Chemico-Biological Interactions 216 (2014) 43–52

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Protective effect of captopril against clozapine-induced myocarditis in rats: Role of oxidative stress, proinflammatory cytokines and DNA damage Basel A. Abdel-Wahab a,⇑, Metwally E. Metwally b,1, Mohamed M. El-khawanki c, Alaa M. Hashim d a

Department of Pharmacology, College of Medicine, Assiut University, Assiut, Egypt Department of Forensic Medicine and Toxicology, College of Medicine, Suez Canal University, Ismailia, Egypt Department of Hematology, College of Medicine, Najran University, Najran, Saudi Arabia d Department of Clinical pathology, College of Medicine, Al-Azhar University, Assiut, Egypt b c

a r t i c l e

i n f o

Article history: Received 1 February 2014 Received in revised form 20 March 2014 Accepted 26 March 2014 Available online 5 April 2014 Keywords: Clozapine Antipsychotics Myocarditis Captopril Oxidative stress DNA damage

a b s t r a c t Clozapine (CLZ) is the most effective therapeutic alternative in the treatment of resistant schizophrenia. However, the cardiotoxicity of CLZ, particularly in young patients, has raised concerns about its safety. Captopril is a well-known angiotensin-converting enzyme inhibitor with antioxidant properties effective in treating hypertension and heart failure. The aim of this study was to investigate the protective effect of captopril against clozapine-induced myocarditis in rats and the possible mechanisms behind this effect. The effect of captopril treatment [5 or 10 mg/kg/d, injected intraperitoneally (i.p.) for 21 days] on the cardiotoxic effect of coadministered CLZ (25 mg/kg/d, i.p.) was assessed. Myocarditis was assessed histopathologically, immunohistochemically and biochemically. Frozen heart specimens were used to determine the amount of lipid peroxides product (MDA), nitric oxide (NO), reduced glutathione (GSH), glutathione peroxidase (GSH-Px) activity, proinflammatory cytokines (TNF-a and IL-10) and DNA degradation product(8-OHdG). Coadministration of captopril with the tested doses of CLZ decreased the histological hallmarks and biochemical markers (CK-MP and LDH) of myocarditis. In addition, captopril attenuated the effects of CLZ on oxidative stress parameters, NO and serum and cardiac 8-OHdG levels. Captopril significantly attenuated the effect of CLZ on all measured parameters in a dose-dependent manner. These results suggested that captopril exerts a protective action against CLZ-induced myocarditis. Multiple mechanisms contribute to this effect, including a decrease in cardiac oxidative stress and proinflammatory cytokines production, modulation of antioxidant status and protection from oxidative DNA damage. Hence, captopril may be effective in reducing the incidence and severity of CLZ-induced myocarditis in humans. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Clozapine (CLZ, piperazinyl-debenzo-[1–4]-diazepine), a tricyclic dibenzodiazepine, is an atypical antipsychotic drug that is very efficacious in treating psychosis, particularly in patients refractory to other agents [66]. CLZ has unique effects on a variety of central nervous system receptors [64]. It also has a strong affinity for D4-dopaminergic receptors and potent serotonergic, noradrener-

⇑ Corresponding author. Current address: Department of Pharmacology, College of Medicine, Najran University, Najran, Saudi Arabia. Tel.: +966 553899185; fax: +966 75442419. E-mail address: [email protected] (B.A. Abdel-Wahab). 1 Current address: Department of Forensic Medicine and Toxicology, College of Medicine, Najran University, Najran, Saudi Arabia. http://dx.doi.org/10.1016/j.cbi.2014.03.012 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

gic, [13] histamine [17] and cholinergic M2 receptor [43] blocking abilities. It differs from traditional antipsychotic drugs in that it has relatively weak D2-receptor activity and few extrapyramidal side effects [9]. The drug is clinically significant because it is the most effective antipsychotic compound in treating therapy-resistant schizophrenia and patients with substance use disorder [63]. It is more effective than other antipsychotics for treating positive symptoms [29], causes a threefold reduction in the risk of suicidal behavior in schizophrenic patients [2] and may be associated with a lower mortality than other antipsychotics [70]. In addition, CLZ can improve cognitive deficits [38]. However, some adverse effects of CLZ have limited its clinical use [14]. One serious health safety concern regarding CLZ stems from reports of myocarditis in some patients treated with the drug

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[60]. Health professionals have warned of the risk of potentially fatal myocarditis, cardiomyopathy and heart failure [50], in addition to pericarditis [45] associated with CLZ therapy. Cardiomyopathy has been reported to occur more often in CLZ-treated patients than in patients treated with other antipsychotic medications or in the general population, and most of the patients were less than 50 years of age [42]. An incentive for early diagnosis of CLZ-induced myocarditis is the potential to avert dangerous complications. Even subclinical CLZ-induced myocarditis may progress to dilated cardiomyopathy [24], a condition characterized by cardiac dysfunction and often symptoms of congestive heart failure. The exact mechanisms of CLZ-myocarditis and cardiomyopathy are not clearly understood [44]. Previous clinical studies have indicated that CLZ-induced myocarditis is accompanied with cardiac and peripheral blood eosinophilia, which indicates a possible IgE-mediated hypersensitivity reaction [76]. In addition, CLZ treatment has been associated with increased levels of the circulating catecholamines norepinephrine and epinephrine [37,15]. A marked increase in blood catecholamine levels has been reported to significantly exacerbate myocarditis in animals and patients [71]. This effect of CLZ on plasma catecholamine levels occurs via enhanced neurotransmitter spillover [16]. Moreover, CLZ-induced myocarditis has been associated with an increased release of proinflammatory cytokines [23]. Angiotensin II, the principal effector of the renin-angiotensin system, has been reported to play a crucial role in the pathogenesis of several cardiovascular injuries [34]. A few studies have suggested that angiotensin-converting enzyme inhibitors (ACEIs) may exert a protective role against cardiotoxicity induced by adriamycin [3], doxorubicin [62] and 5-fluorouracil [1]. The beneficial effects of ACEIs are due to decreases in circulating and tissue angiotensin II (Ang II) and potentiation of the effects of bradykinin [69]. Some clinical studies have indicated that prompt diagnosis in addition to angiotensin-converting enzyme inhibitors or beta blockers may be help limit the cardiac side effects of CLZ, and some studies have demonstrated an electrocardiographic as well as hemodynamic benefit [61]. However, the exact mechanisms of action and efficacies of ACEIs against CLZ-induced cardiotoxicity have yet to be investigated. Hence, the aim of this study was to assess the ability of captopril as an ACEI to protect the myocardium from CLZ-induced myocarditis in rats and investigate the role of proinflammatory cytokines and oxidative stress and the corresponding myocardial cell injury and DNA damage in these protective effects. 2. Materials and methods 2.1. Chemicals CLZ (8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo [b,e] [1,4] diazepine) (Sigma–Aldrich, Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia) was dissolved in 0.1 M HCl and pH balanced in phosphate-buffered saline (PBS). Thiobarbituric acid, reduced glutathione (GSH), Griss reagent, Ellman’s reagent [5,5-dithiobis(2nitrobenzoic acid), DTNB] and bovine serum albumin (BSA) were purchased from Sigma–Aldrich, (Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia). All the reagents used in this study were of analytical grade. 2.2. Animals Male Wistar rats with a body weight of 200–250 g from the animal house of King Saud University, Riyadh, Saudi Arabia were used in this study. The animals were housed in groups of 10 in standard

clear polycarbonate cages with food and water available ad libitum. The animals were kept on a 12-h light–dark schedule (6:00 am– 6:00 pm), and all experimental testing was conducted during the light phase from 9:00 am to 12:00 pm. All experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The Institutional Animal Use and Care Committee (Number NU.MID/10/213) approved the experimental protocol. All efforts were made to minimize animal suffering and reduce the number of animals used. 2.3. Experimental protocol The animals were divided into four groups of ten rats each. For three groups of animals, CLZ was administered in 0.1 ml doses of 25 mg/kg/day intraperitoneally (i.p.) alone or in combination with captopril (5 or 10 mg/kg/d, i.p.) for 21 days. Rats in the fourth group served as controls and were treated with saline. The high dose of CLZ used was based on a previous report [72]. The animals were sacrificed on the last day of treatment. After anesthetizing the rats with 45 mg/kg ketamine and 5 mg/kg xylazine administered i.p. (Sigma Aldrich, Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia), blood was drawn by cardiac puncture. The blood samples were centrifuged at 3000 rpm at 25 °C for 15 min, and serum was obtained. The hearts were excised, washed with ice-cold saline, blotted with a piece of filter paper and divided into two halves. One half of each heart was homogenized in phosphate buffer (pH 7.4). The homogenates were centrifuged at 3000 rpm at 4 °C for 30 min. The supernatants of the homogenates were removed and stored at 80 °C. The serum and supernatants were used for biochemical assays. 2.4. Histopathology The ventricles of the second half of each heart were fixed in 10% neutral formalin, embedded in paraffin, sectioned at a thickness of 5 lm, stained with hematoxylin and eosin (H/E), and examined by light microscopy. The ventricle specimens were evaluated for typical histopathological features associated with CLZ-induced cardiotoxicity (inflammation, myocyte vacuolar degradation, myofiber necrosis, and interstitial fibrosis). Histological evidence of myocarditis was classified in terms of the degree of cellular infiltration and graded on a 5-point scale ranging from 0 to 4+ [47]. Briefly, normal tissue (score 0); mild foci; slight infiltration with damage of one or two myocardial fibers (score 1); moderate-sized foci: aggregated infiltrates compromising three to five muscle fibers (score 2); and intense foci: heavy accumulation of mononuclear cells with destruction of more than five muscle fibers (score 3). In this manner a rat showing three foci of chronic myocarditis, coalescent and extensive lesions (two mild, one moderate) had a score of 4. An experienced pathologist blinded to the study groups examined the sections obtained from cardiac tissues. 2.5. Biochemical assays 2.5.1. Determination of serum LDH activity The lactate dehydrogenase activity in the rat serum was estimated using a commercially available LDH kit (Sigma–Aldrich, Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia), as described previously [73]. In this assay, lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate to lactate in the presence of reduced nicotinamide adenine dinucleotide (NADH) at pH 7.5. The reaction is monitored kinetically at 340 nm using an Optima SP 3000 plus spectrophotometer (Optima Inc., Tokyo, Japan) by the rate of decrease in absorbance resulting from the oxidation of

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NADH to NAD+, which is proportional to the activity of LDH present in the sample.

conjugate (8-OHdGTracer) for a limited amount of 8-OHdG monoclonal antibody.

2.5.2. Determination of serum CK-MB activity The creatine kinase (CK-MB) activity in the serum was estimated using a commercially available CK assay kit (Sigma–Aldrich, Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia), as described by Bishop et al. [6]. This assay is based on the conversion of creatine phosphate and ADP by CK-MB to creatine and ATP. The ATP and glucose are converted to ADP and glucose-6-phosphate by hexokinase (HK), and glucose-6-phosphate dehydrogenase (G6PD) then oxidizes glucose-6-phosphate and reduces nicotinamide adenine dinucleotide (NAD) to NADH. The rate of NADH formation, measured at 340 nm, is therefore directly proportional to CK-MB activity.

2.5.8. Determination of caspase-3 activity Caspase-3 activity was determined in cardiac homogenates using colorimetric assay kit; the procedures were performed according to the manufacturer’s instructions (GenScript, USA). Briefly, spectrophotometric measurement of the chromophore pnitroanilide (pNA) released from DEVD pNA by caspase-3 was performed. The absorbance was measured at 405 nm. The caspase-3 activity can be calculated as pmol of pNA released per minute per mg protein.

2.5.3. Cytokine measurement The cardiac IL-10 and TNF-a levels in the cardiac homogenates were measured using specific ELISA kits (Sigma–Aldrich, Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia), and the results were expressed as pg cytokine/mg wet tissue. 2.5.4. Determination of lipid peroxidation The amount of lipid peroxidation in the cardiac homogenate was determined, as previously described [52]. Briefly, the amount of malondialdehyde (MDA), a measure of lipid peroxidation, was measured by a reaction with thiobarbituric acid at 532 nm using an Optima SP 3000 plus spectrophotometer. 2.5.5. Determination of nitric oxide The level of nitric oxide (NO) in the cardiac homogenate was measured by assaying total nitrate/nitrite, the stable products of NO oxidation, as described by Green et al. [21]. The nitrite concentration was measured spectrophotometrically using the Griss reagent [1% sulphanilamide in 5% phosphoric acid (sulphanilamide solution) and 0.1% N-1-naphthylethylenediamine dihydrochloride in double distilled water]. A standard curve was plotted. The nitrite concentrations in the samples were expressed as micromoles per gram protein (lmol/g protein). 2.5.6. Determination of GSH level and GSH-Px activity To determine the amount of intracellular GSH present in the cardiac homogenate, equal volumes of perchloric acid (1 M) and cardiac homogenate were mixed by vortexing, and the mixture was allowed to stand for 5 min at room temperature. After centrifugation at 3000 rpm for 5 min, the supernatant was collected. The GSH content of the neutralized supernatant was assayed using Ellman’s reagent [5,5-dithiobis-2-nitrobenzoic acid (DTNB solution)] [22]. The GSH-Px activity was measured as previously described [55]. The enzymatic reaction involving b-nicotinamide adenine dinucleotide phosphate (NADPH), GSH, glutathione reductase and a sample or a standard, was initiated by the addition of hydrogen peroxide. The change in absorbance was measured spectrophotometrically, and a standard curve was plotted for each assay. 2.5.7. Determination of 8-OHdG levels Produced by the oxidative damage of DNA by reactive oxygen and nitrogen species, 8-hydroxy-2-deoxy guanosine (8-OHdG) serves as an established marker of oxidative stress. The amount of 8-OHdG in serum and tissue homogenate was determined using an 8-OHdG competitive assay kit (Cayman Chemical Co., MI, USA), according to the manufacturer’s instructions. The assay recognizes both free 8-OHdG and DNA-incorporated 8-OHdG and depends on the competition between 8-OHdG and 8-OHdG-acetylcholinesterase (AChE)

2.5.9. Determination of total protein The total protein in the cardiac homogenates was estimated using the method of Lowry [41]. The absorption was measured spectrophotometrically at 750 nm. BSA was used as a standard. 2.6. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining Apoptotic cardiac muscle cell in vivo were visualized using the ApopTag Fluorescein In Situ Apoptosis Detection Kit according to the manufacturer’s instructions. The deparaffinized and rehydrated heart slices were incubated with proteinase K (20 mg/ml) for 15 min at room temperature. Rinsed with equilibration buffer, the slices were incubated with working-strength terminal deoxynucleotidyl transferase enzyme for 1 h at 37 °C in a humidified chamber. After rinsing in a stop/wash buffer, the sections were incubated with working-strength anti-digoxigenin conjugate at room temperature for 30 min. The slices were stained with 496diamidino-2-phenylindole and viewed under a fluorescence microscope (DM4000B, Leica Wetzlar, Germany). The apoptotic cells were counted with at least 100 cells from four randomly selected fields in each treatment, and the values were expressed as percentages of the total number of cells. 2.7. Statistical analysis All experimental results were expressed as the mean ± SEM, and the data were analyzed using one-way analysis of variance (ANOVA), where appropriate. If any statistically significant difference was detected, post hoc comparisons were performed using the Tuky’s test. A value of p < 0.05 was considered statistically significant. 3. Results 3.1. Effect of captopril on CLZ-induced myocarditis The histopathological study was conducted to examine the cellular changes in the cardiac cells that accompanied CLZ-induced myocarditis and resulted from treatment of animals with CLZ (25 mg/kg/ d, i.p) and its concurrent administration with captopril (5 or 10 mg/ kg/d, i.p) for 21 days. The histological findings in the heart tissues of the control rats were within the normal limits (Fig. 1A). In the CLZtreated group, histological sections revealed interstitial edema, perinuclear vacuolation, evident focal subendocardial fibrosis and disorganization and degeneration of the myocardium. Consistent with myocarditis, inflammatory lesions were found in both the left and right ventricles, mainly in the myocardium below the endocardium of the left ventricle; the posterior papillary muscle of the left ventricle; and the septum (Fig. 1B). In addition, there were significant (p < 0.05) increase in interstitial edema, prenuclear vacuolization, myocardial cell degradation and focal fibrosis in the subendocardial tissues (Table 1). All histopathological changes were less severe in

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Fig. 1. Representative H&E-stained sections (40) of the left ventricle from saline- and clozapine-treated rats. (A) No myocardial inflammation was observed after administration of saline. (B) Inflammatory lesions were observed in the myocardium after intraperitoneal administration of 25 mg/kg/d of clozapine (CLZ) for 21 days (arrows). Fewer inflammatory lesions were observed in the myocardium of animals treated by CLZ with captopril (CAP) in (C) 5 mg/kg/d or (D) 10 mg/kg/d dose for 21 days.

tissues from rats treated with both CLZ and 5 or 10 mg/kg/d captopril compared with tissues from rats treated with CLZ alone (Fig. 1C, D). Detailed findings from the histological studies of the cardiac tissues are listed in Fig. 1 and Table 1. Significant differences were observed in the activity of serum CK–MB and LDH among the tested groups [F(3,39) = 6.566, p = 0.0012 and F(3,39) = 8.2.81, p = 0.003, respectively]. Treatment of rats with CLZ (25 mg/kg/d) for 21 days caused a significant increase in serum levels of both CK-MB (Fig. 2A) and LDH (Fig. 2B) compared with the control group (p < 0.01). Coadministration of 5 or 10 mg/kg/d captopril with CLZ resulted in a significant decrease in the serum CK-MB level compared with treatment with CLZ alone (p < 0.05 for 5 mg/kg/d captopril and p < 0.01 for 10 mg/ kg/d captopril) (Fig. 2A). A decrease in the serum LDH level was also observed in serum from rats treated with either 5 or 10 mg/ kg/d captopril and CLZ compared with serum from rats treated with CLZ alone (p < 0.01) (Fig. 2B).

3.2. Effect of captopril on CLZ-induced changes on myocardial oxidative stress parameters The effects of CLZ and captopril on the levels of MDA, NO, GSH and GSH-Px activity in the cardiac homogenates are shown in Table 2. The results revealed a significant effect of CLZ and captopril treatment on the amount of cardiac MDA [F(3.39) = 6.187, p = 0.0017]. Post hoc analysis indicated that CLZ treatment significantly increased amount of cardiac MDA compared with the amount observed in the controls (p < 0.01). In rats co-injected with captopril and CLZ, there was a significant decrease in the amount of cardiac MDA compared with rats treated with CLZ alone (p < 0.05 for 5 mg/kg/d captopril and p < 0.01 for 10 mg/kg/d of captopril). In addition, CLZ and captopril treatment was associated with significant changes in cardiac nitrite levels among the tested groups [F(3.39) = 6.380, p = 0.0014]. CLZ treatment for 21 days significantly increased the amount of cardiac nitrite relative to that observed in control rats (p < 0.01). In rats coinjected with captopril and CLZ, the amount of cardiac nitrite was significantly less than observed in rats treated with CLZ alone.

Fig. 2. Serum activities of (A) creatine phosphokinase (CK-MB) and (B) lactate dehydrogenase (LDH) in rats after intraperitoneal administration of 25 mg/kg/d clozapine (CLZ) alone or in combination with 5 or 10 mg/kg/d captopril (CAP) for 21 days. Results in each group represent mean ± SEM (n = 10). #p < 0.05 vs. control group. ##p < 0.01 vs. control group. ⁄p < 0.01 vs. CLZ-treated group. ⁄⁄p < 0.001 vs. CLZ-treated group.

GSH, which plays an important role in protecting cells against oxidative damage by scavenging the free radicals, was used as a marker for oxidative stress. A significant difference in cardiac GSH levels was observed among the treated groups [F(3.39) = 5.227, p = 0.0042] (see Table 2). The cardiac GSH level was

Table 1 Effect of clozapine (CLZ, 25 mg/kg/d, i.p) alone and in combination with captopril (CAP, 5 or 10 mg/kg/d, i.p) for 21 days on histopathological changes in cardiac tissues of treated rats. Treatment (mg/kg/d)

Interstitial edema

Myocardial degradation

Prenuclear vacuolization

Focal subendocardial fibrosis

Control CLZ (25) CLZ (25) + CAP (5) CLZ (25) + CAP (10)

0.00 ± 0.00 2.33 ± 0.33 a 1.35 ± 0.23b 1.13 ± 0.21c

0.00 ± 0.00 2.50 ± 0.34a 1.45 ± 0.28b 1.33 ± 0.17c

0.00 ± 0.00 2.83 ± 0.52 a 1.45 ± 0.31b 0.84 ± 0.24c

0.00 ± 0.00 1.97 ± 0.30 a 0.76 ± 0.17b 0.62 ± 0.45b

Results represent mean score recorded ± SEM, (n = 10). a p < 0.05 vs. control group. b p < 0.05 vs. CLZ-treated group. c p < 0.01 vs. CLZ-treated group.

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Table 2 Effect of clozapine (CLZ, 25 mg/kg/d, i.p) alone and in combination with captopril (CAP, 5 or 10 mg/kg/d, i.p) for 21 days on myocardial malondialdehyde (MDA), nitrite, intracellular reduced glutathione (GSH) levels and glutathione peroxidase (GSH-Px) activity in rats. Treatment (mg/kg/d)

MDA (lmol/g protein)

Nitrite (lmol/g protein)

GSH (nmol/g protein)

GSH-Px (IU/g protein)

Control CLZ (25) CLZ (25) + CAP (5) CLZ (25) + CAP (10)

315.62 ± 15.34 412.52 ± 21.32b 332.37 ± 17.25c 325.26 ± 17.25d

3.33 ± 1.14 14.24 ± 1.12b 2.75 ± 3.46d 2.64 ± 2.38d

33.46 ± 3.52 16.85 ± 3.23b 30.33 ± 3.43c 32.45 ± 3.33 c

26.45 ± 3.24 15.45 ± 2.13a 25.34 ± 1.48c 26.34 ± 1.18d

Results in each group represent mean ± SEM (n = 10). a p < 0.05 vs. control group. b p < 0.01 vs. control group. c p < 0.01 vs. CLZ-treated group. d p < 0.001 vs. CLZ-treated group.

significantly lower in the CLZ-treated group than in the control group (p < 0.01). Captopril co-administration with CLZ at the tested doses significantly attenuated the decrease in the cardiac GSH level typically induced by CLZ. GSH-Px is an important enzyme for inactivation of peroxy radicals utilizing GSH. Myocardial GSH-Px activity was significantly reduced in rats treated with CLZ compared with the GSH-Px activity observed in control rats, reflecting the exhaustion of antioxidant enzymes by CLZ [F(3.39) = 6.081, p = 0.0019]. Cardiac GSHPx activity was significantly higher in rats co-administered with CLZ and either 5 mg/kg/d (p < 0.05) or 10 mg/kg/d (p < 0.01) captopril than in rats treated with CLZ alone; the increase in GSH-Px activity due to captopril was dose dependent.

3.3. Effect on cardiac cytokines levels TNF-a is a cytokine involved in systemic inflammation. It is produced chiefly by activated macrophages, although it can be produced by many other cell types. The results revealed significant differences in the serum levels of TNF-a among treatment groups [F(3.39) = 12.472, p = 0.0001]. CLZ treatment significantly increased the TNF-a level relative to that observed in control rats (p < 0.01). The amount of TNF-a was significantly decreased in rats co-administered with CLZ and 5 mg/kg/d captopril (p < 0.05) or 10 mg/kg/d captopril (p < 0.01) compared with that observed in rats treated with CLZ alone (Fig. 3A). IL-10, also known as cytokine synthesis inhibitory factor (CSIF), is an anti-inflammatory cytokine. The results revealed significant differences in serum levels of IL-10 among the treated groups of rats [F(3.39) = 5.364, p = 0.0037]. CLZ treatment significantly reduced the serum level of IL-10 relative to the level observed in control rats. Captopril attenuated this effect in a dose-dependent manner; the amount of serum IL-10 in rats treated with CLZ and 5 mg/kg/d (p < 0.05) or 10 mg/kg/d (p < 0.01) captopril was increased compared with the amount observed in rats treated with CLZ alone (Fig. 3B).

3.4. Effect on serum and cardiac 8-OHdG levels Significant differences were observed in the 8-OHdG levels in both serum [F(3.39) = 12.472, p = 0.0001] and cardiac homogenate [F(3.39) = 6.566, p = 0.0012] among the tested groups. After 21 days treatment with CLZ, the 8-OHdG levels were significantly increased in serum (p < 0.05) and cardiac homogenate (p < 0.01) compared with the levels observed in serum and heart homogenate from control animals, indicating increased oxidative DNA damage in response to CLZ. The levels of 8-OHdG in both serum and cardiac tissues were significantly reduced in rats co-injected with CLZ and either 5 mg/kg/d (p < 0.05) or 10 mg/kg/d (p < 0.01) captopril compared

Fig. 3. Cardiac levels of (A) tumor necrosis factor alpha (TNF-a), (B) interleukin-10 (IL-10) in rats after intraperitoneal administration of 25 mg/kg/d clozapine (CLZ) alone or in combination with 5 or 10 mg/kg/d captopril (CAP) for 21 days. Results in each group represent mean ± SEM (n = 10). #p < 0.05 vs. control group. ##p < 0.01 vs. control group. ⁄p < 0.01 vs. CLZ-treated group. ⁄⁄p < 0.001 vs. CLZ-treated group.

with rats treated with CLZ alone (Fig. 4A, B). This suggests that captopril has a protective effect against cardiac DNA damage. 3.5. Effect of captopril on CLZ-induced apoptotic damage in cardiac tissues Significant differences were observed in the cardiac caspase-3 activity [F(3.39) = 7.458, p = 0.0005] among the tested groups. After 21 days treatment with CLZ, the cardiac caspase-3 activity was significantly (p < 0.01) increased compared with control value. The activity of cardiac caspase-3 in captopril-treated animals significantly (p < 0.05) decreased with the dose 5 mg/kg/d and significantly (p < 0.01) decreased with the dose 10 mg/kg/d compared with CLZ-treated group (Fig. 5). To confirm whether the protective function of captopril against CLZ cardiotoxicity was connected with apoptosis, TUNEL staining was used in rat myocardial tissues. As revealed in Fig. 6B, CLZ-treated animals showed significant increased TUNEL positive cells indicating presence of cellular apoptosis. By contrast, pretreatment with captopril effectively ameliorated CLZ-induced myocardial cell

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Fig. 4. Levels of 8-hydroxy-2-deoxyguanosine (8-OHdG) in (A) serum and (B) myocardium of rats after intraperitoneal administration of 25 mg/kg/d clozapine (CLZ) alone or in combination with 5 or 10 mg/kg/d captopril (CAP) for 21 days. Results in each group represent mean ± SEM (n = 10). #p < 0.05 vs. control group. ## p < 0.01 vs. control group. ⁄p < 0.01 vs. CLZ-treated group. ⁄⁄p < 0.001 vs. CLZtreated group.

Fig. 5. Caspase-3 activity in cardiac tissues of rats after intraperitoneal administration of 25 mg/kg/d clozapine (CLZ) alone or in combination with 5 or 10 mg/kg/d captopril (CAP) for 21 days. Results in each group represent mean ± SEM (n = 10). ## p < 0.01 vs. control group. ⁄p < 0.01 vs. CLZ-treated group. ⁄⁄p < 0.001 vs. CLZtreated group.

apoptosis Fig. 6C and D. The percentage of TUNEL-positive cells was also calculated. As presented in the histogram (Fig. 6E), TUNEL-positive cells treated with CLZ increased to 63.68%, whereas those pretreated with captopril 5 and 10 mg/kg decreased to 34.46% and 23.16% respectively. These results indicated that captopril can protect against CLZ-induced apoptosis in cardiomyocytes.

4. Discussion CLZ continues to be an effective and widely used antipsychotic agent. However, since its introduction, CLZ has been plagued by controversy because of its side-effect profile. Initial studies were primarily concerned with agranulocytosis, but in recent years,

the focus has shifted to its potentially fatal cardiotoxicity [35]. In the present study, treatment with CLZ (25 mg/kg/d) for 21 days induced marked myocarditis and cardiotoxic effects in rats. CLZ-induced cardiotoxicity was manifested by histopathological changes in the heart such as focal subendocardial fibrosis. Marked interstitial edema and perinuclear vacuolation were also observed. Consistent with myocarditis, inflammatory lesions were found in both the left and right ventricles, mainly in the myocardium below the endocardium of the left ventricle; the posterior papillary muscle of the left ventricle; and the septum. Similar histopathological changes have been reported by others [72]. This cardiotoxic effect was confirmed by elevated CK-MB and LDH activity levels in the serum of CLZ-treated rats. Elevation of serum levels of these enzymes is considered important marker of early and late cardiac injury, especially during clinical follow-up of drug-induced cardiotoxicities [31]. All rats treated with CLZ appeared sedated, lethargic and sick for at least 1 h after the injection. Similar lethargy and syncope have been reported in some patients that may be related to CLZ-induced cardiotoxicity [30]. The exact mechanisms of CLZ-induced myocarditis are unclear. Existing evidence points to a multitude of molecular mechanisms involved in CLZ-induced cardiotoxicity. One of the possible mechanisms involves a CLZ-induced cytokine release. CLZ stimulates an in vivo release of TNF-a and various interleukins [23]. In addition, CLZ-induced myocarditis is characterized by inflammation and release of proinflammatory cytokines [59]. The CLZ-induced increase in TNF-a, one of the proinflammatory cytokines induced by CLZ, is dose-dependent [72]. These proinflammatory cytokines have been found to mediate autoimmune myocarditis [12] and may act similarly in CLZ-induced myocarditis. These studies are in agreement with the current study that demonstrated an elevation of cardiac TNF-a and a decrease in IL-10 in CLZ-treated animals. Persistent inappropriate tachycardia has been demonstrated to impair left ventricular function both in animal models and humans [36]. CLZ induces a rise in plasma catecholamines, which correlates with the degree of induced myocardial inflammation and can induce tachycardia [72,8]. Moreover, b-adrenergic blocking agents such as propranolol have been shown to ameliorate the myocardial inflammatory effects of CLZ [72]. The present study demonstrated that CLZ-induced cardiotoxicity was associated with a marked elevation of the myocardial level of the lipid peroxidation product (MDA) and a reduction of GSH content and activity of the antioxidant enzyme GSH-Px. These results support the theory that increased oxidative stress and weakening of antioxidant defenses play an important role in CLZinduced myocarditis [20]. Clinical and experimental investigations have suggested that the increased oxidative stress associated with an impaired antioxidant defense status initiates a cascade of reactions responsible for CLZinduced cardiotoxicity [26]. CLZ induces free radical generation by direct and indirect mechanisms. In a direct manner, CLZ is bioactivated in the myocardial tissues to a chemically reactive nitrenium ion, which stimulates cellular injury, lipid peroxidation and free radical formation [74]. This nitrenium ion also binds with proteins in the myocardium, leading to the formation of an antigenic complex that stimulates an immune response and macrophages [58]. This triggers the release of proinflammatory cytokines such as TNF-a, which mediate cellular inflammation and myocarditis and generate additional free radicals. Increased oxidative stress may attenuate the release of anti-inflammatory cytokines such as IL10, thereby enhancing inflammatory processes in cardiac tissues. This may represent a significant pathway for the development of myocarditis. Indirectly, the above-mentioned CLZ-induced increase in catecholamines triggers tachycardia and increases myocardial oxygen demand by increasing work done and cardiac loads via

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Fig. 6. The immunohistochemical reaction for activated (TUNEL) positive cells in heart. (A) Section from a control rat showing no immunostained cardiac muscle fibres with TUNEL staining cells. (B) Section from rats treated with clozapine in doses 25 mg/kg/d, i.p., for 21 days, showing increased TUNEL positive immunoreactivity showing the apoptotic cells (arrows). (C, D) Cardiac tissues of animals treated with captopril (CAP) 5 and 10 mg/kg/d respectively concurrently with CLZ for 21 days showing decreased number of TUNEL-positive cells. (E) The TUNEL apoptotic index was determined by calculating the ratio of TUNEL positive cells to total cells. Results in each group represent mean ± SEM (n = 10). ##p < 0.05 vs. control. ⁄p < 0.05 vs. clozapine-treated group. ⁄⁄p < 0.01 vs. clozapine-treated group.

catecholamines-induced vasoconstriction [7]. In addition, catecholamines decrease myocardial oxygen perfusion via coronary vasoconstriction [67]. In response to long-term exposure to CLZ, these events can lead to myocardial ischemia and free radical generation. The released free radicals and reactive oxygen species (ROS) can attack membrane phospholipids, increasing lipid peroxidation and damage to the myocardial cell membrane. This process is reflected in the elevated myocardial MDA level observed in this study. In addition, our results demonstrated that CLZ treatment significantly increased the cardiac level of nitrite, a stable end product and indirect marker of NO. Other studies have also demonstrated an increase in cardiac NO levels following exposure to CLZ; this effect may be related to the drug itself or its metabolite N-desmethylclozapine via its agonistic activity to M1 receptors on cardiac vagal preganglionic fibers [27]. NO is a key molecule involved in the pathophysiology of the heart. The dysregulation of NO synthase (NOS) and NO metabolism is a common feature in various cardiac ailments [11]. Evidence demonstrating that CLZ induces nitric oxide synthase (iNOS) expression and NO release in the heart and the ability of NO to promote CLZ redox cycling to produce ROS [26] suggests that NO contributes to CLZ-induced myocarditis. Increased NO formation can be a source of reactive nitrogen species (RNS), which can trigger a number of independent proinflammatory/cytotoxic events, including (i) the initiation of lipid peroxidation, (ii) the inactivation of a variety of enzymes, and (iii) the depletion of GSH. These effects were consistent with the decrease in cardiac GSH level and GSH-Px activity observed in CLZ-treated animals in this study. An increase in ROS and RNS formation and decrease in antioxidant defenses may account for the cellular damage and the observed increase in both serum and cardiac levels of 8-OHdG, a biomarker of DNA damage. A chronic increase in the cellular levels of ROS can lead to the development of myocarditis, cell apoptosis in cardiac muscle and profound cardiomyopathy and heart failure [49]. Coadministration of captopril with CLZ in this study attenuated CLZ-induced myocarditis. This was evidenced by the improvement of the histological changes in cardiac tissues and the significant reduction in the activities of serum cardiac enzymes associated with cardiotoxicity in rats coinjected with CLZ and captopril

compared with rats treated with CLZ alone. These findings confirmed the role of Ang II in CLZ-induced cardiac damage. Consistent with our findings, it has been reported that the angiotensin-converting enzyme (ACE) inhibitors captopril and zofenopril ameliorated the effects of drug-induced cardiac toxicities [62,53]. Clinically, previous studies have reported that ACE inhibitors may limit certain cardiac side effects induced by CLZ [61]. In the current study, coadministration of captopril with CLZ remarkably attenuated the effect of CLZ on biochemical markers of oxidative stress and lipid peroxidation, indicating a profound antioxidant role of CAP. The antioxidant effect of ACE inhibitors has been reported previously [46]. Captopril, as one of the thiolcontaining ACE inhibitors, has been reported to act as a free radical scavenger of various reactive oxygen species and to have excellent antioxidant properties related to its sulfhydryl (SH) group. Sulfhydryl compounds are able to neutralize ROS by either a hydrogendonating or electron-transferring mechanism [65]. Reduction of free radicals protects against lipid peroxidation, as reflected in this study by the decrease in the cardiac level of MDA. In addition, coadministration of captopril with CLZ significantly reduced the cardiac level of nitrites induced by CLZ treatment. Interaction among NO, the angiotensin system, and ROS and RNS is complex. These interacting pathways play an important role in various cardiovascular diseases, including hypertension and heart failure [40]. In this sense, angiotensin II has been reported to augment cytokine-stimulated NO synthesis in rat cardiac myocytes [28]. Angiotensin II has also been shown to stimulate superoxide radical production, which then reacts with NO to form peroxynitrite [25]. The peroxynitrite may contribute to the cellular damage associated with CLZ treatment. A previous study has reported that attenuation of angiotensin II signaling recouples endothelial NO synthase (eNOS) by preventing it from forming superoxide radical ([51]). In addition, angiotensinII receptors (A1) were detected in coronary vessels that mediate vasoconstriction and decrease myocardial oxygen perfusion [75], which may account the formation of free radicals. Moreover, in this study, the cardioprotective action of captopril against CLZ-induced myocarditis was accompanied by an increase in the level of cardiac GSH and activity of GSH-Px, the two

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important components of the antioxidant defense, and a decrease in the level of TNF-a and an increase in the level of IL-10. These results are in agreement with previous results. ACE-inhibitors, particularly captopril, increase GSH level and activity of antioxidant enzymes in the myocardium of hypertensive rats [48]. Furthermore, captopril inhibits the release of the inflammatory cytokine TNF-a and stimulates the release of the anti-inflammatory cytokine IL-10. These effects are partially mediated through the direct effect of captopril on the release of cytokines and indirectly via captopril’s antioxidant effect [4]. The antioxidant effect of captopril is mediated by the direct free radical scavenging effect and by activation of antioxidant defenses. Hence, captopril decreases free radicals generation, which plays an important role in tissue injury, inflammation and release of inflammatory cytokines [32]. The hemodynamic action of captopril cannot be omitted from its cardioprotective mechanisms against CLZ-induced myocarditis. Captopril has been reported to decrease myocardial oxygen demand by decreasing cardiac loads resulting from vasoconstriction and salt/water retention via inhibition of angiotensin-II formation and bradykinin accumulation [39]. These effects antagonize the effects of myocardial ischemia and consequently reduce ROS and RNS production triggered by the CLZ-induced catecholamine release. The renin-angiotensin system is a vital element of the physiological and pathological responses of the cardiovascular system. Its chief effector hormone, Ang II, not only mediates physiological effects such as vasoconstriction and blood pressure regulation but has also been implicated in inflammation, endothelial dysfunction, myocarditis and heart failure [54]. Many of the cellular effects of Ang II appear to be mediated by ROS, which is produced by NAD(P)H oxidase [33]. Renin-angiotensin system consists of two opposing arms. The pressor arm is comprised of the enzyme, ACE; the product, Ang II; and the AT1 receptor, which is the main protein mediating the biological actions of Ang II. The second arm is composed of the monocarboxypeptidase ACE2; Ang (1–7), which are produced through the hydrolysis of Ang II; and the Mas receptor, which is the protein conveying the vasodilator, antiproliferative, antifibrotic, and antithrombotic effects of Ang (1–7) [19]. Inhibition of Ang II formation by ACE inhibitors stimulates the vasodilator and antiproliferative action of Ang (1–7) [57]. Another mechanism by which captopril produces its cardiac antiproliferative and antifibrotic effects involves the ability of captopril to increase plasma and tissue concentrations of N-acetylseryl-aspartyl-lysyl-proline (Ac-SDKP), which is a natural inhibitor of pluripotent hematopoietic stem cell proliferation [5]. This increase is Ac-SDKP results in a decrease in cardiac cell proliferation (probably fibroblasts), inflammatory cell infiltration, and collagen deposition [56]. Under normal conditions, ROS and RNS attack nuclear and mitochondrial DNA, causing oxidized nucleosides and, consequently, mutagenic DNA lesions. One of these lesions is 8-OHdG, the end product of the hydroxylation of guanine. The DNA lesions are consequently removed by the base excision repair (BER) pathway, which prevents replication of DNA lesions. Moreover, ROS and RNS inhibit the BER system through direct interactions with cellular repair proteins [18]. Since the BER pathway removes the mutagenic 8-OHdG lesions, the inhibitory effects of the ROS and RNS pathways on BER activity may potentiate mutagenesis and DNA damage. The ability of captopril to decrease ROS and RNS production via its antioxidant properties and reactivate antioxidant defenses may explain the observed decrease in both serum and cardiac 8-OHdG levels observed in rats coinjected with CLA and captopril compared with rats injected with CLZ alone. In addition, the formation of ROS and its accumulation in the cardiac tissues due to tissue ischemia and attenuation of anti-oxidative capacity initiate mitochondrial apoptotic signaling and activate

number of apoptotic proteins such as Bax on of the BCl-2 family proteins [68]. These proapoptotic factors stimulate Apaf-1 and caspase9 to form apoptosomes and then activates caspase-3 leading to cell death [10]. In our study, both TUNEL positive staining and caspase3 activity significantly increased in CLZ-treated animals, the fact that reflects CLZ-induced cardiac cell injury and apoptosis. However, captopril treated groups showed significant dose-dependent decrease in TUNEL positive cells and significant decrease in caspase-3 activity in the tested doses compared with CLZ-treated animals. Our data suggested that early treatment with captopril might inhibit cardiocyte apoptosis thus providing an antiapoptotic and cardiorpotective effect against CLZ-induced cardiac injury and apoptosis. 5. Conclusions Captopril can protect against myocarditis induced by large dose administration of CLZ in rats. The protective effect of captopril was accompanied with a significant attenuation of CLZ’s effect on oxidative stress parameters (MDA, NO), activation of antioxidant defenses (GSH, GSH-Px), a reduction in the proinflammatory cytokines and oxidative DNA damage, in addition to attenuation of CLZinduced cardyocyte apoptosis. These results reflect the multiple mechanisms in the cardioprotective effect of captopril against CLZ-induced myocarditis. The results of this study suggest a beneficial use for captopril in the protection against CLZ-induced myocarditis in patients with psychiatric illnesses. 6. Conflict of Interest On behalf of the authors, no conflict of interest is found in this work. Transparency Document The Transparency document associated with this article can be found in the online version.

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