TOXICOLOGICAL SCIENCES 97(1), 140–148 (2007)
doi:10.1093/toxsci/kfm024
Advance Access publication February 27, 2007
Zinc Attenuates Malathion-Induced Depressant-like Behavior and
Confers Neuroprotection in the Rat Brain
Patrı́cia S. Brocardo,* Fabrı́cio Assini,† Jeferson L. Franco,‡ Pablo Pandolfo,† Yara M.R. Müller,§
Reinaldo N. Takahashi,† Alcir L. Dafre,‡ and Ana Lúcia S. Rodrigues*,1
*Departamento de Bioquı´mica, †Departamento de Farmacologia, ‡Departamento de Cieˆncias Fisiológicas, and §Departamento de Biologia Celular,
Embriologia e Gene´tica, Centro de Cieˆncias Biológicas, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, Santa Catarina, Brazil
Received October 13, 2006; accepted February 16, 2007
Malathion is an organophosphate widely used as an insecticide
in agriculture and in public health programs, causing risk to
human health. As was recently reported, malathion induces
depressant-like behavior and oxidative damage to the brain of
rodents. Given the relevance of searching for neuroprotective
agents against such damage, this study was therefore undertaken
to investigate the neuroprotective potential of zinc in dealing with
malathion-related toxicity. Female Wistar rats were exposed to
malathion (50 and 100 mg/kg, ip) and/or zinc chloride (ZnCl2;
5 mg/kg, ip) for 3 days. Malathion produced a depressant-like effect,
observed by the increased immobility time in the forced swimming
test (FST), without affecting total locomotor activity and rearing
in the open-field. However, malathion administered at 50 mg/kg
reduced the central time in the arena and at the dose of 100 mg/kg
reduced the central locomotion. These effects were completely
reversed by ZnCl2. Exposure to malathion (50 mg/kg, ip) and/or
ZnCl2 did not affect AChE activity in the hippocampus, cerebral
cortex, and blood. Malathion (50 mg/kg, ip) alone caused some
harmful effects, such as (1) an increase in lipid peroxidation and a
reduction of glutathione peroxidase activity in the cerebral cortex,
(2) reduction of glutathione reductase activity in the hippocampus, and (3) changes in the structure of chromatin in the dentate
gyrus, all effects attenuated by ZnCl2. In conclusion, these results
clearly show that zinc administration is able to attenuate some
neurochemical, morphological, and behavioral effects induced by
malathion, notably the malathion-induced depressant-like effect
in the FST.
Key Words: malathion; zinc; depression; oxidative injury;
pesticides; organophosphates.
Malathion is among the organophosphate (OP) compounds
most widely used as a pesticide in agriculture, in veterinary
practice, and as an ectoparasiticide applied against human body
lice (Maroni et al., 2000). Its widespread use has raised concerns over its potentially adverse effects on the health of humans,
animals, wildlife, and fish (Flessel et al., 1993). Malathion has
1
To whom correspondence should be addressed. Fax: þ55 48 3721-9672.
E-mail: [email protected].
been pointed as one of the main contaminants in cases of OP
intoxication in Santa Catarina, a state in Southern Brazil, according to unpublished data obtained from a Toxicological Information Center (Centro de Informacxões Toxicológicas—CIT)
hosted by the Hospital Universitário, Florianópolis, SC.
Acute toxic effects induced by malathion pesticides are
mainly attributable to the inhibition of acetylcholinesterase
(AChE) in the nervous tissue with a consequent increase in the
levels of acetylcholine, causing several cholinergic symptoms
(Kwong, 2002). It is important to consider that acute OP
poisoning may lead to chronic sequelae, including anxiety and
depression (Stallones and Beseler, 2002), while long-term
exposure to OP may produce neuropsychiatric symptoms,
including depression (Salvi et al., 2003). Preclinical studies
also indicate that malathion, administered acutely (100–250
mg/kg, ip) or repeatedly for 7 days (25–100 mg/kg, ip), causes
depressant-like behavior in the forced swimming test (FST)
(Assini et al., 2005). Another study has shown that malathion
administered chronically for 28 days (25–150 mg/kg, ip) also
caused a depressant-like behavior in the FST (Ramos et al.,
2006). The FST is the most widely used animal test predictive
of antidepressant action (Cryan et al., 2002). Besides this
ability, the FST has been shown to be sensitive to several
experimental models of predisposition to depression, including
the exposure of rodents to chronic mild stress and to substances
that are associated with increased risk to cause depression in
humans, such as amphetamine and interferon-alpha (Cryan
et al., 2003; Makino et al., 2000). These findings raise the
possibility that the increase in the immobility time elicited by
malathion in the rat FST may be of clinical relevance.
Preclinical and clinical studies have shown that depression
can lead to atrophy and cell loss in limbic brain structures,
including the hippocampus (Duman, 2004). Moreover, functional imaging studies reveal that hippocampal volume is
decreased in patients with depression (Duman, 2004; Saylam
et al., 2006), suggesting that neurogenesis is altered in depression. Antidepressant treatment increases adult neurogenesis in
the hippocampus, especially in the dentate gyrus (Dranovsky
and Hen, 2006; Warner-Schmidt and Duman, 2006).
Ó The Author 2007. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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ZINC PROTECTS AGAINST BRAIN DAMAGE CAUSED BY MALATHION
141
Zinc is a trace element present at high concentrations in the
central nervous system, particularly in the hippocampal mossy
fibers (Choi and Koh, 1998). Many studies have been reported
indicating an important role of zinc in the psychopathology and
therapy of depression. In serum, lower zinc concentration has
been found in patients with depression (Maes et al., 1999), and
treatments with antidepressants were able to normalize serum
zincemia (Maes et al., 1997). Moreover, zinc supplementation
enhanced the efficacy of antidepressant therapy (Nowak et al.,
2003). Several studies have shown that acute treatment with
zinc produces an antidepressant-like effect in mice and rats in
the FST (Kroczka et al., 2001; Nowak et al., 2003; Rosa et al.,
2003), in the tail suspension test in mice (Rosa et al., 2003),
and in the olfactory bulbectomy model in rats (Nowak et al.,
2003). Moreover, similarly to antidepressants, chronic administration of zinc hydroaspartate (65 mg/kg, for 14 days) induces
brain-derived neurotrophic factor gene expression in the
cerebral cortex (Nowak et al., 2004) and increases the synaptic
zinc level in the hippocampus of rats (Szewczyk et al., 2006).
Indeed, some studies suggest zinc as a beneficial agent against
brain damage (Aschner et al., 2001; Cabre et al., 1999),
although an excess of zinc may produce cytotoxic effects (Choi
and Koh, 1998; Kim et al., 1999).
Although zinc has been proposed as an antidepressant agent,
to our knowledge, no study was performed until now to
investigate whether zinc produces an antidepressant-like effect
against the depressive-like behavior and also against a morphological damage to the hippocampus induced by a depressogenic
substance. Regarding this issue, malathion was used in this
study to cause such effects, making possible to investigate
a possible antidepressant role of zinc. Neuroprotection afforded
by zinc was searched by investigating the antioxidant defense
system, lipid peroxidation, and the activity of AChE in the
hippocampus and cerebral cortex of rats. We also investigated
whether ZnCl2 produces any protective action against malathioninduced chromatin condensation in dentate gyrus of rats,
considering the involvement of this structure in the pathophysiology of depression.
NADPH); nicotinamide adenine dinucleotide phosphate (oxidized form,
NADPþ); glucose-6-phosphate, tert-butylhydroperoxide (tBOOH), magnesium
chloride, oxidized (GSSG) and reduced (GSH) glutathione; chloro-2,4dinitrobenzene (CDNB); glutathione reductase (GR); acetylthiocholine iodide;
bovine serum albumin (BSA); 5,5#-dithiobis(2-nitrobenzoic acid) (DTNB);
Hoechst 33258; malondialdehyde (MDA); and 2-thiobarbituric acid from
Sigma Chemical Co, St. Louis, MO. Trichloroacetic acid, toluidine blue, and
zinc chloride (ZnCl2) were purchased from Merck (Darmstadt, Hesse,
Germany).
In order to choose a dose of malathion to be used in this study, rats were
treated once a day for 3 consecutive days with an ip injection of malathion (50;
100 or 250 mg/kg). Thirty minutes later, the training session of the FST was
carried out, and 24 h later, rats were submitted to the test session of the FST.
With the purpose to investigate the possible protective effect of ZnCl2
against malathion-induced behavioral alterations, rats received three daily ip
injections of saline (control), malathion (50 or 100 mg/kg) and/or ZnCl2 (5 mg/kg)
in a volume of 1.0 ml/kg body weight. All groups of rats received two
contralateral ip injections. The second injection was given immediately after
the first one. Thus, rats were given saline and saline (control group), ZnCl2 and
saline, malathion and saline, and malathion and ZnCl2. The doses of malathion
were selected based on the dose-response curve in the FST, and the dose of zinc
was chosen based on preliminary experiments in our laboratory. Malathion
doses used did not produce overt signs of toxicity in rats.
Rats were killed by decapitation on the 4th day, 24 h after the last malathion
(50 mg/kg, ip) and/or ZnCl2 (5 mg/kg, ip) injection. One hour after the FST,
hippocampi and cerebral cortices from rats previously exposed to this test were
removed and homogenized 1:2 (wt/vol) in 20mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid buffer, pH 7.4, for biochemical analysis.
For histological analysis, rats treated with malathion (50 mg/kg, ip) and/or
ZnCl2 (5 mg/kg, ip) for 3 consecutive days were anesthetized with sodium
pentobarbital (100 mg/kg) and perfused transcardially, first with physiological
saline and then with a fixative solution containing 4% paraformaldehyde in
0.1M phosphate buffer (pH 7.4). Brains were removed and postfixed in 4%
paraformaldehyde at 4°C for 18–24 h. The tissues were then blocked and
embedded in paraffin according to the standard histological techniques.
All the behavioral, biochemical, and morphological analyses were performed by the staff blind to the dose groups.
MATERIALS AND METHODS
Open-field test. The open-field apparatus consisted of a white-painted
plywood arena measuring 32.5 cm in height and 97 cm in diameter. The floor of
the arena was divided into 19 units by black lines and lighted from above by one
red bulb. In the open-field, each behavioral naive rat was placed in the center of
the open-field and the following variables were recorded, for 5 min: number of
line crossing with four paws (total locomotion), number of central squares (i.e.,
away from the walls) crossed (central locomotion), number of rearing
responses, and time in the central area (central time). The open-field test was
carried out 24 h after the last malathion and/or ZnCl2 dosing. The behavior of
each animal was recorded by video camera positioned above the open-field and
monitored in another room via closed circuit TV camera.
Animals. Naive female Wistar rats with no pregnancy history, approximately 3 months old (weighing 180–220 g) were housed in plastic cages in
a temperature-controlled room (22°C–27°C) with free access to food and water
and maintained on a 12-h light/dark cycle (lights on at 7:00 A.M.). Considering
that the main focus of our study was to investigate the ability of zinc to
attenuate the depressive-like effects of malathion exposure, female rats were
used, taking into account that depression affects more women that men
(Weissman et al., 1996). The rats were randomly distributed into specified
experimental groups. All procedures used in this study were performed after
approval of the protocol by the Ethics Committee of the Institution, and every
effort was made to minimize animal suffering.
Drugs and treatment. The following chemicals were used: commercial
grade malathion, 500 CE (95% purity, CAS 121-75-5, Dipil Chemical
Ind., Brazil); nicotinamide adenine dinucleotide phosphate (reduced form;
Forced swimming test. The studies were carried out on rats according to
the method of Porsolt et al. (1978), with minor modifications. Behavioral naive
rats were individually forced to swim in open plastic cylinders (height 40 cm,
diameter 30 cm) containing 25 cm of water, maintained at 25°C ± 1°C. The
pretest session was carried out 30 min after the last malathion and/or ZnCl2
injection. In the pretest session, rats were allowed to swim for 15 min and then
returned to their home cages. In the test session, 24 h later, rats were again
submitted to the FST, with immobility time measured during 5 min. Each rat
was judged to be immobile when it remained floating motionless in the water,
making only those movements necessary to keep its head above water.
Determination of AChE activity. AChE activity was measured by the
method of Ellman et al. (1961), using acetylthiocholine iodide as a substrate in
homogenates of hippocampus and cerebral cortex centrifuged at 2300 3 g for
15 min. Each sample was taken from one animal and assayed in triplicate. The
rate of hydrolysis of acetylthiocholine iodide was measured at 412 nm through
142
BROCARDO ET AL.
the release of the thiol compound which when reacted with DTNB produced the
color-forming compound thionitrobenzoic acid. For the determination of blood
AChE activity, rats were anesthetized with sodium pentobarbital (100 mg/kg),
and blood was collected from rat liver port vein in heparinized tubes and stored
at 20°C. After thawing, samples were hemolyzed in ice-cold water for 30 min
and centrifuged 10 000 3 g for 10 min. The supernatant was assayed for AChE
activity according the method described by Ellman et al. (1961) and normalized
by hemoglobin (Hb) content. The Hb concentration was measured at 540 nm as
cyano-met-Hb form.
Antioxidant enzyme assays. Glutathione peroxidase (GPx) activity was
measured by the Wendel (1981) method, using tBOOH as a substrate. NADPH
disappearance was monitored by a spectrophotometer at 340 nm. GlutathioneS-transferase (GST) activity was assayed by the procedure of Habig and Jakoby
(1981), using CDNB as a substrate. The assay was conducted by monitoring the
appearance of the conjugated complex of CDNB and GSH at 340 nm. GR
activity in brain homogenates was determined by the method described by
Carlberg and Mannervik (1985). The reduction of GSSG in the presence of
NADPH was measured spectrophotometrically at 340 nm. The activity of
glucose-6-phosphate dehydrogenase (G6PDH) was determined by means of the
absorbance increase induced by the reduction of NADPþ to NADPH at 340 nm
(Glock and McLean, 1953).
Total glutathione assay. Total glutathione (GSH-t) was measured by the
GR-DTNB recycling assay described by Akerboom and Sies (1981). Values
were obtained based on a standard curve with known GSSG concentration.
Measurement of thiobarbituric acid reactive substances levels. As an
index of lipid peroxidation, the formation of thiobarbituric acid reactive
substances (TBARS) was measured in hippocampal and cerebral cortical crude
homogenates by the method of Ohkawa et al. (1979). The results were
expressed in terms of the extent of MDA production.
Protein determination. The protein content in hippocampal and cerebral
cortical homogenate samples was quantified by the method of Bradford (1976),
using BSA as a standard.
Nuclear staining with Hoechst 33258. Fixed hippocampi of treated and
control rats (n ¼ 3) were dehydrated in graded ethanol and embedded in
paraffin. Blocks were oriented and tissue sections cut in the coronal plane at
5 lm thick in series of 10 sections to analyze the apoptotic cells. The sections
were rinsed with phosphate-buffered saline (PBS) and then incubated for
20 min at room temperature with Hoechst 33258 (1 lg/ml in PBS). The sections
were dipped in a 0.1% gelatin-glycerin solution and visualized under
a fluorescence microscope. For histological control, samples were stained with
toluidine blue (1%).
Normal cells were identified by intact round-shaped nuclei with diffuse
fluorescence and apoptotic cells by highly refringent smaller nuclei with
uniformly dispersed chromatin.
FIG. 1. Effect of repeated administration of malathion (50–250 mg/kg, ip)
for 3 days on the immobility time in the FST. Values are expressed as mean ±
SEM of six to eight animals. *p < 0.05 as compared with the saline-treated
control.
result, we choose the lowest doses of malathion for the further
experiment, in which the possible protective effect of ZnCl2
against malathion-induced depressant-like behavior was investigated. Thus, the immobility time in FST of rats treated with
malathion (50 and 100 mg/kg, ip) and/or ZnCl2 (5 mg/kg, ip)
for 3 days is shown in Figure 2. The malathion-induced increase in immobility time was completely prevented by
treatment with ZnCl2.
Open-Field Test
The effects of malathion and/or ZnCl2 on spontaneous
locomotor activity in rats are shown in Figure 3A and 3D.
Malathion and ZnCl2, alone or in combination, did not interfere
with total locomotion and rearing responses. However,
Statistical analysis. All data are expressed as mean ± SEM. Data were
analyzed statistically by one-way ANOVA for the experiments dealing with
malathion effects in the FST and two-way ANOVA for malathion 3 ZnCl2
experiments. When the overall test of significance led to a rejection of the null
hypothesis, a post hoc comparison test was carried out to determine the source
of the effect. This analysis was based on the Duncan’s test. Values were
considered significant when p < 0.05.
RESULTS
Forced Swimming Test
The dose-response curve for malathion administered once
a day for 3 consecutive days is shown in Figure 1. Malathion at
all doses tested produced an increase in the immobility time in
the FST, consistent with a depressant-like effect. Based on this
FIG. 2. Effect of repeated administration of malathion (50 and 100 mg/
kg, ip) and/or ZnCl2 (5 mg/kg, ip) for 3 days on the immobility time in the FST.
Values are expressed as mean ± SEM of seven to nine animals. *p < 0.05 and
**p < 0.01 as compared with the saline-treated control. #p < 0.05 as compared
with the respective malathion-treated group.
ZINC PROTECTS AGAINST BRAIN DAMAGE CAUSED BY MALATHION
143
FIG. 3. Total and central locomotion, time spent in the central area, and rearing responses in the open-field of rats treated with malathion (50 and 100 mg/kg,
ip) and/or ZnCl2 (5 mg/kg, ip) for 3 days. Values are expressed as mean ± SEM of seven to nine animals. *p < 0.05 as compared with the saline-treated control.
#p < 0.05 as compared with the respective malathion-treated group.
malathion administered at 50 mg/kg reduced the central time in
the arena and at the dose of 100 mg/kg reduced the central
locomotion. These effects were completely reversed by ZnCl2
(5 mg/kg, ip).
AChE Activity
Table 1 shows the effect of repeated administration (3 days)
of malathion and/or ZnCl2 on AChE activity. Malathion and
ZnCl2, alone or in combination, did not significantly alter the
activity of AChE in the cerebral cortex, hippocampus, and
blood of rats.
TABLE 1
Effect of the Repeated Administration of Malathion (50 mg/kg, ip)
and/or ZnCl2 (5 mg/kg ip) for 3 days on AChE Activity
in Cerebral Cortex, Hippocampus, and Blood of Rats
Treatment
Saline/saline
ZnCl2/saline
Saline/malathion
ZnCl2/malathion
Cerebral cortex
36.83
34.31
35.00
35.16
±
±
±
±
1.09
3.23
2.56
1.92
Hippocampus
27.15
25.67
24.76
26.48
±
±
±
±
1.34
1.73
1.88
1.92
Blood
22.4
22.0
23.2
22.5
±
±
±
±
5.7
9.3
5.9
5.7
Note. Enzyme activity in the cerebral cortex and hippocampus is expressed
as nmol/min/mg protein and in blood is expressed as nmol/min/g Hb. Values
are given as mean ± SEM (n ¼ 6–7 for cerebral cortex and hippocampus and
n ¼ 4 for blood).
Nuclear Staining in the Dentate Gyrus of Malathion-Treated
Rats
The specific DNA stain, Hoechst 33258, was used to assess
changes in chromatin structure following exposure to malathion and/or ZnCl2. As shown in Figure 4, we qualitatively
demonstrated that nuclei of dentate gyrus in animals treated
with saline (Fig. 4B) observed by fluorescence microscopy
were large and exhibited diffuse staining of the chromatin. The
image for the group treated with ZnCl2 showed few cells with
condensation of nuclear chromatin (Fig. 4C). In contrast, nuclei
of dentate gyrus of animals treated with malathion (Fig. 4D)
showed a variety of abnormal morphology, including highly
condensed and fragmented chromatin, which is a typical
feature of chromatin condensation (i.e., apoptosis). This
morphological profile was attenuated by the treatment of
animals with ZnCl2 (Fig. 4E).
Antioxidant Enzyme Activity and Measurement of GSH-t
Levels
Table 2 shows the influence of repeated administration
(3 days) of malathion and ZnCl2, alone or in coadministration,
on antioxidant enzymes and on the levels of GSH-t in the cerebral cortex and hippocampus of rats. The cerebral cortical
activity of GPx decreased slightly, yet significantly, in the
malathion-treated group, while coadministration of ZnCl2
prevented that effect. In the hippocampus, the decrease in the
144
BROCARDO ET AL.
activity of GR in the malathion-treated group was reversed by
the treatment with ZnCl2.
Malathion, in combination with ZnCl2, significantly reduced
the activity of GST in the hippocampus of rats. Other changes
were not observed in the studied parameters.
TBARS Measurements
Table 3 shows a significant increase in the TBARS levels in
the cerebral cortex of rats treated with malathion, which was
reversed by the treatment with ZnCl2. Malathion and ZnCl2,
alone or in combination, did not significantly alter the levels of
TBARS in the hippocampus of rats.
DISCUSSION
FIG. 4. Representative photomicrograph from a toluidine blue staining
showing the right hippocampal formation of a saline-treated rat (A). The box in
(A) represents the area of the dentate gyrus shown at higher magnification in
(B–E) labeled with the nuclear fluorescent marker Hoechst 33258. Rats were
treated for 3 days with saline (B); ZnCl2 5 mg/kg (C), malathion 50 mg/kg (D),
and coadministration of malathion and ZnCl2 (E). Typical signs of neuronal
apoptosis, such as condensation of nuclear chromatin, were found in the
malathion group and are indicated by arrows (D). This was attenuated by ZnCl2
coadministration (E). In the group exposed to ZnCl2 alone, only few cells with
condensation of nuclear chromatin were observed (C) as compared with salinetreated animals (B). Scale bar: (A) 200 lm, (B–E) 20 lm.
In this study, rats exposed to malathion for 3 days showed
a pronounced increase in immobility time in the FST, an
indication of depressant-like behavior. This result is similar to
previous findings by our group, showing that malathion
administered acutely (100–250 mg/kg, ip) or repeatedly (25–
100 mg/kg, ip) for 7 days caused depressant-like behavior in
the FST (Assini et al., 2005). This result is also similar to the
one reported by Ramos et al. (2006), in which rats exposed
chronically to malathion for 28 days (25–150 mg/kg, ip)
exhibited a depressant-like behavior in the FST. The increase
of immobility time caused by malathion in the FST cannot be
attributed to a locomotor effect caused by this OP since no
changes in total locomotion were apparent in malathionexposed rats in the open-field test. The depressant-like effect
of malathion in the FST is remarkable and may be of
toxicological importance, considering that depression in
TABLE 2
Effect of Malathion (50 mg/kg, ip) and/or ZnCl2 (5 mg/kg, ip) Exposure for 3 days on Antioxidant Enzyme Activity (GR, GPx, GST,
G6PDH) and GSH-t Levels in the Cerebral Cortex and Hippocampus of Rats
Tissue/treatment
Cerebral cortex
Saline
ZnCl2
Malathion
Malathion þ ZnCl2
Hippocampus
Saline
ZnCl2
Malathion
Malathion þ ZnCl2
GR
GPx
GST
G6PDH
GSH-t
22.44
20.84
21.87
21.71
±
±
±
±
0.85
0.43
0.92
1.18
15.23
16.47
13.51
16.32
±
±
±
±
0.78
0.94
1.02a
0.65c
76.47
89.50
80.53
85.06
±
±
±
±
5.97
11.20
5.01
2.19
25.30
29.28
26.13
30.28
±
±
±
±
0.66
3.60
0.90
2.25
1.77
1.63
1.55
1.93
±
±
±
±
0.25
0.14
0.24
0.06
26.05
24.13
20.83
23.73
±
±
±
±
0.78
0.85
0.56b
1.06c
15.99
13.86
13.04
13.79
±
±
±
±
1.06
0.85
0.67
0.67
61.05
57.16
53.56
47.52
±
±
±
±
3.4
3.7
3.9
4.8a
15.16
15.37
13.96
14.01
±
±
±
±
0.62
0.83
0.80
0.63
0.82
0.88
0.89
1.16
±
±
±
±
0.09
0.12
0.11
0.17
Note. Antioxidant enzyme activities are expressed as nmol/min/mg protein and GSH-t levels are expressed as lmol/g wet tissue. Values are expressed as ±
SEM, n ¼ 5–6.
a
p < 0.05 as compared with saline-treated control.
b
p < 0.01 as compared with saline-treated control.
c
p < 0.01 as compared with malathion-treated group.
ZINC PROTECTS AGAINST BRAIN DAMAGE CAUSED BY MALATHION
TABLE 3
TBARS Levels Measured in Cerebral Cortex and Hippocampus of
Animals Treated with Malathion (50 mg/kg, ip) and/or ZnCl2
(5 mg/kg, ip) for 3 days
Treatment
Saline/saline
ZnCl2/saline
Saline/malathion
ZnCl2/malathion
Cerebral cortex
197.4 ± 5.7
204.2 ± 8.6
244.4 ± 9.8a
188.6 ± 7.7b
Hippocampus
174.2
163.2
144.0
154.6
±
±
±
±
8.3
11.2
8.3
11.8
Note. TBARS levels are expressed as nmol/mg wet tissue. Values are
expressed as mean ± SEM; n ¼ 5.
a
p < 0.01 as compared with saline-treated control.
b
p < 0.01 as compared with malathion-treated group.
human beings is reported in many populations exposed to OP
pesticides (Reidy et al., 1992; Stallones and Beseler, 2002) and
is supposed to be a possible cause of suicides by rural workers
exposed to these agents (Parron et al., 1996).
Another important finding of our study is that the depressantlike behavior induced by malathion (50 and 100 mg/kg, ip) was
completely reversed by simultaneous treatment with ZnCl2.
ZnCl2 per se did not cause any alteration of immobility time in
the FST and did not cause behavioral alterations in the openfiled test. It is also in line with data in literature showing that
zinc produces an antidepressant-like effect in rats (Kroczka
et al., 2001) and mice (Kroczka et al., 2001; Rosa et al., 2003).
Moreover, the antidepressant potential of zinc is indicated by
several studies showing that it may have a significant role in the
modulation of depression in humans (Maes et al., 1997, 1999;
Nowak et al., 2003; Nowak and Szewczyk, 2002).
Regarding the results obtained in the open-field test, it is
noteworthy that malathion administered at 50 mg/kg reduced
the central time in the arena and at the dose of 100 mg/kg
reduced the central locomotion. The open-field arena is
generally considered to be a stressful, fear-arousing environment. More anxious, emotional animals tend to ambulate less
and stay away from the central part of the arena in such
conditions (Walsh and Cummins, 1976). These results are
somewhat in accordance with Assini et al. (2005) that showed
the anxiogenic-like responses in the elevated plus-maze after
acute (100 mg/kg, ip) and repeated (25 mg/kg, ip) exposure to
malathion. In our study, malathion-exposed rats exhibited an
anxiogenic-like response in the open-field, as compared to
control rats, an effect completely reversed by ZnCl2. Thus, the
results obtained in the FST and open-field test clearly indicate
a protective effect of zinc regarding malathion-induced behavioral impairments.
OPs are among the most commonly used and most acutely
toxic pesticides. They evoke a consistent pattern of physical
symptoms but also have acute psychological and neurobehavioral effects, such as anxiety, depression, and cognitive
impairments (Stallones and Beseler, 2002). OP inhibits AChE
145
activity in the central and peripheral nervous systems, resulting
in the stimulation of cholinergic synapses (Carr et al., 2001).
Malathion is described as a pesticide of low toxicity. This is
consistent with the fact that no overt signs of cholinergic
toxicity were observed in rats in our study. Moreover, no
alterations in AChE activity in cerebral cortex, hippocampus,
and blood was showed in animals exposed to this pesticide for
3 days, which impacts the current view that OP toxicity is
based on AChE inhibition (Kwong, 2002; Ramos et al., 2006).
The absence of AChE inhibition in the repeated exposure at
relatively low doses of malathion employed in the present
study, associated with a clear depressant-like effect, may
suggest that other biochemical targets may be responsible for
the observed effects (Fortunato et al., 2006) and does not
support the notion that increased immobility time in the FST
caused by malathion is associated with the activation of
cholinergic receptors by accumulated acetylcholine following
AChE inhibition (Ramos et al., 2006). However, it cannot be
excluded that AChE inhibition is a contributing factor in
malathion toxicity. Indeed, as previously reported, a single
dose of malathion (250 mg/kg, ip) was able to decrease the
AChE activity in the cerebral cortex and hippocampus
(Brocardo et al., 2005) and in the forebrain (Assini et al.,
2005) of rats. Malathion treatment for longer periods of time as
compared to the present study has also been shown to decrease
brain AChE activity (Assini et al., 2005; Ramos et al., 2006),
whereas short-time treatment, as the present one, seems to fall
within an adaptive response in the AChE. This statement is
supported by results from our group (unpublished data). It
appears that this putative adaptive response is overcome after
more extended exposure.
Antioxidant status is one possible alternative target of OP
toxicity. It is clearly demonstrated in literature that exposure to
malathion and other OP increases the levels of lipid peroxidation end products (TBARS) in erythrocytes, liver, and brain
of rats (Akhgari et al., 2003; Brocardo et al., 2005; Fortunato
et al., 2006; Goel et al., 2005), which is in agreement with the
findings of this study. OP exposure also leads to a clear
imbalance in the antioxidant status found in the brain
(Brocardo et al., 2005; Sharma et al., 2005), liver (Goel
et al., 2005), and erythrocytes (John et al., 2001). Lower GST
activity was also found in the brain of OP-treated animals
(Brocardo et al., 2005; Timur et al., 2003). These data are in
agreement with our observation that GPx in the cerebral cortex
and GR in the hippocampus showed a mild reduction in activity
after malathion exposure. In our protocol, GST activity in the
hippocampus was significantly diminished only when ZnCl2
and malathion were coadministered, indicating that ZnCl2,
when in association to malathion, can produce some adverse
effect, at least regarding GST activity. As expected for our
protocol, 3-day exposure to malathion (50 mg/kg, ip) produced
less pronounced effects than found previously with an acute
exposure to a higher dose of malathion (250 mg/kg, ip)
(Brocardo et al., 2005). Despite the less prominent effects in
146
BROCARDO ET AL.
the antioxidant status, this study points to the role of
mechanisms other than AChE inhibition, which should be
further considered in regard to malathion neurotoxicity and the
neuroprotective effects of zinc. However, our results do not
allow us to establish a clear relationship between the reversal of
the malathion-induced behavioral effects by zinc with the
antioxidant status in the hippocampus. Indeed, a clearer neuroprotection afforded by zinc concerning oxidative stress was
observed in the cerebral cortex.
The protective effect of ZnCl2 is in line with the fact that zinc
can protect against oxidative damage, although other mechanisms cannot be ruled out. The most remarkable examples were
found in hepatic tissue. Livers of Wilson’s disease patients
benefit from zinc treatment by attenuating lipid peroxidation
and improving oxidative markers (Farinati et al., 2003). In
animals, hepatic protection has been clearly shown in OP (Goel
et al., 2005) and alcohol (Kang and Zhou, 2005) exposure, but
few studies (Brocardo et al., 2005) have addressed the neuroprotective effect of zinc in vivo.
Although acute treatment with ZnCl2 (5 mg/kg, ip) produced
some adverse effects, such as decreased AChE, GST, and GR
activity (Brocardo et al., 2005), when animals were treated for
3 days using identical ZnCl2 concentration, no changes were
observed in the biochemical parameters evaluated. These
results suggest that repeated treatment produced lower toxicity
than acute protocols. An interaction between malathion and
ZnCl2, leading to lower GST activity in the hippocampus, and
a slight zinc-induced increase in the chromatin condensation,
were the only ZnCl2 adverse effects observed. On the contrary,
ZnCl2 afforded protection, reversing the biochemical alterations induced by malathion exposure.
The hippocampus, especially in the dentate gyrus region, has
been identified as a major target site for numerous initiators of
damage caused by various forms of insult, including ischemia,
disease processes, excitotoxicity, and environmental factors
(Harry and Lefebvre d’Hellencourt, 2003). Within the hippocampal formation, the dentate gyrus is one of the few brain
structures where the production of new neurons occurs even in
the adult mammalian brain (Eriksson et al., 1998). Moreover,
the behavioral changes in depression are associated with
alterations in the hippocampal function, and treatment with
antidepressants has been shown to induce neurogenesis in the
dentate gyrus (Duman, 2004). In the present study, degenerating neurons in the dentate gyrus region were found in rats
exposed to malathion. In line with our findings, Abdel-Rahman
et al. (2004) reported that malathion caused neuronal degeneration in the hippocampus of rats exposed to this pesticide
chronically (30 days to 44.4 mg/kg of malathion by dermal
route). The malathion-induced morphological alterations in the
dentate gyrus observed in our study were attenuated in the
group exposed to malathion/ZnCl2, although ZnCl2 per se
produced a mild damage. It is possible that a higher dose of
ZnCl2 would produce a stronger damage since some studies in
the literature report that excess of zinc may be cytotoxic (for
review, see Choi and Koh, 1998). However, the morphological
findings clearly suggest that zinc has a neuroprotective effect
against malathion-induced damage.
In summary, ZnCl2 completely reversed the malathioninduced depressant-like effect in rats without changes in the
locomotor activity, which can be considered as an antidepressant effect. In addition, malathion-exposed rats exhibited an
anxiogenic-like response in the open-field that was also
reversed by ZnCl2. Regarding the biochemical analysis, the
effect of malathion was associated with no alteration in AChE
activity in the cerebral cortex, hippocampus, and blood of rats
exposed to this pesticide for 3 days. Therefore, we suggest that
the cholinergic system may not be involved in the depressantlike effect of malathion in the FST. The pro-oxidant effect of
malathion was also attenuated by ZnCl2, restoring TBARS
levels and GPx activity in the cerebral cortex and GR activity in
the hippocampus. The morphological alterations in the dentate
gyrus were attenuated by ZnCl2. Based on these findings, it is
tempting to speculate that zinc may be a useful agent in the
neuroprotection against brain damage caused by malathion.
ACKNOWLEDGMENTS
This study was supported by grants from Plano Sul Pesquisa e PósGraduac
xão (PSPPG; 520684/99-0); Coordenadoria de Aperfeic
xoamento de
Pessoal de Nı́vel Superior (CAPES), Conselho de Desenvolvimento Cientı́fico e
Tecnológico (CNPq), Brazil, and International Foundation for Science. The
authors also wish to thank Barbara Uecker who kindly revised the English
manuscript.
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