Toxicology in Vitro 21 (2007) 403407

www.elsevier.com/locate/toxinvit

Captopril ameliorates toxicity induced by paraquat in mitochondria

isolated from the rat liver
M. Ghazi-Khansari
a

a,

, A. Mohammadi-Bardbori

a,b

Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Poursina Avenue, P.O. Box 13145-784, Tehran, Iran
b
Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
Received 25 June 2006; accepted 1 October 2006
Available online 10 October 2006

Abstract
The aim of the present study was to show the abilities of captopril as a thiol ACEi (angiotensin converting enzyme inhibitor), on mitochondria toxicity due to paraquat. Mitochondrial isolation from rat liver was divided into 4 groups. Group 1 was considered as control,
group 2 received paraquat (5 mM), group 3 received captopril (0.08 mM) and group 4 received paraquat (5 mM) + captopril (0.08 mM).
Lipid peroxidation, catalase activity, GSH (reduced glutathione) and GSSG (oxidized glutathione) concentrations were determined in
isolated rat liver mitochondria. Simultaneous treatment of mitochondria with captopril (0.08 mM) + paraquat (5 mM) signiWcantly ameliorate the mitochondria toxicity induced by paraquat (5 mM) alone. The results conWrm antioxidant eVect of captopril. This eVect
appears to be attributable to the Sulfhydryl Groups (SH) in the compound which may be due to captopril abilities to scavenge reactive
oxygen species. The results indicate that captopril may prevent oxidative stress induced by paraquat.
2006 Elsevier Ltd. All rights reserved.
Keywords: Captopril; Paraquat; Rat liver mitochondria; Antioxidant

1. Introduction
Mitochondria are candidate targets of paraquat (N,Ndimethyl 4,4-bipyridium) toxicity in animal tissues and
plants (Taylor et al., 2002). Many cases of acute poisoning
and deaths have been reported over the past decade (Bruyndonckx et al., 2002). Mitochondria are considered to be
the major source of reactive oxygen species in cells (Cadenas and Davies, 2000; Ramasarma, 1982; Lenaz, 1998).
Components of the electron transport chain (e.g. Xavoproteins, ubisemiquinone) are known to undergo auto-oxidation and generate reactive oxygen species in mitochondria
(Turrens and Boveris, 1980; Cadenas et al., 1977). Inhibition of the electron transport chain can increase the steadystate levels of these auto-oxidizable components and conse-

Corresponding author. Tel./fax: +98 21 6640 2569.

E-mail addresses: ghazikha@sina.tums.ac.ir, khansagm@yahoo.com
(M. Ghazi-Khansari).
0887-2333/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2006.10.001

quently increase reactive oxygen species generation by

mitochondria (Han et al., 2001; Boveris, 1977).
Paraquat is very toxic, with putative toxicity mechanisms associated to mitochondrial redox systems (Lambert
and Bondy, 1989). The mechanisms of paraquat toxicity are
frequently related to the generation of the superoxide
anion, which can lead to the formation of more toxic reactive oxygen species, e.g., superoxide anion, often taken as
the main toxicant (Farrington et al., 1973).
Angiotensin-converting enzyme (ACE) inhibitors, which
have both an antihypertensive and a cardioprotective action,
are commonly used in the treatment of hypertension and
most forms of heart failure (Brunner et al., 1979; Kiowski
et al., 1991; Konstam et al., 1992). The beneWcial eVects of
ACE inhibitors were thought to be primarily due to the
inhibition of angiotensin II formation. A number of studies
have shown that ACE inhibitors improve oxidant stress
and Wbrosis. Treatment with captopril showed to increase
antioxidant enzymes and nonenzymatic antioxidant defenses
in several mouse tissues (De Cavanagh et al., 1997). Some

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M. Ghazi-Khansari, A. Mohammadi-Bardbori / Toxicology in Vitro 21 (2007) 403407

in vitro studies, indicate that, sulfhydryl containing (i.e.

captopril) can scavenge free radicals (Ghazi-Khansari et al.,
2005). In the present study, the inhibitory eVect of captopril, on the mitochondrial toxicity of PQ have been assessed
in isolated rat liver mitochondria.
2. Materials and methods
2.1. Chemicals
Paraquat dichloride salt, MTT, were purchased from the
Sigma Chemical Co. (St. Louis, Mo). Janus green was
obtained from Merck (Germany). All other chemicals were
obtained from the Sigma Chemical Co.
2.2. Animals
Male Wistar rats (180220 g) were housed environmentally (t D 25 C) and air humidity controlled room (60%)
and kept on standard laboratory diet and were maintained
on a 12 h lightdark cycle. Animals were fasted overnight
before mitochondrial isolation. The Animal Ethics Committee of the Tehran University of Medical Sciences, School of
Medicine, Education Section of Basic Sciences approved the
experiment protocol (132/11737, March 18, 2006).

Fig. 1. EVect of diVerent concentrations of captopril (0.5, 0.08, 0.1 and

1 mM) on percent viabilities of rat liver mitochondria suspension. All data
are given as mean SEM, 6 duplicate mitochondria suspension per groups.

(p < 0.05), (p < 0.01) signiWcantly diVerent when compared with control.

and 1 mM) was used and the submaximal concentration was

determined to be 0.08 mM (Fig. 1). Mitochondria suspensions
were divided into 4 groups of six duplicate samples. Group 1
was considered as control, group 2 received paraquat (5 mM),
group 3 received captopril (0.08 mM) and group 4 received
paraquat (5 mM) + captopril (0.08 mM). Mitochondria suspensions were incubated in vitro for 1 h at 37 C.

2.3. Preparation of mitochondria

2.5. MTT assay
The liver was removed with small scissor and minced in
a cold manitol solution containing 0.225 M D-manitol,
75 mM sucrose and 0.2 mM ethylenediaminetetraacetic acid
(EDTA). Approximately 30 g of minced liver was gently
homogenized in a glass homogenizer with a TeXon pestle
and then centrifuged at 700g for 10 min at 4 C to remove
nuclei, unbroken cells and other non-subcellular tissues.
The supernatants were centrifuged at 7000g for 20 min.
These second supernatant were pooled as the crude microsomal fraction and the pale loose upper layer, which was
rich in swollen or broken mitochondria, lysosomes and
some microsomes, of sediments was washed away. The dark
packed lower layer (heavy mitochondrial fraction) was
resuspended in the manitol solution and recentrifuged twice
at 7000g for 20 min. The heavy mitochondrial sediments
were suspended in Tris solution containing 0.05 M Tris
HCl buVer (pH 7.4) 0.25 M sucrose, 20 mM KCl, 2.0 mM
MgCl2 and 1.0 mM Na2HPO4 at 4 C before assay.
2.4. Experimental design
In order to determine the LC50 of paraquat and the submaximal concentration of captopril (the concentration without any eVect on mitochondrial viability), mitochondrial
viability was investigated by 3-(4,5-dimethylthiazol-2-yl)2,5diphenyl tetrazolium bromide (MTT) assay. DiVerent concentrations of paraquat (1, 5, 10, 50 and 100 mM) was used
and LC50 of paraquat was determined to be 5.01(3.11
6.91) mM. Various concentrations of captopril (0.05, 0.8, 0.1

This assay is a quantitative colorimetric method to determine cell viability. It utilizes the yellow tetrazolium salt
(MTT) which is metabolized by mitochondrial dehydrogenase enzyme from viable cells to yield a purple formazan
reaction product which was determined spectrophotometrically at wavelength of 570 nm (Mosmann, 1983).
2.6. Lipid peroxidation
Lipid proxidation was evaluated as thiobarbituric acid
reactive products. Measurement of TBA reactive compounds was performed after mixing 1 ml of the mitochondrial suspension with 2 ml of TBA reagent containing 0.5 M
HCL, TCA15%, TBA 0.3%. This mixture was heated at
95 C for 20 min, after cooling in tap water, 2 ml of n-butanol were added and the mixture shaken vigorously, then a
centrifugation at 3500 rpm for 15 min was performed, the
n-butanol layer was taken for spectrophotometric measurement. The amount of reactive products formed was calculated by using an extinction coeYcient of 165 mM1 cm1
at 530 nm (Babincova et al., 2002).
2.7. Determination of GSH and GSSG
Aliquots of isolated mitochondria homogenates were
deproteinized with 20% (w/v) trichloroacetic acid and
centrifuged at 10,000g for 20 min. The supernatant was analyzed for reduced glutathione (GSH) by the 5,5-dithiobis-2-

M. Ghazi-Khansari, A. Mohammadi-Bardbori / Toxicology in Vitro 21 (2007) 403407

405

nitrobenzoic acid (DTNB) recycling procedure (Tietze,

1969). GSSG (oxidized glutathione) + GSH were determined
in supernatant after mixing with 1 mL of 5% sodium borohydride (NaBH4), a reducing agent, and then incubated at 45 C
for 60 min. The mixture was neutralized with 0.5 mL of 2.7 N
HCl and the resulting sulfhydryl groups of GSH were
assayed as described above. The absorbance at 412 nm was
measured immediately after mixing and the GSH values were
determined by extrapolation from a standard curve (Zachrias et al., 1992) and GSSG expressed as GSH equivalents.
2.8. Catalase assay
The Cayman Chemical Catalase assay Kit utilizes the
peroxidation of CAT for determination of enzyme activity.
The method is based on the reaction of the enzyme with
methanol in the presence of an optimal concentration of
H2O2. The catalase activity was measured spectrophotometrically with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole
(Purpald) as the chromogen (Johansson and Borg, 1988;
Wheeler et al., 1990).
2.9. Protein concentration

Fig. 2. EVect of treatment with captopril on paraquat induced change in

mitochondrial GSH after 1 h. All data are given as mean SEM, 6 duplicate mitochondria suspension per groups. (p < 0.001) signiWcantly diVerent when compared with paraquat 5 mM alone. 9 (p < 0.001) signiWcantly
diVerent when compared with control.

Mitochondrial protein concentrations were determined

using the method developed by Bradford (1979).
2.10. Statistical analysis
All values were expressed as mean standard error
(SEM) of 6 samples. Analysis of variance (ANOVA) followed by student NewmansKeuls test was used to evaluate
the signiWcance of the results obtained. All computations
were performed using SPSS software.
3. Results
3.1. GSH and GSSG concentration
As shown in Fig. 2, mitochondrial GSH levels of paraquat-treated rats were signiWcantly lower than those of
controls and PQ + captopril treated group (p < 0.001).
Mitochondrial GSSG levels of paraquat-treated rats were
signiWcantly higher than those of controls and PQ +
captopril treated p < 0.001 (Fig. 3). GSSG expressed as
GSH equivalents p < 0.001 (Figs. 2 and 3).

Fig. 3. EVect of treatment with captopril on paraquat induced change in

mitochondrial GSSG after 1 h. All data are given as mean SEM, 6
duplicate mitochondria suspension per groups. (p < 0.001) signiWcantly
diVerent when compared with paraquat 5 mM alone. 9 (p < 0.001) signiWcantly diVerent when compared with control. GSSG expressed as GSH
equivalents.

3.2. Catalase activity

Data have shown that the paraquat treatment and the
administration of captopril provided protection against
paraquat-induced oxidative stress, p < 0.001 (Fig. 4).
3.3. Lipid peroxidation
The TBARs concentration was signiWcantly greater in
mitochondria suspension treated with paraquat alone com-

pared to controls and PQ + captopril treated groups

p < 0.001 (Fig. 5).
4. Discussion
Studies have demonstrated that oxidative stress plays an
important role in the pathogenesis of diseases such as cancer, diabetes, cardiovascular diseases, Parkinsons disease,
schizophrenia, atherosclerosis, lung diseases, cataracts, etc.

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M. Ghazi-Khansari, A. Mohammadi-Bardbori / Toxicology in Vitro 21 (2007) 403407

Fig. 4. EVect of treatment with captopril on paraquat induced change in

Catalase activity after 1 h. All data are given as mean SEM, 6 duplicate
mitochondria suspension per groups. (p < 0.001) signiWcantly diVerent
when compared with paraquat 5 mM alone. 9 (p < 0.001) signiWcantly
diVerent when compared with control.

Fig. 5. EVect of treatment with captopril on paraquat induced change

TBARs after 1 h. All data are given as mean SEM, 6 duplicate mitochondria suspension per groups. (p < 0.001) signiWcantly diVerent when
compared with paraquat 5 mM alone. 9 p < 0.001) signiWcantly diVerent
when compared with control.

(Halliwell and Gutteridge, 1999). Biological eVect of ROS

are controlled by a wide spectrum of enzymatic and nonenzymatic defense mechanisms such as catalase (CAT), glutathione peroxidase, and total-SH protein. GSH is the
major low molecular weight antioxidant in cells and its levels are often decreased during oxidative stress (DeLeve and
Kaplowitz, 1991). Since GSH is known to play an important role in protecting mitochondria from oxidant-induced
injury, levels of GSH as well as GSSG in the isolated mitochondria were measured in this study. Among the oxidative

stress agents paraquat is a thiol-oxidizing agent resulting in

fast oxidation of GSH to GSSG. GSH is considered the
principal mitochondrial antioxidant and its depletion markedly enhances the sensitivity of the mitochondrial structure
to the ROS-mediated injury the sensitivity of the mitochondrial structure to the ROS-mediated injury (FernandezCheca et al., 1998). Our data demonstrate that treatment
with captopril, ameliorate the decreased total GSH and
increased GSSG due to paraquat. Since both the GSH and
GSSG represents speciWc marker of oxidant stress (Jaeschke,
1990), therefore captopril may be able to aVect the glutathione cascade (Figs. 2 and 3).
Membranes are highly permeable to H2O2 (Chance et al.,
1979). Paraquat toxicity has been assigned to H2O2 production (Farrington et al., 1973; Hassan and Fridovich, 1979).
Catalase is known to protect paraquat toxicity as well. Our
data indicate that captopril may interfere with the H2O2
production therefore decreased catalase activity due to
paraquat (Fig. 4).
Lipid peroxidation has been implicated in a number of
deleterious eVects such as increased membrane rigidity,
osmotic fragility, decreased cellular and subcellular components, reduced mitochondrial survival and lipid Xuidity
(Aydin et al., 2004). Our results suggest that captopril may
exert its eVect by inhibiting membrane lipid peroxidation
mediated by superoxide anion (Fig. 5).
Captopril was eVective in ameliorating mitochondrial
toxicity by paraquat. Our pervious observation suggests
that the presence of a thiol group in the ACEi structure
may be determinant for the antioxidant properties (GhaziKhansari et al., 2005). Under normal physiological conditions, antioxidants protect against reactive oxygen species
(ROS) generated in the mitochondria. Numerous studies
have employed antioxidants, such as vitamin E (GarciaEstrada et al., 2003; Yilmaz and Yilmaz, 2006) and melatonin, as treatments for excessive ROS production. The beneWcial eVect of captopril on paraquat toxicity appears to be
through enhancement of the endogenous antioxidant system preventing the lung Wbrosis (De Cavanagh et al., 2001).
Captopril is a thiol compound which can react with superoxide anion radical acting as a scavenger, or with hydroxyl
radical (Al-Shabanad et al., 1998; Aruoma et al., 1991;
Bartoze et al., 1997; Benzie and Tommilson, 1998). Antioxidant eVect of captopril as shown by its eVect on GSH,
GSSG, lipid peroxidation and catalase activity suggest that
exogenously administrated antioxidants such as captopril
may either make their way to mitochondria or improve the
cells antioxidant status, thus protecting these organelle
from oxidant damage. More studies are needed to elucidate
the exact mechanism by which captopril ameliorate the
paraquat mitochondrial toxicity.
Acknowledgement
This study was supported by a grant from Vice Chancellor of Research of Tehran University of Medical Sciences
(132/11737, March 18, 2006).

M. Ghazi-Khansari, A. Mohammadi-Bardbori / Toxicology in Vitro 21 (2007) 403407

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