Behavioral and biochemical effects of neonicotinoid thiamethoxam on the cholinergic system in rats

Article (PDF Available)inEcotoxicology and Environmental Safety 73(1):101-7 · June 2009with105 Reads
DOI: 10.1016/j.ecoenv.2009.04.021 · Source: PubMed
Abstract
Thiamethoxam is a neonicotinoid insecticide, a group of pesticides that acts selectively on insect nicotinic acetylcholine receptors (nAChRs), with only a little action on mammalian nAChRs. Nevertheless, the selectivity of neonicotinoids for the insect nAChRs may change when these substances are metabolized. Therefore, we aimed to determine the potential effects of thiamethoxam on mammalian brain, testing the performance in the open field and elevated plus-maze of rats exposed to this insecticide and, in order to establish the neurochemical endpoints, we measured the acetylcholinesterase activity in different brain regions (hippocampus, striatum and cortex) and the high-affinity choline uptake (HACU) in synaptosomes from rat hippocampus. Treated animals received thiamethoxam (25, 50 or 100mg/kg) for 7 consecutive days. The results showed that treatment with thiamethoxam induced an increase in the anxiety behavior at two doses (50 or 100mg/kg). Moreover, there was a significant decrease in both HACU and acetylcholinesterase activity. Our hypothesis is that thiamethoxam (or its metabolites) could be acting on the central rats nAChRs. This would produce an alteration on the cholinergic transmission, modulating the anxiety behavior, acetylcholinesterase levels and HACU.
Behavioral and biochemical effects of neonicotinoid thiamethoxam on the
cholinergic system in rats
Rodrigues K.J.A.
a
, Santana M.B.
a
, Do Nascimento J.L.M.
a
, Picanc- o-Diniz D.L.W.
a
, Maue
´
s L.A.L.
a
,
Santos S.N.
a,b
, Ferreira V.M.M.
b
, Alfonso M.
c
, Dura
´
nR.
c
, Faro L.R.F.
c,
a
Departamento de Fisiologia, Centro de Cie
ˆ
ncias Biolo
´
gicas, Universidade Federal do Para
´
, Bele
´
m-PA, Brazil
b
Departamento de Farma
´
cia, Faculdade de Cie
ˆ
ncias da Sau
´
de, Universidade de Brası
´
lia, Brası
´
lia-DF, Brazil
c
Department of Functional Biology and Health Sciences, Faculty of Biology, University of Vigo, Campus As Lagoas-Marcosende, 36310 Vigo, Spain
article info
Article history:
Received 19 June 2008
Received in revised form
20 April 2009
Accepted 25 April 2009
Available online 29 May 2009
Keywords:
Thiamethoxam
Acetylcholinesterase
Anxiety
Rats
Sodium-dependent choline transporter
abstract
Thiamethoxam is a neonicotinoid insecticide, a group of pesticides that acts selectively on insect
nicotinic acetylcholine receptors (nAChRs), with only a little action on mammalian nAChRs.
Nevertheless, the selectivity of neonicotinoids for the insect nAChRs may change when these
substances are metabolized. Therefore, we aimed to determine the potential effects of thiamethoxam
on mammalian brain, testing the performance in the open field and elevated plus-maze of rats exposed
to this insecticide and, in order to establish the neurochemical endpoints, we measured the
acetylcholinesterase activity in different brain regions (hippocampus, striatum and cortex) and the
high-affinity choline uptake (HACU) in synaptosomes from rat hippocampus. Treated animals received
thiamethoxam (25, 50 or 100 mg/kg) for 7 consecutive days. The results showed th at treatment with
thiamethoxam induced an increase in the anxiety behavior at two doses (50 or 100 mg/kg). Moreover,
there was a significant decrease in both HACU and acetylcholinesterase activity. Our hypothesis is that
thiamethoxam (or its metabolites) could be acting on the central rats nAChRs. This would produce an
alteration on the cholinergic transmission, modulating the anxiety behavior, acetylcholinesterase levels
and HACU.
& 2009 Elsevier Inc. All rights reserved.
1. Introduction
Thiamethoxam is a synthetic organic insecticide included in
the class of neonicotinoids, the most important new class of
insecticides developed in the past three decades. Since their
introduction onto the market in 1991, neonicotinoids have been
the fastest growing class of insecticides, due to their putative
moderate toxicity to mammals and their advantage in combating
insects that are resistant to other pesticide classes (Bingham et al.,
2008). The annual sales of this class of chemicals reach nearly a
billion dollars, corresponding to 11–15% of the total insecticide
market (Tomizawa and Casida, 2003), sharing preference with the
pyrethroid insecticides. Thiamethoxam is included in the thiani-
cotinil class, being commercialized by the name Actara
s
for foliar
application, and Cruiser
s
for seed treatment.
The insecticidal activity of neonicotinoids is due to their action
on nicotinic acetylcholine receptors (nAChRs), which are members
of a superfamily of ligand-gated ion channels responsible for rapid
excitatory cholinergic neurotransmission (Karlin, 2002; Tomizawa
and Casida, 2005). Neonicotinoids act as agonists at the
postsynaptic insect nAChRs with much higher affinity (Tomizawa
and Casida, 2003), and the toxicity induced by these substances is
considered to be centrally mediated because the symptoms of
poisoning are similar to those of nicotine (Tomizawa and Casida,
2005).
Mammalian nAChRs exist as heteromeric complexes, compris-
ing
a
(
a
2–
a
6) and
b
(
b
2–
b
4) subunits, or homomeric complexes,
comprising
a
(
a
7–
a
9) subunits (Romanelli and Gualtieri, 2003).
However, a few subtypes of nAChRs predominate, notably
a
4–
b
2
and
a
7, which are widespread in the vertebrate central nervous
system (CNS) (Seguela et al., 1993; Zoli et al., 2002). The toxicity
induced by neonicotinoids in mammals correlates with agonist
action and binding affinity at the
a
4
b
2 nAChRs, the primary target
for neonicotinoids in the brain (Tomizawa and Casida, 2005).
Although it has been shown that neonicotinoids act as agonists
on insect and mammal nAChRs (particularly the
a
4
b
2 subtype)
(Tomizawa and Casida, 2003), several pieces of evidence in the
literature show that neonicotinoid insecticides have a higher
selectivity for the insect nAChRs than the mammalian ones. This
selectivity is due to the electronegative nature of their nitrogua-
nidine, nitromethylene, and cyanamine moiety, which is a key
determinant in the selective recognition by a specific subsite
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journal hom epage: www.elsevier.com/locate/ecoenv
Ecotoxicology and Environmental Safety
0147-6513/$ - see front matter & 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2009.04.021
Corresponding author. Fax: +34 986812556.
E-mail address: lilianfaro@uvigo.es (L.R.F. Faro).
Ecotoxicology and Environmental Safety 73 (2010) 101–107
unique in insect nAChRs (Tomizawa et al., 2003; Tomizawa and
Casida, 2005). Thus, the presence of a nitroguanidine moiety in
the thiamethoxam structure gives this substance a negative
characteristic that confers a higher selectivity for insect nAChRs
than those of vertebrates (Tomizawa and Casida, 2005; Tomizawa
et al., 2003; Kanne et al., 2005). However, the selectivity of
neonicotinoids for the insect nAChRs may undergo profound
alterations when these substances are metabolized. Today, the
participation of the hepatic microsomal enzyme CYP3A4 and
cytosolic aldehyde oxidase (AOX) in the metabolism of these
pesticides is clear (Honda et al., 2006).
Consequently, systemic administration of neonicotinoids to
mammalians generates a very large number of metabolites, with
differential effects, affinity, or potency for the nAChRs ( Roberts
and Hutson, 1999; Schulz-Jander et al., 2002). Systemic adminis-
tration of thiamethoxam (20 mg/kg) in mice has shown that at
least 44% of this substance is metabolized and that its metabolites
are found in the brain (Ford and Casida, 2006). In this
metabolization process, the nitroguanidine moiety of thiamethox-
am is enzymatically broken down and then reduced to nitroso-
guanidine and aminoguanidine (Yokota et al., 2003). The
metabolites produced are now cationic in nature and conse-
quently selective for the mammalian nAChRs, making their
potential effects in mammalian CNS of considerable interest.
Therefore, the neonicotinoid insecticides are currently used in
great amounts, but this can be a problem when the possible risks
of occupational and environmental contamination are taken into
account. Although the data about the action of neonicotinoid
pesticides on insect nAChRs are clear, there are a few works that
demonstrate possible in vivo effects of this group of pesticides on
the mammalian biological system.
The purpose of this study was to determine the potential
effects of neonicotinoid thiamethoxam on the mammalian brain,
testing the performance (in the open field and elevated plus-
maze) of rats exposed to this insecticide, and with regard to the
neurochemical endpoints of the action of thiamethoxam, we
measured the acetylcholinesterase (AChE) activity in different
brain regions and high-affinity choline uptake (HACU) in synapto-
somes from rat hippocampus.
2. Materials and methods
2.1. Chemicals
Thiamethoxam [3-2-Cloro-1,3-tiazol-5-ilmetil-1,3,5-oxadiazinan-4-ilideno
(nitro) anima] (99.7%) was purchased from Fluka-Sigma-Aldrich (USA), [
3
H]Cho-
line chloride (specific activity, 83.0 Ci/mmol) was purchased from Amersham
International plc (Buckinghamshire, England). Choline chloride (499%), propionyl
thiocholine iodide (499%), POP-POPOP were acquired from Sigma (St Louis, USA).
All other chemicals and reagents were of analytical grade.
2.2. Thiamethoxam treatment
Male Wistar rats, weighing 230–280 g, 2.5 months old, were housed in groups
of four to five animals in each polycarbonate cage, under controlled conditions of
temperature (2371 1C) and photoperiod (light:dark 12:12 h), with free access to
food and water. Treated animals received, by subcutaneous (s.c.) route, different
doses (25, 50 or 100 mg/kg) of thiamethoxam, dissolved in saline 0.9%, for 7 days.
Control animals received vehicle (saline) in the same volume as treated animals.
The oral LD
50
value of thiamethoxam was 1563 mg/kg (Maienfisch et al., 1999). All
experiments were conducted between 08:00 and 13:00 h in order to reduce
circadian influences. All procedures were carried out in accordance with the
Brazilian Society for Neuroscience and Behavior animal care guidelines and
European Community animal care guidelines, and were approved by our
institutional ethics committee.
2.3. Behavioral tests
Open field: The open field was utilized to measure exploratory activity. The
apparatus consists of a black wooden arena (60 60 35 cm
3
) enclosed by
wooden walls and divided into nine equal squares by white lines. The locomotion
was considered when each animal placed all four paws into a single square. The
open field was placed inside a light- and sound-attenuated room. Each animal was
placed in the center of the arena, and the number of squares crossed with all four
paws was counted for a period of 5 min.
Elevated plus-maze: This is a simple and well-validated model for studying
anxiety, and it is useful for assessing anxiogenic and anxiolytic states in rodents.
This test was utilized to evaluate anxiety-related behaviors induced by
thiamethoxam. The apparatus was made of wood painted in black and consisted
of two opposite open arms, 50 10 cm, and two opposite enclosed arms,
50 10 40 cm
3
, elevated to a height of 50 cm above the floor. The junction area
of the four arms (central platform) measured 10 10 cm
2
. The floor of the maze
was painted with impermeable dark epoxy resin, to avoid urine impregnation.
Each animal was placed on the center platform of the maze facing the enclosed
arms, and for 5 min the number of entries and the time spent in the open and
enclosed arms were recorded. Anxiogenic effect was defined as a decrease in the
proportion of open-arm entries and/or the time spent in open arms relative to the
total time spent in both arms. After each trial, the maze was cleaned with ethanol
solution (10% v/v).
2.4. Biochemical analysis
Sample preparation: Hippocampus, striatum and cortex were homogenized
separately in 20 vol (w/v) of ice-cold 0.32 M sucrose solution with a potter-type
glass-Teflon homogenizer. These homogenates were first centrifuged at 1000g for
10 min at 4 1C and then the supernatants were recentrifuged at 17,800g for 15 min
at 4 1C to obtain the synaptosomal pellets (Simon et al., 1976). The synaptosomal
pellets were gently suspended in 30 vol of ice-cold 0.32 M sucrose and used
immediately in the experiment of choline uptake (hippocampal samples only) or
stored at 80 1C until assay of AChE activity.
Acetylcholinesterase activity: AChE activity in the synaptosomal pellets was
measured using the Ellman spectrophotometric assay (Ellman et al., 1961). Briefly,
at 2 h or 7 days after the last thiamethoxam injection, animals were euthanized by
cervical dislocation and the hippocampus, striatum and cortex were homogenized
to obtain the synaptosomes, as described above. The activity of AChE in the
homogenate was assayed with 1.5 mM propionyl thiocholine iodide (PthCh) as
substrate in an incubation time of 4 min at 25 1C. The solution was read by a
Microplate Reader (Model 450 Bio-Rad) at a wavelength of 412 nm. The AChE
activity was expressed as nmol thiocholine (ThCh) formed/mg tissue/min.
Assays of HACU: Synaptosomes (50
m
L) were preincubated for 2 min at 37 1Cin
Krebs Ringer solution (pH 7.3; 126 mM NaCl, 4.75 mM KCl, 1.27 mM CaCl
2
, 15.8 mM
Na
2
HPO
4
, 1.42 mM MgCl
2
in final concentrations) plus 2 mg/mL of D(+) glucose.
Next, choline (0.04–2
m
M) and [
3
H]Choline (0.4
m
Ci) were added to obtain a 500
m
L
final reaction volume and consequently the kinetic of the choline uptake was
allowed to run for 4 min. For the low-affinity choline uptake (sodium indepen-
dent), a parallel tube where the sodium-containing reagents were replaced by
252 mM sucrose and 15.8 mM Tris (hidroximetil) aminometano was assayed. Upon
completion of the incubation time, the reaction was stopped by adding ice-cold
sodium-free Ringer solution and then the synaptosomes were collected in fiber
glass filters (0.45
m
m in diameterSigma) through a vacuum filter. Each filter was
washed three times with 1 ml of ice-cold sodium-free Ringer and placed in a vial.
The filters were then left to dry for 30 min, at a temperature of 80 1C. Finally, 10mL
of scintillation cocktail (POP-POPOP diluted in toluene) was added into each vial
and the sample radiation was counted by a scintillation spectrometer (TRI-CARB
2100 TR, HP). The total radioactivity, measured in counting per minute (c.p.m.) was
converted to pmols of [
3
H]Choline. HACU was obtained by subtracting the values
of total choline uptake (using Ringer media with sodium) from the values of
sodium-independent choline uptake (in the sodium-free Ringer media). For the
measurement of HACU in control and thiamethoxam-treated animals, we chose a
fixed point of final choline concentration (0.4
m
M) that was used in the
experiments. The amount of protein in the samples was measured by the method
of Lowry et al. (1951), which uses bovine serum albumin as a standard.
2.5. Statistical analysis
Behavioral data were analyzed using one-way analysis of variance (ANOVA).
Post hoc comparisons were performed using the Dunnett test, which is specifically
designed for situations where all groups are compared against one Reference
group, and also show a result that is much more homogenous. Values of po0.05
were considered significant. The data from AChE activity and HACU, which are
presented as mean7SEM values, were analyzed using analysis of variance
followed by Fisher’s post hoc test, with the level of significance set at po0.05.
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Rodrigues et al. / Ecotoxicology and Environmental Safety 73 (2010) 101–107102
3. Results
3.1. Signs of toxicity
Thiamethoxam was administered in 25, 50 or 100 mg/kg s.c.
doses, which were below the lethal value. Repeated exposure to
thiamethoxam at the doses tested did not produce seizures,
tremors, weight loss, or other types of apparent behavioral or
physiological dysfunctions.
3.2. Spontaneous motor activity
Thiamethoxam treatment produced significant effects on the
spontaneous motor activity at a dose of 100 mg/kg. Fig. 1 shows
that the thiamethoxam treatment reduced, in a statistically
significant way, the motor activity (control: 53.3875.06;
100 mg/kg: 33.2576.79; F ¼ 5.6457; po0.05), while 25 and
50 mg/kg did not produce any significant changes when
compared to the control animals.
3.3. Elevated plus-maze
Fig. 2 shows the data obtained in the elevated plus-maze.
Thiamethoxam treatment produced a significant anxiogenic effect
that was dose-dependent. Thus, animals treated with 50 or
100 mg/kg of thiamethoxam for 7 days spent significantly less
time in the open arms (7.8872.93, F ¼ 4.64; po0.05, and
6.4072.12, F ¼ 6.42, po0.05, respectively) when compared to
the control group (19.0775.92). However, we did not find any
differences in relation to the frequency of entries in open arms of
the maze compared to the control group. Regarding all arms of the
maze (open and enclosed), administration of 25 mg/kg did not
produce any change in total entries.
3.4. Acetylcholinesterase activity
For this set of experiments, animals were treated with 25, 50 or
100 mg/kg of thiamethoxam for 7 days and the effects of this
insecticide on AChE activity and HACU were measured. The effects
of thiamethoxam on AChE activity (nmol ThCh/mg protein min) in
the hippocampus, striatum and cortex in animals euthanized 2 h
and 7 days after the last pesticide administration are shown in Fig.
3. As illustrated in Fig. 3A, administration of 50 or 100 mg/kg of
thiametoxam for 7 days decreased AChE activity in the
hippocampus when it was measured 2 h after the last injection
of insecticide (control: 6.7970.08; 50 mg/kg: 4.8970.44,
F ¼ 21.00; po0.05; 100 mg/kg: 5.4170.25, F ¼ 21 . 1 7; po0.05).
When this parameter was measured 7 days after the last injection,
only the higher concentration (100 mg/kg) of thiamethoxam was
able to maintain the low level of AChE activity (5.5770.25,
F ¼ 18.03, po0.05).
Fig. 3B
shows that administration of 50 or
100 mg/kg of thiametoxam for 7 days decreased AChE activity in
the cortex when measured at 2 h (control: 5.1370.31; 50 mg/kg:
3.7970.28, F ¼ 7.08; po0.05; 100 mg/kg: 4.0770.5, F ¼ 4.11;
po0.05) or 7 days after the last injection of insecticide (control:
5.0470.3; 50 mg/kg: 3.8170.1, F ¼ 19.27; po0.05; 100 mg/kg:
3.3270.4, F ¼ 11.08; po0.05). In the striatum we may observe the
same pattern observed with the cortex, where both treatments
were able to decrease the AChE activity at 2 h (control:
27.7271.50; 50 mg/kg: 19.8372.57, F ¼ 6.21; po0.05; 100 mg/
kg: 21.1171.77, F ¼ 8.95; po0.05) or 7 days after the end of the
thiamethoxam administration (control: 28.0771.00; 50 mg/kg:
23.8072.00, F ¼ 3.69; po0.05; 100 mg/kg: 21.1871.50, F ¼ 7.63;
po0.05) (Fig. 3C). In contrast, the dose of 25 mg/kg did not have
any effect on the AChE activity in all studied regions.
3.5. High-affinity choline uptake
HACU was obtained through the subtraction of the values of
total choline uptake (in the presence of sodium ion) from the
values of the choline uptake without the related ion in the
incubation media. Our assay of the kinetic of choline transporters
(data not shown) is in accordance with the data already provided
in the literature (Simon et al., 1976). In this kinetic, it can be
observed, as already well described, that the sodium-dependent
choline transporter has the characteristic of suffering saturation
when it is close to the concentration of 2
m
M choline, while the
low-affinity choline uptake is virtually nonsaturable (Cooper et al.,
1991). This assay also helped us in the choice of a fixed point of
the choline concentration to be used in the experiments with the
pesticide. The effects of 25, 50 or 100 mg/kg of thiamethoxam on
hippocampal HACU were determined in animals 2 h after
pesticide administration. Fig. 4 shows that treatment with
50 mg/kg thiamethoxam for 7 days was able to decrease the
HACU compared with the control group (10.9271.5 and
8.5770.7 pmol
+3
H-Choline/ mg protein/min, respectively;
F ¼ 4.57; po0.05). Thiamethoxam at a dose of 100 mg/kg also
significantly decreased hippocampal HACU compared with the
control group (10.9271.5 and 6.3870.35, respectively; F ¼ 6.83;
po0.05). The administration of 25 mg/kg was without any effect
on hippocampal HACU.
4. Discussion
Systemic administration of 50 or 100 mg/kg of thiamethoxam
resulted in an anxiogenic-like effect that was dose-dependent,
this behavioral deficit being observed in the absence of any
measures of systemic toxicity. Thus, administration of 50 or
100 mg/kg thiamethoxam produced a decreased time in the open
arms of the elevated plus-maze apparatus and it is consistent with
an often-reported anxiogenic effect (Rodgers et al., 1992; Da Silva
et al., 1996), since none of the doses tested produced a change in
the number of enclosed arm entries.
Treatment with a higher dose (100 mg/kg) of thiamethoxam
decreased the locomotor activity. There is therefore a possibility
that the reduction of the time in the open arms may be secondary
to a nonspecific decrease in general activity. However, the
behavioral specificity of the anxiogenic-like effect of thiamethox-
am was confirmed by the absence of any significant alteration in
the enclosed arm entries, the parameter considered to be a valid
ARTICLE IN PRESS
0
10
20
30
40
50
60
70
Control
Locomotion
*
100 mg/kg50 mg/kg25 mg/kg
Fig. 1. Spontaneous locomotor activity of rats in the open-field apparatus. The rat’s
behavior was evaluated in an arena divided into nine equal squares. The results are
shown as the mean7SEM of nine animals per group. *po0.05 represents a
significantly different value in relation to control animals treated with saline
(ANOVA, Dunnet’s test).
Rodrigues et al. / Ecotoxicology and Environmental Safety 73 (2010) 101–107 103
index of locomotor activity in the plus-maze (File, 1992; Rodgers
and Johnson, 1995).
The elevated plus-maze is undoubtedly one of the most widely
used animal models in contemporary preclinical research on
anxiety (Walf and Frye, 2007). This test derives from the early
observation that, in mazes consisting of open and closed alleys,
rats consistently show higher levels of exploration in enclosed
alleys and, when faced with a choice of alley type, typically avoid
those without walls. On the other hand, anxiety is an observed
sign in cases of exposure to pesticides as suggested by epidemio-
logical data in humans (Peres et al., 2001; Salvi et al., 2003;
Solomon et al., 2007) and animal models (Lo
´
pez-Crespo et al.,
2007; Cloutier et al., 2006; Righi and Palermo-Neto, 2003), signs
that may be related to the results observed here.
Stimulation of nAChRs situated presynaptically by nicotinic
agonists modulates the release of acetylcholine and other
neurotransmitters (Wonnacott, 1997; Severance and Cuevas,
2004). This enhanced release of neurotransmitters (acting at
various postsynaptic receptors) is the usual mechanism by which
nicotine and analogs modulate this behavior of anxiety (Kenny et
al., 2000). Several works point to the fact that systemic
administration of high doses of nicotine can produce an
anxiogenic effect through an increase of serotonin release in
dorsal hippocampus, which is probably mediated by nAChR
activation (Sprouse and Aghajanian, 1987; Cheeta et al., 2001;
Tomizawa and Casida, 2005). Moreover, evidence in the literature
has suggested an involvement of cholinergic systems in the
hippocampus and the modulation of anxiety-related behaviors
(File et al., 1998a, b; Engin and Treit, 2007). However, the
mechanism by which thiamethoxam produces the anxiogenic
effect is unknown.
With the results we obtained, it is reasonable to hypothesize
that thiamethoxam (or its metabolites) acts as a nicotinic agonist,
activating nAChRs and increasing serotonin release, and that this
increase is responsible for the anxiogenic effect observed in the
elevated plus-maze test. In view of this background, in this work
we also attempted to associate behavioral changes observed with
the open field and elevated plus-maze to alterations on the
cholinergic system by studying the effects of thiametoxam (50
and 100 mg/kg) on AChE activity in the hippocampus, striatum
and cortex , and HACU in the hippocampus. AChE is a specific
cholinergic marker protein for the functional state of cholinergic
neurons, which can play a key role in the maintenance of
acetylcholine levels at the cholinergic neurons (Eckenstein and
Sofroniew, 1983), being responsible for the degradation of
acethylcholine to acetate and choline in the synaptic cleft.
We showed that thiamethoxam treatment changed AChE
activity in the three analyzed regions. Thus, 50 mg/kg of
thiamethoxam administered for 7 days decreased AChE activity
in the hippocampus, cortex, and striatum when measured 2 h after
the end of insecticide treatment, and this decrease could still be
observed in the cortex 1 week after the last administration of the
drug (Fig. 3B). The higher dose of thiamethoxam (100 mg/kg) also
decreased AChE activity in the three cerebral regions when
measured 2 h after the last administration of the pesticide. The
inhibition of AChE activity induced by thiamethoxam was long-
standing, being observed 1 week after the end of treatment. These
results suggest that exposure to high doses of thiamethoxam
produces AChE inhibition that persists some days after exposure
ceases and that it is accompanied by deficits in behavioral
performance.
Despite the fact that our results demonstrate a decrease in
AChE activity induced by thiamethoxam, they do not provide any
information as to whether this is due to a direct action of this
insecticide on AChE or an indirect effect through an action on
nA
ChRs. However, it is likely that the decreased enzyme activity
observed after subchronic exposure to thiamethoxam reflects a
secondary response of the neurons to the insecticide: the
thiamethoxam or its metabolites could be acting on the rat
central nAChRs, producing an imbalance in the pattern of
cholinergic neurotransmission; in this case, the cell would seek
compensatory mechanisms, e.g. changes in AChE activity and
HACU, in order to reestablish its normal activity.
In this work, the thimethoxam was systemically administered
for 1 week. So, it is expected that a great amount of the
thiamethoxam continuously administered to the rats could be
affecting metabolization negatively and that these metabolites
could be acting on the nAChRs, altering the cholinergic transmis-
sion as shown in the results here obtained. In an attempt to
recover the normal activity pattern, some regulatory mechanisms
such as alteration of the phosphorylation state or expression of
nAChRs, AChE, and choline transporter could be brought into
action.
ARTICLE IN PRESS
0
5
10
15
20
25
30
35
40
Control
% Open arm entries
0
5
10
15
20
25
30
Control
% Open arm time
*
*
0
1
2
3
4
5
6
7
8
9
10
Enclosed arm entries
100 mg/kg50 mg/kg25 mg/kg
100 mg/kg50 mg/kg25 mg/kg
Control 100 mg/kg50 mg/kg25 mg/kg
Fig. 2. Anxiety behavior evaluated in the elevated plus-maze. Panel A represents
the percentage of entries into the open arms; panel B represents the percentage of
time the animals spent in the open arms; and panel C represents the frequency of
permanence in the enclosed arms. The results are shown as the mean7SEM of
nine animals per group. *po0.05 represents a significantly different value in
relation to control animals treated with saline (ANOVA, Dunnet’s test).
Rodrigues et al. / Ecotoxicology and Environmental Safety 73 (2010) 101–107104
Our results are compatible with that observed by Chang et al.
(1973). According to the authors, nicotine was able to produce a
long-standing action, for the assay done 12 h after the last
administration, and supposedly little or even nothing of nicotine
would be present in the brain. In our study, the subcutaneous
administration of 1 mg/kg of nicotine, five times a day during
8–16 weeks, resulted in a significant decrease of AChE activity in
the rat brain. Another possibility for these findings is that
thiamethoxam, besides acting on nAChRs, also acts as a direct
inhibitor of AChE in a similar manner to that of carbamate
insecticides (Volpe et al., 1985; Lockhart et al., 2001; Tomizawa
and Casida, 2005) and other cholinesterase drugs that act as
nAChR and AChE inhibitors. However, we did not check this
hypothesis.
The present study also demonstrated that thiamethoxam
treatment decreased hippocampal HACU at both doses used.
Choline uptake is generally believed to be the rate-limiting step in
acetylcholine synthesis. This uptake is highly localized in
ARTICLE IN PRESS
2 h after
0
1
2
3
4
5
6
7
8
9
AChE activity
(nmol AthCh/mg protein/min)
7 d after
*
*
*
0
1
2
3
4
5
6
7
AChE activity
(nmol AthCh/mg protein/min)
2 h after
2 h after
7 d after
7 d after
*
*
*
*
0
5
10
15
20
25
30
35
AChE activity
(nmol AthCh/mg protein/min)
*
*
*
100 mg/kg50 mg/kg25 mg/kgControl100 mg/kg50 mg/kg25 mg/kgControl
100 mg/kg50 mg/kg25 mg/kgControl100 mg/kg50 mg/kg25 mg/kgControl
100 mg/kg50 mg/kg25 mg/kgControl100 mg/kg50 mg/kg25 mg/kgControl
Fig. 3. Acetylcholinesterase activity in the rat hippocampus (A), cortex (B), and striatum (C) samples. Left side: Results of experiments from the samples collected 2 h after
the end of the treatment with thiamethoxam. Right side: Results of experiments from the samples collected 7 days after the end of the treatment. The results are shown as
the mean7SEM of five animals per group. *po0.05 represents a significantly different value in relation to control animals treated with saline.
Rodrigues et al. / Ecotoxicology and Environmental Safety 73 (2010) 101–107 105
cholinergic neurons, and can be utilized as a relative measure of
the activity of cholinergic nerve terminals in vivo (Simon et al.,
1976). Although the nature of the effects of thiamethoxam on this
cholinergic process is unclear, it is known that treatments that
reduce neuronal activity also reduce HACU through changes in
impulse-flow in vivo (Atweh et al., 1975). Moreover, from the
results obtained, we can hypothesize that the change of uptake
induced by thiamethoxam is secondary to an altered intraterminal
acetylcholine content following a decreased release, for there
much evidence to suggest that intraterminal acetylcholine levels
might regulate choline uptake (Tue
`
ek, 1985; Collier, 1988).
5. Conclusion
Ours results showed that administration of the neonicotinoid
insecticide thiamethoxam may provoke alterations on the choli-
nergic system of rats, producing biochemical and behavioral
effects that can be correlated to the toxicity produced by other
kinds of pesticides that are linked to the development of
neurodegenerative diseases such as Alzheimer’s type dementia.
Several works show that Alzheimer’s disease is a consequence of
neuron loss, decrease of choline acetyltransferase (ChAT), sodium-
dependent choline transporter, and AChE activities, and a
decreased level of acetylcholine in the hippocampal and cortical
regions of the brain, all of which may be related with the results
we obtained.
Acknowledgment
Klebson Rodrigues acknowledges CAPES (Brazil) for a research
grant.
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    • "These pesticides were previously thought to be relatively harmless to mammalian species because of their low binding affinities to mammalian nAChRs ( Casida, 1999, 2005). However, recent in vivo and in vitro studies have reported that neonicotinoids possess sufficient binding affinity and agonistic potential for mammalian nAChRs to exert nicotine-like effects that are stronger than originally believed (de Oliveira et al., 2010; Rodrigues et al., 2010; Li et al., 2011; Kimura-Kuroda et al., 2012). Neonicotinoids such as acetamiprid (ACE), imidacloprid, and clothianidin can bind to the α 4 and β 2 subunits of mammalian nAChRs (Tomizawa and Casida, 1999; Li et al., 2011; Kimura-Kuroda et al., 2012). "
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    • "The insecticidal activity of neonicotinoids is caused by their modes of action on nicotinic acetylcholine receptors (nAChRs). Neonicotinoids are active as acetylcholine agonists at the postsynaptic insect nAChRs with much higher affinity, and the toxicity of these compounds plays a major role in pest control (Muccio et al., 2006; Rodrigues et al., 2010; Tomizawa & Casida, 2005; Van Scoy et al., 2010). However, the neurotoxicity of these compounds also affects useful insects such as bees (EFSA, 2010), and this non-targeted property may constitute a fatal flaw of the neonicotinoid insecticides. "
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