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Nonthiol ACE inhibitors, enalapril and lisinopril are unable to protect mitochondrial toxicity due to paraquat
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 89 (2007) 163–167 www.elsevier.com/locate/ypest
Nonthiol ACE inhibitors, enalapril and lisinopril are unable to protect mitochondrial toxicity due to paraquat Afshin Mohammadi-Bardbori a
a,b
, Mahmoud Ghazi-Khansari
a,*
Department of Pharmacology, School of Medicine, Medical Sciences/University of Tehran, P.O. Box 13145-784, Tehran, Iran b Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran Received 6 March 2007; accepted 11 June 2007 Available online 15 June 2007
Abstract Angiotensin-converting enzyme inhibitors (ACEi) were shown to ameliorate endothelial dysfunction in various human diseases and some of these inhibitors have been reported to enhance antioxidant defenses. The objective of the present study was to shown the abilities of enalapril and lisinopril as two nonthiol ACEi on mitochondrial toxicity due to paraquat. In this study, mitochondrial isolation from rat liver was divided into six groups. Group 1 was considered as control, group 2 received paraquat (5 mM), group 3 received enalapril (0.25 mM), group 4 received lisinopril (0.01 mM), group 5 received paraquat (5 mM) + enalapril (0.25 mM), and group 6 received paraquat(5 mM) + lisinopril (0.01 mM). Viability, lipid peroxidation, catalase activity, GSH (reduced glutathione) and GSSG (oxidized glutathione) concentrations were also determined. Simultaneous treatment of mitochondria with enalapril (0.25 mM) + paraquat (5 mM) and lisinopril (0.0.01 mM) + paraquat (5 mM) did not significantly ameliorate the mitochondrial toxicity induced by paraquat (5 mM) alone (p > 0.05). However, the nonthiol ACEi, enalapril showed to partially improve target of lipid peroxidation due to paraquat. In conclusion, nonthiol ACEi treatment did not improve the increased oxidative stress and the decreased antioxidant mechanisms. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Nonthiol ACEi; Isolated rat liver mitochondria; Paraquat; Lisinopril; Enalapril
1. Introduction Some of the beneficial effects of ACEi (angiotensinconverting enzyme inhibitor) occur independently of the ability of this drug to reduce arterial blood pressure [1– 3]. The beneficial effects of ACE inhibitors were thought to be primarily due to the inhibition of angiotensin II formation. However, a number of studies have shown to improve oxidant stress and fibrosis. ACEi may act as ‘‘magic bullets’’ against oxidative stress and this may explain some of the beneficial effects of ACEi that cannot be associated to their action on blood pressure [4]. Treatment with enalapril or captopril showed to increase antioxidant enzymes and non-enzymatic antioxidant defenses in several mouse tissues [5]. ACEi could also limit super*
Corresponding author. Fax: +98 21 6640 2569. E-mail addresses: [email protected], [email protected] (M. Ghazi-Khansari). 0048-3575/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.06.001
oxide generation, and could modulate reactive oxygen and nitrogen species generation [6,7]. ACEi can enhance the endogenous antioxidant defenses and suggest that this induction can protect cells from oxidant stress [5]. The potential ability of ACEi to scavenge reactive oxygen and nitrogen species (RONS) has produced conflicting results [8,9]. Some studies, mainly done in vitro, indicate that both, sulfhydryl-containing (i.e., captopril) and non-sulfhydryl-containing ACEi (i.e., enalapril and lisinopril) can scavenge free radicals [9]. By contrast, other reports show that only sulfhydryl-containing ACEi are effective free radical/oxidant scavengers [10,11]. Paraquat is very toxic, with putative toxicity mechanisms associated to mitochondrial redox systems [12]. 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 [13].
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A. Mohammadi-Bardbori, M. Ghazi-Khansari / Pesticide Biochemistry and Physiology 89 (2007) 163–167
In this report, we studied the role of two nonthiol ACE inhibitors, enalapril and lisinopril to protect mitochondrial toxicity due to paraquat.
Paraquat dichloride salt, MTT and all other chemicals were obtained from the Sigma Chemical Co.
april and 0.01 mM lisinopril were selected doses from our previous work [14]. Mitochondrial suspensions were divided into six groups of six duplicate samples. Group 1 was considered as control, group 2 received paraquat (5 mM), group 3 received enalapril (0.25 mM), group 4 received lisinopril (0.01 mM) group 5 received paraquat (5 mM) + enalapril (0.25 mM) and group 6 received paraquat (5 mM) + lisinopril (0.01 mM). Mitochondrial suspensions were incubated in vitro for 1 h at 37 °C.
2.2. Animals
2.5. MTT assay
Male Wistar rats (180–220 g) were housed environmentally (t = 25 °C) and air humidity controlled room (60%) and kept on standard laboratory diet and were maintained on a 12-h light–dark 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).
This assay is a quantitative colorimetric method to determine cell viability [15]. In our experiments, MTT assay was for rat liver mitochondria suspension in tubes. Mitochondrial suspension was mixed with MTT yielding the final concentration of MTT 0.2 mg/ml and final concentration of mitochondria 0.2 mg/ml in the sample. In this assay, incubation of mitochondria loaded with MTT lasted 2 h. In the first time, mitochondrial suspension was mixed with different concentration of toxicant. Finally, solution was gently removed. To eliminate the toxicant is necessary to wash mitochondria with the washing solution with centrifuged at 7000g for 20 min, and mixed with MTT and produced formazan crystals were dissolved in 200 ll of DMSO. After a few minutes at room temperature to ensure that all crystals were dissolved the plates. Then the plate was read on a multiplate reader, using a test wavelength 570 nm a reference wavelength of 630 nm, and a calibration setting of 1.99 [16].
2. Materials and methods 2.1. Chemicals
2.3. Preparation of mitochondria 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 Teflon 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 buffer (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 enalapril and lisinopril (the concentration without any effect on mitochondrial viability), mitochondrial viability was investigated by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay (data not shown). Different concentrations of paraquat (1–100 mM) was used and LC50 of paraquat was determined to be 5.01(3.11–6.91)mM. Various concentrations of enalapril (0.25–1 mM) and lisinopril (0.01–0.1 mM) were investigated. In the present study, 5 mM paraquat, 0.25 mM enal-
2.6. Lipid peroxidation Lipid peroxidation 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, TCA 15%, 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 coefficient of 156000 M 1cm 1 at 535 nm [17]. 2.7. Determination of GSH and GSSG Aliquots of isolated mitochondria homogenates were acidified with 20% (w/v) trichloroacetic acid and centrifuged at 10,000g for 20 min. The supernatant was analyzed for total glutathione (GSH) by the 5,5-dithiobis nitrobenzoicacid, DTNB recycling procedure [18]. 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.
A. Mohammadi-Bardbori, M. Ghazi-Khansari / Pesticide Biochemistry and Physiology 89 (2007) 163–167
165
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 [19]. 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 [20,21]. 2.9. Protein concentration Mitochondrial protein concentrations were determined using the method developed by Bradford [22]. 2.10. Statistical analysis
Fig. 1. Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria. Mitochondrial viability is shown. All data are given as means ± SEM, 6 mitochondria suspension per groups. ***p < 0.01, significantly different when compared with control.
All values were expressed as means ± SEM of six samples. Analysis of variance (ANOVA) followed by student Newmans–Keuls test was used to evaluate the significance of the results obtained. All computations were performed using SPSS 13.0 for windows software (Chicago, Illinois, USA). 3. Results 3.1. Viability assay Simultaneous treatment of mitochondria with Lisinopril (0.01 mM) + paraquat (5 mM) and enalapril (0.25 mM) + paraquat 5(mM) did not significantly changed the mitochondrial toxicity by paraquat 5 mM (p > 0.05, Fig. 1). 3.2. Lipid peroxidation The TBARs concentration was significantly greater in mitochondria suspension treated with paraquat and paraquat + lisinopril compared to controls. PQ + enalapril treated group were significantly lower than those of paraquat alone (p < 0.05, Fig. 2).
Fig. 2. Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria. Lipid peroxidation is shown. All data are given as means ± SEM, 6 mitochondria suspension per groups. **p < 0.01, significantly different when compared with control. p < 0.05, significantly different when compared with paraquat 5 mM alone.
3.3. Catalase activity Data have shown that the paraquat treatment and the administration of enalapril or lisinopril cannot provided protection against paraquat-induced oxidative stress (p < 0.05, Fig. 3).
cantly lower than those of controls. Mitochondrial GSSG levels of paraquat-treated and paraquat + enalapril or lisinopril were significantly higher than those of controls (p < 0.05, Table 1). 4. Discussion
3.4. GSH and GSSG concentration As shown in Table 1 mitochondrial GSH levels of paraquat-treated and PQ + enalapril or lisinopril were signifi-
In this study however, the nonthiol ACEi enalapril also have to partially ameliorate target of lipid peroxidation but we cannot find any changes in the other targets due to
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Fig. 3. Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria. Catalase activity is shown. All data are given as means ± SEM, 6 mitochondria suspension per groups. ***p < 0.001, significantly different when compared with control.
paraquat (Figs. 1–3 and Table 1). These findings and the results of the past study therefore show that among the thiol and nonthiol ACEi, captopril was able to quench ROS generation from isolated mitochondria [23]. Captopril, the first described, is a thiol compound which can react with superoxide anion radical acting as a scavenger, or with hydroxyl radical [24–27]. Considering the chemical structure of most of the ACEi used, the latter assessment seems to be the correct one, since non-sulfhydryl-containing ACEi molecules lack chemical groups that can scavenge radicals. Biological effect of ROS is controlled by a wide spectrum of enzymatic and non-enzymatic defense mechanisms. Among the oxidative stress agent such as 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 [28]. GSH is the major low molecular weight antioxidant in cells and its levels are often decreased during oxidative stress [29]. Since GSH is known to play an important role in protecting mitochondria from oxidant-induced injury, level of GSH as well as GSSG in the isolated mitochondria
were measured in this study. Since both the GSH and GSSG represents specific marker of oxidant stress [30], therefore enalapril or lisinopril cannot able to affect the glutathione cascade (Table 1). Lipid peroxidation has been implicated in a number of deleterious effects such as increased membrane rigidity, osmotic fragility, decreased cellular and subcellular components, reduced mitochondrial survival and lipid fluidity [31]. Our results suggest that enalapril may exert its effect by inhibiting membrane lipid peroxidation mediated by superoxide anion, but lisinopril, which failed to decrease MDA concentration (Fig. 2). Fig. 3 shows the nonthiol ACE inhibitors; enalapril and lisinopril are unable to protect mitochondrial toxicity due to paraquat by decreased in catalase activity. Owing that membranes are highly permeable to H2O2 [32]. Paraquat toxicity has been assigned to H2O2 production [13,33], therefore catalase should protect against paraquat toxicity. This is shown by decreased in catalase activity due to paraquat. Our data indicates that enalapril and lisinopril cannot interfere with the H2O2 production therefore prevented the decreased in catalase activity due to paraquat. However the partially antioxidant effect properties of certain non-sulfhydryl-containing ACEi, enalapril might be related to unexpected antioxidant actions, various studies suggest that these agents may affect on RAS, antioxidant effect through the free radical scavenger action [34–37] or ACEi can provide tissue protection [38], via inhibition of molecules adhesion [39]. The mechanism of this specific protection is not yet understood although some ACE inhibitors have been described as enhancers of endogenous antioxidant defenses [5,40]. We found significant differences among three assayed ACEi in mitochondrial toxicity induced by paraquat [14,23]. Captopril was the most effective in decreasing mitochondrial toxicity by paraquat. Our observations suggest that the presences of a thiol group in the ACEi structure may be determinant for the antioxidant properties. Acknowledgment This study was supported by a grant from Vice Chancellor of Research of Tehran University of Medical Sciences (132/2801, March 18, 2006).
Table 1 Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria Group
GSH (nmol/mg protein)
GSSG (nmol/mg protein)
Total (nmol/mg protein)
GSH/GSSG ratio
Control Enalapril (0.25 mM) Lisinopril (0.01 mM) Paraquat 5 mM PQ + enalapril PQ + lisinopril
13.00 ± 0.16 12.98 ± 0.25 13.04 ± 0.02 11.42 ± 0.18*** 11.78 ± 0.12 10.99 ± 0.11
0.45 ± 0.02 0.45 ± 0.04 0.47 ± 0.01 2.54 ± 0.18*** 2.77 ± 0.12 3.02 ± 0.50
13.44 ± 0.10 13.45 ± 0.10 13.51 ± 0.01 13.05 ± 0.11** 14.55 ± 0.14 14.01 ± 0.35
29.15 ± 0.36 28.91 ± 0.53 27.75 ± 31 4.52 ± 0.14*** 4.25 ± 0.08 3.7 ± 0.33
GSH, GSSG, GSSG + GSH and GSH/GSSG ratio are shown. GSSG expressed as GSH equivalents. All data are given as means ± SEM, 6 mitochondria suspension per groups. ** p < 0.01, ***p < 0.001, significantly different when compared with control.
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Pesticide Biochemistry and Physiology 89 (2007) 163–167 www.elsevier.com/locate/ypest
Nonthiol ACE inhibitors, enalapril and lisinopril are unable to protect mitochondrial toxicity due to paraquat Afshin Mohammadi-Bardbori a
a,b
, Mahmoud Ghazi-Khansari
a,*
Department of Pharmacology, School of Medicine, Medical Sciences/University of Tehran, P.O. Box 13145-784, Tehran, Iran b Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran Received 6 March 2007; accepted 11 June 2007 Available online 15 June 2007
Abstract Angiotensin-converting enzyme inhibitors (ACEi) were shown to ameliorate endothelial dysfunction in various human diseases and some of these inhibitors have been reported to enhance antioxidant defenses. The objective of the present study was to shown the abilities of enalapril and lisinopril as two nonthiol ACEi on mitochondrial toxicity due to paraquat. In this study, mitochondrial isolation from rat liver was divided into six groups. Group 1 was considered as control, group 2 received paraquat (5 mM), group 3 received enalapril (0.25 mM), group 4 received lisinopril (0.01 mM), group 5 received paraquat (5 mM) + enalapril (0.25 mM), and group 6 received paraquat(5 mM) + lisinopril (0.01 mM). Viability, lipid peroxidation, catalase activity, GSH (reduced glutathione) and GSSG (oxidized glutathione) concentrations were also determined. Simultaneous treatment of mitochondria with enalapril (0.25 mM) + paraquat (5 mM) and lisinopril (0.0.01 mM) + paraquat (5 mM) did not significantly ameliorate the mitochondrial toxicity induced by paraquat (5 mM) alone (p > 0.05). However, the nonthiol ACEi, enalapril showed to partially improve target of lipid peroxidation due to paraquat. In conclusion, nonthiol ACEi treatment did not improve the increased oxidative stress and the decreased antioxidant mechanisms. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Nonthiol ACEi; Isolated rat liver mitochondria; Paraquat; Lisinopril; Enalapril
1. Introduction Some of the beneficial effects of ACEi (angiotensinconverting enzyme inhibitor) occur independently of the ability of this drug to reduce arterial blood pressure [1– 3]. The beneficial effects of ACE inhibitors were thought to be primarily due to the inhibition of angiotensin II formation. However, a number of studies have shown to improve oxidant stress and fibrosis. ACEi may act as ‘‘magic bullets’’ against oxidative stress and this may explain some of the beneficial effects of ACEi that cannot be associated to their action on blood pressure [4]. Treatment with enalapril or captopril showed to increase antioxidant enzymes and non-enzymatic antioxidant defenses in several mouse tissues [5]. ACEi could also limit super*
Corresponding author. Fax: +98 21 6640 2569. E-mail addresses: [email protected], [email protected] (M. Ghazi-Khansari). 0048-3575/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2007.06.001
oxide generation, and could modulate reactive oxygen and nitrogen species generation [6,7]. ACEi can enhance the endogenous antioxidant defenses and suggest that this induction can protect cells from oxidant stress [5]. The potential ability of ACEi to scavenge reactive oxygen and nitrogen species (RONS) has produced conflicting results [8,9]. Some studies, mainly done in vitro, indicate that both, sulfhydryl-containing (i.e., captopril) and non-sulfhydryl-containing ACEi (i.e., enalapril and lisinopril) can scavenge free radicals [9]. By contrast, other reports show that only sulfhydryl-containing ACEi are effective free radical/oxidant scavengers [10,11]. Paraquat is very toxic, with putative toxicity mechanisms associated to mitochondrial redox systems [12]. 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 [13].
164
A. Mohammadi-Bardbori, M. Ghazi-Khansari / Pesticide Biochemistry and Physiology 89 (2007) 163–167
In this report, we studied the role of two nonthiol ACE inhibitors, enalapril and lisinopril to protect mitochondrial toxicity due to paraquat.
Paraquat dichloride salt, MTT and all other chemicals were obtained from the Sigma Chemical Co.
april and 0.01 mM lisinopril were selected doses from our previous work [14]. Mitochondrial suspensions were divided into six groups of six duplicate samples. Group 1 was considered as control, group 2 received paraquat (5 mM), group 3 received enalapril (0.25 mM), group 4 received lisinopril (0.01 mM) group 5 received paraquat (5 mM) + enalapril (0.25 mM) and group 6 received paraquat (5 mM) + lisinopril (0.01 mM). Mitochondrial suspensions were incubated in vitro for 1 h at 37 °C.
2.2. Animals
2.5. MTT assay
Male Wistar rats (180–220 g) were housed environmentally (t = 25 °C) and air humidity controlled room (60%) and kept on standard laboratory diet and were maintained on a 12-h light–dark 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).
This assay is a quantitative colorimetric method to determine cell viability [15]. In our experiments, MTT assay was for rat liver mitochondria suspension in tubes. Mitochondrial suspension was mixed with MTT yielding the final concentration of MTT 0.2 mg/ml and final concentration of mitochondria 0.2 mg/ml in the sample. In this assay, incubation of mitochondria loaded with MTT lasted 2 h. In the first time, mitochondrial suspension was mixed with different concentration of toxicant. Finally, solution was gently removed. To eliminate the toxicant is necessary to wash mitochondria with the washing solution with centrifuged at 7000g for 20 min, and mixed with MTT and produced formazan crystals were dissolved in 200 ll of DMSO. After a few minutes at room temperature to ensure that all crystals were dissolved the plates. Then the plate was read on a multiplate reader, using a test wavelength 570 nm a reference wavelength of 630 nm, and a calibration setting of 1.99 [16].
2. Materials and methods 2.1. Chemicals
2.3. Preparation of mitochondria 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 Teflon 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 buffer (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 enalapril and lisinopril (the concentration without any effect on mitochondrial viability), mitochondrial viability was investigated by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay (data not shown). Different concentrations of paraquat (1–100 mM) was used and LC50 of paraquat was determined to be 5.01(3.11–6.91)mM. Various concentrations of enalapril (0.25–1 mM) and lisinopril (0.01–0.1 mM) were investigated. In the present study, 5 mM paraquat, 0.25 mM enal-
2.6. Lipid peroxidation Lipid peroxidation 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, TCA 15%, 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 coefficient of 156000 M 1cm 1 at 535 nm [17]. 2.7. Determination of GSH and GSSG Aliquots of isolated mitochondria homogenates were acidified with 20% (w/v) trichloroacetic acid and centrifuged at 10,000g for 20 min. The supernatant was analyzed for total glutathione (GSH) by the 5,5-dithiobis nitrobenzoicacid, DTNB recycling procedure [18]. 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.
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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 [19]. 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 [20,21]. 2.9. Protein concentration Mitochondrial protein concentrations were determined using the method developed by Bradford [22]. 2.10. Statistical analysis
Fig. 1. Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria. Mitochondrial viability is shown. All data are given as means ± SEM, 6 mitochondria suspension per groups. ***p < 0.01, significantly different when compared with control.
All values were expressed as means ± SEM of six samples. Analysis of variance (ANOVA) followed by student Newmans–Keuls test was used to evaluate the significance of the results obtained. All computations were performed using SPSS 13.0 for windows software (Chicago, Illinois, USA). 3. Results 3.1. Viability assay Simultaneous treatment of mitochondria with Lisinopril (0.01 mM) + paraquat (5 mM) and enalapril (0.25 mM) + paraquat 5(mM) did not significantly changed the mitochondrial toxicity by paraquat 5 mM (p > 0.05, Fig. 1). 3.2. Lipid peroxidation The TBARs concentration was significantly greater in mitochondria suspension treated with paraquat and paraquat + lisinopril compared to controls. PQ + enalapril treated group were significantly lower than those of paraquat alone (p < 0.05, Fig. 2).
Fig. 2. Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria. Lipid peroxidation is shown. All data are given as means ± SEM, 6 mitochondria suspension per groups. **p < 0.01, significantly different when compared with control. p < 0.05, significantly different when compared with paraquat 5 mM alone.
3.3. Catalase activity Data have shown that the paraquat treatment and the administration of enalapril or lisinopril cannot provided protection against paraquat-induced oxidative stress (p < 0.05, Fig. 3).
cantly lower than those of controls. Mitochondrial GSSG levels of paraquat-treated and paraquat + enalapril or lisinopril were significantly higher than those of controls (p < 0.05, Table 1). 4. Discussion
3.4. GSH and GSSG concentration As shown in Table 1 mitochondrial GSH levels of paraquat-treated and PQ + enalapril or lisinopril were signifi-
In this study however, the nonthiol ACEi enalapril also have to partially ameliorate target of lipid peroxidation but we cannot find any changes in the other targets due to
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Fig. 3. Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria. Catalase activity is shown. All data are given as means ± SEM, 6 mitochondria suspension per groups. ***p < 0.001, significantly different when compared with control.
paraquat (Figs. 1–3 and Table 1). These findings and the results of the past study therefore show that among the thiol and nonthiol ACEi, captopril was able to quench ROS generation from isolated mitochondria [23]. Captopril, the first described, is a thiol compound which can react with superoxide anion radical acting as a scavenger, or with hydroxyl radical [24–27]. Considering the chemical structure of most of the ACEi used, the latter assessment seems to be the correct one, since non-sulfhydryl-containing ACEi molecules lack chemical groups that can scavenge radicals. Biological effect of ROS is controlled by a wide spectrum of enzymatic and non-enzymatic defense mechanisms. Among the oxidative stress agent such as 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 [28]. GSH is the major low molecular weight antioxidant in cells and its levels are often decreased during oxidative stress [29]. Since GSH is known to play an important role in protecting mitochondria from oxidant-induced injury, level of GSH as well as GSSG in the isolated mitochondria
were measured in this study. Since both the GSH and GSSG represents specific marker of oxidant stress [30], therefore enalapril or lisinopril cannot able to affect the glutathione cascade (Table 1). Lipid peroxidation has been implicated in a number of deleterious effects such as increased membrane rigidity, osmotic fragility, decreased cellular and subcellular components, reduced mitochondrial survival and lipid fluidity [31]. Our results suggest that enalapril may exert its effect by inhibiting membrane lipid peroxidation mediated by superoxide anion, but lisinopril, which failed to decrease MDA concentration (Fig. 2). Fig. 3 shows the nonthiol ACE inhibitors; enalapril and lisinopril are unable to protect mitochondrial toxicity due to paraquat by decreased in catalase activity. Owing that membranes are highly permeable to H2O2 [32]. Paraquat toxicity has been assigned to H2O2 production [13,33], therefore catalase should protect against paraquat toxicity. This is shown by decreased in catalase activity due to paraquat. Our data indicates that enalapril and lisinopril cannot interfere with the H2O2 production therefore prevented the decreased in catalase activity due to paraquat. However the partially antioxidant effect properties of certain non-sulfhydryl-containing ACEi, enalapril might be related to unexpected antioxidant actions, various studies suggest that these agents may affect on RAS, antioxidant effect through the free radical scavenger action [34–37] or ACEi can provide tissue protection [38], via inhibition of molecules adhesion [39]. The mechanism of this specific protection is not yet understood although some ACE inhibitors have been described as enhancers of endogenous antioxidant defenses [5,40]. We found significant differences among three assayed ACEi in mitochondrial toxicity induced by paraquat [14,23]. Captopril was the most effective in decreasing mitochondrial toxicity by paraquat. Our observations suggest that the presences of a thiol group in the ACEi structure may be determinant for the antioxidant properties. Acknowledgment This study was supported by a grant from Vice Chancellor of Research of Tehran University of Medical Sciences (132/2801, March 18, 2006).
Table 1 Effect of lisinopril (0.01 mM) and enalapril (0.25 mM) on paraquat toxicity (5 mM) isolated rat liver mitochondria Group
GSH (nmol/mg protein)
GSSG (nmol/mg protein)
Total (nmol/mg protein)
GSH/GSSG ratio
Control Enalapril (0.25 mM) Lisinopril (0.01 mM) Paraquat 5 mM PQ + enalapril PQ + lisinopril
13.00 ± 0.16 12.98 ± 0.25 13.04 ± 0.02 11.42 ± 0.18*** 11.78 ± 0.12 10.99 ± 0.11
0.45 ± 0.02 0.45 ± 0.04 0.47 ± 0.01 2.54 ± 0.18*** 2.77 ± 0.12 3.02 ± 0.50
13.44 ± 0.10 13.45 ± 0.10 13.51 ± 0.01 13.05 ± 0.11** 14.55 ± 0.14 14.01 ± 0.35
29.15 ± 0.36 28.91 ± 0.53 27.75 ± 31 4.52 ± 0.14*** 4.25 ± 0.08 3.7 ± 0.33
GSH, GSSG, GSSG + GSH and GSH/GSSG ratio are shown. GSSG expressed as GSH equivalents. All data are given as means ± SEM, 6 mitochondria suspension per groups. ** p < 0.01, ***p < 0.001, significantly different when compared with control.
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