APPENDIX
II-AO: Mun, et al, Effects
of Cypermethrin on the Dopaminergic Neurons in the Progressive Hemiparkinsonian
Rats, Toxicology
Mechanisms and Methods, 15:6, 399 - 404
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Effects of
Cypermethrin on the Dopaminergic Neurons
in the Progressive Hemiparkinsonian Rats
Ji
Young Mun a; Won Yong Lee b; Sung Sik Han a
a Lab. of Cell Engineering
& 3-D Structure, School of Life Sciences and
Biotechnology,
Korea University. Seoul. Korea
b Department of Neurology,
Samsung Medical Center, Sungkyunkwan University
School
of Medicine. Seoul. Korea
To cite this Article:
Mun, Ji Young, Lee, Won Yong and Han, Sung Sik , 'Effects of
Cypermethrin
on the Dopaminergic Neurons in the Progressive Hemiparkinsonian
Rats', Toxicology
Mechanisms and Methods, 15:6, 399 - 404
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Toxicology Mechanisms and Methods, 15: 399–404, 2005
Copyright c _ Taylor & Francis LLC
ISSN: 1537-6524 print / 1537-6516 online
DOI: 10.1080/15376520500194742
Effects of Cypermethrin on the Dopaminergic
Neurons
in the Progressive Hemiparkinsonian Rats
Ji Young Mun
Lab. of Cell Engineering & 3-D Structure, School of Life
Sciences and Biotechnology,
Korea University, Seoul 136-701, Korea
Won Yong Lee∗
Department of Neurology, Samsung Medical Center, Sungkyunkwan
University School of Medicine,
Seoul 120-752, Korea
Sung Sik Han∗
Lab. of Cell Engineering & 3-D Structure, School of Life
Sciences and Biotechnology,
Korea University, Seoul 136-701,
Korea
Cypermethrin is a potent pesticide
derived from natural
pyrethrin of the chrysanthemum plant. Cypermethrin has been
known to modulate the blood-brain barrier and induce oxidative
stress in rats. The oxidative stresses leading to increased
reactive
oxygen species generation have been identified within the
degeneration
of the dopaminergic (DA) neuron. However, in testing cypermethrin
for its relationship to the degeneration of DA neurons, an
experimental study has not yet been done.
This study was designed to investigate
the effects of cypermethrin
on the DA neurons in the substantia nigra of normal and
progressive hemiparkinsonian rats. The degree of degeneration of
DA neurons was evaluated by
tyrosine hydroxylase (TH) immunohistochemistry
and forepaw adjusting step (FAS) test.
The administration of cypermethrin
(15 and 75 mg/kg/day) to
the normal rats for 15 days did not decrease the number of
THimmunopositive
(TH-IP) DA neurons in the substantia nigra. However,
the low dose (15 mg/kg/day) of cypermethrin enhanced the
rate of decline ofDAneurons in the substantia nigra of
hemiparkinsonian
rats at 10 days and 3 weeks (p < 0.05). Also, the number of
FAS in cypermethrin-treated
hemiparkinsonian rats was reduced
more rapidly than that of cypermethrin not-treated hemiparkinsonian
rats at 10 days, 3 weeks, and 6 weeks (p < 0.05).
These results suggest that
cypermethrin per se cannot directly
induce the degeneration of DA neurons but can accelerate a toxic
∗These two authors contributed equally to this work.
This work was supported by a
grant from the
grant No. R01-2001-000-00344-0 from the
Foundation.
Address correspondence to Won
Yong Lee, M.D. and Ph.D., Department
of Neurology,
wylee@smc.samsung.co.kr. Sung Sik Han, Ph.D., Cell Engineering
& 3-D Structure Laboratory,
and
sshan@korea.ac.kr
effect on the degeneration of DA neurons in the progressive
hemiparkinsonian
rats.
INTRODUCTION
Cypermethrin is a potent
pesticide derived from natural
pyrethrin of the chrysanthemum plant (Crawford et al. 1981).
Due to its effective insecticidal
properties and low mammalian
toxicity as a result of rapid metabolism, cypermethrin is
currently
widely used (Hayes et al. 1991). However, a liberal use of
pyrethroids increases the risk of intoxication to nontarget
organisms
(Crawford et al. 1981; Malaviya
et al. 1993). Cypermethrin
is essentially a sodium channel toxin. Modified channels through
cypermethrin cause stable repetitive firing, depolarization, and
conduction block (Narahashi et al. 1992). Furthermore, it
antagonizes
γ -aminobutyric acid
(GABA)—mediated inhibition,
modulates nicotinic cholinergic transmission, and markedly
enhances
adrenaline and noradrenalin release (Aldridge 1990).
Cypermethrin induces the
blood-brain barrier permeability of
neonatal rats, and it takes a long period of time, after its
withdrawal,
for the rats to completely recover (Gupta et al. 1999).
Additionally, cypermethrin has
been reported to induce oxidative
stress in rats (Kale et al. 1999; Giray et al. 2001).
Parkinson’s disease (PD) is a
commonmovement disorder associated
with degeneration of dopaminergic neurons in the substantia
nigra and a corresponding loss of dopamine in the basal
ganglia (Bernheimer et al. 1973; Hornykiewicz and Kish 1986).
Although the cause of PD has yet to be accurately discovered,
the environmental or endogenous agents leading to oxidative
damage might contribute to the degeneration of dopaminergic
(DA) neurons. The oxidative
stresses leading to increased reactive
oxygen species generation have been identified within the
399
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400 MUN ET AL.
degeneration of the DA neuron (Sian et al. 1994). Specifically,
the idea of environmental involvement comes from experimental
and epidemiological research that suggests pesticides might play
a role in the neurodegenerative process leading to idiopathic PD
(Bocchetta and Corsini 1986; Barbeau et al. 1987; Koller et al.
1990; Semchuk et al. 1992, 1993;
Hubble et al. 1993; Beate and
Fei 2000; Betarbet et al. 2000). However, in testing cypermethrin
for its relationship to the degeneration of DA neurons, an
experimental study has not yet been done.
6-Hydroxydopamine (6-OHDA) is a
hydroxylated analog of
the natural dopamine neurotransmitter, which induces the motor
impairment of PD by the degeneration of DA neurons in animals
(Ungerstedt 1968;Kostrzewa et al.
1974). Aecent study reported
that intrastriatal injection of 6-OHDA could produce the
progressive
degeneration of DA neurons in rat models (Sauer and
Oertel 1994; Przedborski et al.
1995; Lee et al. 1996). 6-OHDA
induces nigrostriatal dopaminergic lesions via the generation
of reactive oxygen species (ROS) (Kumar et al. 1995).
Additionally,
the enzymatic and nonenzymatic self auto-oxidation
responses to decrease ROS further induce oxidative stress (Hall
et al. 1992).
In order to examine the effects
of cypermethrin on the degeneration
of DA neurons, the degeneration ratio of DA neurons
and the behavior of the experimental rats were examined after
an administration of cypermethrin to normal and progressive
hemiparkinsonian rats induced by 6-OHDA. For the behavioral
experiment, we used the non–drug-induced forepaw adjusting
step (FAS) test, which reflects akinesia of PD and has been
successfully
applied in pharmacological, transplantation, and gene
therapy studies (Olsson et al. 1995, Chang et al., 1999, Lee et
al.
2000, Kirik et al. 2002). The
purpose of this experiment is to see
if there is a direct relationship between cypermethrin and the
degeneration of DA neurons by using progressive hemiparkinsonian
rats.
MATERIALS AND METHODS
Animals
The in vivo protocols were
approved by the Institutional Animal
Care and Use Committee of the
Korea University. Male
Sprague–Dawley rats weighing about 200 to 250 g were used
as subjects (N=84).
Animals had free access to food and water
through the experiment and were maintained on a 12 h light-dark
cycle.
Progressive Hemiparkinsonian Rat
Model
For dopamine depletion, rats (N=48) were anesthetized with
a mixture of ketamine (75 mg/kg), acepromazine (0.75 mg/kg),
and xylazine (3.8 mg/kg). In order to spare noradrenergic neurons,
a desipramine (12.5 mg/kg) (Sigma, USA) was injected
intraperitoneally
30 minutes prior to lesioning. To
produce the progressive
degeneration of DA neurons in rat models, we injected
6-hydroxydopamine (6-OHDA; Sigma, USA) into the striatum.
As previously described for the
intrastriatal lesion (Przedborski
et al. 1995), 17.5 µg (free base weight) of 6-OHDA was infused
unilaterally in 3.5 µL of normal saline at a rate of 0.75 µL/min
at the following coordinates: incisor bar −3.3 mm, AP 0.5 mm,
ML 2.5 mm relative to bregma, and
−5.5 mm from the dura
(Paxinos and Watson 1998).
Administration of Cypermethrin
In the first set of experiments,
the effect of cypermethrin on
healthy DA neurons was investigated with normal nonlesioned
rats (N=36).
Cypermethrin was dissolved in corn oil (Cantalamessa
et al. 1998) and administered orally via intragastric
tube to normal nonlesioned rats with two different doses of 15
mg/kg/day (1/50 of LD50, N=12) or 75 mg/kg/day (1/10 of
LD50, N=12) for 15 days. For the control, rats of the same age
(N=12) were
administered with an equal amount of corn oil
without cypermethrin.
In the second set of experiments,
the effects of cypermethrin
on the previously insultedDAneurons by6-OHDA, which correspond
to the case of Parkinson’s disease, was investigated with
the progressive hemiparkinsonian rat model (N=48). Cypermethrin
15 mg/kg/day (1/50 of LD50, N=24) was given to
the progressive hemiparkinsonian rats a day after lesioning (the
injection of 6-OHDA). The cypermethrin-treated hemiparkinsonian
rats were divided into four groups according to duration of
cypermethrin administration: 0, 10 days, 3 weeks, and 6 weeks
(N=6 for each). For the control, the hemiparkinsonian rats were
administered with an equal amount of corn oil without cypermethrin
(N=6 for
each, same as cypermethrin-treated group).
Behavioral Analysis: Forepaw
Adjusting Step Test
For the behavioral experiment, we
used the non–druginduced
forepaw adjusting step (FAS) test, which reflects akinesia
of PD (Olsson et al. 1995, Chang et al. 1999). For the FAS
test, the experimenter held the rat’s hindlimbs and one forepaw,
so that the animal must bear its weight solely with the strength
of its opposing forepaw. As the treadmill moved (in a lateral
direction with respect to the anterior-posterior orientation of
the
animal), the rat adjusted its free forepaw, or “step,” in order to
maintain its balance. Steps were counted manually for each
interval,
as described in previous studies (Chang et al. 1999; Lee
et al. 2003). One cycle using our motorized treadmill is set to
90 cm/ 12 seconds (Boowon
Instrument, Seoul, Korea). The
stepping numbers over five cycles were then averaged for each
forepaw.
FAS test was done at 0 and 15
days after cypermethrin administration
for the first set of experiments, and 0, 10 days, 3
weeks, and 6 weeks after cypermethrin administration for the
second set of experiments.
Tyrosine Hydroxylase (TH)
Immunohistochemistry
Rats were anesthetized at 0, 15 days (for first set of
experiments),
or 0, 10 days, 3 weeks, and 6 weeks (for second set of
experiments) after cypermethrin administration. Anesthetized
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DA NEURON’S DEGENERATION BY
CYPERMETHRIN 401
rats were transcardially perfused with 125 mL of normal saline
followed by 250 mL of ice-cold 4% paraformaldehyde. The
perfusion-fixed brain was quickly removed and further fixed in
4 _ for 15 to 24 h. The fixed material was treated with a 30%
sucrose solution to prevent ice crystal formation, embedded in
OCT compound (Sakura, Torrance, CA) and quickly frozen.
Sections in 30µmthickness were made using a
rotary microtome
HM 340E (Microm International GmbH, Germany). Immunohistochemistry
of the DA neurons was performed as follows
(Towle et al. 1984): Following
two rinses in tris-saline (TS), the
endogenous peroxidase activity was quenched for 15 minutes
in 0.6% H2O2. After rinsing in TS, sections
were preincubated
for 30 minutes in 0.6% Triton X-100 with 1% bovine serum
albumin and transferred to a 1:200 dilution of primary mouse
antityrosine hydroxylase (TH; Chemicon, CA). Following a 12-
to 18-h incubation at 4 _, the sections were rinsed in TS, incubated
in a 1:200 dilution of antimouse IgG (Sigma, MO) for
1h, rinsed repeatedly and
incubated in ABC solution (DAKO,
Carpinteria, CA) for 20 minutes. Finally, 3-3’-diaminobenzidine
(Sigma, MO) served as a chromogen
in the subsequent visualization
reaction.
Tyrosine
Hydroxylase-Immunopositive Cell Count
Thirty serial sections (30 µmthickness) within the substantia
nigra in the midbrain (Paxinos andWatson 1998) were used for
cell counting. The tyrosine hydroxylase-immunopositive (THIP)
neurons were counted by three experimenters individually
and averaged. Only one experimenterwas aware of the treatment
condition. The TH-IP neurons, in which the nucleus is totally
visible, were counted (Dwight et al. 1993) using an Axioskope
2 light microscope (Carl Zeiss
Jena GmbH, Germany). There
was no significant individual variation.
TABLE 1
The number of TH-IP neurons in
the substantia nigra of normal
nonlesioned rats (N=12 for each group)
Control Cypermethrin Cypermethrin
(corn oil) 15 mg/kg 75 mg/kg
Rat 1 2386 2399 2372
Rat 2 2466 2428 2578
Rat 3 2310 2379 2446
Rat 4 2444 2447 2410
Rat 5 2377 2369 2389
Rat 6 2412 2417 2412
Rat 7 2421 2399 2383
Rat 8 2425 2438 2394
Rat 9 2389 2394 2392
Rat 10 2391 2368 2380
Rat 11 2411 2432 2361
Rat 12 2395 2378 2371
Mean ± SEM
2402.2 ± 11.22 2404 ±
8.03 2407.3 ± 16.84
∗Cell numbers represent the bilateral number of cells; 30 serial
sections
(30 µm
thick) were used for cell counting.
Statistical Analysis
All data represented the mean ±
standard error of mean
(SEM). SPSS (version 9) program
forWindows was used for the
statistical analysis.To compare the FAS and number of dopaminergic
neurons in the two sides of the brain came from the same
animal, we used the paired t test. The t test was used in comparing
cypermethrin-treated hemiparkinsonian rats and nontreated
hemiparkinsonian rats. Post hoc, multiple comparisons between
FAS means were performed using
Dunnet’s test. The individual
p value was adjusted by multiplying the comparison number to
correct type 1 error rate (Rosner 1995). The adjusted p value of
<0.05 was defined as significant.
RESULTS
Effects of Cypermethrin on the
Healthy DA Neurons of
Nonlesioned Rats
The average number of tyrosine hydroxylaseimmunopositive
(TH-IP) neurons in the control
rats, cypermethrin
15 mg/kg-treated rats, and cypermethrin 75 mg/kgtreated
rats was 2402.2 ± 11.22
(mean ± SEM), 2404 ±
8.03,
and 2407.3 ± 16.84,
respectively (Table 1). The FAS test was
not different among the groups (data were not shown).
These results suggest that even
high doses of cypermethrin
per se cannot directly induce the degeneration of DA neurons.
We also investigated the number
of Purkinje cells in the cerebellar
hemisphere by using paraffin section and HE staining.
There was no difference of the
number of Purkinje cells between
control and cypermethrin-treated groups (data were not
shown). Additionally, in high doses (75 mg/kg) of
cypermethrintreated
groups, 2 or 3 hours after treatment, abnormal reactions
such as severe convulsive movement, salivation, and unstable
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402 MUN ET AL.
TABLE 2
The number of TH-IP neurons in the substantia nigra of the hemiparkinsonian
rats at
different time points after cypermethrin
Cypermethrin not-treated rats
Cypermethrin-treated rats
Ipsilateral Contralateral Ipsilateral Contralateral
0 week 1223.7 ±
13.03 1238.8 ± 14.32 1216.3 ± 20.71
1226.3 ± 24.24
10 days 968.2 ± 14.47
1279.2 ± 16.61 797.8 ±
35.2∗ 1188 ± 38.0
3 weeks 754.5 ±
12.21 1144.7 ± 28.6 731.5 ± 13.63∗
__________1184 ± 21.07
6 weeks 725 ± 9.21 1225
± 16.0 683.5 ±
26.23 1233 ± 29.13
Data represent mean ± SEM.
∗p < 0.05
; t test, Dunnet’s post hoc test, compared with cypermethrin not-treated rats.
posture began to appear, but most reactions stopped after 4 to 5
hours.
Effects of Cypermethrin on the DA
Neurons of the
Progressive Hemiparkinsonian Rats
In the control (not
cypermethrin-treated rats), the number of
TH-IP neurons in the ipsilateral
side to 6-OHDA injection was
gradually decreased in process of time (1223.7±13.03 at 0 days,
968.2 ±
14.47 at 10 days, 754.5 ± 12.21 at 3 weeks, and 725
±
9.21 at 6 weeks). Furthermore, in the cypermethrin-treated
group, the number of TH-IP neurons in the ipsilateral side to 6-
OHDA injection was more rapidly
decreased with time course
(1216.3 ± 20.71 at
0 days, 797.8 ± 35.2 at 10 days, 731.5 ±
13.63 at 3 weeks, and 683.5 ±
26.23 at 6 weeks). The number
FIG. 1. The effect of cypermethrin on
forepaw adjusting steps in hemiparkinsonian rats. The numbers of steps were
counted over 90 cm in 12 seconds. The
time means a week. Cy ipsi, ipsilateral side of the
cypermethrin-treated parkinsonian rat; Cy contra, contralateral side of the
cypermethrin-treated parkinsonian rat;
PD ipsi, ipsilateral side of the cypermethrin not-treated
hemiparkinsonian rat; PD contra, contralateral side of the cypermethrin
not-treated hemiparkinsonian rat.
∗p < 0.05;
t test, Dunnet’s post hoc test, compared with cypermethrin not-treated rats.
of TH-IP neurons in the ipsilateral side to 6-OHDA injection
at 10 days and 3 weeks was significantly less in
cypermethrintreated
group than those in the control (p <0.05, Table 2). However,
the number of TH-IP neurons in the contralateral side to
6-OHDA injection was not
different between the control and
cypermethrin-treated group (Table 2).
The number of FAS in the controlwas 18.40±0.40 at 0 days,
16.32 ±
0.17 at 10 days, 12.90 ± 0.22 at 3 weeks, and 10.63
±0.45 at 6 weeks. Comparatively, cypermethrin-treated parkinsonian
rats had the following results: 18.88 ±
0.15 at 0 days,
14.98 ±
0.59 at 10 days, 10.77 ± 0.71 at 3 weeks, and 10.54
±
0.52 at 6 weeks. Compared with the control group, the
number
of FAS in cypermethrin-treated groups had reduced more
rapidly, especially at 10 days and 21 days (p < 0.05, Fig. 1).
Otherwise, the number of FAS in
relation to the side of the brain
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DA NEURON’S DEGENERATION BY
CYPERMETHRIN 403
unaffected by 6-OHDA was not different between the control
and cypermethrin-treated group (p >0.05, Fig. 1).
These results suggest that
cypermethrin can enhance the rate
of decline of DA in the progressive hemiparkinsonian rats.
Administration of cypermethrin
did not result in any body
weight loss of the animals.
DISCUSSION
In this study, in order to
examine if cypermethrin can independently
induce the degeneration of dopaminergic (DA) neurons,
nonlesioned normal rats were treated with cypermethrin for 15
days, and then the number of DA neurons in the substantia nigra
(SN) was compared with that of
the cypermethrin nontreated
rats. However, there were no significant differences between
them. These results suggested that cypermethrin per se could
not induce the degeneration of healthy dopaminergic neurons.
On the other hand, in the study
on the effect of cypermethrin
on the previously insulted dopamine neurons by 6-OHDA, the
number of DA neurons in the SN of the cypermethrin-treated
hemiparkinsonian rats was more rapidly decreased with time
course. Moreover, compared to the cypermethrin nontreated
hemiparkinsonian rats, the forepaw adjusting step number of the
cypermethrin-treated hemiparkinsonian rats was more reduced.
These behavioral deficits were
corresponding to the degeneration
ofDAneurons, suggesting the progress of PD. These results
offer that cypermethrin could accelerate the degeneration of DA
neurons combined with oxidative stress induced by 6-OHDA.
Indeed, a modulation of the
blood-brain barrier by cypermethrin
could easily induce changes that might influence the
integrity of DA and other neuron types throughout the brain.
Condes-Lara et al. reported that
repeated exposure to cypermethrin
(300 mg/kg i.p. dose, belowtheLD50value) could cause
paroxysmal epileptic activity (Condes-Lara et al. 1999). However,
there was no report that repeated convulsion derives the
degeneration of dopaminergic neurons, and any correlation of
epilepsy and Parkinson’s disease has not been reported yet. Our
study showed that even high doses (75 mg/kg/day) of cypermethrin
did not damage the dopaminergic neurons in normal
nonlesioned rats even though they showed convulsive movement.
Therefore, we assume that
cypermethrin-induced convulsive
movement could not induce the degeneration of dopaminergic
neurons.
In this study, although
cypermethrin could not directly degenerate
healthy DA neurons, the administration of cypermethrin to
the progressive hemiparkinsonian rats accelerated the degeneration
of previously insulted DA neurons.
To date, it has been found that
when adult rats are treated with
cypermethrin, oxidative stress in the central nervous system is
induced even after cerebral development has terminated, which
can generate motor disturbances (Giray et al. 2001). These results
suggest that cypermethrin could induce the degeneration
of cerebral tissue and cells having dopamine receptors.
Because of the high basal ROS
level in DA neurons
by 6-OHDA or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) (Rios and Tapia 1987; Hall et al. 1992; Hirsch et al.
1997), monoamine oxidases induce
the formation of hydrogen
peroxide, and the nonenzymatic auto-oxidation of dopamine
produces the formation of neuromelanin, which potentiates hydrogen
peroxide formation when combined with iron (Jenner et
al. 1992). Therefore, oxidative stress has often been put forward
as the major cause of DA neuron degeneration.
These results suggest that cypermethrin
cannot directly induce
the degeneration of DA neurons but can accelerate a toxic
effect on the degeneration of DA neurons in the progressive
hemiparkinsonian rats. It is not clear whether cypermethrin
could accelerate the toxic effects of 6-OHDA or if cypermethrin
could damage only the ongoing degenerating neurons.
In conclusion, we assume that the
oxidative stress induced
by cypermethrin can cause the degeneration of dopaminergic
neurons not in healthy individuals, but in the case of
degenerative
diseases such as Parkinson’s disease. To determine the precise
mechanism of partial toxic effect of cypermethrin, further study
will be needed.
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