Toxicology in Vitro 21 (2007) 403–407www.elsevier.com/locate/toxinvit

Captopril ameliorates toxicity induced by paraquat in mitochondriaisolated from the rat liver

M. Ghazi-Khansari a,¤, A. Mohammadi-Bardbori a,b

a Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Poursina Avenue, P.O. Box 13145-784, Tehran, Iranb Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

Received 25 June 2006; accepted 1 October 2006Available 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 mito-chondria 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 inisolated rat liver mitochondria. Simultaneous treatment of mitochondria with captopril (0.08 mM) + paraquat (5 mM) signiWcantly ame-liorate the mitochondria toxicity induced by paraquat (5 mM) alone. The results conWrm antioxidant eVect of captopril. This eVectappears to be attributable to the Sulfhydryl Groups (SH) in the compound which may be due to captopril abilities to scavenge reactiveoxygen 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,N�-dimethyl 4,4�-bipyridium) toxicity in animal tissues andplants (Taylor et al., 2002). Many cases of acute poisoningand deaths have been reported over the past decade (Bru-yndonckx et al., 2002). Mitochondria are considered to bethe major source of reactive oxygen species in cells (Cade-nas and Davies, 2000; Ramasarma, 1982; Lenaz, 1998).Components of the electron transport chain (e.g. Xavopro-teins, ubisemiquinone) are known to undergo auto-oxida-tion and generate reactive oxygen species in mitochondria(Turrens and Boveris, 1980; Cadenas et al., 1977). Inhibi-tion of the electron transport chain can increase the steady-state 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 bymitochondria (Han et al., 2001; Boveris, 1977).

Paraquat is very toxic, with putative toxicity mecha-nisms associated to mitochondrial redox systems (Lambertand Bondy, 1989). The mechanisms of paraquat toxicity arefrequently related to the generation of the superoxideanion, which can lead to the formation of more toxic reac-tive oxygen species, e.g., superoxide anion, often taken asthe main toxicant (Farrington et al., 1973).

Angiotensin-converting enzyme (ACE) inhibitors, whichhave both an antihypertensive and a cardioprotective action,are commonly used in the treatment of hypertension andmost forms of heart failure (Brunner et al., 1979; Kiowskiet al., 1991; Konstam et al., 1992). The beneWcial eVects ofACE inhibitors were thought to be primarily due to theinhibition of angiotensin II formation. A number of studieshave shown that ACE inhibitors improve oxidant stressand Wbrosis. Treatment with captopril showed to increaseantioxidant enzymes and nonenzymatic antioxidant defensesin several mouse tissues (De Cavanagh et al., 1997). Some

404 M. Ghazi-Khansari, A. Mohammadi-Bardbori / Toxicology in Vitro 21 (2007) 403–407

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 capto-pril, on the mitochondrial toxicity of PQ have been assessedin isolated rat liver mitochondria.

2. Materials and methods

2.1. Chemicals

Paraquat dichloride salt, MTT, were purchased from theSigma Chemical Co. (St. Louis, Mo). Janus green wasobtained from Merck (Germany). All other chemicals wereobtained from the Sigma Chemical Co.

2.2. Animals

Male Wistar rats (180–220 g) were housed environmen-tally (tD25 °C) and air humidity controlled room (60%)and kept on standard laboratory diet and were maintainedon a 12 h light–dark cycle. Animals were fasted overnightbefore mitochondrial isolation. The Animal Ethics Commit-tee of the Tehran University of Medical Sciences, School ofMedicine, Education Section of Basic Sciences approved theexperiment protocol (132/11737, March 18, 2006).

2.3. Preparation of mitochondria

The liver was removed with small scissor and minced ina 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 gentlyhomogenized in a glass homogenizer with a TeXon pestleand then centrifuged at 700g for 10 min at 4 °C to removenuclei, unbroken cells and other non-subcellular tissues.The supernatants were centrifuged at 7000g for 20 min.These second supernatant were pooled as the crude micro-somal fraction and the pale loose upper layer, which wasrich in swollen or broken mitochondria, lysosomes andsome microsomes, of sediments was washed away. The darkpacked lower layer (heavy mitochondrial fraction) wasresuspended in the manitol solution and recentrifuged twiceat 7000g for 20 min. The heavy mitochondrial sedimentswere suspended in Tris solution containing 0.05 M Tris–HCl buVer (pH 7.4) 0.25 M sucrose, 20 mM KCl, 2.0 mMMgCl2 and 1.0 mM Na2HPO4 at 4 °C before assay.

2.4. Experimental design

In order to determine the LC50 of paraquat and the sub-maximal concentration of captopril (the concentration with-out any eVect on mitochondrial viability), mitochondrialviability was investigated by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay. DiVerent con-centrations of paraquat (1, 5, 10, 50 and 100mM) was usedand LC50 of paraquat was determined to be 5.01(3.11–6.91)mM. Various concentrations of captopril (0.05, 0.8, 0.1

and 1mM) was used and the submaximal concentration wasdetermined to be 0.08 mM (Fig. 1). Mitochondria suspensionswere divided into 4 groups of six duplicate samples. Group 1was considered as control, group 2 received paraquat (5 mM),group 3 received captopril (0.08 mM) and group 4 receivedparaquat (5mM)+ captopril (0.08mM). Mitochondria sus-pensions were incubated in vitro for 1h at 37°C.

2.5. MTT assay

This assay is a quantitative colorimetric method to deter-mine cell viability. It utilizes the yellow tetrazolium salt(MTT) which is metabolized by mitochondrial dehydroge-nase enzyme from viable cells to yield a purple formazanreaction product which was determined spectrophotometri-cally at wavelength of 570 nm (Mosmann, 1983).

2.6. Lipid peroxidation

Lipid proxidation was evaluated as thiobarbituric acidreactive products. Measurement of TBA reactive com-pounds was performed after mixing 1 ml of the mitochon-drial suspension with 2 ml of TBA reagent containing 0.5 MHCL, TCA15%, TBA 0.3%. This mixture was heated at95 °C for 20 min, after cooling in tap water, 2 ml of n-buta-nol were added and the mixture shaken vigorously, then acentrifugation at 3500 rpm for 15 min was performed, then-butanol layer was taken for spectrophotometric measure-ment. The amount of reactive products formed was calcu-lated by using an extinction coeYcient of 165 mM¡1 cm¡1

at 530 nm (Babincova et al., 2002).

2.7. Determination of GSH and GSSG

Aliquots of isolated mitochondria homogenates weredeproteinized with 20% (w/v) trichloroacetic acid andcentrifuged at 10,000g for 20 min. The supernatant was ana-lyzed for reduced glutathione (GSH) by the 5,5�-dithiobis-2-

Fig. 1. EVect of diVerent concentrations of captopril (0.5, 0.08, 0.1 and1 mM) on percent viabilities of rat liver mitochondria suspension. All dataare given as mean§ SEM, 6 duplicate mitochondria suspension per groups.¤ (p < 0.05), ¤¤ (p < 0.01) signiWcantly diVerent when compared with control.

M. Ghazi-Khansari, A. Mohammadi-Bardbori / Toxicology in Vitro 21 (2007) 403–407 405

nitrobenzoic acid (DTNB) recycling procedure (Tietze,1969). GSSG (oxidized glutathione)+ GSH were determinedin supernatant after mixing with 1 mL of 5% sodium borohy-dride (NaBH4), a reducing agent, and then incubated at 45 °Cfor 60 min. The mixture was neutralized with 0.5 mL of 2.7NHCl and the resulting sulfhydryl groups of GSH wereassayed as described above. The absorbance at 412 nm wasmeasured immediately after mixing and the GSH values weredetermined by extrapolation from a standard curve (Zach-rias et al., 1992) and GSSG expressed as GSH equivalents.

2.8. Catalase assay

The Cayman Chemical Catalase assay Kit utilizes theperoxidation of CAT for determination of enzyme activity.The method is based on the reaction of the enzyme withmethanol in the presence of an optimal concentration ofH2O2. The catalase activity was measured spectrophotomet-rically 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

Mitochondrial protein concentrations were determinedusing 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) fol-lowed by student Newmans–Keuls test was used to evaluatethe signiWcance of the results obtained. All computationswere performed using SPSS software.

3. Results

3.1. GSH and GSSG concentration

As shown in Fig. 2, mitochondrial GSH levels of para-quat-treated rats were signiWcantly lower than those ofcontrols and PQ + captopril treated group (p < 0.001).Mitochondrial GSSG levels of paraquat-treated rats weresigniWcantly higher than those of controls and PQ +captopril treated p < 0.001 (Fig. 3). GSSG expressed asGSH equivalents p < 0.001 (Figs. 2 and 3).

3.2. Catalase activity

Data have shown that the paraquat treatment and theadministration of captopril provided protection againstparaquat-induced oxidative stress, p < 0.001 (Fig. 4).

3.3. Lipid peroxidation

The TBARs concentration was signiWcantly greater inmitochondria suspension treated with paraquat alone com-

pared to controls and PQ + captopril treated groupsp < 0.001 (Fig. 5).

4. Discussion

Studies have demonstrated that oxidative stress plays animportant role in the pathogenesis of diseases such as can-cer, diabetes, cardiovascular diseases, Parkinson’s disease,schizophrenia, atherosclerosis, lung diseases, cataracts, etc.

Fig. 2. EVect of treatment with captopril on paraquat induced change inmitochondrial GSH after 1 h. All data are given as mean § SEM, 6 dupli-cate mitochondria suspension per groups. ¤ (p < 0.001) signiWcantly diVer-ent when compared with paraquat 5 mM alone. 9 (p < 0.001) signiWcantlydiVerent when compared with control.

Fig. 3. EVect of treatment with captopril on paraquat induced change inmitochondrial GSSG after 1 h. All data are given as mean § SEM, 6duplicate mitochondria suspension per groups. ¤ (p < 0.001) signiWcantlydiVerent when compared with paraquat 5 mM alone. 9 (p < 0.001) signiW-cantly diVerent when compared with control. GSSG expressed as GSHequivalents.

406 M. Ghazi-Khansari, A. Mohammadi-Bardbori / Toxicology in Vitro 21 (2007) 403–407

(Halliwell and Gutteridge, 1999). Biological eVect of ROSare controlled by a wide spectrum of enzymatic and non-enzymatic defense mechanisms such as catalase (CAT), glu-tathione peroxidase, and total-SH protein. GSH is themajor low molecular weight antioxidant in cells and its lev-els are often decreased during oxidative stress (DeLeve andKaplowitz, 1991). Since GSH is known to play an impor-tant role in protecting mitochondria from oxidant-inducedinjury, levels of GSH as well as GSSG in the isolated mito-chondria were measured in this study. Among the oxidative

Fig. 4. EVect of treatment with captopril on paraquat induced change inCatalase activity after 1 h. All data are given as mean § SEM, 6 duplicatemitochondria suspension per groups. ¤ (p < 0.001) signiWcantly diVerentwhen compared with paraquat 5 mM alone. 9 (p < 0.001) signiWcantlydiVerent when compared with control.

Fig. 5. EVect of treatment with captopril on paraquat induced changeTBARs after 1 h. All data are given as mean § SEM, 6 duplicate mito-chondria suspension per groups. ¤ (p < 0.001) signiWcantly diVerent whencompared with paraquat 5 mM alone. 9 p < 0.001) signiWcantly diVerentwhen compared with control.

stress agents paraquat is a thiol-oxidizing agent resulting infast oxidation of GSH to GSSG. GSH is considered theprincipal mitochondrial antioxidant and its depletion mark-edly enhances the sensitivity of the mitochondrial structureto the ROS-mediated injury the sensitivity of the mitochon-drial structure to the ROS-mediated injury (Fernandez-Checa et al., 1998). Our data demonstrate that treatmentwith captopril, ameliorate the decreased total GSH andincreased GSSG due to paraquat. Since both the GSH andGSSG represents speciWc marker of oxidant stress (Jaeschke,1990), therefore captopril may be able to aVect the glutathi-one cascade (Figs. 2 and 3).

Membranes are highly permeable to H2O2 (Chance et al.,1979). Paraquat toxicity has been assigned to H2O2 produc-tion (Farrington et al., 1973; Hassan and Fridovich, 1979).Catalase is known to protect paraquat toxicity as well. Ourdata indicate that captopril may interfere with the H2O2production therefore decreased catalase activity due toparaquat (Fig. 4).

Lipid peroxidation has been implicated in a number ofdeleterious eVects such as increased membrane rigidity,osmotic fragility, decreased cellular and subcellular compo-nents, reduced mitochondrial survival and lipid Xuidity(Aydin et al., 2004). Our results suggest that captopril mayexert its eVect by inhibiting membrane lipid peroxidationmediated by superoxide anion (Fig. 5).

Captopril was eVective in ameliorating mitochondrialtoxicity by paraquat. Our pervious observation suggeststhat the presence of a thiol group in the ACEi structuremay be determinant for the antioxidant properties (Ghazi-Khansari et al., 2005). Under normal physiological condi-tions, antioxidants protect against reactive oxygen species(ROS) generated in the mitochondria. Numerous studieshave employed antioxidants, such as vitamin E (Garcia-Estrada et al., 2003; Yilmaz and Yilmaz, 2006) and melato-nin, as treatments for excessive ROS production. The bene-Wcial eVect of captopril on paraquat toxicity appears to bethrough enhancement of the endogenous antioxidant sys-tem preventing the lung Wbrosis (De Cavanagh et al., 2001).Captopril is a thiol compound which can react with super-oxide anion radical acting as a scavenger, or with hydroxylradical (Al-Shabanad et al., 1998; Aruoma et al., 1991;Bartoze et al., 1997; Benzie and Tommilson, 1998). Antioxi-dant eVect of captopril as shown by its eVect on GSH,GSSG, lipid peroxidation and catalase activity suggest thatexogenously administrated antioxidants such as captoprilmay either make their way to mitochondria or improve thecells antioxidant status, thus protecting these organellefrom oxidant damage. More studies are needed to elucidatethe exact mechanism by which captopril ameliorate theparaquat mitochondrial toxicity.

Acknowledgement

This study was supported by a grant from Vice Chancel-lor of Research of Tehran University of Medical Sciences(132/11737, March 18, 2006).

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