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To cite this article: Milad Vahidirad, Milad Arab-Nozari, Hamidreza Mohammadi, Ehsan Zamani
& Fatemeh Shaki (2017): Protective effect of captopril against diazinon induced nephrotoxicity
and neurotoxicity via inhibition of ROS-NO pathway, Drug and Chemical Toxicology, DOI:
10.1080/01480545.2017.1391830
Download by: [University of New England] Date: 08 November 2017, At: 10:55
DRUG AND CHEMICAL TOXICOLOGY, 2017
https://doi.org/10.1080/01480545.2017.1391830
RESEARCH ARTICLE
and 100 mg kg1) and positive control (N-acetylcysteine 200 mg kg1) were injected intraperitoneally KEYWORDS
30 min before Dz. After 24 h, animals were anesthetized and the brain and kidney tissues were sepa- Captopril; diazinon;
rated. Then oxidative stress factors were evaluated. Also, blood was collected for assessment of blood neurotoxicity; nephrotox-
urea nitrogen (BUN), creatinine (Cr) and nitric oxide (NO) levels. Dz significantly increased oxidative icity; oxidative stress
stress markers such as reactive oxygen species (ROS), lipid peroxidation, and protein carbonyl as well as
glutathione (GSH) oxidation in both tissues. Increased levels of the BUN, Cr and NO were observed after
Dz injection. Interestingly, captopril administration significantly decreased ROS production in both tis-
sues. Captopril significantly protected kidney and brain against lipid peroxidation and GSH oxidation.
Administration of captopril could markedly inhibit protein carbonyl production in kidney and brain after
Dz injection. Furthermore, captopril ameliorated the increased level of BUN, Cr and NO. These results
suggested that captopril can prevent Dz-induced oxidative stress, nephrotoxicity and neurotoxicity
because of its antioxidant activity.
Introduction Boroushaki et al. 2013). On the other hand, the brain is the
most vulnerable organ to xenobiotic-induced oxidative dam-
Diazinon (Dz) is an organophosphate (OP) insecticide that
age because of high polyunsaturated fatty acids (PUFAs) con-
widely used in agriculture and households products to con-
tent, low levels of antioxidants and also a high rate of
trol insects (Oruç and Usta 2007, Jafari et al. 2012).
oxygen consumption (Pizzurro et al. 2014). Several studies
Nowadays, pesticide poisoning is one of the most common
have pointed to the potential role of oxidative stress in OPs
causes of poisoning in the world, as WHO reported about
neurotoxicity (Giordano et al. 2007, Lukaszewicz-Hussain
3 million cases of pesticide poisoning per year which lead to
more than 250,000 deaths annually (Hamad et al. 2016). 2010, Rashedinia et al. 2016).
Although OPs are primarily known as acetylcholinesterase Increased ROS production after Dz exposure can attack to
inhibitors, induction of oxidative stress by OPs has been cellular macromolecules and leads to lipid peroxidation, pro-
reported as one of the main mechanism of tissue toxicity tein oxidation and DNA damage (Rashedinia et al. 2016).
for OPs poisoning in both humans and animals (Colovi c et al. On the other hand, endogenous nitric oxide (NO) is formed
2015). from L-arginine by nitric oxide synthetase (NOS) enzyme
Oxidative stress is the imbalance between the rate of (Tutanc et al. 2012). The excessive production of NO is linked
reactive oxygen species (ROS) production and the protective to the oxidative stress and tissue damage (Peresleni et al.
effects of antioxidants (Ahangar et al. 2017). 1996). The role of NO in Dz-induced toxicity showed by
Previous studies revealed the role of oxidative stress in the numerous studies (Beydilli et al. 2015, Abdel-Daim 2016).
pathogenesis of OPs-induced acute and chronic intoxication Failure of antioxidant defense system in attenuation of
in clinical and experimental studies (Giordano et al. 2007, oxidative damage leads to initiation of cell death signaling
Lukaszewicz-Hussain 2010). Exposure to Dz could lead to ser- and finally tissue toxicity (Rashedinia et al. 2016). Therefore,
ious histopathological lesions in various tissues such as kid- compounds with radical scavenging or antioxidant properties
ney, brain and liver (Tsitsimpikou et al. 2013, Boussabbeh can be beneficial for ameliorating tissue toxicity of Dz
et al. 2016). (Boroushaki et al. 2013, Salem et al. 2015).
Nephrotoxicity of Dz via oxidative damage has been Captopril is an angiotensin-converting enzyme inhibitor
well shown in previous studies (Yavuz et al. 2003, that is frequently used to treatment of hypertension disease
CONTACT Fatemeh Shaki fshaki.tox@gmail.com Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical
Sciences, Sari, Iran
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
2 M. VAHIDIRAD ET AL.
(Hosseinimehr and Karami 2005). It was shown that com- spectrophotometer at 485 nm excitation and 520 nm emission
pounds with sulfhydryl group have antioxidant effects wavelength the results were expressed as fluorescent inten-
(Banerjee et al. 2010, Kasperczyk et al. 2014, Ortiz et al. 2016). sity per 1 mg protein (Gao et al. 2009).
On this basis, the radical scavenging effect of captopril is
probably linked to a terminal sulfhydryl group in its structure Measurement of GSH content
(Hosseinimehr and Karami 2005).
Interestingly, several studies showed that captopril GSH content was determined using 5,50 -dithiobis-(2-nitroben-
increased enzymatic and non-enzymatic antioxidants levels zoic acid) (DTNB) as the indicator and the developed yellow
and also decreased lipid peroxidation, protein oxidation in color was read at 412 nm on a spectrophotometer (UV-
various animal models (Yavuz et al. 2003, Ghazi-Khansari et al. 1601 PC, Shimadzu, Japan). GSH content was expressed as n
2005, Kalia et al. 2007). mol l1 (Sadegh and Schreck 2003).
Thus, the aim of the current study was to test the effect of
captopril treatment against DZ-induced nephrotoxicity and Measurement of lipid peroxidation
neurotoxicity, focusing on its inhibitory effects on oxidative
stress. The content of MDA was determined using dithiobarbituric
acid (TBA) as an indicator. Briefly, 0.25 ml sulfuric acid
(0.05 mol l1) was added to 0.2 ml samples (1 mg protein
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Methods ml1) afterwards, with the addition of 0.3 ml 0.2% TBA. All
the microtubes were placed in a boiling water bath for
Animal treatment
30 min. At the end, the tubes were shifted to an ice-bath and
Male Wistar rats (200–250 g) were kept in an air-conditioned 0.4 ml n-butanol was added to each tube. Then, they were
room with a controlled temperature of 22 ± 2 C and main- centrifuged at 3500 g for 10 min. The amount of MDA
tained on a 12:12 h light cycle with free access to food and formed in each of the samples was assessed by measuring
water. All experimental procedures were conducted according the absorbance of the supernatant at 532 nm with an
to the ethical standards and protocols approved by the ELISA reader (Tecan, Rainbow Thermo, and Austria).
Committee of Animal Experimentation of Mazandaran Tetrametoxypropane was used as standard and MDA content
University of Medical Sciences, Sari, Iran. All efforts were was expressed as l mol l1 (Zhang et al. 2008).
made to minimize the number of animals and their suffering.
Animals were randomly divided into five groups (n ¼ 30) Measurement of protein carbonyl
and the groups were as follows: negative control group
(received olive oil), DZ group (received 150 mg kg1 Dz intra- Determination of protein carbonyl by spectrophotometric
peritoneally), captopril groups (that received 60 and 100 mg method, briefly 200 ll of brain and kidney tissues are needed
kg1 captopril intraperitoneally) and positive control group that to homogenate. Samples are extracted in 500 ll of 20% (w/v)
received N-acetylcysteine (NAC) (200 mg kg1). After 30 min, DZ TCA. Then, samples placed at 4 C for 15 min. The precipitates
(150 mg kg1) that previously diluted with olive oil, injected to are treated with 500 ll of 0.2% 2,4-dinitrophenylhydrazine
captopril and NAC groups intraperitoneally. After 24 h, rats (DNPH) and 500 ll of 2 mol l1 HCL for the control group, and
were anesthetized and their brain and kidney tissues were samples are incubated at room temperature for 1 h with vortex-
removed. After homogenization of tissues, oxidative damage ing at 5 min intervals. Then proteins are precipitated by adding
factors such as protein carbonyl, malondialdehyde (MDA), gluta- 55 ll of 20% TCA. The microtubes are centrifuged and washed
thione (GSH), and reactive oxygen spices (ROS) were evaluated. three times with 1000 ll of the ethanol-ethyl acetate mixture.
Furthermore, blood samples were collected for blood urea And the microtubes are dissolved in 200 ll of 6 mol l1 guan-
nitrogen (BUN) and creatinine (Cr) and NO levels assessment. idine hydrochloride. The carbonyl content is determined by
reading the absorbance at 365 nm wavelength (Aebi 1984).
one-way ANOVA test, followed by the post-hoc Tukey test. The result showed that GSH levels significantly decreased in
Statistical significance was set at p < 0.05. rats that received DZ compared to negative control group in
both tissues. In addition, GSH concentration was increased
Results (p < 0.05) in the brain of rats that received 100 mg kg1 of
captopril as compared to DZ group (Figure 3(A)). While GSH
Effects of captopril on ROS formation in rat brain and levels elevation were not significant after administration of
kidney both doses of captopril (60 and 100 mg kg1) in kidney tissue
ROS formation is an indicator of oxidative stress that signifi- (Figure 3(B)).
cantly (p < 0.001) were increased in rats that received DZ as
compared to negative control group in both kidney and brain
tissues. Data showed that ROS formation markedly was Effects of captopril on protein carbonyl in rat brain and
decreased in groups that received both doses of captopril (60 kidney
and100 mg kg1) as compared to the group that received DZ Protein carbonyl was increased (p < 0.001) in the group that
in brain tissue. While in kidney tissue, administration of cap- received DZ as compared to negative control group in both
topril at the dose of 100 mg kg1 inhibited Dz-induced ROS brain and kidney. As shown in Figure 4, the elevation of pro-
formation as well as NAC group (p < 0.001) (Figure 1). tein carbonyl content was significantly decreased in group
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Figure 1. Effects of captopril on ROS formation in rat brain and kidney. ROS formation was determined in N.C (negative control), Dz (diazinon), Dz þ CP (captopril)
and Dz þ NAC (positive control) groups in the (A) brain and (B) kidney tissue, using DCFH-DA as described in ‘Methods’ section. Values represented as mean ± SD
(n ¼ 6). aaaSignificantly different compared to the NC group (p < 0.001), bbsignificantly different compared to Dz group (p < 0.01), bbbsignificantly different compared
to Dz group (p < 0.001), csignificantly different between two captopril groups (p < 0.05), ddsignificantly different compared to NAC group (p < 0.01), ns: non-
significant.
4 M. VAHIDIRAD ET AL.
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Figure 2. Effects of captopril on lipid peroxidation in rat brain and kidney. Lipid peroxidation was determined in N.C (negative control), Dz (diazinon), Dz þ CP (cap-
topril) and Dz þ NAC (positive control) groups in the (A) brain and (B) kidney tissue, using TBA as described in ‘Methods’ section. Values represented as mean ± SD
(n ¼ 6). aaaSignificantly different compared to the NC group (p < 0.001), bsignificantly different compared to Dz group (p < 0.05), bbsignificantly different compared
to Dz group (p < 0.01), ns: non-significant.
Figure 3. Effects of captopril on GSH concentration in rat brain and kidney. GSH concentration was determined in N.C (negative control), Dz (diazinon), Dz þ CP
(captopril) and Dz þ NAC (positive control) groups in the (A) brain and (B) kidney tissue, using DTNB as described in ‘Methods’ section. Values represented as
mean ± SD (n ¼ 6). aaSignificantly different compared to the N.C group (p < 0.01), aaasignificantly different compared to the N.C group (p < 0.001), bsignificantly dif-
ferent compared to Dz group (p < 0.05), ns: non-significant.
dose of 100 mg kg1 before Dz exposure could significantly to organ toxicity via induction of oxidative stress (Yavuz et al.
ameliorate these parameters as well as NAC group (Table 2). 2003, Lukaszewicz-Hussain 2010, Tsitsimpikou et al. 2013). So,
it is necessary to identify the effective and beneficial com-
Discussion pounds to the prevention of this unpleasant effect.
In our study, Dz administration led to increase of oxidative
Pesticide poisoning is a concern for WHO that estimated 3 stress markers such as ROS, lipid peroxidation, protein car-
million poisoning cases per year. Dz is one of the most com- bonyl and GSH oxidation in both kidney and brain tissues.
monly used pesticide in the world (Hamad et al. 2016), that Induction of oxidative stress by Dz was shown by previous
notable cases of poisoning were reported worldwide. Dz poi- studies that reported GSH oxidation (Ghazi-Khansari et al.
soning leads to damage to several organs in the body, 2005, Heo et al. 2008) and MDA elevation (Boroushaki et al.
including kidney, brain and liver (Tsitsimpikou et al. 2013, 2013, Abdel-Daim 2016) in the brain and kidney tissues
Boussabbeh et al. 2016). Despite its major inhibitory effect on of rats after exposure to Dz, which confirmed the data of
acetylcholinesterase, it is observed that Dz exposure can lead our study.
DRUG AND CHEMICAL TOXICOLOGY 5
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Figure 4. Effects captopril on protein carbonyl in rat brain and kidney. Protein carbonyl was determined in N.C (negative control), Dz (diazinon), Dz þ CP (captopril)
and Dz þ NAC (positive control) groups in the (A) brain and (B) kidney tissue, using DNPH as described in ‘Methods’ section. Values represented as mean ± SD
(n ¼ 6). aaaSignificantly different compared to the N.C group (p < 0.001), bsignificantly different compared to Dz group (p < 0.05), bbsignificantly different compared
to Dz group (p < 0.01), dSignificantly different compared to NAC group (p < 0.05), ns: non-significant.
Table 1. Effect of in vivo administration of captopril and Table 2. Effect of in vivo administration of captopril and diazinon on blood
diazinon on serum nitric oxide (NO) concentration in rats. urea nitrogen (BUN) and serum creatinine levels in rats.
Animal groups NO (mM) Animal groups BUN (mg/dl) Creatinine (mg/dl)
N.C 5.20 ± 0.1 N.C 23 ± 1.2 0.6 ± 0.08
Dz 79.53 ± 11.91aaa Dz 68 ± 6a 1.23 ± 0.18a
Dz þ CP (60 mg kg1) 53.38 ± 7.1b Dz þ CP (60 mg kg1) 46 ± 5 0.97 ± 0.1
Dz þ CP (100 mg kg1) 27.36 ± 7.8bbb,c Dz þ CP (100 mg kg1) 33 ± 3b 0.65 ± 0.15b
Dz þ NAC (200 mg kg1) 33.43 ± 11.25bbb Dz þ NAC (200 mg kg1) 28 ± 4b 0.71 ± 0.13b
N.C: negative control; Dz: Diazinon; Dz þ CP: captopril; N.C: negative control; Dz: Diazinon; Dz þ CP: captopril; and Dz þ NAC: positive
Dz þ NAC: positive control groups. control groups.
Values are expressed as mean ± SD for six rats in each Values are expressed as mean ± SD for six rats in each group.
group. a
Significantly different compared to the N.C group (p < 0.05),
aaa
Significantly different compared to the N.C group b
significantly different compared to Dz group (p < 0.05).
(p < 0.001), bsignificantly different compared to Dz group
(p < 0.05), bbbsignificantly different compared to Dz group
(p < 0.001), csignificantly different compared to (Dz þ CP
60 mg kg1) group (p < 0.05). attenuate Dz-induced oxidative stress in the testis tissue of
rat by elevation in GSH level and reduction of MDA content
(Oksay et al. 2013). In another study, NAC could prevent GSH
Because of high lipid content and also the low capacity of depletion, lipid peroxidation and caused a significant increase
antioxidants, brain is one of the most susceptible organs to in endogenous antioxidant levels in liver oxidative damage
oxidative damage (Pizzurro et al. 2014). In addition, free radi- induced by malathion (Lasram et al. 2014).
cals can disrupt kidney function by the same way Captopril is an angiotensin-converting enzyme inhibitor
(Tsitsimpikou et al. 2013). that recently gained attention because of its antioxidant
So, compounds with antioxidant properties may be benefi- activity (Ghazi-Khansari et al. 2005, Kalia et al. 2007). The pro-
cial to prevent oxidative damage of Dz. Previous studies tective effect of captopril in pathological conditions that oxi-
showed the protective role of antioxidant agents against Dz dative stress is involved were shown in previous studies
toxicity. For example, aqueous extracts of Matricaria Recutita (Yavuz et al. 2003, Ghazi-Khansari et al. 2005, Kalia et al.
L. and Asparagus officinalis could ameliorate Dz-induced tox- 2007). For example, Ghazi et al. showed that captopril could
icity through the MDA level reduction and elevation of GSH prevent liver oxidative damage induced by paraquat (Kalia
content (Sulak et al. 2005). Also, pomegranate seed oil et al. 2007).
reduced Dz-induced nephrotoxicity via inhibition of oxidative It is suggested that Dz toxicity is due to generation of free
stress in the kidney of rats (Boroushaki et al. 2013). radicals such as hydroxyl, superoxide and etc. (Ghazi-Khansari
It is imagined that Dz toxicity might be due to binding et al. 2005). In this study, we showed that captopril could
ability of Dz to sulfhydryl groups of GSH in the tissues (Kalia prevent ROS production in both kidney and brain tissues.
et al. 2007). Several studies indicated that compounds with Free radicals can attack to lipid membranes because of
sulfhydryl groups in their structures can bind to free radicals the PUFAs available in the cell membranes, are very suscep-
and scavenge them and finally, reduce their destructive tible to oxidation by ROS. Lipid peroxidation is a harmful out-
effect. For example, Oksay et al. showed that NAC can come of free radicals that can result in cell or organelle
6 M. VAHIDIRAD ET AL.
membrane damage (Noctor et al. 2015). Our research indi- for improving the pathological condition that oxidative stress
cated that administration of captopril caused a significant is involved.
decrease in MDA level in both tissues.
Also, ROS can target other cellular macromolecules such
Disclosure statement
as proteins. This damage is generally associated with
increased level of carbonylated proteins that are good No potential conflict of interest was reported by the authors.
markers for protein oxidation (Sharma et al. 2012).
Captopril could reduce the protein carbonyl levels in both
Funding
kidney and brain tissues. It means that probably captopril has
a potential effect of inhibition of protein oxidation induced This study was extracted from MSc thesis of Milad Vahidirad and sup-
by Dz. ported by a grant from the Pharmaceutical Sciences Branch, Islamic Azad
University, Tehran, Iran.
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oxidant defense system by direct interaction with ROS. GSH
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