chemicals (L'isle d'Abeau Chesnes, France). Experiments were carried out at room temperature (21°C). Data were expressed as mean ± SE when quantified.
Effect of DMDS on cellular respiration
D. MELANOGASTER S2 CELL LINE. The S2 cell line was cultured in Shields and Sang M3 insect medium. For respiratory measurements, cells in the exponential phase of growth were collected with culture medium, concentrated by centrifugation (1000 rpm), and finally suspended in a small volume of medium and kept on ice until experiment. Oxygen consumption was monitored with an oxygen electrode (Oxytherm, Hansatech, King's Lynn, UK) at 30°C in 1 ml of culture medium. Cells (100 µl; 6.8 x 106 cells) were added to the reaction chamber previously equilibrated with 0.9 ml of culture medium. Inhibitors were added as required and indicated in the corresponding figures and legends.
ISOLATED PLANT MITOCHONDRIA. Mitochondria were isolated from 22
h imbibed pea (Pisum sativum) seeds and purified using Percoll
(Amersham Pharmacia Biotech) gradients using general method for plant
mitochondria (Douce et al.
1987). The mitochondria were highly purified according to marker
enzyme analysis for cytosol, plastid, peroxysome, and electron microscopy
(results not shown). Oxygen consumption was monitored with the oxygen
electrode at 25°C in 1 ml of reaction medium containing 0.6 M mannitol, 20
mM MOPS, pH 7.5, 10 mM KH2PO4, 10 mM KCl, 5 mM
MgCl2, and 0.1% (wt/vol) BSA. Mitochondria, substrates, cofactors,
and inhibitors were added as required and indicated in the corresponding
figures and legends. The analysis of electron transfer from exogenous NADH to
cytochrome c was followed spectrophotometrically with a microplate reader
(Spectramax plus, Molecular Devices). Reactions were carried out at 25°C
in a volume of 250 µl of reaction medium supplemented with 0.04% Triton
X100 (vol/vol), 1 mM KCN, 80 µM horse cytochrome c (Sigma), and
mitochondria (0.8 mg protein/ml). The reaction was initiated by the addition
of 2 mM NADH, and reduction of cytochrome c was followed through its
absorbance at 550 nm. A cytochrome oxidase assay based on a classical protocol
(Trounce et al. 1996) was
adapted to the microplate format that allows the simultaneous recording of
multiple absorbance and spectrum. The reaction mixture (270 µl) contained
50 µM MOPS, pH 7.4, 0.05% (vol/vol) Triton X100, 40 µM reduced
cytochrome c (stock solution reduced by a crystal of sodium dithionite), and
mitochondria (15 µg protein/ml). The reaction was incubated at 25°C
inside the microplate reader and absorbance at 550 nm was recorded every 5 s.
After 3 min of reaction, cytochrome c spectra were recorded simultaneously on
all samples. Protein concentrations were determined by Bradford bioassay
(Biorad, UK) using BSA as a standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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For the three insect species, the rates of mortality increased with the DMDS concentrations, but their susceptibility to this sulfur compound was different (Fig. 1A). Adults D. basalis were very susceptible to DMDS since the LC50 was 0.31 µl/l air. A dose of 0.66 µl/l air was enough to cause 100% mortality. DMDS was also tested on adult C. maculatus (Fig. 1A). They were less susceptible than adults D. basalis since a dose of 0.65 µl/l air was required for LC50. All insects were killed after a 24-h exposure to a dose of 1.33 µl/l air. For comparison, the LC50 estimated for adults P. americana was 1.01 µl/l air. To obtain 100% mortality 1.66 µl/l air DMDS was needed. These results indicated that after a 24-h DMDS exposure, the cockroaches were 3.3- and 1.5-fold less susceptible to DMDS than D. basalis and C. maculatus, respectively. In parallel, we performed experiments to determine if mammals were also susceptible to DMDS vapors. Figure 1B illustrates that all adult mice were dead when they were exposed to DMDS doses ranging from 5 to 10 µl/l air. The mice were not killed for lower doses (from 0.1 to 1 µl/l air). For concentrations ranging from 1.5 to 1.9 µl/l air (in 0.1 µl/l concentration increment), the rate of mortality was strongly increased from 43.5 ± 2.0% to 78.6 ± 1.7%, respectively. The 24-h LC50 value was estimated at 1.5 µl/l air and a concentration of 5 µl/l air DMDS was needed to achieve 100% kill.
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Because DMDS exhibited highest bioactivity against insects, we examined further the effectiveness of DMDS on the insect CNS as a potent insect neurotoxic agent.
Effects of DMDS on the cockroach CNS
Because one of the most attractive aspects of using DMDS is its ability to
exhibit insecticide neurotoxicity, we performed a new series of experiments on
the cockroach CNS to obtain more insight into its insecticide mode of action.
As already indicated above, the cockroach CNS possesses a number of
interesting features which make it suitable for studying neurotoxicity of
insecticides. The neurotoxic effect of DMDS were studied on three distinct
preparations, including 1) isolated giant axon
(Pelhate et al. 1990),
2) cercal afferent/giant interneuron synapses in the terminal
abdominal ganglion (Hue and Callec
1990), and 3) short-term cultured neurosecretory cells
identified as DUM neurons (Grolleau and
Lapied 2000; Wicher et al.
2001). The effects of DMDS was first studied on the isolated axon.
Both action potentials and ionic currents (i.e., inward sodium and outward
potassium currents) recorded under current- and voltage-clamp conditions,
respectively (Pelhate et al.
1990) were not affected at 100 µM DMDS (data not shown). This
indicated that the voltage-dependent ionic channels underlying cockroach
axonal electrical activity could not be proposed as responsible for the toxic
effect of DMDS.
Consequently, we wanted to determine whether DMDS could affect the
cockroach synaptic transmission between sensory fibers which originate from
mechanoreceptors of the cerci and GIs (Hue
and Callec 1990). Previous findings have reported that the
electrical stimulation delivered on cercal nerve XI evokes composite
excitatory postsynaptic potentials (cEPSP) mainly due to the activation of
postsynaptic nicotinic acetylcholine receptors (nAChRs)
(Hue and Callec 1990). In this
study, the superfusion of the experimental chamber with saline containing DMDS
(100 µM) induced postsynaptic depolarization (about 5 mV, not shown)
together with a gradual decrease (15% and 90% within a period of time of
10–60 min, respectively) of both cEPSP
(Fig. 2A, a and
b) and random unitary EPSPs
(Fig. 2B, a and
b). The synaptic transmission did not recover after DMDS
application. Although it was not possible to obtain a subthreshold cEPSP by
increasing the presynaptic cercal nerve stimulation during DMDS treatment,
ionophoretic microapplication (300 ms in duration, 300 µA) of CCh could
evoke postsynaptic potential with an amplitude very similar to that of
recorded in control (Fig. 2C, a
and b). It is interesting to mention that the DMDS
effects were mimicked by 10 µM 2,4-dinitrophenol (2,4-DNP, a nonspecific
mitochondrial uncoupler, data not shown). These results suggested that
postsynaptic AChRs located on the GI dendritic tree cannot be considered as a
synaptic target for DMDS molecules. By contrast, these experiments led us to
conclude that DMDS could interfere with presynaptic processes depending on ATP
known to be involved in synthesis and/or release of the neurotransmitter as it
was previously demonstrated with threonine-6-bradykinin
(Hue and Piek 1989). Finally,
the lack of effects of 100 µM DMDS (Fig.
2D, a and b) observed on action potential
elicited in the GI axonal membrane by passing depolarizing square current
pulse (5 ms in duration, 8 nA in amplitude) confirmed the results obtained in
both current- and voltage-clamp conditions on isolated giant axon. From these
results, it is tempting to suggest, among other possibilities, that the
alteration of the neurotransmitter release depending on intracellular ATP
observed at presynaptic level might reflect an alteration in the oxydative
phosphorylation process induced by DMDS.
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To substantiate this hypothesis, isolated short-term cultured DUM neurons
were used to facilitate the study of the neurotoxic effect of DMDS on the
electrophysiological properties of individual cells
(Fig. 3). The somata of DUM
neurons generate overshooting sodium-dependent action potentials and are
characterized by a membrane potential depending on the external concentration
of both sodium and potassium (Grolleau and
Lapied 2000). When isolated DUM neuron cell body was superfused
with 100 µM DMDS, two distinct effects depending on time of application
were observed. As illustrated in Fig.
3Aa, the amplitude of the action potentials, triggered by
a depolarizing current pulse (0.7 nA, 50 ms in duration) slightly decreased 10
min after applying 100 µM DMDS. This effect was associated with a
hyperpolarization of the membrane potential (23.6 ± 2.8 mV, n
= 6) obtained 16 min after DMDS treatment
(Fig. 3Aa). Because it
is known that the sodium inward current is responsible for the depolarizing
phase of action potentials (Lapied et al.
1990), we tested DMDS (100 µM) on the inward sodium current
under voltage-clamp condition (Fig.
3B). As expected from current-clamp experiments, the
amplitude of the inward sodium current elicited by a 30-ms depolarizing pulse
to 0 mV from a holding potential of –90 mV was also reduced (21.7
± 2.4%, n = 7). This effect correlated well with the reduction
of the action potential amplitude. To ensure that the hyperpolarization
observed was due to an activation or an inhibition of the resting conductances
(Grolleau and Lapied 2000), we
tested the effect of DMDS on the DUM neuron input resistance. As shown in
Fig. 3Ab, 100 µM
DMDS produced a decrease in the input resistance (by 32 ± 4%,
n = 6) in response to a hyperpolarizing current pulse (400 ms in
duration). This indicated that the hyperpolarization was due to the activation
of a hyperpolarizing conductance and not to the loss of a depolarizing resting
conductance (Grolleau and Lapied
2000). In other words, the activation of potassium channels
involved in the maintenance of the membrane potential could accounted for the
membrane hyperpolarization observed in the presence of DMDS. Because this last
effect represents an unusual neurotoxic effect for such compounds exhibiting
insecticide activity, the remaining part of this study will mainly be focused
on the mode of action of DMDS on the membrane potential. To express more
quantitatively the effect of DMDS on the membrane potential, isolated DUM
neuron cell bodies were exposed to various concentrations of DMDS
(Fig. 3C). Mean values
of hyperpolarization were plotted against the logarithm of the noncumulative
concentration of DMDS. The threshold concentration inducing hyperpolarization
was about 500 nM, and this hyperpolarization became more important with
increasing DMDS concentration (Fig.
3C). The sigmoid curve corresponded to the best fit
(r = 0.998) according to the Hill equation. The EC50 value
estimated for DMDS (i.e., the concentration of DMDS that produces 50% increase
of the hyperpolarization) was 8.7 µM. For comparison, the corresponding
concentration calculated from the LC50 (1.01 µl/l air) estimated
from the study of the in vivo toxicity performed with adult cockroaches was
11.2 µM (DMDS density 1.046). The maximum hyperpolarization was obtained at
a concentration of 1 mM. As indicated above, because it appeared that the
potassium channels could be suspected to be involved in the hyperpolarization
produced by DMDS, different potassium channel blockers were tested
(Fig. 4A). We first
examined the effect of the most commonly used blocker, TEA-Cl, known to
inhibit potassium channels in DUM neurons
(Grolleau and Lapied 2000). As
illustrated in Fig.
4A, TEA-Cl (5 mM) was a weak inhibitor of the
hyperpolarization observed after application of 100 µM DMDS. Similar
effects were obtained with higher TEA-Cl concentrations (i.e., 10 mM, data not
shown). By contrast, sulfonylurea drugs glibenclamide and tolbutamide, known
to block ATP-sensitive potassium (KATP) channels
(Inagaki et al. 1996) strongly
reduced the effect of DMDS. As indicated in
Fig. 4A, bath
application of DMDS in the presence of 100 µM glibenclamide and 100 µM
tolbutamide only induced a small hyperpolarization (17.5 ± 2.3% and
12.4 ± 4.5%, respectively, n = 4) compared with control
(100%). This suggested that KATP channels could be involved in the
hyperpolarization induced by DMDS. The following experiments were designed to
reinforce this hypothesis. In all the DUM neuron tested, glibenclamide (100
µM) alone caused an important depolarization (more than 25 mV), suggesting
that these KATP channels were activated under resting condition and
contributed to the membrane potential of DUM neurons. Interestingly, under
this experimental condition, DMDS only produced a small conductance change
(i.e., 2–4 mV of hyperpolarization,
Fig. 4B). As
illustrated in the inset of Fig.
4B, the decrease in input resistance provoked by 100
µM DMDS (see Fig.
3Ab) can be counteracted by glibenclamide (100 µM).
Finally, for comparison, it should be noted that 100 µM diazoxide, a
well-known KATP channel opener
(Ashcroft and Gribble 2000),
induced a similar DUM neuron hyperpolarization to that of DMDS
(Fig. 4A). Because
KATP channels are closed for high concentration of cytoplasmic ATP
and are open when the ATP concentration decreases below a threshold (e.g.,
Ashcroft and Gribble 1998), DUM
neuron KATP channels can also be identified by their sensitivity to
various intracellular ATP concentrations. Using the conventional whole cell
recording configuration, we examined the DMDS sensitivity of the DUM neuron
KATP channels by combining incubation of isolated cell bodies with
100 µM DMDS with dialysis of different ATP concentration pipette solutions.
After 4 min of dialysis, stable DMDS-induced hyperpolarizations were obtained
depending on the ATP concentration pipette solutions.
Figure 4C shows the
corresponding construction of the ATP-dose inhibition curve. The DMDS-induced
hyperpolarization almost was fully inhibited by internal ATP concentrations
above 10 mM and was approximately half-maximal at 5 mM. At low ATP
concentration (10 µM), the effect of DMDS on the membrane potential was
maximum. The apparent IC50 for DMDS-induced hyperpolarization
inhibition by internal ATP was 5.6 mM, according to the Hill equation
(r = 0.999).
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It is well known in the literature that KATP channels are an important class of ionic channels, linking bioenergetic metabolism to membrane excitability. Furthermore, KATP channels were sometimes associated with metabolic dysfunction since they were opened or closed in response to decrease and increase internal ATP concentrations, respectively. Consequently, intracellular factors such as mitochondrial dysfunction are believed to play an important role in the alteration of the KATP channels activity. In our context, it was tempting to suggest that the DUM neuron KATP channels might be one the targets acting as direct functional response element to DMDS-induced mitochondrial dysfunction, which thereby produced changes in intracellular ATP concentration. To substantiate this hypothesis, we first applied DMDS intracellularly (i.e., trough the intrapipette solution) or extracellularly (i.e., through the bathing solution superfusing the DUM neuron cell body). In both cases (Fig. 4D), DMDS (100 µM) induced an important hyperpolarization of the membrane potential [from –50.2 ± 1.2 (n = 5) to –71.9 ± 1.1 (n = 7) and from –49.3 ± 2.8 (n = 5) to –65.8 ± 1.5 (n = 6), respectively]. These results indicated that DMDS could cross the membrane to exert its neurotoxic effect on the KATP channels. Then we compared the effect of DMDS to oligomycin (100 µM; a mitochondrial ATPase inhibitor) and 2,4-DNP (100 µM), which were introduced into the cell body by diffusion through the patch pipette. Figure 4D illustrates that both compounds were capable of producing a hyperpolarization of the membrane potential [from –48.3 ± 3.8 (n = 5) to –68.6 ± 3.5 (n = 5) and from –55.2 ± 3.5 (n = 5) to –95.4 ± 5.6 (n = 6), respectively]. They also indicated that a decrease in intracellular ATP concentration, following mitochondrial dysfunction, could activate DUM neuron KATP channels. It should be noted that we never observed any effect of bath applied DMDS using oligomycin in the patch pipette (data not shown). Together these results seemed to indicate that DMDS could indirectly activate KATP channels, leading to a hyperpolarization, via an inhibition of mitochondrial respiration that thereby decreased intracellular ATP concentration. To further understand this unusual neurotoxic mechanisms, we decided to examine further the effect of DMDS on the different mitochondrial respiratory chain complexes.
Effects of DMDS on cellular respiration
The effect of DMDS on animal cellular respiration was first investigated
with cultured D. melanogaster S2 cells
(Towers and Sattelle 2002),
which gave us higher cell density than isolated DUM neurons for such
investigation. The oxygen consumption of S2 cells in the exponential phase of
growth was measured with an oxygen electrode. The respiration rate was
constant and totally inhibited by 1 mM cyanide (results not shown). Addition
of increasing concentrations of DMDS inhibited progressively the oxygen
consumption (Fig. 5), which was
reduced to 25% of the initial rate in the presence of 30 µM DMDS [i.e.,
from 11.2 nmolO2/ml (control) to 2.7 nmolO2/ml; see
Fig. 5]. The residual oxygen
consumption was abolished by 1 mM cyanide. When the cells were preincubated
for 10 min with DMDS, the inhibition appeared stronger (i.e., 56% inhibition
obtained with only 10 µM DMDS, not shown). This strong inhibition suggests
that the existence of a diffusion barriers in intact cell prevents the rapid
diffusion of DMDS toward its site of action. The inhibition of S2 cells
respiration led us to further directly investigate the DMDS effect on the
respiratory metabolism of isolated plant mitochondria.
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EFFECTS OF DMDS ON PLANT MITOCHONDRIA. The effect of DMDS on the
respiratory metabolism was tested using plant mitochondria isolated from
imbibed pea (P. sativum L.) seeds. The great advantage of plant
mitochondria with respect to their animal counterparts is that the respiratory
electron-transport pathways comprise the cyanide-sensitive cytochrome pathway
(like in animals) and the cyanide insensitive alternative pathway that
consists of only one protein the alternative oxidase (AOX); beyond the branch
point (ubiquinone, see for details Fig.
8A), the alternative pathway does not contribute to the
generation of a proton-motive force, in contrast to the cytochrome oxidase
pathway (Moller and Rasmusson
1998; Vanlerberghe and
McIntosh 1997). Furthermore, in plant mitochondria, the NAD(P)H
dehydrogenases is distinct from complex I
(Moller and Rasmusson 1998).
These additional features make plant mitochondria a suitable model to better
identified which complex could be specifically affected by DMDS using one of
the two pathways available in plant and not in animal mitochondria.
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The function of isolated mitochondria was assessed by measurement of oxygen consumption using different energy substrates in control and in the presence of DMDS (see Fig. 6). Isolated mitochondria will exhibit an initial slow rate of oxygen consumption in the presence of energy substrate. The addition of ADP will stimulate electron transport chain activity and will initiate a rapid consumption of oxygen. Consequently, using pea seed–purified mitochondria, we analyzed the effects of DMDS on the oxidation of various substrates that were monitored with the oxygen electrode. DMDS (10 µM) was diluted in the electrode buffer just before starting the experiment and placed in the reaction chamber containing mitochondria. The oxidation of succinate, measured as the state III rate (in the presence of ADP), was strongly reduced by 10 µM DMDS (44% inhibition; Fig. 6, A and B), suggesting an inhibition of electron transfer. The decreased rate was likely not due to an inhibition on the ATP synthetase since a strong uncoupler like P-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) did not reverse the DMDS inhibition (data not shown). A similar effect of DMDS was observed on the oxidation of exogenous NADH (51% of inhibition, Fig. 6, C and D) and pyruvate (40% of inhibition, Fig. 6, E and F). The oxidation of these three substrates (i.e., succinate, pyruvate, and NADH) involves different dehydrogenases that feed a common electron pathway from ubiquinones to complex III, cytochrome c, and complex IV. Therefore we thought that DMDS could either exert a general effect on all components or inhibit a specific component in the common pathway (i.e., cytochrome oxidase pathway). Careful observation of the pyruvate oxidation graph (Fig. 6, E and F) shows that the DMDS effect was less pronounced (21% of inhibition) on the respiration rate after cyanide addition. In fact, in the presence of pyruvate and when complex IV was blocked by cyanide, the electrons from reduced ubiquinones were directly transferred to oxygen through the cyanide-insensitive AOX pathway (Vanlerberghe and McIntosh 1998; Fig. 8A). This cyanide-insensitive respiration shown in Fig. 6, E and F, was inhibited by propylgallate, which is an inhibitor of the AOX pathway. The weaker inhibitory effect of DMDS on this cyanide-resistant pathway suggested that its site of action was more likely localized in the complexes III-IV segment of the electron transfer chain than in the set of initial dehydrogenases.
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To evaluate a possible effect at the level of complex III, we analyzed the effect of DMDS on the electron transfer from exogenous NADH to cytochrome c, thus bypassing complex IV. This was made by following spectrophotometrically the NADH-dependent reduction of exogenous cytochrome c after blocking complex IV with cyanide and rupturing the outer membrane with a calibrated amount of Triton X 100. Even at 40 µM, DMDS was not able to inhibit the electron transfer from NADH to cytochrome c since the initial rate of reduction was almost identical to the control (Fig. 7A). The highest value obtained for the plateau is due to small variations in the cytochrome c concentration in the experiment. Since all these results focused on complex IV as the site of action of DMDS, we analyzed its impact on cytochrome oxidase activity. Mitochondrial membranes were solubilized by Triton X 100, and oxidation of reduced cytochrome c by cytochrome oxidase was followed spectrophotometrically. The spectra shows that 40 µM DMDS totally blocked the cytochrome oxidase, yielding the same effect with 0.8 mM cyanide (Fig. 7B). Lower concentrations of 4 and 0.4 µM were still effective in inhibiting the electron transfer through complex IV.
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Taken together, these results obtained on plant mitochondria that display two respiratory electron-transport pathways including the cyanide-sensitive cytochrome pathway (common to animal) and the cyanide-insensitive alternative oxidase pathway allowed us to precisely identified within the electron transport chain the target site of DMDS. This sulfur compound, in the micromolar range, is a powerful inhibitor of complex IV (cytochrome oxidase).
EFFECTS OF DMDS ON DUM NEURON RESPIRATION. Because it was necessary to determine if DMDS neurotoxicity observed in DUM neurons occurred through mitochondrial respiratory chain complex IV dysfunction, we tested on isolated cell body KCN, which is commonly used in vitro as a rapid specific inhibitor cytochrome oxidase, the terminal enzyme of the electron-transport chain that catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. As illustrated in Fig. 7C, bath application of 10 µM KCN produced an important hyperpolarization of the membrane potential [from –55.1 ± 0.6 mV (n = 4) to –82.1 ± 1.3 mV, n = 4]. It is interesting to note that KCN did not produce any significant effect when DUM neurons were pretreated with 100 µM DMDS (Fig. 7C). These results strongly suggest that DMDS might affect the complex IV of the DUM neuron electron-transport chain.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sinakevitch et
al. 1996), DMDS, undoubtedly will produce crucial downstream
consequences in insect. Moreover, because insect neuronal KATP
channels have never been characterized before this study, our results suggest
that these channels might correspond to a novel target site for such
insecticides acting through mitochondrial dysfunction. This is reinforced by
the fact that DMDS effects occur similarly following an external as well as an
internal application mode. This might suggest that DMDS is highly membrane
permeable, easy to diffuse over the cells and to penetrate into mitochondria.
Such properties give to DMDS advantage for proposal as a good candidate as
insecticidal compound. Mitochondria enzymes as insecticide target
Many insecticidal activities occur due to the opening and closing of
specific ion channel proteins embedded within the nerve membrane. The insect
voltage-gated sodium channel is the well-established target of a variety of
insecticides including DDT, pyrethroids, N-alkylamides, and the recently
introduced oxadiazine Indoxacarb (Lapied
et al. 2001; Zlotkin
1999). A large subtype diversity of cholinergic or GABA cell
membrane receptors are also altered by other classes of insecticidally active
molecules such as neonicotinoids and phenylpyrazoles
(Bloomquist 2001;
Nauen et al. 2001). Beside
these most extensively known insecticidal targets, the mitochondria, which is
responsible for most ATP production, is also targeted by pesticides
(Schuler and Casida 2001). The
disruption of energy metabolism usually results of either an inhibition of the
electron transport system or an uncoupling of the transport system from ATP
production. Some compounds block the production of ATP through an inhibition
of the electron transport system and causes a decrease in oxygen consumption
by the mitochondria. The common binding site in the electron transport chain
is the complex I (NADH/ubiquinone oxidoreductase), which catalyzes the
transfer of electrons from NADH to coenzyme Q through flavin mononucleotide.
For instance, a number of insecticide or miticide synthetic compounds such as
rotenone, pyridaben, fenazzaquin, and fenpyroximate act as complex I
inhibitors. Furthermore, a small group of molecules called uncoupling agents
(e.g., the original uncoupler dinitrophenol) are able to dissociate the
oxidation of substrates from the manufacture of ATP. In this case, the
transport system is not affected, the oxygen consumption increases but a
dissipation of the proton gradient across the inner mitochondrial membrane
does not allow a normal production of ATP. The newly developed compound
chlofenapyr acts similarly, offering promise for further development of
uncoupler for pest control. Finally, except for methyl bromide
(CH3Br) for which an effect on cellular respiration is only
suspected, a number of fumigants including hydrogen phosphide (PH3
or phosphine), are chemicals also known to induce mitochondrial dysfunction
(Price 1985). However, as
indicated below, such compounds display strong secondary effects and/or
numerous disadvantages.
Can DMDS be used as fumigant?
Although fumigation has become an endangered technology due to pressures
regarding environmental contamination and health concerns, it still remains
one of the most effective methods for the protection of stored food against
insect infestation. At present only two fumigants are still in use:
CH3Br and phosphine. However, as indicated in the Introduction,
CH3Br presents many disadvantages. As a consequence, the use of
another fumigant such as phosphine was proposed. However, although phosphine
does not interfere with germination
(Sittisuang and Nakakita
1985), many investigations pointed out major disadvantages
including the time required to eliminate the target pest (ranging from 3 to 7
days), the development of resistance in several species of stored product
insects (Price 1991;
Zettler 1991;
Zettler et al. 1989) and the
important oxidative damage observed in mammals. In this context, volatil
Allium sulfurs such as DMDS is expected to increase in use due to the
limited duration of legal use of both CH3Br and phosphine. DMDS is
highly volatile, mixes readily with air, and acts in gaseous state. Because of
its small molecule, DMDS diffuses quickly and penetrates commodities more
quickly than other fumigants. Furthermore, this study shows, for the first
time, that DMDS, decreases ATP production via an inhibition of the
mitochondrial respiratory chain complex IV (cytochrome oxidase). The fact that
DMDS alters the electron transfer chain in a manner distinct from that of most
of the compounds used (see Schuler and
Casida 2001), it might be anticipated that resistant insects would
show low cross-resistance to DMDS.
However, although one of the most attractive aspects of using DMDS as crop
protectant is its high toxicity against a range of insect pests including the
eggs, larvae, and adults in comparison with the well-known fumigants
CH3Br or thiosulfinates (Auger
et al. 1994; Dugravot et al.
2002), it should be pointed out that DMDS also showed a mammalian
toxicity. Previous findings established that DMDS was the causative agent in
kale poisoning of cattle. Typical signs of kale poisoning were circulating of
DMDS in the blood, Heinz body formation in the erythrocytes, and loss of body
weight (Steven et al. 1981).
Our study also reveals that the CL50 for the most susceptible
insect (D. basalis) is only five times lower than that measured for
mice. Consequently, for the strategy of insecticide research, all together
these results may implicate to look for DMDS derivatives with higher
insecticidal activity. Finally, although the specific effect of DMDS as
complex IV inhibitor make it ideal probe in the dissection of the function of
the mitochondrial electron transport chain, another interesting facet of our
study is that this toxic effect observed in pacemaker DUM neurons is
associated with the KATP channel activation inducing
hyperpolarization. It is known that chronic KATP channel activity
could have fatal consequences particularly at neuronal level. Several
compounds, which inhibit the mitochondrial electron transport chain, were
considered probable suspect of nigral pathology in humans such as Parkinson's
disease. For example, chronic brain infusion of low doses of the complex I
inhibitor rotenone gives rats a Parkinsonian syndrome
(Jenner 2001;
Liss and Roeper 2001). Among
the pathophysiological response to mitochondrial dysfunction previously
observed, it seems now evident that the complex I inhibition is not the only
reason for the vulnerability of neuronal cells to neurodegenerative process.
Complex IV inhibition and its potential downstream consequences like
KATP activation could also represent an epidemiological linkage
between insecticide and the incidence of Parkinson's disease. Because previous
studies indicated that aminergic pacemaker DUM neurons
(Grolleau and Lapied 2000)
were considered as interesting neuronal biomedical model for investigating the
neurotoxic effect of anticancer agent
(Grolleau et al. 2001), they
could represent an exciting alternative way for studying, in our context, the
place of complex IV deficiency in the neurodegenerative mechanism.
In conclusion, our results show that DMDS is a potent specific complex IV inhibitor in DUM neurons. Since alteration of the mitochondrial electron transport is a very generalized action, it seems conceivable to assume that other cells and/or physiological mechanisms are also affected (i.e., cockroach synaptic transmission between sensory fibers and GIs). It is also probable that DMDS has only little phytotoxicity since we showed that plant possess a DMDS-insensitive oxidative pathway. However, the mammal toxicity observed requires serious attention regarding a possible use of DMDS for pest control. More generally, research on new natural molecules for crops protection or pest management is becoming of growing interest in view of health hazard to humans, environmental insecurity as well as prevalence of insect resistance accounting for existing insecticides. Based on its great potential as fumigant, pest control using DMDS might be feasible and future work is highly recommended in this respect.
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FOOTNOTES |
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Address for reprint requests: B. Lapied, Laboratoire de Neurophysiologie UPRES EA 2647—RCIM, Université d'Angers, UFR Sciences, 2 boulevard Lavoisier, F-49045 Angers Cedex 01, France (E-mail: bruno.lapied{at}univ-angers.fr).
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REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ashcroft FM and Gribble F. Correlating structure and function in ATP-sensitive K+ channels. Trends Neurosci 21: 288–294, 1998.[ISI][Medline]
Ashcroft FM and Gribble F. New windows on the mechanism of action of KATP channel openers. TIPS 21: 439–445, 2000[Medline]
Auger J, Ferary S, and Huignard J. A possible new class of natural sulfur pesticide for fumigation. Ecology 25: 27–35, 1994.
Auger J, Lecomte C, and Thibout E. Leek odour analysis by gaz chromatography and identification of the most active substance for the leek moth Acrolepiopsis assectella. J Chem Ecol 15: 1847–1854, 1989.
Bell CH. Fumigation in the 21st century. Crop Protect 19: 563–569, 2000.
Bloomquist JR. GABA and glutamate receptors as biochemical sites for insecticide action. In: Biochemical Sites of Insecticide Action and Resistance, edited by Ishaaya I. Berlin: Springer-Verlag, 2001, p. 17–41.
Brattsen LB. Metabolic defenses against plant allelochemicals. In: Herbivores: Their Interaction with Secondary Plant Metabolites, edited by Rosenthal GA and Janzen DH. New York: Academic, 1992, p. 175–241.
Buckingham SD, Lapied B, LeCorronc H, Grolleau F, and Sattelle DB. Imidacloprid actions on insect neuronal acetylcholine receptors. J Exp Biol 200: 2685–2692, 1997.[Abstract]
Carlini CR and Grossi-de-sa MF. Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. Toxicon 40: 1515–1539, 2002.[Medline]
Douce R, Bourguignon J, Brouquisse R, and Neuburger M. Isolation of plant mitochondria: general principles and criteria of integrity. Methods Enzymol 148: 403–415, 1987.[ISI]
Dugravot S, Sanon A, Thibout E, and Huignard J. Susceptibility of Callosobruchus maculatus (Coleoptera: Bruchidae) and its parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) to sulphur-containing compound: consequences on biological control. Environ Entomol 31: 550–557, 2002.
Feyereisen R. Insect P450 enzymes. Annu Rev Entomol 44: 507–33, 1999.[ISI][Medline]
Finney DL. Probit Analysis, 3rd ed. Cambridge, UK, 1971.
Grolleau F,
Gamelin L, Boisdron-Celle M, Lapied B, Pelhate M, and Gamelin E. A
possible explanation for a neurotoxic effect of the anticancer agent
oxaliplatin on neuronal voltage-gated sodium channels. J
Neurophysiol 85:
2293–2297, 2001.
Grolleau F and Lapied B. Dorsal unpaired median neurones in the insect central nervous system: towards a better understanding of the ionic mechanisms underlying spontaneous electrical activity. J Exp Biol 203: 1633–1648, 2000.[Abstract]
Hue B and Callec JJ. Electrophysiology and pharmacology of synaptic transmission in the central nervous system of the cockroach. In: Cockroaches as Models for Neurobiology: Application in Biomedical Research, edited by Huber I, Masler EP, and Rao BR. Boca Raton, FL: CRC Press, 1990, p. 149–168.
Hue B and Piek T. Irreversible presynaptic activation-induced block of transmission in the insect CNS by hemicholinium-3 and threonine-6-bradykinin. Comp Biochem Physiol 93C: 87–89, 1989.
Inagaki N, Gonoi T, Clement JP, Wang CZ, Aguilar-Bryan L, Bryan J, and Seino S. A family of sulfoxylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16: 1011–1017, 1996.[ISI][Medline]
Jenner P. Parkinson's disease, pesticides and mitochondrial dysfunction. Trends Neurosci 24: 245–246, 2001.[ISI][Medline]
Lapied B, Grolleau F, and Sattelle DB. Indoxacarb, an oxadiazine insecticide, blocks insect neuronal sodium channels. Br J Pharmacol 132: 587–595, 2001.[ISI][Medline]
Lapied B,
Malécot CO, and Pelhate M. Ionic species involved in the electrical
activity of single aminergic neurones isolated from the sixth abdominal
ganglion of the cockroach Periplaneta americana. J Exp
Biol 144:
535–549, 1989.
0
Lapied B,
Malécot CO, and Pelhate M. Patch clamp study of the properties of
the sodium current in cockroach single isolated adult aminergic neurones.
J Exp Biol 151:
387–403, 1990.
Lindroth RL. Hydrolysis of phenolic glucosides by midgut
-glucosidases in Papilio glaucus. Insect Biochem
18: 789–792,
1988.
Liss B and
Roeper J. ATP-sensitive potassium channels in dopaminergic neurons:
transducers of mitochondrial dysfunction. News Physiol
Sci 16:
214–217, 2001.
Moller IM and Rasmusson AG. The role of NADP in the mitochondrial matrix. Trends Plant Sci 3: 21–27, 1998.
Nauen R, Ebbinghaus-Kintscher U, Elbert A, Jeschke P, and Tietjen K. Acetylcholine receptors as site for developing neonicotinoid insecticides. In: Biochemical Sites of Insecticide Action and Resistance, edited by Ishaaya I. Berlin: Springer-Verlag, 2001, p. 77–105.
Ouedraogo AP, Sou S, Sanon A, Monge JP, Huignard J, Tran MD, and Credland PF. Influence of temperature and humidity on population of C. Maculatus (Coleoptera:Bruchidae) and its parasitoid D. Basalis (Pteromalidae) in two zones of Burkina Faso. Bull Entomol Res 86: 695–702, 1996.
Pelhate M, Pichon Y, and Beadle DJ. Cockroach axons and cell bodies: electrical activity. In: Cockroaches as Models for Neurobiology: Application in Biomedical Research, edited by Huber I, Masler EP, and Rao BR. Boca Raton, FL: CRC Press, 1990, p. 131–148.
Price NR. The mode of action of fumigants. J Stored Prod Res 21: 157–164, 1985.
Price NR. Enzymatic factors in the resistance of stored-product pests to insecticides. Biochem Soc Trans 19: 759–762, 1991.[ISI][Medline]
Rauscher MD. Natural selection and the evolution of plant-insect interactions. In: Insect Chemical Ecology, edited by Roitberg BD and Isman MB. New York: Chapman and Hall, 1992, p. 20–88.
Schuler F and Casida JE. The insecticide target in the PSST subunit of complex I. Pest Manag Sci 57: 932–940, 2001.[ISI][Medline]
Sinakevitch IG, Geffard M, Pelhate M, and Lapied B. Anatomy and targets of dorsal unpaired median neurones in the terminal abdominal ganglion of the male cockroach Periplaneta americana L. J Comp Neurol 367: 147–163, 1996.[ISI][Medline]
Sittisuang P and Nakakita H. The effect of phosphine and methyl bromide on germination of rice and corn. J Pestic Sci 10: 461–468, 1985.
Sogorb MA and Vilanova E. Enzymes involved in the detoxification of organophosphorus, carbamate and pyrethroid insecticides through hydrolysis. Toxicol Lett 128: 215–228, 2002.[ISI][Medline]
Steven FS, Griffin MM, and Smith RH. Disulphide exchange reactions in the control of enzymic activity. Evidence for the participation of dimethyl disulphide in exchanges. Eur J Biochem 119: 75–78, 1981.[ISI][Medline]
Taylor RW. Commodity fumigation beyond the year 2000. Pestic Outlook 7: 31–34, 1996.
Towers PR and Sattelle DB. A Drosophila melanogaster cell line (S2) facilitates post-genome functional analysis of receptors and ion channels. BioEssays 24: 1066–1073, 2002.[ISI][Medline]
Trounce A, Kim YL, Jun AS, and Wallace DC. Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol 264: 484–509, 1996.[Medline]
Vanlerberghe GC and McIntosh L. Alternative oxidase: from gene to function. Annu Rev Plant Physiol Plant Mol Biol 48: 703–734, 1997.[ISI][Medline]
Watson RT. Methyl bromide. Science and technology and economic synthesis report. Fumigant Pheromone 29: 11–24, 1992.
Wicher D, Walther CH, and Wicher C. Non-synaptic ion channels in insects— basic properties of currents and their modulation in neurons and skeletal muscles. Prog Neurobiol 64: 431–525, 2001.[ISI][Medline]
Zettler JL. Pesticide resistance in Tribolium castaneum and T. confusum (Coleoptera: Tenebrionidae) from flour mills in the United States. J Econ Entomol 83: 1677–1681, 1991.
Zettler JL, Halliday WR, and Arthur FH. Phosphine resistance in insects infecting stored peanuts in the southeastern United States. J Econ Entomol 82: 1508–1511, 1989.
Zlotkin E. The insect voltage-gated sodium channel as target of insecticides. Annu Rev Entomol 44: 429–455, 1999.[ISI][Medline]
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