Food and Chemical Toxicology xxx (2010) xxx–xxx

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Diazinon-induced oxidative stress and renal dysfunction in rats

Muhammad Dawood Shah, Mohammad Iqbal ⇑
Biotechnology Research Institute, Universiti Malaysia Sabah, Locked Bag No. 2073, 88999 Kota-Kinabalu, Sabah, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: Diazinon (O,O-diethyl-O-[2-isopropyl-6-methyl-4-pyrimidinyl] phosphoro thioate), an organo-phos-

Received 14 April 2010 phate insecticide, has been used worldwide in agriculture and domestic for several years, which has
Accepted 1 September 2010 led to a variety of negative effects in non target species including humans. However, its nephrotoxic
Available online xxxx
effects and mechanism of action has not been fully elucidated so far. Therefore, the present study was
aimed at evaluating the nephrotoxic effects of diazinon and its mechanism of action with special refer-
Keywords: ence to its possible ROS generating potential in rats. Treatment of rats with diazinon significantly
Diazinon
enhances renal lipid peroxidation which is accompanied by a decrease in the activities of renal antioxi-
Oxidative stress
Antioxidant enzymes
dant enzymes (e.g. catalase, glutathione peroxidise, glutathione reductase, glucose-6-phosphate dehy-
Renal dysfunction drogenase, glutathione S-transferase) and depletion in the level of glutathione reduced. In contrast, the
activities of renal c-glutamyl transpeptidase and quinone reductase were increased. Parallel to these
changes, diazinon treatment enhances renal damage as evidenced by sharp increase in blood urea nitro-
gen and serum creatinine. Additionally, the impairment of renal function corresponds histopathological-
ly. In summary, our results indicate that diazinon treatment eventuates in decreased renal glutathione
reduced, a fall in the activities of antioxidant enzymes including the enzymes involved in glutathione
metabolism and excessive production of oxidants with concomitant renal damage, all of which are
involved in the cascade of events leading to diazinon-mediated renal oxidative stress and toxicity. We
concluded that in diazinon exposure, depletion of antioxidant enzymes is accompanied by induction of
oxidative stress that might be beneficial in monitoring diazinon toxicity.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
Pesticides are used extensively in agriculture to enhance the
food production by controlling the unwanted insects and disease
vectors. The wide spread use of pesticides in public health and
agricultural programs has caused severe environmental pollution
and potential health hazards including severe acute and chronic
cases of human poisonings and therefore, are cause of concern
(Abdollahi et al., 1999; Ellenhorn et al., 1997). WHO estimates that
the incidence of pesticide poisonings in developing countries has
doubled during the past 10 years. It was estimated in 1982 that
while developing countries accounted for only 15% of the world-
wide use of pesticides, over 50% of the pesticide poisoning cases
occurred in these countries due to misuse (WHO, 1997). The impli-
cations of pesticide residues on the status of human health yet to
be comprehensively documented.
Among pesticides, organophosphates (OPs), which contain
phosphorous derived from phosphoric acid, are commonly used
as insecticides, and are generally the most toxic of all pesticides
to vertebrate animals. Residual amounts of OPs pesticide have
⇑ Corresponding author. Tel.: +60 88 320000; fax: +60 88 320993.
E-mail addresses: miqbal2k2008@hotmail.com, miqbal@ums.edu.my (M. Iqbal).
been detected in the soil, water bodies, vegetables, grains, and
other food products (John et al., 2001). Due to the wide availability
of OPs, toxic effects in human have been shown (De-Bleecker et al.,
1993). Their mechanism of action is based on the inhibition of ace-
tylcholinesterase activity through covalent binding to its serine
residues, thus producing a detention of the nerve impulses that
leads to death (Abu-Quare and Abou-Donia, 2001). Acute poisoning
by OPs causes stimulation of muscarinic receptors and abdominal
pain, diarrhea, hypersalivation, sweating, increased bronchial
secretion and other signs and symptoms are manifested (Buyukok-
uroglu et al., 2008). Stimulation of nicotinic receptors at neuro-
muscular junctions of skeletal muscles causes involuntary
twitching, weakness and paralysis (Olson, 2004). On the other
hand, studies have revealed that oxidative stress could be an
important component of mechanism of OPs compound poisoning
(Abdollahi et al., 2004; Altuntas et al., 2004). Toxicity of OPs pesti-
cides also causes adverse effects on many organs (Sultatos, 1994).
Other systems that could be affected by OPs intoxicant are immune
system (Neishabouri et al., 2004) urinary system (Rodrigo et al.,
2001), reproductive system (Joshi et al., 2003), hematological and
biochemical changes (de Blaquiere et al., 2000). Moreover, the lipo-
philic nature of OPs facilitated their interaction with cell mem-
brane and led to perturbations of phospholipids bilayer structure

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.09.003

Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
doi:10.1016/j.fct.2010.09.003
2 M.D. Shah, M. Iqbal / Food and Chemical Toxicology xxx (2010) xxx–xxx
of most visceral organs (Videria et al., 2001). Several factors includ-
ing dose, route of exposure, physiochemical property and rate of
metabolism play a role in severity and duration of poisoning (Kara-
lliedde et al., 2003). Every year there are million cases of severe
poisoning and 220 000 deaths; the majority of these poisoning
and 99% of the resulting deaths occur in third word (Tinoco and
Halperin, 1998).
Amongst the most frequently used OPs insecticides, diazinon
(O,O-diethyl-O-[2-isopropyl-6-methyl-4-pyrimidinyl] phosphoro
thioate), a synthetic chemical substance, is used worldwide with
applications in agriculture and horticulture for controlling insects
in crops, ornamentals, lawns, fruit and vegetables (Baily et al.,
2000). The most important feature of diazinon is related to their
irreversible cholinesterase inhibition, which at high doses could
lead to animal death (Davies and Holub, 1980). It can be absorbed
through the digestive system, the skin, or via the respiratory tract
when inhaled. Although it is mainly eliminated by the kidney,
microsomal enzymes in the liver oxidize diazinon producing more
potent acetylcholinesterase inhibitors, such as diazoxon, hydrox-
ydiazoxon and hydroxydiazinon (WHO, 1998). Diazinon affects
mitochondrial membrane transportation in rat liver (Nakagawa
and Moore, 1999). Furthermore, it disturbs cytochrome P450 sys-
tem in human liver (Sams et al., 2003). Meanwhile, diazinon causes
toxic effects on other organisms (Keizer et al., 1995). Diazinon is
classified as moderately hazardous class-II organophosphorus
insecticide. Treatment of rats with diazinon also resulted in hyper-
glycemia, depletion of glycogen from the brain and peripheral tis-
sues accompanied with increased activity of glycogen
phosphorylase in the brain and liver and increased activity of the
hepatic gluconeogenic enzyme, phosphoenolpyruvate carboxyki-
nase (Matin et al., 1990). It has also been shown that 335 mg/kg
body weight of diazinon caused an increase in lipid peroxidation
in rat erythrocytes (Sutcu et al., 2007). However, its nephrotoxic ef-
fects and mechanism of action has not been fully elucidated so far.
Therefore, the present study was aimed at evaluating the toxic ef-
fects of diazinon and its mechanism of action in rats with special
reference to its possible reactive oxygen species (ROS) generating
potential in kidney, which caused biochemical, hematological
and histopathological alterations. Our results indicate that diaz-
inon treatment eventuates in decreased renal glutathione reduced,
a fall in the activities of antioxidant enzymes including the en-
zymes involved in glutathione metabolism and excessive produc-
tion of oxidants with concomitant renal damage, all of which are
involved in the cascade of events leading to diazinon-mediated re-
nal oxidative stress and toxicity.
2. Materials and methods
2.1. Chemicals
Tris–HCl, thiobarbituric acid (TBA), oxidized and reduced glutathione, b-nico-
tinamide adenine dinucleotide phosphate reduced (NADPH), glucose-6-phosphate,
1-chloro-2,4-dinitrobenzene (CDNB), glutathione reductase, 5,50 -dithio-bis-2-nitro-
benzoic acid (DTNB), sulfosalicylic acid (SSA), bovine serum albumin (BSA), hydro-
gen peroxide (H2O2), flavin adenine dinucleotide (FAD), 2,6-dichloroindophenol,
diacetylmonoxime, urea, picric acid, sodium tungstate, trichloroacetic acid (TCA),
tween 20, sodium hydroxide (NaOH), ethylenediamine tetraacetic acid (EDTA), cre-
atinine, glycyl glycine, sodium azide and L-c-glutamyl-p-nitroaniline were pur-
chased from either Sigma Chemical Company, St Louis, MO, USA or Aldrich, USA.
All other solvents and chemicals used were either of analytical grade or the highest
purity commercially available.
2.2. Animals
Adult Sprague Dawley male rats (4–8 weeks old) weighing 150–200 g were ob-
tained from Tes Jaya Laboratory Services, Pulau Pinang, Malaysia. All these animals
were acclimatized for one week before the onset of experiment. All these animals
were treated in humane manner and well maintained under standard ethical prin-
ciples as per the university regulations and of the Federal laws for experiment on
Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
doi:10.1016/j.fct.2010.09.003
animals. Animals were housed in plastic (polypropylene) cages using paddy husk
bedding at room temperature (25 ± 1 °C) and 50 ± 5% humidity. Animals were al-
lowed free access to water and chow diet until the start of the experiment.
2.3. Experimental protocol
For studying, the effect of diazinon on renal oxidative stress, antioxidant en-
zymes and histopathological studies, thirty adult Sprague Dawley male rats (4–
8 weeks old) weighing 150–200 g were taken and divided randomly into five groups
having six animals in each. The animals of group I (n = 6) was treated with saline
and served as control. Whereas the animals of group II was treated with corn oil
(vehicle of diazinon) daily for a period of 8 weeks via oral (gavage) and also served
as control. The animals of group III, IV, and V (n = 18 having 6 animals in each
group) was treated with diazinon in corn oil via oral (gavage) accordingly to the se-
lected doses (10 mg/kg body weight, 15 mg/kg body weight and 30 mg/kg body
weight respectively) daily for a period of 8 weeks. The selection of dose regimen
was based on previously published data which indicate substantial alterations in
many of the biochemical parameters at these doses [19–26]. All of these animals
were killed 24 h after the last dose of diazinon or saline with in a period of 1 h.
Blood and kidney of these animals were taken quickly, cleaned free of extraneous
material and perfused immediately with ice-cold saline (0.85% w/v, sodium chlo-
ride) for biochemical, hematological and histopathological investigations to assess
the derangement in the functioning of kidney.
2.4. Preparation of post-mitochondrial supernatant
The standard procedure modified from Mohandas et al. (1984), was adopted for
the preparation of tissue fractionations for all biochemical estimations as described
by Iqbal et al. (1999). Kidneys were quickly removed, cleaned free of extraneous
material and perfused immediately with ice-cold saline (0.85% w/v, sodium chlo-
ride). Kidneys were homogenized in chilled phosphate buffer (0.1 M, pH 7.4) con-
taining KCl (1.17% w/v) using homogenizer (Polytron PT 1200E, Switzerland). The
homogenate was centrifuged at 2000  g for 15 min at 4 °C in an refrigerated cen-
trifuge (model Avanti J-E) to separate the nuclear debris. The aliquot so obtained
was centrifuged at 12 000  g for 30 min at 4 °C to obtained post-mitochondrial
supernatant (PMS) which was used for the determination of malondialdehyde
and glutathione reduced content as well as a source of enzymes.
2.5. Biochemical assays
2.5.1. Determination of glutathione reduced
Glutathione reduced in kidney was determined by the method of Jollow et al.
(1974). An aliquot of 1.0 ml of renal PMS (10% w/v) was precipitated with 1.0 ml
of sulfosalicylic acid (4% w/v). The samples were kept at 4 °C for at least 1 h and
then subjected to centrifugation at 2000  g for 30 min at 4 °C. The assay mixture
contained 0.2 ml filtered aliquot, 2.6 ml phosphate buffer (0.1 M, pH 7.4) and
0.2 ml DTNB (4 mg/ml of phosphate buffer 0.1 M, pH 7.4) in a total volume of
3.0 ml. The yellow color developed was read immediately at 412 nm on a spectro-
photometer (model 4001/4). Results were expressed as micromoles of glutathione
reduced per gram of tissue.
2.5.2. Determination of lipid peroxidation
Renal lipid peroxidation in post-mitochondrial supernatant was done following
the method of Buege and Aust (1978), as described by Iqbal et al. (1999), by mea-
suring the rate of production of TBARS (expressed as malondialdehyde equivalents).
One volume of post-mitochondrial supernatant was mixed with 0.5 volume of tri-
chloroacetic acid (10% w/v) and centrifuged at 2000  g for 30 min. Supernatant
(1.0 ml) was mixed with 1.0 ml thiobarbituric acid (0.67% w/v), prepared by dis-
solving 0.67 g of TBA in warm distilled water. All the tubes were placed in a boiling
water bath for a period of 30 min. At the end, the tubes were shifted to an ice bath
and centrifuged at 12 000  g for 30 min at 4 °C using cold centrifuge (model Avanti
J-E). The results were expressed as amount of malonaldehyde (MDA) formed in each
of the samples and was assessed by measuring the optical density of the superna-
tant at 535 nm using spectrophotometer (model 4001/4) at 37 °C and it was calcu-
lated by using a molar extinction coefficient of 1.56  105 M1cm1.
2.5.3. Determination of glutathione peroxidase activity
Glutathione peroxidase activity was measured according to the procedure of
Mohandas et al. (1984), as described by Iqbal et al. (1998). The reaction mixture
consisted of 1.44 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (0.5 mM),
0.1 ml sodium azide (1.0 mM), 0.05 ml glutathione reductase (1.0 EU/ml), 0.1 ml
GSH (1.0 mM), 0.1 ml NADPH (0.1 mM), 0.1 ml hydrogen peroxide (0.019 M) and
0.025 ml of renal PMS (10% w/v) in a total volume of 2.0 ml. Enzyme activity was
calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coef-
ficient of 6.22  103 M1cm1.
 M.D. Shah, M. Iqbal / Food and Chemical Toxicology xxx (2010) xxx–xxx
2.5.4. Determination of glucose-6-phosphate dehydrogenase activity
Glucose-6-phosphate dehydrogenase activity was assayed by the method of
Zaheer et al. (1965), as described by Iqbal et al. (1998). The reaction mixture in a
total volume of 2.0 ml consisted of 0.45 ml tris–HCl buffer (0.05 M, pH 7.6),
0.05 ml NADP (0.1 mM), 0.05 ml glucose-6-phosphate (0.8 mM), 0.25 ml MgCl2
(8 mM), 0.2 ml of renal PMS (10% w/v) and 1.0 ml distilled water. The changes in
absorbance were recorded at 340 nm and the enzyme activity was calculated as
nmol NADP reduced/min/mg protein using a molar extinction coefficient of
6.22  103 M1cm1.
2.5.5. Determination of glutathione reductase activity
Glutathione reductase activity was determined by the method of Carlberg and
Mannervik (1975), as described by Iqbal et al. (1998). The reaction mixture con-
sisted of 1.7 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml EDTA (0.5 mM), 0.05 ml
oxidized glutathione (1 mM), 0.1 ml NADPH (0.1 mM) and 0.05 ml of renal PMS
(10% w/v) in a total volume of 2.0 ml. Enzyme activity was quantitated by measur-
ing the disappearance of NADPH at 340 nm and was calculated as nmol NADPH oxi-
dized/minute/mg protein using molar extinction coefficient of 6.22  103 M1cm1.
2.5.6. Determination of catalase activity
Catalase activity was determined by the method of Claiborne (1985), as de-
scribed by Iqbal et al. (1998). Briefly, the assay mixture consisted of 0.95 ml phos-
phate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen peroxide (0.019 M), and 0.05 ml of
renal PMS (10% w/v) in a total volume of 2.0 ml. The changes in absorbance were
recorded at 240 nm and enzyme activity was calculated as nmol H2O2 consumed/
min/mg protein using a molar extinction coefficient of 6.4  103 M1cm1.
2.5.7. Determination of glutathione S-transferase activity
Glutathione S-transferase activity was determined by the method of Habig et al.
(1974)), as modified by Athar and Iqbal (1998), using 1-chloro 2,4 dinitrobenzene as
a substrate. The reaction mixture consisted of 2.75 ml phosphate buffer (0.1 M, pH
6.5), 0.1 ml reduced glutathione (1.0 mM), 0.05 ml CDNB (1.0 mM), and 0.1 ml of re-
nal PMS (10% w/v) in a total volume of 3.0 ml. The changes in absorbance were re-
corded at 340 nm and enzyme activity was calculated as nmol CDNB conjugate
formed/min/mg protein using a molar extinction coefficient of 9.6  103 M1cm1.
2.5.8. Determination of NAD(P)H: quinone oxidoreductase activity
The activity of quinone reductase was determined by the method of Benson
et al. (1980), as modified by Iqbal et al. (1999). The assay system consisted of
2.1 ml of Tris–HCl buffer (0.025 M, pH 7.4), 0.7 ml BSA (1 mg/ml), 0.1 ml FAD
(150 lV), 0.02 ml NADPH (0.1 mM), 0.02 ml Tween-20 (1% w/v), 0.1 ml of renal
PMS (10% w/v), and 0.05 ml of 2,6-dichlorophenolindophenol (2.4 mM) in a final
volume of 3.0 ml. The enzyme activity was quantitated by measuring the disappear-
ance of 2,6-dichlorophenolindophenol at 600 nm for 3 min at 30 s intervals. The en-
zyme activity was calculated as nmoles of 2,6-dichlorophenolindophenol reduced/
minute/mg protein using molar extinction coefficient of 2.1  104 M1cm1.
2.5.9. Determination of c-glutamyl transpeptidase activity
The activity of c-glutamyl transpeptidase was determined by the method of
Orlowski and Meister (1973), as modified by Iqbal et al. (1996), using c-glutamyl
p-nitroanilide as substrate. The reaction mixture in a total volume of 1.0 ml con-
tained 0.2 ml PMS (10% w/v) which was incubated with 0.8 ml substrate mixture
(containing 4 mM c-glutamyl p-nitroanilide, 40 mM glycylglycine and 11 mM
MgCl2 in 185 mM tris–HCl buffer, pH 8.25) at 37 °C. Ten minutes after initiation
of the reaction, 1.0 ml of TCA (25% w/v) was added and mixed to terminate the reac-
tion. The solution was centrifuged and supernatant fraction was read at 405 nm. En-
zyme activity was calculated as nmol p-nitroaniline formed/minute/mg protein
using molar extinction coefficient of p-nitroaniline as 1.74  103 M1 cm1.
2.5.10. Determination of blood urea nitrogen
Blood urea nitrogen was determined by the diacetyl monoxime method of Kan-
ter (1975). Protein free filtrate was prepared. To 0.5 ml of protein free filtrate, 3.5 ml
of distilled water, 0.8 ml diacetylmonoxime (20% w/v) and 3.2 ml of sulfuric acid/
phosphoric acid reagent (the reagent was prepared by mixing 150 ml of 85% w/v
phosphoric acid to 140 ml of water and 50 ml of conc. H2SO4) were added. The reac-
tion mixture was placed in a boiling water bath for 30 min and then cooled. The
absorbance was recorded at 480 nm. Results were expressed in mg/dl of blood.
2.5.11. Determination of creatinine
Creatinine was be determined by the alkaline picrate method of Hare (1950).
Protein free filtrate was prepared. To 1.0 ml of plasma/serum, 1.0 ml of sodium
tungstate (5% w/v), 1.0 ml of sulfuric acid (0.6 N) and 1.0 ml of distilled water were
added and mixed thoroughly and centrifuged at 2000  g for 15 min. The superna-
tant was added to a mixture containing 1.0 ml of picric acid (1.05% w/v) and 1.0 ml
of sodium hydroxide (0.75 N). The absorbance was recorded exactly after 20 min at
520 nm. Results were expressed in mg/dl of blood.
Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
doi:10.1016/j.fct.2010.09.003
3
2.5.12. Histopathological assessments
For histopathological studies, few-millimeter–thick-mid-sections of kidneys ex-
cised from each group were processed for light microscopy to substantiate bio-
chemical findings and to ascertain the cause of renal cell death. The process
involving fixing of tissue specimens in 10% neutral buffered formalin solution, pre-
paring the blocks in paraffin, cutting sections 5–6 lm in thickness and staining the
sections with hematoxylin and eosin stain (H&E). The sections were scanned and
analyzed by an expert pathologist who was not aware of sample assignment to
experimental groups for the pathological symptoms of nephrotoxicity such as
necrosis, fatty infiltration, fibrosis, lymphocyte infiltration, etc.
2.5.13. Determination of protein
Protein concentration in all samples was determined by the method of Aitken
et al. (1996) using bovine serum albumin as a standard.
2.5.14. Statistical analysis
The statistical analysis were carried out by using SPSS 17.0 windows statistical
package (SPSS Inc., Chicago IL). Differences between groups were analyzed using
analysis of variance (ANOVA) followed by Dunnet’s multiple comparisons test. All
data points are presented as the treatment group mean ± standard error of the
mean (S.E.). A P values < 0.05 were regarded as significant.
3. Results
Since glutathione reduced is an important endogenous antioxi-
dant whose nucleophillic and reducing properties play a central
role in metabolic pathway, as well as in antioxidant system of aer-
obic cells (Jollow et al., 1974). Enhanced generation of ROS are ex-
pected to consume the endogenous tissue antioxidants such as
glutathione reduced, thereby depleting its concentration within
the tissue. Therefore, we evaluated the effect of diazinon adminis-
tration on glutathione reduced in kidney. As shown in fig. 1, treat-
ment of animals with diazinon has resulted a dose-dependent
depletion of glutathione reduced in kidney. At the lower dose of
diazinon 10 mg/kg body weight, the depletion was around 82% of
saline treated control, while at the higher dose of diazinon
30 mg/kg body weight, the depletion was around 53%. Corn oil
alone treatment to animals was without any significant effect on
glutathione reduced in kidney.
Since ROS are highly reactive and can oxidize cellular macro-
molecules (e.g. lipids, DNA, nucleic acid and proteins) which may
lead to genetic alterations. Lipid peroxidation is linked with exces-
sive generation of ROS, which may be contributed by exogenous or
endogenous sources and is one of the most commonly used marker
for the evaluation of oxidative stress and is thought to be impli-
cated in mutagenesis and carcinogenesis (Iqbal et al., 1999). There-
fore, we studied the effect of diazinon administration on renal lipid
peroxidation. As shown in Fig. 2, diazinon administration induces
lipid peroxidation in a dose-dependent manner, reflecting the for-
mation of activated species in rat kidney. At the lower dose of diaz-
inon 10 mg/kg body weight, about 1.1-fold increase in lipid
peroxidation occurred compared to saline treated control. How-
ever, at the higher dose of diazinon 30 mg/kg body weight, the in-
crease in lipid peroxidation was around 1.3-fold. Corn oil alone
treatment to animals was without any significant effect and did
not cause any induction on renal lipid peroxidation.
Because studies have shown that antioxidant enzymes inside
the cells are important defense against ROS (Sun, 1990). These en-
zymes function to protect the cells against toxic oxygen radical
produced during normal metabolism and after oxidative insult
(Sun, 1990). Inactivation and removal of ROS depends on the reac-
tion involving antioxidant defense system (Sun, 1990). Therefore,
we evaluated the effect of diazinon administration on renal antiox-
idant enzymes. Data in Table 1 show dose-depenedent effects of
diazinon administration on renal antioxidant enzymes. The level
of antioxidant enzymes were found to decrease after diazinon
administration at all doses studied. The maximal decrease in the
level of antioxidant enzymes occurred at the higher dose of diaz-
inon 30 mg/kg body weight, as shown in Table 1. The observed
4 M.D. Shah, M. Iqbal / Food and Chemical Toxicology xxx (2010) xxx–xxx

decrease in the activities for glutathione reductase, glucose-6-
phosphate dehydrogenase, catalase and glutathione peroxidase
were 12%, 36%, 23%, and 16% as compared to saline treated control,
respectively. However, at the lower dose of diazinon 10 mg/kg
body weight, the observed decrease in the activities for glutathione
reductase, glucose-6-phosphate dehydrogenase, catalase and glu-
tathione peroxidase were 2%, 16%, 20%, and 5% as compared to sal-
ine treated control, respectively as shown in Table 1. Treatment of
animals with corn oil alone was without any significant effect on
renal antioxidant enzymes activities.
Studies have shown that glutathione S-transferase and quinone
reductase are important members of phase II-metabolizing en-
zymes which are involved in detoxification of ROS and carcinogen-
esis (Habig et al., 1974; Benson et al., 1980; Sun, 1990). Therefore,
we analyzed the effect of diazinon treatment on renal phase II-
metabolizing enzymes and results are shown in Table 2. Diazinon
treatment resulted in a dose-dependent decrease in glutathione
S-transferase activity at all doses studied. At the lower dose of
diazinon 10 mg/kg body weight, depletion was 91% of saline trea-
ted control, whereas at the higher dose of diazinon 30 mg/kg body
weight, depletion could be recorded up to 80% in glutathione S-
transferase activity. In contrast, quinone reductase activity was in-
creased after diazinon administration at all doses studied. At the
lower dose of diazinon 10 mg/kg body weight, the induction in qui-
none reductase activity was around 110% of the saline treated con-
trol, while at the higher dose of diazinon 30 mg/kg body weight,
the induction was around 120%. Treatment of animals with corn
oil alone was without any significant effect on renal phase II-
metabolizing enzymes activities.
Serum creatinine and blood urea nitrogen is a useful and inex-
pensive method of evaluating of renal dysfunction (Iqbal et al.,
1999). Therefore, we analyzed the effect of diazinon administration

3
Reduced GSH (micromole/g tissue)

2.5

2
* Control saline

1.5 * Control corn oil

1 Diazinon 10 mg/kg b.w.

Diazinon 15 mg/kg b.w.

0.5
Diazinon 30 mg/kg b.w.

Fig. 1. Dose-dependant effect of diazinon administration on renal glutathione reduced in rats. Each value represents mean ± SE of six animals. Saline treated animals served
as control. Dose regimen, treatment protocols and other details are described in text. Values marked with asterisks differ significantly from the corresponding values for
saline treated control (*p < 0.05).

Fig. 2. Dose-dependant effect of diazinon administration on renal lipid peroxidation in rats. Each value represents mean ± SE of six animals. Saline treated animals served as
control. Dose regimen, treatment protocols and other details are described in text. Values marked with asterisks differ significantly from the corresponding values for saline
treated control (*p < 0.05).

Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
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Table 1
Dose-dependant effect of diazinon administration on renal antioxidant enzymes in rats.

Experimental Glutathione reductase (nmol NADPH Glucose-6-phosphate dehydrogenase Catalase (nmol H2O2 Glutathione peroxidase (nmol
groups oxidized/min /mg protein) (nmol NADPH formed/min/mg protein) consumed/min/mg NADPH oxidized/min/mg protein)
protein)
Saline control 50.11 ± 1.12 28.52 ± 0.86 78.06 ± 6.08 81.37 ± 1.54
Corn oil alone 49.66 ± 0.71 28.10 ± 0.76 78.64 ± 4.46 81.98 ± 1.17
Diazinon 49.31 ± 0.71 24.12 ± 0.51 62.70 ± 3.05 77.80 ± 1.71
(10 mg/kg
b.w.)
Diazinon 47.01 ± 0.40* 22.22 ± 0.76* 60.73 ± 5.68* 75.56 ± 1.03*
(15 mg/kg
b.w.)
Diazinon 44.59 ± 0.28* 18.49 ± 0.56* 60.23 ± 2.46* 68.60 ± 1.87*
(30 mg/kg
b.w.)

Each value represents mean ± SE of six animals. Saline treated animals served as control. Dose regimen, treatment protocols and other details are described in text. Values
marked with asterisks differ significantly from the corresponding values for saline treated control (*p < 0.05).

inine were around 1.2-fold and 1.1-fold, respectively, of the saline

Table 2 treated control values. Corn oil alone treatment to animals was
Dose-dependant effects of diazinon administration on renal phase II-metabolizing
enzymes in rats.
without any significant effect and did not cause any induction in
the value of blood urea nitrogen and serum creatinine.
Experimental Glutathione S-transferase Quinone reductase The dose-dependent effect of diazinon treatment on renal c-
groups activity (nmol CDNB conjugate activity (nmol 2,6 DCP
formed/min/mg protein) reduced/min/ mg protein)
glutamyl transpeptidase activity is shown in Fig. 3. Diazinon treat-
ment resulted in a dose-dependent increase in renal c-glutamyl
Saline 20.93 ± 0.27 6.63 ± 0.23
transpeptidase activity at all doses studied. At the higher dose of
control
Corn oil 20.26 ± 0.39 6.76 ± 0.08 diazinon 30 mg/kg body weight, the induction was around 1.4-fold
alone of saline treated control value, whereas at the lower dose of diaz-
Diazinon 19.13 ± 0.39* 7.33 ± 0.13* inon 10 mg/kg body weight, it reached to a value of 1.1-fold that of
(10 mg/kg
saline treated control. Similarly, corn oil alone treatment to ani-
b.w.)
Diazinon 18.94 ± 0.33* 7.54 ± 0.29* mals was without any significant effect and did not cause any
(15 mg/kg induction on renal c-glutamyl transpeptidase activity.
b.w.) Histopathological studies on rats treated with diazinon were
Diazinon 16.78 ± 0.35* 7.96 ± 0.08* performed to ascertain the cause of renal cell death and to substan-
(30 mg/kg
tiate the biochemical findings. Our histopatholgical findings sup-
b.w.)
port biochemical studies. These biochemical modifications
Each value represents mean ± SE of six animals. Saline treated animals served as induced by diazinon correspond histopathologically. The presence
control. Dose regimen, treatment protocols and other details are described in text.
of renal injuries in the kidney of rats treated with diazinon at all
Values marked with asterisks differ significantly from the corresponding values for
saline treated control (p < 0.05). doses was revealed by histopathological studies as shown in
Fig. 4. The treatment of animals with diazinon at all doses induces
kidney swelling with obliteration of space in Bowman’s capsule,
nuclear pycnosis, degeneration of tubular epithelial cells, necrosis
Table 3
Dose-dependant effect of diazinon administration on serum creatinine and blood urea of proximal tubules, flattened epithelium and congested blood ves-
nitrogen in rats. sels. Corn oil alone treatment to animals was without any signifi-
cant effect and does shows normal histopathology.
Experimental groups Serum creatinine Blood urea nitrogen
(mg/dl) (mg/dl)
Saline control 0.70 ± 0.01 23.88 ± 2.02 4. Discussion
Corn oil alone 0.71 ± 0.01 23.68 ± 1.26
Diazinon (10 mg/kg b.w.) 0.76 ± 0.02 28.67 ± 1.26
Diazinon (15 mg/kg b.w.) 0.81 ± 0.01* 37.75 ± 2.17* The potentially reactive derivatives of oxygen, ascribed as ROS
Diazinon (30 mg/kg b.w.) 0.93 ± 0.36* 40.93 ± 3.75* such as O2 H2O2 and OH, are continuously generated inside the
human body as a consequences of exposure to a plethora of
Each value represents mean ± SE of six animals. Saline treated animals served as
control. Dose regimen, treatment protocols and other details are described in text. exogenous chemicals in our ambient environment and/or a num-
Values marked with asterisks differ significantly from the corresponding values for ber of endogenous metabolic processes involving redox enzymes
saline treated control (*p < 0.05). and bioenergetic electron transfer (Sun, 1990). Under normal cir-
cumstances, the ROS generated are detoxified by the antioxi-
dants present in the body and there is equilibrium between
on serum creatinine and blood urea nitrogen. The effect of diazinon the ROS generated and the antioxidants present (Sun, 1990).
administration on the levels of blood urea nitrogen and serum cre- Detrimental effects caused by ROS occur as a consequence of
atinine are shown in Table 3. A dose-dependent increase in the lev- an imbalance between the formation and inactivation of these
els of blood urea nitrogen and serum creatinine was observed after species (Sun, 1990). However, owing to ROS overproduction
diazinon treatment. At the higher dose of diazinon 30 mg/kg body and/or inadequate antioxidant defense, this equilibrium is ham-
weight, the levels of blood urea nitrogen and serum creatinine pered favoring the ROS upsurge that culminates in oxidative
were around 1.7-fold and 1.3-fold, respectively, of the saline trea- stress (Sun, 1990). Oxidative stress is through a series of events,
ted control values. However, at the lower dose of diazinon 10 mg/ dysregulate cellular physiology and its sustained presence may
kg body weight, the levels of blood urea nitrogen and serum creat- lead to pathogenesis of several chronic ailments (Hogg, 1998).

Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
doi:10.1016/j.fct.2010.09.003
6 M.D. Shah, M. Iqbal / Food and Chemical Toxicology xxx (2010) xxx–xxx
Fig. 3. Dose-dependant effect of diazinon administration on c-glutamyl transpeptidase activity in rats. Each value represents mean ± SE of six animals. Saline treated animals
served as control. Dose regimen, treatment protocols and other details are described in text. Values marked with asterisks differ significantly from the corresponding values
for saline treated control (*p < 0.05).
The ROS readily attack and induce oxidative damage to various
biomolecules including proteins, lipids, mitochondria, lipopro-
teins and DNA (Faber, 1994). This oxidative damage is a crucial
etiological factor implicated in several chronic human diseases
such as diabetes mellitus, cancer, atherosclerosis, arthritis, neu-
rodegenerative diseases and also in the aging process (Hogg,
1998). Oxidative stress affects many cellular functions by various
mechanisms such as alteration in gene expression through acti-
vation of transcription factor NF-kB or induction of permeability
transition in mitochondria with lethal consequences (Kaplowitz
and Tsukamoto, 1996). Renal injury caused by diazinon is attrib-
uted to oxidative stress.
The present study demonstrates that diazinon treatment even-
tuates in decreased renal glutathione reduced, a fall in the activi-
ties of antioxidant enzymes including the enzymes involved in
glutathione metabolism and excessive production of oxidants with
concomitant renal damage, all of which are involved in the cascade
of events leading to diazinon-mediated renal oxidative stress and
toxicity. Diazinon is an organophosphate insecticide has been
widely used in industrial agriculture worldwide that would be
potentially an exposure risk to workers in this field and the public
(Baily et al., 2000). Although several reports about the toxicity of
diazinon have been published, little study has been performed
about its nephrotoxic effects and mechanism of action in rats with
special reference to its possible ROS generating potential. Our re-
cent finding indicates that toxic manifestations induced by diaz-
inon may be associated with an enhanced production of ROS.
Diazinon administration causes a significant increase in the levels
of serum markers that are associated with renal dysfunction (Ta-
ble 3) along with depletion in the level of glutathione reduced
(Fig. 1). It should be noted that oxidative stress is often counter-
acted by glutathione reduced resulting in its depletion (Jollow
et al., 1974). This indicates that renal injury induced by diazinon
is the result of oxidative stress that arise as a result of excessive
generation of ROS, which have been reported to attack various bio-
logical molecules including lipids and causing lipid peroxidation.
The activities of antioxidant enzymes including the enzymes in-
volved in glutathione metabolism were also perturbed in diazinon
treated group (Tables 1 and 2). Results indicate the involvement of
oxidative stress in diazinon-mediated renal injury. These results
are consistent with the literature (Abdollahi et al., 2004; Altuntas
Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
doi:10.1016/j.fct.2010.09.003
et al., 2004) and point towards the role of ROS in diazinon-medi-
ated renal injury and toxicity.
A growing body of evidence indicates that glutathione reduced
plays a vital role in cellular function. It detoxifies toxic metabolites
of drugs, regulates gene expression, apoptosis, and transmembrane
transport of organic solutes (Lauterberg, 2002). Glutathione re-
duced, which constitutes one of the physiologically important
mechanisms to curtail progression of tissue damage, is generally
affected under the conditions of oxidative stress (Jollow et al.,
1974). Therefore, we studied the effect of diazinon administration
on glutathione reduced in kidney. Depletion of glutathione reduced
levels in the kidney after the administration of diazinon as ob-
served in the present study, makes tissue susceptible to damage,
indicating the occurrence of free radical reactions in diazinon-
mediated renal injury. These effects are consistent with earlier
studies that have shown the effect of diazinon on glutathione re-
duced (Abdollahi et al., 2004; Altuntas et al., 2004). Glutathione re-
duced levels reflect the summation of a number of processes.
Glutathione reductase maintains glutathione in reduced form
whereas glutathione peroxidise utilizes it for the decomposition
of organic hydroperoxide as well as hydrogen peroxide. c-Glutam-
yl transpeptidase catalyses the transfer of c-glutamic acid of re-
duced glutathione to a recipient peptide molecules. Glutathione
S-transferase conjugate reduced glutathione with various xenobi-
otics forming water soluble products which are more readily ex-
creted (Habig et al., 1974). Glucose-6-phosphate dehydrogenase,
although not directly involved in glutathione metabolism, provides
NADPH needed for reduction of oxidized glutathione. The activities
of all these enzymes determine tissue levels of reduced
glutathione.
Since ROS are highly reactive and can oxidize cellular macro-
molecules (e.g. lipids, DNA, nucleic acid and proteins) which may
lead to genetic alterations. Lipid peroxidation is linked with exces-
sive generation of ROS, which may be contributed by exogenous or
endogenous sources and is the most destructive process in the liv-
ing cells has been implicated in causing a wide range of biological
effects such as increase membrane rigidity, osmotic fragility, de-
creased cellular deformation, reduced erythrocyte survival, and
membrane fluidity (Hogg, 1998; Kaplowitz and Tsukamoto,
1996). Lipid peroxidation products, such as malonaldehyde and
4-hydroxy-2-nonenal (the most cytotoxic) cross link the mem-
 M.D. Shah, M. Iqbal / Food and Chemical Toxicology xxx (2010) xxx–xxx 7

Fig. 4. Dose-dependant effect of diazinon administration on renal histopathological alterations in rats. Saline treated animals served as control. Dose regimen, treatment
protocols and other details are described in text. (a) Saline treated control (b) corn oil treated (c) diazinon treated (10 mg/kg body weight) (d) diazinon treated (15 mg/kg
body weight) (e) diazinon treated (30 mg/kg body weight). Specimens stained with hematoxylin and eosin. (a, b, c, d & e)  20.

brane, damage the DNA and are mutagenic leading to functional
changes (Iqbal et al., 1999). Therefore, we used lipid peroxidation
as a marker of oxidative stress and studied the effect of diazinon
administration on renal lipid peroxidation. Treatment of animals
with diazinon lead to the induction of lipid peroxidation dose
dependently, as monitored by measuring the rate of production
of TBARS (expressed as malondialdehyde equivalents), reflecting
the formation of activated species in rat kidney. These results are
in line with the observation of previous researcher (Akturk et al.,
2006). Accumulation of lipid peroxide is believed to be a major
contributor to the loss of cell function under oxidative stress con-
ditions (Iqbal et al., 1999).This further indicate that renal injury in-
duced by diazinon in present study is the result of oxidative stress
that arise as a result of excessive generation of ROS, which have
been reported to attack various biological molecules including lip-
ids and causing lipid peroxidation.
Because studies have shown that antioxidant enzymes inside
the cells are important defense against ROS (Sun, 1990). These en-
zymes function to protect the cells against toxic oxygen radical
produced during normal metabolism and after oxidative insult
(Sun, 1990). Inactivation and removal of ROS depends on the reac-
tion involving antioxidant defense system (Sun, 1990). Therefore,
we assessed the effect of diazinon administration on renal antiox-
idant enzymes. Diazinon administration to rats depletes enzymatic
and non-enzymatic antioxidant armory in the kidney including
catalase, glutathione reductase, glutathione S-transferase, glutathi-
one peroxidase and glutathione reduced levels. In addition, it also
diminishes the activity of glucose-6-phosphate dehydrogenase.
Therefore, decreased reduced glutathione following diazinon treat-
ment may arise in part from impaired glutathione reductase activ-
ity as well as diminished availability of NADPH. The decrements in
glutathione peroxidase and glutathione S-transferase following
exposure to diazinon may lead to accumulation of peroxides. We
should note that similar alterations in activities of antioxidant en-
zymes including the enzyme involved in glutathione metabolism
and associated oxidative stress have been reported previously fol-
lowing diazinon administration to rats (Akturk et al., 2006). The
turnover of glutathione reduced depends on the activities of vari-
ous antioxidant enzymes including the enzymes involved in gluta-
thione metabolism. The activities of all these enzymes may,

Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
doi:10.1016/j.fct.2010.09.003
8 M.D. Shah, M. Iqbal / Food and Chemical Toxicology xxx (2010) xxx–xxx
therefore, be one of the factors detrimental to the tissue level of re-
duced glutathione and in turn the level of oxidants present in the
cell (Sun, 1990). Thus, diazinon administration leads to glutathione
reduced depletion and inhibition of antioxidant enzymes including
the enzymes involved in glutathione metabolism. Furthermore, the
observed elevations in c-glutamyl transpeptidase may further de-
pletes reduced glutathione, leading to oxidative stress. Finally it is
well known that diazinon catalyzes the generation of highly reac-
tive oxidants (Akturk et al., 2006). Thus diazinon continues to
cause renal damage as evidenced by several fold increase in blood
urea nitrogen and serum creatinine in the present study. Therefore,
the diazinon-mediated renal damage may be due to decrease in tis-
sue levels of reduced glutathione, a fall in the activities of antiox-
idant enzymes including the enzymes involved in glutathione
metabolism and simultaneous generation of various oxidants
including lipid peroxide and other ROS. The kinetics of depletion
of reduced glutathione, a fall in the activities of antioxidant en-
zymes including the enzymes involved in glutathione metabolism
and enhancement of oxidant generation is paralleled by increase in
blood urea nitrogen and serum creatinine, markers for renal dys-
function further suggesting a role of oxidative stress in diazinon-
mediated renal injury.
Because quinone reductase diverts potentially active electro-
philes from damaging interactions with nucleophillic group of
DNA and ultimately protect tissue against carcinogenic/mutagenic
and toxic compounds (Benson et al., 1980; Riley and Workman,
1992). Therefore, our next experiment were directed towards eval-
uating the effect of diazinon administration on renal quinone
reductase activity. Quinone reductase also prevents the formation
of semiquinones by one electron reduction and in turn reduces the
generation of free radical from the autooxidation of semiquinones
(Lind et al., 1981). Hence, an increase of the specific activity of the
quinone reductase enzyme in diazinon treated animals suggest an
enhanced regeneration and maintenance of the antioxidative form
of coenzyme Q which might have imparted a protection to cellular
membranes. Quinone reductase is also induced in response to wide
range of antioxidants, such as t-butyl hydroquinone, t-butyl 4-
hydroxyanisole, and t-butyl 4-hydroxytoluene (Benson et al.,
1980; Talalay and Benson, 1981). In addition, tumor promoter such
as 12-O-tetradecanoyl phorbol 13-acetate, hydrogen peroxide, ion-
izing radiation, etc. also induce this enzyme (Boothman et al.,
1993). It, therefore, seems that this enzyme is a chemopreventive
enzyme, which also acts as an in situ antioxidant to protect against
the oxidative stress response (Talalay et al., 1988). This further
indicate that renal injury induced by diazinon in present study is
the result of oxidative stress that arise as a result of excessive gen-
eration of ROS. It should be emphasized that glutathione S-trans-
ferase and quinone reductase are important members of phase II
enzymes which are involved in detoxification of ROS (Habig
et al., 1974; Benson et al., 1980).
In our next experiments were directed towards further evaluat-
ing the effect of diazinon administration on renal histopathological
alterations to ascertain the cause of renal cell death and to sub-
stantiate the biochemical findings. Our histopatholgical findings
support biochemical studies. These biochemical modifications in-
duced by diazinon correspond histologically. In fact, histological
changes, seen in the kidney of rats treated with diazinon are char-
acterized by a narrowed Bowman’s space, degeneration of tubular
epithelial cells and widened tubular lumen. Besides these, exten-
sive necrosis in renal proximal tubules was observed. Our results
confirmed previous findings of Sulak et al. (2005) and Kalender
et al. (2007) who had found degenerative changes in kidney of rats
exposed to methidathion and methyl parathion (OPs pesticides).
Moreover severe interstitial mononuclear cell’s infiltration (Sulak
et al., 2005; Betrosian et al., 1995), hyperplasia and hypertrophy
of tubular cells (Kalender et al., 2007) had been also observed.
Please cite this article in press as: Shah, M.D., Iqbal, M. Diazinon-induced oxidative stress and renal dysfunction in rats. Food Chem. Toxicol. (2010),
doi:10.1016/j.fct.2010.09.003
Reviewing all the observations, our results indicate that diaz-
inon treatment eventuates in decreased renal glutathione reduced,
a fall in the activities of antioxidant enzymes including the en-
zymes involved in glutathione metabolism and excessive produc-
tion of oxidants with concomitant renal damage, all of which are
involved in the cascade of events leading to diazinon-mediated re-
nal oxidative stress and toxicity. We concluded that in diazinon
exposure, depletion of antioxidant enzymes is accompanied by
induction of oxidative stress that might be beneficial in monitoring
diazinon toxicity.
Conflict of Interest
A conflicting interest exists when professional judgement con-
cerning a primary interest (such as patient’s welfare or the validity
of research) may be influenced by a secondary interest (such as
financial gain or personal rivalry). It may arise for the authors
when they have financial interest that may influence their inter-
pretation of their results or those of others. Examples of potential
conflicts of interest include employment, consultancies, stock
ownership, honoraria, paid expert testimony, patent applications/
registrations, and grants or other funding.
Acknowledgments
Authors are thankful to Ministry of Higher Education, Malaysia
for providing grant–in-aid No.FRG166-SP-2008 for scientific re-
search to support these studies. MDS is also grateful to Islamic
Development Bank for providing research fellowship (ID No: AF/
2008/001).
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