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Biokemistri
Nigerian Society for Experimental Biology
ISSN: 0795-8080
Vol. 20, Num. 2, 2008, pp. 93-98
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Biokemistri, Vol. 20, No. 2, Dec, 2008, pp. 93-98
Oxidative stress
and superoxide dismutase activity in brain of rats fed with diet containing permethrin
Olawale
Otitoju1, Ikechukwu N.
E. Onwurah2*, Grace T. O. Otitoju3
and Chidiebere E.
Ugwu4
1Department of Biochemistry, Faculty of
Basic Medical Sciences, University of Uyo, Uyo, Nigeria
2 Pollution Control and Biotechnology Unit,
Department of Biochemistry, University of Nigeria, Nsukka, Enugu State, Nigeria
3Department of Food
Science, Nutrition and Dietetics, University of Nigeria, Nsukka, Enugu State, Nigeria
4Department of Biochemistry, Kogi State University, Anyigba, Nigeria
*To
whom correspondence should be addressed. E-mail:iyknuelo@yahoo.com, Tel: 08034006429
Received 20 September 2008
Code Number: bk08014
Abstract
Many pesticides have been
reported to cause a lot of health problems in workers, users and non-target
organisms in the environment. Permethrin
containing insect pesticide has been reported to be toxic to non-target
organisms. However, the underlying mechanism involved in the toxicity is not
well understood. The present study was envisaged to investigate the possible
role of oxidative stress in permethrin neurotoxicity
and to evaluate the protective effect of superoxide dismutase (SOD) activity in
brain homogenates of Wistar rats. Oxidative stress measured as thiobarbituric
acid reacting substances (TBARS) was found to significantly increase (p <
0.05) in all the experimental groups compared with their parallel controls.
Concomitantly, the activity of SOD was found to decrease or increase
significantly (p < 0.05) in the experimental groups compared with
their controls. Our result also showed that activity of SOD was aged and
concentration dependent. Hence, the newly weaned rats appear to be more
susceptible to the pesticide contaminated diet because the SOD activity
decreased more in the brain homogenates compared with the middle aged rats or aged
rats. Observed aggressive behaviour was noticed in the pesticide exposed rats,
hence a possible neurobehavioral effect. The result demonstrated that the
pyrethroid insect powder exerts its toxic effect by promoting oxidative stress
in the brain and this may affect normal brain functioning and growth.
Keywords: pesticides, reactive oxygen species,
superoxide dismutase, lipid peroxidation
INTRODUCTION
Pesticide
use in developing nations like Africa has increased dramatically in recent
times the adverse effects in humans and other non-target organisms1.
Although relative to global utilization of pesticides, African pesticides use
may be considered as low, but the ways in which they are being used are causing
serious environmental and health problems. In most developing nations safety
equipment is rarely used and in some cases completely lacking, storage methods
are unsafe and the instructions for pesticide use are not always understood
since most farmers or pesticide users are uneducated hence, increased risk of
exposure.
The
adverse effect of some pesticides has necessitated legislation in most
developed countries against potentially damaging ones2. However, in
most developing nations, regulatory laws governing pesticide production and
utilization are almost completely lacking or where available these laws are
poorly implemented thereby having a far-reaching effect on the ecosystem3.
The
use of pesticides has been widely expanded as a consequence of increased
availability and improved prices4. However, the benefits of
pesticides use must be balanced in regards to environmental degradation, worker
safety and public health. Environmental contamination of pesticide is a threat
to life and the hazard goes beyond the farming community because pesticide
residues are found in the food chain, soil and water supplies.
Numerous
pesticides such as paraquat, DDT, PCB, Arochlor® etc have been used as model
factors inducing oxidative stress both in-vivo and in vitro5,6.
The conversion of pesticides to free radicals or via the formation of
superoxide radical is a by-product of their metabolism and this is regarded as
one of the basic mechanisms of tissue damage. Several studies have pointed out
the risk of carcinogenic effect, neurological actions and brain damage in
living organisms exposed to various concentrations of xenobiotics in the
environment4. In other to ameliorate these damages, organisms have
evolved mechanisms to control the amount of hydroxyl and superoxide radicals
generated. These fragments are quickly scavenged by natural protective
molecules in the cells called antioxidants7. Antioxidants are
intimately involved in the prevention of cellular damage the common pathway
for cancer, aging and a variety of diseases4. They safely interact
with free radicals and terminate the chain reactions before vital molecules are
damaged, antioxidants could be enzymatic or non- enzymatic, the latter include
glutathion reductase, glutathion-S-transferase, glutathion peroxidase,
superoxide dismutase (SOD) and catalase8.
SOD
is an important enzyme family in living cells for maintaining normal
physiological conditions and for coping with stress. The action of SOD
therefore is to protect the biological integrity of the cells and tissues
against harmful effects of superoxide free radical9. Humans and
other non-target organisms are sensitive to a great number of pollutants in the
environment such as pesticides and heavy metal. Bioaccumulation of these
pollutants in the biological system may pose a serious challenge to public
health, hence the aim of this study.
MATERIALS
AND METHODS
Test
samples:
The
pesticide used for this experiment was permethrin, formulated as Rambo insect
powder. It is a product of Gogoni Co. limited Nigeria.
Treatment
formulation
Commercial
animal feed was contaminated with the pesticide to give the formulation of 1%,
5% or 10% (w\w) contamination (Table 1). The control groups were not given
contaminated diet.
Table
1: Formulation of experimental diet
Formulation
control |
Pesticide
qty (g) |
Weight of feed (g) |
Total
(g) |
1% |
1 |
99 |
100 |
5% |
5 |
95 |
100 |
10% |
10 |
90 |
100 |
Lipid
Peroxidation
Lipid
peroxidation was assayed as thiobabituric acid reacting substances (TBARS)
using the method described by Wallin et al10. The differences
in absorbance at 350 nm and 600 nm were read in Sp 500 spectrophotometer
against the blank.
Superoxide
Dismutase Assay
An
indirect method of inhibiting auto-oxidation of epinephrine to its adrenochrome
was used to assay SOD activity in the blood plasma11. The
auto-oxidation was monitored in a Spectrophotometer (Sp 500) at 480nm every 30
secs for 5min. A graph of absorbance against time was plotted for each
absorbance, and the initial rate of auto-oxidation calculated. One unit of SOD
activity was defined as the concentration of the enzyme (mg protein/ml) in the
plasma that caused 50% reduction in the auto-oxidation of epinephrine12.
Statistical
analysis
Mean
values (±SD) of replicate experiment with quadruplet sampling
(N=2x4) were taken for each analysis. Significantly different results were
established by one-way ANOVA and differences between groups, and concentrations
were determined by DUNCAN multiple range tests21. The accepted level
of significance was p<0.05.
RESULTS
Inhibition
study on the autoxidation of epinephrine on brain homogenates of rats exposed
to various concentrations of pesticides is shown in Table 2. The result showed
that percent inhibition of SOD in the brain was significantly different (p<0.05)
within the exposed groups when compound with their parallel control groups. The
trend was such that the newly
weaned rat(NWR) groups showed marked decrease in percent
inhibition while the middle
aged rats(MAR) and aged rat (AR)groups showed significant increase when compared with
their control groups.
SOD
activity result (Table 3) showed that in the NWR group, there was a significant
decrease (p<0.05) in activity but MAR and AR groups showed
significant induction in SOD activity compared with their respective controls.
Lipid
peroxidation value was determined as thiobarbituric acid reacting substances
(TBARS). The result showed that the levels of liquid peroxidation increased
significantly (p<0.05) in all the pesticide exposed groups when
compared with their controls (Table 2).
Table 2: Rate of Autoxidation
of Epinephrine in Rats Exposed To Insecticide-Contaminated Diet
Auto-oxidation
mixture |
Autoxidation
rate
(Unit/min)
|
% inhibition |
AM
+ 1.0ml distilled water |
0.0035
± 0.00005 |
- |
AM
+ 1.0ml BH NWR 1 % |
0.0073
± 0.00001 |
212.08 ± 3.51 |
AM
+ 1.0ml BH NWR 5 % |
0.0108
± 0.00005 |
311.64 ± 3.07 |
AM
+ 1.0ml BH NWR 10 % |
0.0121
± 0.00000 |
349.37 ± 6.51 |
AM
+ 1.0ml BH NWR Control |
0.0224
± 0.00020 |
646.64 ± 18.07 |
AM
+ 1.0ml BH MAR 1 % |
0.0122
± 0.00060 |
352.02 ± 10.84 |
AM
+ 1.0ml BH MAR 5 % |
0.0164
± 0.00230 |
474.50 ± 59.79 |
AM
+ 1.0ml BH MAR 10 % |
0.0150
± 0.00010 |
434.92 ± 9.21 |
AM
+ 1.0ml BH MAR Control |
0.0036
± 0.00005 |
103.15 ± 1.56 |
AM
+ 1.0ml BH AR 1 % |
0.0039
± 0.00005 |
110.00 ± 1.43 |
AM
+ 1.0ml BH AR 5 % |
0.0236
± 0.00030 |
684.33 ± 18.62 |
AM
+ 1.0ml BH AR 10 % |
0.0265
± 0.00025 |
766.93 ± 18.36 |
AM
+ 1.0ml BH AR Control |
0.0125
± 0.00005 |
360.97 ± 6.67 |
Table 3: SOD
activity and lipid peroxidation products in brain homogenates of rats exposed o
insecticide-contaminated diet
GROUP |
SOD
activity
(Unit
/ ml)
|
Lipid peroxidation
(nMol / ml)
|
AM
+ 1.0ml BH NWR 1 % |
4.24
± 0.07 |
2.85
± 0.15 |
AM
+ 1.0ml BH NWR 5 % |
6.23
± 0.06 |
5.13
± 0.13 |
AM
+ 1.0ml BH NWR 10 % |
6.99
± 0.13 |
6.10
± 0.10 |
AM
+ 1.0ml BH NWR Control |
12.93
± 0.36 |
0.53
± 0.03 |
AM
+ 1.0ml BH MAR 1 % |
7.04
± 0.22 |
5.60
± o.60 |
AM
+ 1.0ml BH MAR 5 % |
9.49
± 1.20 |
5.59
± 0.09 |
AM
+ 1.0ml BH MAR 10 % |
8.69
± 0.19 |
5.43
± 0.58 |
AM
+ 1.0ml BH MAR Control |
2.06
± 0.03 |
1.25
± 0.25 |
AM
+ 1.0ml BH AR 1 % |
2.20
± 0.03 |
2.43
± 0.43 |
AM
+ 1.0ml BH AR 5 % |
13.69
± 0.38 |
11.83
± 0.18 |
AM
+ 1.0ml BH AR 10 % |
15.34
± 0.37 |
9.25
± 0.25 |
AM
+ 1.0ml BH AR Control |
7.22
± 0.13 |
1.38
± 0.38 |
DISCUSSION
The
use of pesticides in field and domestic pest control programs seems to have
produced many physiological biochemical and behavioural changes in man and
other non-target organism by influencing both the activities of many enzymes
and other cellular processes. All pesticides must be toxic or poisonous to be
effective against the pest they are intended to control they are also
potentially hazardous to humans and pets. During application they may
contaminate food or bioaccumulate in crops or in food chain causing liver
damage or become carcinogenic after uptake1, 13.
The
brain is an important part of the biological system whose function helps to
regulate other parts of the body. However, any damage or form of stress
experienced in this part of the body may have serious impact on the entire
organism.Many studies have shown that the mechanism of pesticide action in
animals is associated with the production of reactive oxygen species (ROS)1.
This study showed that lipid peroxidation activity increased significantly in
the brain homogenate of rats. This increase may be associated with possible
damage to the brain cells. The increase in lipid peroxidation was found to be
concentration dependent. This is in agreement with earlier work by Gangadaharam
et al.14, who reported increased lipid peroxidation in
incubated goats sperm cells. A similar result was also obtained by
Latchoumycandane et al15, who reported an increase in lipid
peroxidation in the testes of rats exposed to different concentration of
metoxyclor and showed that the level of lipid peroxidation was age-dependent as
NWR group showed decrease activity of SOD compared to their parallel control.
Organisms have evolved mechanism to counteract the effect of radicals generated
in the biological membrane. This
mechanism involves antioxidant system such as glutathione reductase, glutathione
peroxidase, superoxide dismutase(SOD) e.t.c. The function of antioxidant
systems is to modify the highly reactive oxygen species to form less reactive
intermediate which no longer pose a threat to the cell8.
However,
there must be a balance between oxidation and antioxidants level in the system
for healthy biological integrity to be maintained. Oxidants such as superoxide
anions (O.2) Hydroxyl radical (HO.) may
attack the membranes of the brain cells thereby causing oxidative stress our
observation has shown that (SOD) activity in brain homogenate of NWR group
decreased significantly compared with parallel control group this decrease may
be as a result of imbalance between oxidants and antioxidants level in favour
of the oxidants (Rambo pesticides). SOD is considered to be one of the most
active enzymes whose activity is sufficient for dismutation of superoxide
anions produced during oxidative stress in cells16. The MAR and AR
groups showed increased SOD activity compared with the control groups this
increase in SOD activity may suggest a possible survival mechanism for the
organism in order to reduce possible neurobehavioural effects such as
aggression and body tremor.
Although, metabolism in the brain has been
associated with ROS generation17, significant Increase compared with
the control group
may be associated with permethrin
containing pesticide poisoning. Oxidative metabolism of xenobiotics or endogenous
compounds is the most important source for ROS18. The control of ROS
by antioxidant defense systems appears to maintain low concentrations rather
than complete elimination. Oxidative stress occurs in a cell or tissue when the
concentration of ROS generated exceeds the antioxidants capability of that cell19.
Hence we suggest that NWR group experiences oxidative stress.
Permethrin,
a pyrethroid is used in pest control in agriculture and public health programs.
This study has shown that permethrin pesticide formulation could have negative
health impact on the brain and nervous system of humans as handlers and
producers, therefore adequate precaution must be taken especially among the
children population. Soderlund et al.6 has reported that
neurotoxin effect of permethrin include tremors in coordination, hyperactivity,
increase activity like chewing, aggressive behaviours, resistance to been
capture and disruption in learning. Similar observations were recorded in this
study in the entire group 1% 5% or 10% irrespective of age. Although SOD
activity was high in MAR and AR some of these behavioral effects we noticed but
not as high as in NWR groups.
However,
increase in lipid peroxidation has been reported in several cases of toxicity
induced by ethanol, heavy metals and some xenobiotics20. The
increase in lipid per oxidation products observed in brain homogenates of the
exposed rats might be associated with toxicity and tissue damage. This may
depend on a lot of factors such as age, sex and concentration. Children exposed
to insecticide contaminated diet or environment may be at higher health risk in
that their internal organs and tissues are still developing. They also have
higher feeding metabolic rate hence, increased the generation of oxygen free
radicals which may attack or damage cell membranes. The overall consequence is
breakdown in membrane integrity and membrane dependent functions.
However,
due to selective vulnerability of neurons, the brain contain in addition to
antioxidant defense, the blood brain barriers which controls the entry of many
types of solutes from general circulation to the cerebral parenchyma. It is
possible that the blood brain barrier of the NWR group is not well forms or may
be the Rambo pesticide is lipophilic thereby crossing the blood brain barriers
into the brain to elicit peroxidative activity on the polyunsaturated fatty
acids of the neuronal membranes. This may be a possible mechanism by which permethrin
elicits its neuronal or behavioral effects especially the young ones or
neonates.
We
therefore suggest that this pesticide be kept out of the reach of children or
from pregnant mothers. Although the MAR and AR groups showed significant
increase in SOD activity, within the experimental regime possible exposure may
be toxic to non-target organisms irrespective of their age or sex differential.
Proper disposal method should be adopted in disposing the carcasses of rats,
cockroaches and other pests treated with Rambo so that non-target organisms
(domestic fowls, cats and dogs) may not be affected as the residues flow
through the food chain.
Developing
nations must learn to balance pesticide use with environmental degradation as
well as health safety especially in humans and other non-target organisms.
Indiscriminate short term exposure may not have immediate obvious health effect
but in the long-run the health impact if not taken serious by producers,
marketers and users most especially may be very heinous.
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© 2008 Nigerian Society for
Experimental Biology
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