Chlorpyrifos-Induced Neuro-Oxidative Damage in Bee

Shafiq-ur-Rehman1,2, Shaheen Rehman1

& M. I. S. Waliullah2
1

Laboratory of Environmental Health and Toxicology, Division of

Environmental Sciences
2
Division of Entomology, S. K. University of Agricultural Sciences
and Technology of Kashmir, Srinagar, J&K, India
Correspondence and requests for materials should be addressed
to Shafiq-ur-Rehman (shafiqurrehman11@yahoo.com)
Received 27 October 2011 / Received in revised form 28 December 2011
Accepted 2 March 2012
DOI 10.1007/s13530-012-0114-9
The Korean Society of Environmental Risk Assessment and
Health Science and Springer 2012

Abstract
Complaints regarding excessive use of chlorpyrifos
and consequent poisoning to non target pollinators
have increased throughout the world. Loss of honeybee has been observed in the Kashmir valley of India,
too. The lipid peroxidation, known to cause oxidative
stress/damage, was found to be increased in nervous
system of Apis mellifera exposed to chlorpyrifos. Further exacerbation of chlorpyrifos-induced oxidative
damage was observed in OH-generated H2O2 system. The OH radical scavenger, DMSO, mitigated
the initiation of lipid peroxidation mediated by either
H2O2 or CPF. The DMSO also repressed the combined oxidative effect of H2O2 and chlorpyrifos on
the nervous system. Findings suggest that oxidative
stress/damage caused by chlorpyrifos in honeybee
nervous system accomplished the toxic OH build
up, which successively provides a possible mechanism for chlorpyrifos neurotoxicity and its mitigation
by OH scavenging biomolecules. Elevated malondialdehyde may possibly serve as an indicator of neurooxidative stress in bees and their loss due to chlorpyrifos-contaminated environment.
Keywords: Apis mellifera, Chlorpyriphos neurotoxicity, Dimethylsulfoxide; Honeybee, Hydrogen peroxide, Hydroxyl radical, Lipid peroxidation, Malondialdehyde, Oxidative damage

Introduction
In modern agriculture, pesticides play an important
role by providing dependable, persistent and relatively

complete control against harmful pests with less expense and efforts. They have, no doubt, increased crop
yields by killing different types of pests that are known
to cause substantial or total crop damages. At the same
time, these chemicals are considerably potent environmental pollutants, and they produce undesirable toxic
effects on non-target organisms, including humans1-3.
Bees can be also suffered with serious adverse effects
from toxic chemicals in their environments. In recent
years, indiscriminating evidences have shown enormous decline in bees population across U.S., France,
Germany, and other parts of the world. Researchers in
Germany and France have indicated over 30 percent
decline of honeybee population due to insecticide application on crops4. These investigations highlighted
that 80 per cent of fruits and vegetables, requiring pollination, may not make it to the market. The reports
further emphasized that decline of pollinators would
upset the world economy by loss around 350 million
U.S. dollars per annum. Organophosphate (OP) insecticides, accounting for up to 50% of the global insecticidal use, were predominately related to incidents of
poisoning bees5-7. The emerging evidences revealed
that CPF was highly toxic to honeybees, besides other
non target species such as birds, fish and even aquatic
invertebrates1,8,9.
Chlorpyrifos [CPF, Molecular formula: C9H11Cl3
NO 3PS, Molecular mass: 350.6, Chemical Name
(IUPAC): diethoxy-sulfanylidene-(3, 5, 6-trichloropyridin-2-yl) oxy-phosphorane, or chlorpryphosethyl
(O,O-diethyl O-[3, 5, 6-trichloro-2-pyridyl] phosphorothionate)], a broad spectrum chlorinated OP, is
the most widely used insecticide, largely due to its
greater stability, persistence, and deep effectiveness
against a wide range of plant-eating insect pests. However, the broad-spectrum insecticides common use (or
abuse) were often, as toxic to beneficial insects as,
they are to the target species10. CPF was metabolically
activated by oxidative desulfuration to a short-lived
metabolite CPF oxon, which inhibited the acetylcholinesterase (AChE) through phosphorylation of its
serine site11. Virtually, all types of OPs were considered to have a common mechanism of toxicity, whereby the initial step in a cascade of reactions was to inhibit AChE12-14. However, evidences against a common
mechanism of toxicity were mounting. Fundamentally,
AChE inhibition was non-specific and affected the
whole body systems through the cholinergic, muscari-

Neuro-Oxidative Damage of Chlorpyriphos

MDA (mol formed/30 min)

5.5
Regression analysis: MDA versus CPF
=1.58985+1.88404 CPF
MDA=
=0.991
Multiple R-Sq=
1.2

4.5

1.6

0.8

3.5

radical damage30. Because, ferrous iron was known to

interact with endogenous H2O2 to generate extremely
reactive toxic hydroxyl radical (OH), which could
react at high rate with most molecules in the cell including DNA, proteins, lipids, amino acids, and could damage them34. The OH, as one of the most destructive
ROS, could cause indiscriminate toxicity and cell death. The OH was also shown to cause significant reduction in AChE activity in the rat brain, which may
be partly due to the induction of oxidative stress35,36.
These highly reactive pro-oxidant species seemed to
put forth its dynamic role in chlorpyrifos induced neurotoxicity in terms of the oxidative stress in exposed
organisms. For these reasons, the present investigation
was undertaken to assess whether the toxic OH could
offer a mechanism in CPF-induced oxidative damage
of nervous tissues and could provide an alternate mechanism of CPF neurotoxicity (other than AChE) and its
mitigation as well.

Results
Effects of Increasing Exposure Levels of CPF
on the Formation of the Malondialdehyde
Relationships between the impact of different levels
5
Lipid peroxidation
(MDA, n mol formed/30 min)

nic and nicotinic receptor pathways. Nonetheless, the

affected systems were basically the central nervous
system and the autonomic nervous system, as well as
peripheral muscular pathways15. Furthermore, there
was worldwide concern regarding OPs involvement
in carcinogenesis, birth defects, malfunctioning immune system, neurobehavioural disorders, and neurotoxicity15-18. It seemed that unknown OP targets could
exist, and therefore, the initiation of toxicity would not
only be the inhibition of AChE, but also interaction
between OP and non-AChE targets19. These concerns
were focusing on CPFs independent neurotoxicity
from cholinergic inhibition20-22.
The reactive oxygen species (ROS) or free radicals,
which were generated during xenobiotics attack, could
indiscriminately interfere with cellular constituents and
could cause oxidative damage to lipids, proteins and
nucleic acids, and eventually could result in cell death.
The peroxidative damage to membrane lipids by free
radicals simultaneously produce thio-barbituric acid
reactive substances, such as malondialdehyde, leading
to lipid peroxidation. The role of lipid peroxidation as
indicator of oxidative damage in living tissues, received considerable attention for its potential pathophysiological events and toxicological risks from certain
metals and pesticides exposure23-28. Increasing evidences revealed association of oxidative damage in CPFinduced neurotoxicosis22,29-33. Moreover, the exacerbation of pro-oxidative manifestation of Fe2+ in nervous
tissues of bee by CPF indicated a risk of highly toxic

31

**cc
**cc

3
*c
2

0.4

Control

2.5

H2O2

CPF

CPF+H2O2

Groups
0.2

1.5
0

Chlorpyrifos (g)

Figure 1. Effect of increasing concentrations of Chlorpyrifos

on oxidative stress in nervous system. Different concentrations
of Chlorpyrifos (0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 g) were tested
for malonaldialdehyde formation as the index of lipid peroxidation to evaluate the oxidative stress in the nervous tissues
of Apis mellifera. Data were expressed as meanSE. The
Oxidative injuries showed a linear pattern of regression test as,
=1.58985+1.88404 CPF with a multiple R-Sq=
=0.991
MDA=
with the significant level (P) less than 0.0001.

Figure 2. Chlorpyrifos response with hydroxyl radical generator in nervous tissue of honeybee. Oxidative toxicity of
Chlorpyrifos (1.0 g) exposure was assessed alone or in presence of hydroxyl radical generator H2O2 (10 mM) in the Apis
mellifera nervous tissue homogenates. Malonaldialdehyde
formation was assayed to evaluate the oxidative injuries in
the nervous tissues. Data are presented as lipid peroxidation
(MDA nmol formed (meanSE) for each experiment comprising six replicate per treatment group and analyzed with
ANOVA. Pair samples t-test between control (C) or corresponding control (CC, i.e. CPF group) and the treatment groups
was performed. Asterisk indicates statistically significant differences from the control (or corresponding control), *P0.01
and **P0.001.

32

Toxicol. Environ. Health. Sci. Vol. 4(1), 30-36, 2012

Lipid peroxidation
(MDA, n mol formed/30 min)

3.5
*cc
2.5

*c
**cc

1.5

*c

Co

ntr

ol

SO
DM

O2
H2

SO
M
+D

O2
H2

F
CP

SO
M
+D
F
CP

Groups

Figure 3. Effect of hydroxyl radical scavenger on Chlorpyrifos-induced oxidative stress in nervous tissue of honeybee.
Oxidative toxicity of Chlorpyrifos (1.0 g) exposure was assessed alone or in presence of hydroxyl radical scavenger
DMSO (0.5%) in the Apis mellifera nervous system. Data are
presented as lipid peroxidation (MDA nmol formed (mean
SE) for each experiment comprising six replicate per treatment group and analyzed with ANOVA. Pair samples t-test
between control (C) or corresponding control (CC referred as
H2O2 for H2O2+DMSO or as CPF for CPF+DMSO) and the
treatment groups was performed. Histograms marked with
asterisk indicate statistically significant differences from the
control (or corresponding control), *P0.01 and **P0.001.

of CPF exposure (0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 g) and
the formation of MDA in the nervous tissues showed
=1.58985+
a linear pattern of regression test as, MDA=
=0.991, and signi1.88404 CPF with a multiple R-Sq=
ficant level less than 0.0001. The result of experimental study, displaying a trend of oxidative damage over
exposure levels with statistical topography, was represented in a regression chart (Figure 1). One-way
ANOVA demonstrated statistically significant differences in the malondialdehyde levels among different
experimental groups (P0.0001).

Enhancement of Malondialdehyde in
Nervous Tissues by CPF and Hydroxyl ROS
Generator H2O2
The results showed that the formation of malondialdehyde in nervous tissues of honeybees was significantly increased (P0.01) by H2O2. The production of
malondialdehyde was observed significantly higher
(P0.001) in the nervous tissues of honeybees, following the exposure of CPF as compared with controls. The CPF-induced significant increase (P0.001)
of malondialdehyde formation was aggravated significantly (P0.001) by the OH radical generator H2O2
(Figure 2).

Lipid peroxidation
(MDA, n mol formed/30 min)

4.9

**c

**cc

4.4

3.9

**cc

3.4

CPF

CPF+H2O2

CPF+H2O2+DMSO

Groups

Figuer 4. Function of hydroxyl radical initiator and scavenger in Chlorpyrifos neurotoxicity. Chlorpyrifos (1.0 g) exposure was evaluated for the oxidative toxicity in the Apis mellifera nervous tissue homogenates in presence of hydroxyl
radical generator H2O2 (10 mM) and scavenger DMSO (0.5%)
alone or in combination. Data are presented as lipid peroxidation (MDA nmol formed (meanSE) for each experiment
comprising six replicate per treatment group and analyzed
with ANOVA. Pair samples t-test between corresponding control (CC referred as CPF for CPF+H2O2 or as CPF+H2O2 for
CPF+H2O2+DMSO) and the treatment groups was performed.
Histograms marked with asterisk indicate statistically significant differences from the corresponding control, **P0.001.

Reduction of CPP-Induced Formation of

Malondialdehyde in Nervous Tissues by ROS
Radical Scavenger DMSO
DMSO, which reacted with lipid peroxidation process by OH free radical scavenging mechanism, reduced the formation of malondialdehyde in honeybee
nervous tissues (P0.01), in comparison with controls. The H2O2-mediated formation (P0.01) of malondialdehyde in nervous tissues of bees was significantly alleviated (P0.001) from addition of DMSO.
Performing in similar fashion, CPF induced significant
production (P0.001) of malondialdehyde in honeybee nervous tissues, and DMSO significantly suppressed it (P0.01) (Figure 3).
Involvement of OH Radical in CPF-Induced
Oxidative Damage of Apis mellifera Nervous
System
The results showed that the CPF-induced (P0.001)
malondialdehyde formation was aggravated significantly (P0.001) by the OH radical generator H2O2 in
Apis mellifera nervous system. Further observation
indicated that the OH radical scavenger, DMSO, significantly mitigated (P0.001) the exacerbated formation of the lipid peroxidation, from the combined
action of CPF and H2O2 (P0.001) (Figure 4).

Neuro-Oxidative Damage of Chlorpyriphos

Discussion
Among different pesticides, organophosphate insecticides were predominately related to incidents of poisoning bee6. Chlorpyrifos for widespread agricultural
usage, was highly toxic to birds, fish, aquatic invertebrates and honey bees1,8. The symptoms of dying bees
were typical for intoxication to CPF exposure7. Our
preliminary investigation revealed for the first time
that CPF neurotoxicity increased malondialdehyde
formation in honeybees30. Malondialdehyde, a thiobarbituric acid reactive substance (TBARS) and a marker of lipid peroxidation of damaged membrane/cell,
was the end product of oxidative stress in biological
systems, following pathophysiology or xenobiotics
toxicity. Lipid peroxidation was linked with degradation of phospholipids in the rat brain by toxic chemicals, like lead exposure23. In the present study, significant increases in malondialdehyde levels in honeybee
nervous system by different levels of CPF exposures
showed a dose dependent slope, signifying that apparent oxidative stress was taking place by the action of
the insecticide. Earlier studies with pro-oxidant system
(Fe II) also revealed the formation of lipid peroxidation
products in CPF toxicity, suggesting oxidative damage
in the honeybee nervous system30. Studies suggested
that ROS may be involved in the oxidative stress in rat
organs, following CPF toxicity38,39. Other investigations reported that toxicity caused by CPF may be due
to induction of oxidative stress in the central nervous
system22,31,33,40-44. These studies also suggested that
CPF could induce ROS production in nervous tissues
of bees, resulting in neurotoxic effects through a highly
toxic OH radical mechanism instead of cholinesterase inhibition. For this reason, we examined OH
radical generator (H2O2) and scavenger (DMSO) systems to ascertain the role of OH radical in the neurooxidative damage caused by CPF neurotoxicity in the
bees.
H2O2, a non radical derivative of oxygen, was a well
established reactive oxygen specie (ROS). It could cross
cell membranes easily, and hence could attack the
target directly45. Studies revealed that elevated formation and build-up of H2O2 within the cells often could
cause DNA damage, although H2O2 did not directly
reacted with DNA or membrane lipids34. Nevertheless,
H2O2 demonstrated its ability to be involve in the processes of oxidative stress by converting to the indiscriminately reactive OH radical, in turn causing molecular damages and cell death46. The mode of action of
H2O2 in a biological system followed OH mechanism of lipid peroxidation process, whereas highly reactive OH radicals could frequently attack biological
molecules (including membrane lipids) by abstracting

33

hydrogen from them. This represented a mechanism

of initiating the process of lipid peroxidation. The oxidative stress in a system could lead to cell damage,
which consequently could influence into lipid peroxidation. Therefore, lipid peroxidation appeared to be a
highly significant consequence of oxidative stress in
patho-toxicity events. Nervous tissues were particularly highly sensitive to oxidative damage from lipid peroxidation, due to its high oxygen consumption coupled with low levels of anti-oxidant defence system
and high membrane constituents with polyunsaturated
lipids, prone to oxidation47. In the present study, exposure of H2O2 increased the generation of malondialdehyde molecules in the nervous tissues, hinting a
dependency on OH radicals in the lipid peroxidation
process. CPF individually caused the significant formation of high level of MDA molecules in the bee
nervous tissues. Furthermore, we observed that CPF
potentiated TBARS production during H2O2 stimulated
lipid peroxidation in the nervous tissues. This showed
a mediatory role of the OH radical in the oxidative
damage in the nervous system of honeybees from
CPF toxicosis. These evidences supported our earlier
demonstration of CPF-induced lipid peroxidation,
which was aggravated by a pro-oxidant Fe II system30. The Fe2+ ions, which were present within the
biological system, reacted with H2O2 molecules to
generate a bunch of very active OH species that subsequently took part in the process of chain reactions of
the lipid peroxidation.
It was acknowledged that one possible candidate for
triggering lipid peroxidation was theOH. Suppression
of the OH formation could alleviate oxidative propagation of the critical chain reaction of the lipid peroxidation with subsequent reductions in toxic build up,
as end-products in the system. With this scheme, the
proxidative chain reactions, resulting from xenobiotic
attack, could be inhibited by the scavenger of free radicals to avert the cell damage. So, the validation ofOH
participation of CPF-mediated oxidative damage in the
honeybee nervous system was reaffirmed in the presence of DMSO, a well-established inhibitor of OH
species. Our findings, therefore, indicated that DMSO
possed an ability of significantly reducing the CPFinduced formation of malondialdehyde in the nervous
system. Moreover, the exaggerated production of the
malondialdehyde caused by CPF action during H2O2stimulated lipid peroxidation in nervous tissues of the
bee was inhibited by the OH scavenger DMSO.
Hence, the OH scavenger showed its capability of
alleviating the exaggerated TBARS production in the
nervous tissues, caused by the oxidative stress potentiated by H2O2 and CPF toxicity. This apparently revealed the involvement of OH in CPF toxicity, as a

34

Toxicol. Environ. Health. Sci. Vol. 4(1), 30-36, 2012

contributory factor to the oxidative stress in the bee

nervous system from the lipid peroxidation definitely.
Therefore, biological scavengers, competently inhibiting the OH radicals, could mitigate the lipid peroxidative damage of the nervous system following CPF
toxicity.

Conclusions
The increased presence of highly poisonous CPF
insecticide in the agricultural and contiguous environment placed entire pollinators population into surviving edge. The observed oxidative stress in the honeybee
nervous system by CPF toxicity was measured to be
elevated from the increased levels of malondialdehyde.
Further evidences obtained from the present observations with ROS generator and inhibitor systems supported the involvement of most damaging OH radicals by initiating the lipid peroxidation process in the
nervous system of bee from CPF toxicity. The study
suggested that the elevation of malondialdehyde levels
in nervous tissues could be considered as an indicator
of CPF poisoning in honeybees and their population
loss. The anti-oxidative intervention strategies could
also play a potential role for ameliorating the CPF
neurotoxicity.

Materials and Methods

Chemicals
Chlorpyrifos-ethyl [CPF; O,O-diethyl O-(3, 5, 6-trichloro-2-pyridyl) phosphorothionate] and 2-thiobarbituric acid were from Sigma (USA). Other chemicals
and reagents used in this study were of purified grade.
Dissection of Nervous System and Tissue
Preparation
Apis mellifera was ventrally dissected out for nervous system30. The nervous system tissues were pooled
in a Petri dish on an ice bath. Nervous ganglion was
homogenized in chilled 0.10 M KCl, using Teflon pestle to obtain a 3% w/v homogenate for in vitro studies.
Formation of Malondialdehyde in Nervous
Tissues Homogenate Exposed to Increasing
Concentration of CPF
The observation of formation of malondialdehyde
in the nervous system of Apis mellifera was performed
with different exposure levels of CPF (ranging 0.2-2.0
g) in tissue homogenates. For the control, experiment
was performed without addition of the CPF.

Formation of Malondialdehyde in Nervous

Tissues Exposed to CPF under ROS
(H2O2 System)
The toxic competency of CPF, as provoker of oxidative stress, was evaluated under pro-oxidation environment in the Apis mellifera nervous system. Dose of
CPF (1 g) was considered to be a minimal dose to
reach the nervous ganglia30. For interaction studies, the
tissue homogenates were treated with H2O2 (10 mM),
CPF (1 g), CPF (1 g) plus H2O2 (10 mM), or without
any addition of stimulator. The malondialdehyde formation was assayed, as described below.
Effect CPF-Induced Formation of
Malondialdehyde in Nervous Tissues under
ROS Radical Scavenger (DMSO System)
Effect of anti-oxidant DMSO was tested for malondialdehyde formation in the nervous tissues of Apis
mellifera exposed to CPF. The nervous ganglia homogenates were incubated in presence of DMSO (0.5%)
alone or in combination with CPF (1 g). The malondialdehyde formation was assayed, as described below.
Involvement of OH Radical in CPF-Induced
Oxidative Damage of Apis mellifera Nervous
System
The validation of involvement of highly toxic OH
ROS in the oxidative damage of the nervous system
of Apis mellifera was examined in the following experiments: the homogenates consisted H2O2 (10 mM)
alone, or H2O2 (10 mM) and DMSO (0.5%) combination, CPF (1 g) and H2O2 (10 mM) combination, or
CPF (1 g), H2O2 (10 mM) and DMSO (0.5%) combination. Experimental groups [H2O2 (10 mM); CPF (1
g) plus H2O2 (10 mM)] were considered, as the corresponding controls. The malondialdehyde formation
was assayed accordingly, as described below.
Lipid Peroxidation (Molecular Formation of
Malondialdehyde) Assay
The quantitative assay of malondialdehyde formation
was performed by the 2-thiobarbituric acid (TBA)
reaction method, as described by Shafiq-ur-Rehman23
and Shafiq-ur-Rehman et al.24. For the purpose, 1 mL
of nervous tissue homogenate of Apis mellifera in a
test tube was aerobically incubated at 37
C in a water
bath-cum-metabolic shaker (180 strokes/min of 2 cm
amplitude) for 2 h. The test tube was immediately
cooled with tap water, and 1 mL cold 10% w/v trichloroacetic acid was added. The solution was thoroughly
mixed on vortex-mixer and centrifuged at 2000 rpm
for 10 min. One mL of supernatant was transferred to
another test tube and allowed to react with an equal
volume of 0.67% w/v TBA for 10 min in a boiling

Neuro-Oxidative Damage of Chlorpyriphos

water bath. The tube was cooled with tap water, and
the mixture was diluted with 1 mL of double distilled
water. The TBA reactive substance (TBARS), malondialdehyde, thus formed was measured at 535 nm. The
result was expressed as n mol of malondialdehyde
formed per 30 min; the molecular extension coefficient
of malondialdehyde expressed as E535=1.56105 37.

Statistical Analysis
The statistical analysis and presentation were performed with statistical software (SPSS version 11 or
Minitab version 13). Values were expressed as mean+
standard error (SE). Relationships between CPF exposure and MDA formation levels were tested by linear
regression. Significant differences among groups were
evaluated using one-way analysis of variance (ANOVA)
followed by Turkeys pair-wise multiple comparisons
post hoc test. Comparison between control/corresponding control and treatment groups were analysed by
Students paired t-test. Values of P less than 0.05 (or
otherwise mentioned) were considered statistically
significant.

Acknowledgements
I am highly thankful to Prof. Anwar Alam, ViceChancellor, and Prof. A. R. Trag, Director Research
(now Vice-Chancellor of the Islamic University, Srinagar) for their continuous encouragement and support.
The work was carried out from our own available resources and facilities at the university.

References
1. Kidd, H. & James, D. R. in The Agrochemicals Handbook, 3rd Edn. (Royal Society of Chemistry Information Services, Cambridge, 1991).
2. Ellenhorn, M. J., Schonwald, S., Ordog, G. & Wassergerger, J. in Ellenhorns medical toxicology: diagnosis and treatment of human poisoning. (Williams &
Wilkins, Maryland, 1997).
3. Jalali, N., Pajoumand, A., Abdollahi, M. & Shadnia, S.
Epidemiological survey of poisoning mortality in Tehran during 1997-1998. Toxicol. Letts.116, 84 (2000).
4. Yadav, R. http://www.dw.de/dw/article/0,,4188187,
00.html (2009).
5. Casida, J. E. & Quistad, G. B. Organophosphate toxicology: safety aspects of non-acetylcholinesterase
secondary targets. Chem. Res. Toxicol. 17, 983-998
(2004).
6. Fletcher, M. & Barnett, L. Bee pesticide poisoning
incidents in the United Kingdom. Bull. Insectol. 56,
141-145 (2003).
7. Kamler, F., Tit e ra, D., Pi s kulov, J., Hajslov, J. &

35

Mastovsk, K. Intoxication of honeybees on chemical

treated winter rape: Problem of its verification. Bull.
Insectol. 56, 125-127 (2003).
8. United States Environmental Protection Agency
(USEPA). Pesticide fact sheet number 37, Chlorpyrifos. Office of Pesticides and Toxic Substances, Washington DC (1984).
9. Reigart, J. R. & Roberts, J. R. in Recognition and
management of pesticide poisonings, 5th Edn (United
States Environmental Protection Agency, Office of
Prevention, Pesticides and Toxic Substances, Office
of Pesticide Programs, US Government Printing Office,
Washington DC, 1999).
10. Johansen, C. A. & Mayer, D. F. in Pollinator protection: a bee and pesticide handbook (Wicwas Press,
Connecticut, 1990).
11. Eto, M. in Organic and biological chemistry (ed Eto,
M.) 387 (CRC Press, Cleveland, 1974).
12. McDonough, J. H. & Shih, T. M. Neuropharmacological mechanisms of nerve agent-induced seizure and
neuropathology. Neurosci. Biobehav. Rev. 21, 559579 (1997).
13. Mileson, B. E. et al. Common mechanism of toxicity:
a case study of organophosphorous pesticides. Toxicol.
Sci. 41, 8-20 (1998).
14. Pope, C. N. Organophosphorus pesticides: do they all
have the same mechanism of toxicity? J. Toxicol.
Environ. Health 2, 161-181 (1999).
15. The BMA. Guide to Pesticides, Chemical and Health.
(Edward Arnold, London, 1992).
16. Robbins, C. in Poison Harvest: A Consumers Guide
to Pesticide Use and Abuse (Victor Gollancz Ltd.,
London, 1991).
17. Rauh, V. A. et al. Impact of prenatal chlorpyrifos
exposure on neurodevelopment in the first 3 years of
life among inner-city children. Pediatrics 118, 18451859 (2006).
18. Whyatt, R. M. et al. Renatal insecticide exposures
and birth weight and length among an urban minority
cohort. Environ. Health Perspect. 112, 1125-1132
(2004).
19. Duysen, E. G. et al. Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to
vx lethality. J. Pharmacol. Exp. Therap. 299, 528-535
(2001).
20. Slotkin, T. A. Cholinergic systems in brain development and disruption by neurotoxicants: nicotine, environmental tobacco smoke, organophosphates. Toxicol.
Appl. Pharmacol. 198, 132-151 (2004).
21. Slotkin, T. A. in Toxicity of Organophosphate and
Carbamate Pesticides (ed Gupta, R.C.) 293 (Elsevier
academic press, San Diego, 2005).
22. Slotkin, T. A., MacKillop, E. A., Ryde, I. T. & Seidler,
F. J. Ameliorating the developmental neurotoxicity of
chlorpyrifos: a mechanisms-based approach in PC12
cells. Environ. Health Perspect. 115, 1306-1313 (2007).
23. Shafiq-ur-Rehman. Lead-induced regional lipid per-

36

Toxicol. Environ. Health. Sci. Vol. 4(1), 30-36, 2012

oxidation in brain. Toxicol. Letts. 21, 333-338 (1984).

24. Shafiq-ur-Rehman, Rehman, S., Chandra, O. & Abdulla, M. Evaluation of malondialdehyde as an index of
lead damage in rat brain homogenates. BioMetals 8,
275-279 (1995).
25. Shafiq-ur-Rehman. Endosulfan toxicity and its reduction by selenium: a behavioral, hematological and peroxidative stress evaluation. Internet J. Toxicol. 3, 1-12
(2006).
26. Dib, M., Garrel, C., Favier, A., Robin, V. & Desnuelle,
C. Can malondialdehyde be used as a biological marker of progression in neurodegenerative disease? J.
Neurol. 249, 367-374 (2002).
27. Shafiq-ur-Rehman, Mahdi, A. A. & Hasan, M. Trace
metal-induced lipid peroxidation in biological system.
Soc. Free Rad. Res. India Bull. 2, 12-18 (2003).
28. Abdollahi, M., Ranjbar, A., Shadnia, S., Nikfar, S. &
Rezaiee, A. Pesticides and oxidative stress: a review.
Med. Sci. Monit. 10, RA141-RA147 (2004).
29. Ambali, S. F. Ameliorative effect of vitamins C and
E on neurotoxicological, hematological and biochemical changes induced by chronic chlorpyrifos in Wistar
rats (PhD Dissertation, Ahmadu Bello University,
Zaria, Nigeria, 2009).
30. Shafiq-ur-Rehman. Evaluation of malonaldialdehyde
as an index of chlorpyrifos insecticide exposure in
Apis mellifera: Ameliorating role of melatonin and atocopherol against oxidative stress. Toxicol. Environ.
Chem. 91, 1135-1148 (2009).
31. Giordano, G. et al. Organophosphorus insecticides
chlorpyrifos and diazinon and oxidative stress in neuronal cells in a genetic model of glutathione deficiency. Toxicol. Appl. Pharmacol. 219, 181-189 (2007).
32. Slotkin, T. A., Oliver, C. A. & Seidler, F. J. Critical
periods for the role of oxidative stress in the developmental neurotoxicity of chlorpyrifos and terbutaline,
alone or in combination. Dev. Brain Res. 157, 172180 (2005).
33. Qiao, D., Seidler, F. J. & Slotkin, T. A. Oxidative mechanisms contributing to the developmental neurotoxicity of nicotine and chlorpyrifos. Toxicol. Appl. Pharmacol. 206, 17-26 (2005).
34. Halliwell, B. & Gutteridge, J. M. in Free Radicals in
Biology and Medicine, 3rd Edn. (Oxford University
Press, England, 1999).
35. Iwalokun, B. A., Adelakun, O. M, Magbagbeola, O. A.,
Afolabi, A. S. & Akinwande, A. I. Effect of aqueous
extracts of Allium sativum on some parameters of oxi-

dative stress in mice brain. Nigerian J. Nat. Prod.

Med. 8, 13-18 (2004).
36. Tsakiris, S., Angelogianni, P., Schulpis, K. H. & Stavridis, J. C. Protective effect of L-phenylalanine on rat
brain acetylcholinesterase inhibition induced by free
radicals. Clin. Biochem. 33, 103-106 (2000).
37. Utely, H. C., Bernheim, F. & Hochstein, P. Effect of
sulfhydryl reagents on peroxidation in microsomes.
Arch. Biochem. Biophys. 118, 29-32 (1967).
38. El-Kashoury, A. A. & El-Din, H. A. T. Chlorpyrifos
(from different sources): effect on testicular biochemistry of male albino rats. J. Am. Sci. 6, 252-261 (2010).
39. Verma, R. S. & Srivastava, N. Effect of chlorpyriphos
on thio-barbituric acid reactive substances, scavenging
enzymes and glutathione in rat tissues. Indian J. Biochem. Biophys. 40, 423-428 (2003).
40. Ambali, S. F., Ayo, J. O., Ojo, S. A. & Esievo, K. A. N.
Ameliorative effect of vitamin C on chronic chlorpyrifosinduced erythrocyte osmotic fragility in Wistar rats.
Hum. Exp. Toxicol. 30, 19-24 (2011).
41. Bebe, F. N. & Panemangalore, M. Exposure to low
doses of endosulfan and chlorpyrifos modifies endogenous antioxidants in tissues of rats. J. Environ. Sci.
Health Part B 38, 349-363 (2003).
42. Crumpton, T. L., Seidler, F. J. & Slotkin, T. A. Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos? Dev. Brain Res. 121, 189-195
(2000).
43. Garcia, S. J., Seidler, F. J., Crumpton, T. L. & Slotkin,
T. A. Does the developmental neurotoxicity of chlorpyrifos involve glial targets? Macromolecule synthesis,
adenylyl cyclase signaling, nuclear transcription factors, and formation of reactive oxygen in C6 glioma
cells. Brain Res. 891, 54-68 (2001).
44. Yu, F., Wang, Z., Ju, B., Wang, Y., Wang, J. & Bai, D.
Apoptotic effect of organophosphorus insecticide
chlorpyrifos on mouse retina in vivo via oxidative
stress and protection of combination of vitamins C
and E. Exp. Toxicol. Pathol. 59, 415-423 (2008).
45. Halliwell, B. & Gutteridge, J. M. C. in Free radicals
in biology and medicine. 2nd Edn. (Clarendon Press,
Oxford, 1989).
46. Halliwell, B. & Aruoma, O. I. DNA damage by oxygenderived species: its mechanism and measurement in
mammalian systems. FEBS Letts. 281, 9-19 (1991).
47. Floyd, R. A. Antioxidants, oxidative stress and degenerative neurological disorders. Proc. Soc. Exp. Biol.
Med. 222, 236-245 (1999).