Indian Journal of Experimental Biology
Vol. 44, July 2006, pp. 580-583
Erythrocyte antioxidant enzymes in toxicological evaluation of commonly used
organophosphate pesticides
Mohan Singh, Rajat Sandhir & Ravi Kiran*
Department of Biochemistry, Panjab University, Chandigarh, 160 014, India
Received 18 November 2005; revised 13 April 2006
Erythrocytes are excellent models for the study of interactions of xenobiotics with biomembranes. Present work is
designed to study the in vitro effects of some organophosphates (ethion, chlorpyrifos, dimethoate and monocrotophos) on rat
erythrocytes. Treatment of erythrocytes with organophosphates resulted in decreased erythrocyte glucose-6-phosphate
dehydrogenase (G-6-PD) activity, whereas activities of glutathione-s-transferase (GST) and glutathione reductase (GR) were
increased. Reduced Glutathione (GSH) content of RBCs was decreased after treatment with the pesticides. Increased
activities of GST and GR were due to induction of natural defense mechanism of erythrocytes against the toxicity of the
pesticides. Membrane bound enzymes like acetylcholinesterase (AChE), Na+-K+-ATPase and Ca2+-ATPase were also
inhibited. Altered activities of these enzymes along with decreased GSH content indicate increased oxidative stress in
erythrocytes after treatment with organophosphates.
Keywords: Acetylcholinesterase, Erythrocytes, Glucose-6-phosphate dehydrogenase, Glutathione reductase, Glutathiones-transferase.
Pesticides are used worldwide in agriculture in vast
amounts each year. The more commonly used
organophosphate pesticides are ethion, dimethoate,
monocrotophos and chlorpyriphos. The widespread
use of synthetic organic pesticides over decades has
led to their frequent exposure in the environment1.
Also, acute and chronic exposures of humans to
pesticides occur during their commercial production
and their application2. Synthetic pesticides are
deliberately sprayed on crops or agricultural land to
increase food production but these agrochemicals are
not very selective in producing their effects3,4. They
are toxic to many non-target species and contaminate
the environment. Putative detrimental effects on life
caused by pesticides should be known and minimized,
most especially because of the extensive
environmental distribution of these xenobiotics.
Exposure to organophosphates leads to change in
membrane permeability of erythrocytes5. To assess the
detrimental effect of commonly used pesticides, antioxidant
enzymes
like
glucose-6-phosphate
dehydrogenase (G-6-PD), glutathione-s-transferase
(GST),
glutathione
reductase
(GR),
______________
*Correspondent author
Phone: (0172) 2534133
Fax: (0172) 2541022
E-mail: [email protected]
acetylcholinesterase (AChE), Na+-K+ and Ca2+ATPases and reduced glutathione content of RBCs
have been studied in male Wistar rats.
Materials and Methods
Ethion,
dimethoate,
monocrotophos
and
chlorpyriphos (technical grade) were gifted by Punjab
Pesticides Industrial Co-operative Society, Kharar
(Punjab). Most of the biochemicals used in the study
were purchased from Sigma Chemical Company, St.
Louis, MO, USA or Sisco Research Laboratories Pvt
Ltd., Bombay, India. All other chemicals used were of
analytical grade obtained locally.
Male albino rats (Wistar Strain) weighing 105120g were procured from the Central Animal House
of the University. The blood was drawn from the tail
vein of the animals. Blood samples from 6 animals
were used to test the action of each pesticide and
same number of matched controls were used. All the
experiments performed were according to guidelines
for use and care of laboratory animals and were
approved by the ethical committee of the University.
Preparation
of
erythrocyte
hemolysate––
Erythrocyte hemolysate was prepared by the method
of Lohr and Waller6. Blood (0.5 ml) was taken from
the tail vein of rat into a graduated centrifuge tube
containing 0.5 ml of anticoagulant. Tube was
centrifuged at 1000 g, the buffy coat was removed
SINGH et al.: ANTIOXIDANT ENZYMES IN TOXICOLOGICAL EVALUATION
and the cells were washed twice with 5ml
physiological saline. The sediment obtained was
suspended in 1ml physiological saline and was
properly mixed by rotation; 1ml of erythrocyte
suspension was pipetted into a centrifuge tube and
was mixed with 1ml double distilled water, 0.7 ml
triethanolamine buffer (pH 7.5) and 0.3 ml digitonin
solution (1g/100ml). The mixture was allowed to
stand for 15 min in a refrigerator at 4°C and was then
centrifuged at 1000 g for 15 min. The insoluble
material was discarded.
Glucose-6-phosphate dehydrogenase (G-6-PD),
glutathione-s-transferase (GST) and glutathione
reductase (GR) were assayed by the standard
methods. Reduced Glutathione (GSH) content was
measured by the method of Jollow et al7.
Erythrocyte membranes were prepared by
hemolysing the erythrocytes in distilled water and
centrifuging at 20,000 g. Post hemolytic residue was
washed twice with 1 mM Tris EDTA (pH 7.4).
Erythrocyte membranes were kept frozen at -80°C.
Acetylcholinesterase(AChE)8, Na+-K+ and Ca2+ATPases9 activities were assayed in the membrane
preparation. Protein content was measured by the
method of Lowry et al10.
Statistical analysis––Data presented in this study
was statistically evaluated by Student’s t test.
Differences between two comparable sets of results
were considered significant when P≤0.05.
Results and Discussion
AChE activity is useful as an indicator of the
inhibition by organophosphates and in vitro treatment
of organophosphates in the present study resulted in
significant decrease in the erythrocyte AChE activity.
Treatment of dimethoate, chlorpyrifos, ethion and
monocrotophos resulted in approximately 90%
decrease in AChE activity in each case (Table 1). The
decrease was found to be statistically significant
(P≤0.001).The inhibition in the activity of AChE in
vitro may be due to direct interaction of pesticide with
the enzyme as is known in the case of ethion,
chlorpyrifos, dimethoate and monocrotophos. In vitro
inhibition of AChE by pesticides is well
documented11-13. There are cases of in vitro inhibition
of AChE from four marine species by
organophosphates14.
Results of in vitro treatment of various
organophosphates on Na+-K+-ATPase have been
presented in Table 1. In vitro treatment of rat
erythrocytes with all the four organophosphates resulted
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INDIAN J EXP BIOL, JULY 2006
in decreased activity of Na+-K+-ATPase. A decrease
ranging from 12 to 75% in the enzyme activity was
observed in case of different pesticides. The decrease
was found to be statistically significant for
dimethoate, chlorpyrifos and ethion. Earlier work
from our laboratory showed that in vivo
administration of various pesticides decreases the
activity of Mg2+-ATPase15,16.
In the case of Ca2+-ATPase 22 to 57 % decrease
was observed in the enzyme activity by dimethoate,
monocrotophos, chlorpyrifos and ethion treatment
(Table 1). All the results were found to be statistically
significant (P≤0.001). Yang et al.17 reported the
inhibition of Ca2+-ATPase in the muscle cells
following dimethoate exposure. It has been shown
that Ca2+-ATPase of human erythrocytes is inhibited
with various compounds of toxicological significance
mostly chlorides18.
The results of various organophosphates treatment
on erythrocyte G-6-PD activity have been presented
in Table 1. Dimethoate, ethion, chlorpyrifos and
monocrotophos treatment resulted in 26 to 40 %
decrease in G-6-PD activity. The decrease was found
to be statistically significant (P≤0.001). G-6-PD is an
important enzyme of hexose monophosphate shunt
and its function in the mature RBCs is to generate
NADPH, which is required for the conversion of
oxidized glutathione to reduced glutathione, that in
turn is necessary for membrane integrity of
erythrocyte membranes19. This might be the possible
reason for the increased fragility of erythrocytes upon
treatment with different organophosphates as reported
during the earlier work done in our laboratory that in
vivo administration of various pesticides results in
decrease in the activity of G-6-PD15, 16.
The data presented in Table 1 demonstrated the
effect of various organophosphates on GR activity of
rat erythrocyte hemolysate. It was observed that the
administration of dimethaote, chlorpyrifos, ethion and
monocrotophos resulted in 16 to 46% increased
activity of erythrocytes GR. The increase was
statistically significant, for dimethoate, chlorpyrifos
and ethion. Thus increased activity of this enzyme
appears to be defense against pesticide toxicity in
erythrocytes. Earlier work showed that in vivo
administration of various pesticides results in
decrease in the activity of GR15, 16.
The results presented in Table 1 depict the effect of
the in vitro treatment of various organophosphates on
erythrocyte GST activity. It is clear from the data that
[
treatment with all the pesticides results in increased
GST activities. The increase was statistically
significant in the case of chlorpyrifos and dimethoate.
GSH and glutathione dependent enzyme systems
provide major protection against toxic agents20.
Among them GST is the most important phase II drug
metabolizing enzyme21.
Effects
of
administration
of
various
organophosphates on erythrocyte GSH content has
been presented in Table 1. In vitro treatment of
erythrocytes with dimethoate, chlorpyrifos, ethion and
monocrotophos resulted in decreased GSH content in
all the cases. Decreased GSH content might be
attributed to the decrease in G-6-PD activity of the
erythrocytes observed in the present study. Decrease
G-6-PD activity results in the decreased synthesis of
NADPH, which in turn results in the low levels of
GSH. It is well documented in literature that in vivo
administration of various xenobiotics results in
decrease in GSH content of erythrocytes22-23, but not
many reports are available about in vitro effects of
pesticide on glutathione content of erythrocytes.
Based on our results it is concluded that the
organophosphates tested exert differential effect on
the activity of antioxidant enzymes, glutathione and
membrane bound enzymes which may be useful in
their toxicological evaluation.
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