Mol Biol Rep (2013) 40:6533–6545 DOI 10.1007/s11033-013-2673-5

(2)-Epigallocatechin-gallate (EGCG) stabilize the mitochondrial enzymes and inhibits the apoptosis in cigarette smoke-induced myocardial dysfunction in rats Gokulakrishnan Adikesavan • Magendira Mani Vinayagam • Liyakath Ali Abdulrahman • Thirunavukkarasu Chinnasamy

Received: 16 January 2013 / Accepted: 14 September 2013 / Published online: 7 November 2013  Springer Science+Business Media Dordrecht 2013

Abstract The present study brings out the preventive role of (-)-epigallocatechin-gallate (EGCG) on cardiac mitochondrial metabolism and apoptosis in cigarette smoke (CS)-exposed rats. The CS-exposed rats showed significantly decreased activities of TCA cycle enzymes and mitochondrial enzymatic antioxidants, on the other hand, mitochondrial lipid peroxidation was increased and GSH level was decreased. Further, CS exposure was found to induce cardiac apoptosis through release of cytochrome c into the cytosol, cleavage of pro-caspase-3 to active caspase-3, up-regulation of pro-apoptotic (Bax) and downregulation of antiapoptotic (Bcl-2) molecules. The CSinduced apoptosis was further confirmed by mitochondrial and nuclear ultra structural apoptotic features as evaluated by electron microscopic studies. EGCG supplementation shelters the activities of TCA cycle enzymes and antioxidant enzymes, with concomitant decrease in lipid peroxidation and increase in GSH level. EGCG administration inhibited apoptosis through the inhibition of cytochrome c release into cytosol, activation of pro-caspase-3, down regulation of Bax and significant up regulation of Bcl-2. EGCG reversed the ultra structural apoptotic alterations of mitochondria and nucleus. The present study has provided

G. Adikesavan
T. Chinnasamy (&) Department of Biochemistry, Periyar University, Salem 636016, Tamil Nadu, India e-mail: [email protected] G. Adikesavan
M. M. Vinayagam
L. A. Abdulrahman Department of Biochemistry, Islamiah College, Vaniyambadi 635753, Tamil Nadu, India T. Chinnasamy Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry 605014, India

experimental evidences that the EGCG treatment enduring to cardio protection at mitochondrial level. Keywords Antioxidants
Apoptosis
Cigarette smoke
EGCG Abbreviations CVD Cardiovascular disease CS Cigarette smoke EGCG (-)-Epigallocatechin-gallate ROS Reactive oxygen species

Introduction Cigarette smoking is one of the most prevalent social habits practiced world wide and remains a major cause of cardiovascular disease (CVD)-related morbidity and mortality. A growing body of evidence has confirmed the notion that cigarette smoking adversely affects the structure and function of cardiovascular system [1, 2]. Furthermore, epidemiological studies have shown that passive cigarette smoking increases the risk of coronary heart disease, myocardial infarction, stroke and cardiac arrest [3]. The degree of this risk is proportional to the frequency of smoking [4]. Although the specific mechanisms underlying these changes have not been completely understood, they are likely to be related to the fact that cigarette smoke (CS) exposure increases vascular inflammation, oxidative stress and mitochondrial damage [3, 5, 6]. Since mitochondria control cellular energy levels, reactive oxygen species (ROS) production, detoxification and apoptosis, their dysfunction is a major contributor to CS-induced cardiac injury [6]. Smoking reduces arterial oxygen carrying capacity through increased serum

123

6534

carboxyhemoglobin and causes oxidative phosphorylation dysfunction in cardiac cells [7]. Exposure to passive tobacco smoke from two cigarettes per day for 2 months have severely damaged myocardial oxidative phosphorylation function in rats [8]. Smoking has been shown to inhibit mitochondrial enzyme activity in platelets, lymphocytes and cause mitochondrial dysfunction in alveolar macrophages [9, 10]. These observations clearly show that mitochondria play an important role in CS-induced cardiac damage. Apoptosis, a physiological program of cellular death has been shown to play an important role in the pathogenesis of heart failure of various etiologies like chronic pressure overload, ischemia reperfusion injury and congestive heart failure [11]. Apoptosis or programmed cell death is a tightly regulated process that consists of complex biochemical cascades involving the activation of pro-caspases [12]. Caspases are a class of cysteine proteases involved in the initiation and execution of apoptosis and are activated through either intrinsic or extrinsic apoptosis pathways. The intrinsic pathway is triggered by signals that cause the release of cytochrome c, Apaf-1 and other proteins from mitochondria. These proteins then form an apoptosome with procaspase-9, resulting in the formation of active caspase-9. Caspase-9 then cleaves procaspase-3, resulting in active caspase-3, the major apoptosis executor [12]. Several studies have demonstrated that exposure to CS results in cardiac remodeling and impaired ventricular function through increased oxidative stress, apoptosis and inflammation [13, 14]. CS is a complex mixture of over 4,800 identified constituents, which are able to accumulate in mitochondria and may disturb the functions of the mitochondrial respiratory chain and cellular ATP production, leading to myocardial contractile dysfunction and cardiac fibrosis [2, 4, 6, 14, 15]. Studies have shown that the impairment of mitochondrial function and programmed cell death pathways can be inhibited by antioxidants [9, 16]. In this view, one of the natural antioxidant such as (-)-epigallocatechin-gallate (EGCG) is an attractive agent that could defend against cardiovascular injury. EGCG, the most abundant and potent antioxidant of green tea has been proved to have cardio protective effect in experimental studies [17, 18]. Sheng et al. [19] reported that green tea extract and EGCG protected cardiomyocytes against ischemia/reperfusioninduced apoptotic cell death both in vitro and in vivo. Recently we reported that the administration of EGCG prevents CS-induced cardiac dysfunction through its antioxidative, anti-inflammatory and lipid lowering effects [5]. The present study was carried out to gain in-depth evidence of preventive role of EGCG in CS-induced cardiac dysfunction by evaluating the effect of EGCG on the mitochondrial function and apoptosis in CS-induced myocardial dysfunction.

123

Mol Biol Rep (2013) 40:6533–6545

Materials and methods Chemicals and their sources EGCG, Tris–HCl, Triton X-100, nicotinamide adenine dinucleotide (NAD) were purchased from M/s. Sigma Chemical Co., St. Louis, USA. Monoclonal antibodies of cytochrome c, caspase-3, caspase 9, Bax, Bcl-2 and secondary antibodies were purchased from Santa Cruz Biotechnology and Cell Signaling Technologies, USA. All other chemicals, including solvents, were of high purity and analytical grade procured from SD Fine Chemicals, Mumbai and Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Experimental animals Adult male albino rats of Wistar strain, weighing approximately 140–160 g, were obtained from Saveetha University, Chennai 600077, Tamil Nadu, India. They were acclimatized to animal house conditions for a week, fed commercial pellet rat chow (Hindustan Lever Ltd., Bangalore, India) and water, ad libitum. The experiments were conducted according to the ethical norms approved by Ministry of Social Justice and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines of Saveetha University (IAEC No. PU/001/10). Experimental design The animals were divided into four groups of six animals each: • • • •

Group I: Control rats (without any treatment). Group II: Rats exposed to CS for a period of 12 weeks. Group III: Rats administered with EGCG (20 mg/kg body weight/day). Group IV: Rats exposed to CS for a period of 12 weeks and simultaneously administered with EGCG (20 mg/kg b.w./day).

EGCG was dissolved in physiological saline (0.89 % of NaCl) and administered for 12 weeks by oral gavage. The dose was equivalent to the EGCG content in approximately 2–3 cups (one cup = 120 ml; 185 mg of EGCG per cup) of green tea consumption everyday in humans (approximately 70 kg weight). Groups II and IV rats were exposed to CS for a period of 12 weeks as described by Anbarasi et al. [20]. Briefly, the rats were placed in a whole body smoke exposure chamber, which contains two holes of about 3 cm in diameter. Smoke from a lighted cigarette was introduced through one hole and air through the other. The cigarette was fixed away from the chamber and smoke was drawn in by slow

Mol Biol Rep (2013) 40:6533–6545

suction with the help of a tube and an aerator, so that there was no temperature change within the chamber. The animals were exposed to side stream CS twice daily, the duration of each exposure was 3 h with an interval of 10 min between each cigarette, using eight to ten cigarettes per day. The same brand of locally available cigarette (Scissors Standard) was used throughout the experiment. Control animals were subjected to the same handling and time in the smoke exposure chamber with air in the place of smoke/air mixture. After the experimental period (12 weeks), the animals were anesthetized with pentobarbital sodium (35 mg/kg body weight, i.p.) and sacrificed by cervical dislocation. The heart was dissected out immediately and washed in ice-cold saline. The whole organ was weighed. A portion of heart tissue was used for mitochondrial isolation and a portion of tissue was stored for electron microscopic studies. Determination of ratio of heart weight to body weight After sacrifice, the hearts from the experimental animals were quickly excised and weighed. The ratio of heart weight to body weight was determined for each rat. Isolation of heart mitochondria Mitochondria were isolated from the heart tissue by the method of Jhonson and Lardy [21]. The heart was washed, minced well in ice-cold saline and homogenized in 0.25 M ice-cold sucrose solution at 4 C. This homogenate was centrifuged at 500 g for 10 min to remove nuclear fraction and the broken cell debris. The supernatant was then centrifuged in a refrigerated REMI C-24 centrifuge at 12,0009g for 12 min. The pellet was taken as the mitochondrial pellet and suspended in 0.25 M sucrose containing 10 mM Tris–HCl buffer, pH 7.4 and 1 mM EDTA to a known volume. This was gently homogenized and used to assess the antioxidant status and the activities of mitochondrial enzymes. Biochemical analysis Lipid peroxides in cardiac mitochondria were determined as thiobarbituric acid reactive substances (TBARS) by the methods of Ohkawa et al. [22] and reduced glutathione (GSH) was measured by the method of Ellman [23]. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) were assayed by the method of Misra and Fridovich [24], Sinha [25], Rotruck et al. [26], Habig and Jacoby [27] and Carlberg and Mannervik [28], respectively. The activities of isocitrate

6535

dehydrogenase (ICDH) [29], a-ketoglutarate dehydrogenase (a-KGDH) [30], succinate dehydrogenase (SDH) [31], malate dehydrogenase (MDH) [32], NADH dehydrogenase [33] and cytochrome-c-oxidase [34] were estimated by standard methods. Immunoblotting Western blot technique was employed to quantify the abundance of the cytochrome c, caspase-3, caspases-9, Bax and Bcl-2. Heart of control and experimental rats were homogenized in buffer containing 135 mM NaCl, 20 mM Tris–HCl, 2 mM EDTA and 1 mM PMSF (pH 7.4). The volume of homogenization buffer was 1 ml per 100 mg tissue sample. The homogenates were centrifuged (15 min, 10,000 rpm at 4 C) and the protein content of the supernatant was determined according to method of Lowry et al. [35] with BSA as standard. Supernatant aliquots (20 lg total protein) were boiled for 5 min in sample buffer (0.2 M Tris–HCl buffer, 10 % glycerol, 2 % SDS, 0.02 % b-mercaptoethanol). Proteins were separated by Tris–glycine–SDS discontinuous 10 % polyacrylamide gel and electroblotted on to Immobilon-P membranes (Millipore Corp., Bedford, MA) using a semidry transfer system [36]. After protein transfer, the membrane was incubated for 2 h at room temperature in blocking buffer [0.05 % Tween-20, 5 % non-fat dried milk, Tris-buffered saline (TBS), pH 7.5]. After blocking, the membrane was rinsed for 5 min with washing buffer [TBS containing 0.05 % Tween-20 (TBST)], then incubated for 16 h at 4 C with primary antiserum diluted (1:1,000) with TBS containing 0.05 % Tween-20 and 1.0 % non-fat dried milk. After over night incubation of primary antibody, the membrane was washed thrice (5 min each) with TBST. Then it was incubated with respective secondary antibodies (1:50,000 dilution) for 1 h at room temperature. Specific signals were detected by chemiluminescence using luminol substrate. Electron microscopic studies Small pieces of heart were rinsed in 0.1 M phosphate buffer, pH 7.2, trimmed into *1 mm heart pieces and immediately fixed into 3 % ice-cold glutaraldehyde in 0.1 M phosphate buffer and kept overnight at 4 C. Then tissue was processed for electron microscopic studies. The grids containing sections were stained with 2 % uranyl acetate and 0.2 % lead acetate. The sections were examined on a transmission electron microscope. Sirius red staining Heart tissues were fixed in 4 % formalin, embedded in paraffin and sectioned. Slides were hydrated through a

123

Mol Biol Rep (2013) 40:6533–6545 4

8

NS

3.5

*

*

7

*

3 6

2.5

NS

2

5

1.5

*

*

NS

Units

Heart weight/body weight ratio (mg/g)

6536

1 0.5

4

*

3

0 Group I

Group II

Group III

Group IV

Fig. 1 Effect of EGCG on the ratio of heart weight to body weight in control and experimental rats. Results are expressed as mean ± SD, for six animals in each group. Statistical significant variations are compared as follows: control versus CS exposed and EGCG alone treated; CS exposed versus EGCG ? CS exposed. *p \ 0.05, NS non significant

series of graded alcohols (100, 95, and 75 %), 15 min each. The slides were stained with Weigert’s hematoxylin (Sigma-Aldrich, MO, USA) for 8 min, washed and restained with picro-Sirius red (Sigma) for 1 h. After gently rinsing with water, slides were dehydrated through graded alcohols for 15 min each, cleared in xylene and cover slipped. The sections were examined under microscope.

2

1

0 TBARS

GSH

Group I

Group III

Group II

Group IV

Fig. 2 Effect of EGCG on the levels of mitochondrial lipid peroxidation and reduced glutathione (GSH) in control and experimental groups. Units: nmol of TBARS formed/100 mg of mitochondrial protein for LPO; nmol/100 mg of mitochondrial protein for GSH. Results are expressed as mean ± SD, for six animals in each group. Statistical significant variations are compared as follows: control versus CS exposed and EGCG alone treated; CS exposed versus EGCG ? CS exposed. *p \ 0.05, NS non significant

Statistical analysis All the grouped data were evaluated with SPSS/10 software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference (LSD) test. P values of less than 0.05 were considered to indicate statistical significance. All these results were expressed as mean ± SD for six animals in each group.

with control rats (Group I) (Fig. 1). In rats administrated with EGCG and simultaneously exposed to CS (Group IV), there was a significant increase (p \ 0.05) in the ratio of heart weight to body weight when compared to CS-exposed rats (Group III), demonstrating the growth-impeding effect of CS and the counteracting action of EGCG against CS. No significant difference was observed in control rats treated with EGCG alone (Group III) when compared to control rats (Group I).

Results Effect of EGCG on the CS-induced body and heart weight During treatment, all animals appeared active and no mortality was encountered in either of the groups. Among the four groups of animals, the CS-exposed animals (Group II) showed less body weight and heart weight gain when compared to control (Group I) (data not shown). The ratio of heart weight to body weight was decreased significantly (p \ 0.05) in CS-exposed (Group II) rats when compared

123

Effect of EGCG on CS-induced mitochondrial oxidative stress The levels of LPO and GSH of heart mitochondria in control and experimental animals are shown in Fig. 2. CS exposure (Group II) showed a significant increase in mitochondrial LPO level with a concomitant reduction (p \ 0.05) in GSH when compared to control rats (Group I). EGCG treatment to CS-exposed rats showed a significant decrease in LPO with a corresponding increase in mitochondrial GSH when compared to those of CS-exposed rats

Mol Biol Rep (2013) 40:6533–6545

6537

Table 1 Effect of EGCG on the activities of heart mitochondrial antioxidant enzymes in control and experimental groups Particulars

SOD

Group I

12.36 ± 0.57

CAT

GPx

GST

GR

1.78 ± 0.096

3.96 ± 0.61

68.43 ± 3.71

4.82 ± 0.35

1.28 ± 0.11*

1.86 ± 0.34*

42.84 ± 4.62*

2.43 ± 0.62*

12.22 ± 0.68NS

1.82 ± 0.082NS

3.92 ± 0.42NS

67.68 ± 6.63NS

4.71 ± 0.61NS

11.12 ± 0.92*

1.66 ± 0.12*

3.21 ± 0 .57*

59.27 ± 5.36*

4.21 ± 0.52*

Group II

5.86 ± 0.89*

Group III Group IV

Units Activities are expressed as units/min/100 mg of mitochondrial protein for SOD; nmol H2O2 consumed/min/mg of mitochondrial protein for CAT; nmol of GSH consumed/min/100 mg of mitochondrial protein for GPx; nmol of 1-chloro-2,4-dinitrobenzene (CDNB) conjugated/min/ 100 mg of mitochondrial protein for GST; nmol of NADH oxidized/min/100 mg of mitochondrial protein for GR. Results are expressed as mean ± SD, for six animals in each group. Statistical significant variations are compared as follows: control versus CS exposed and EGCG alone treated; CS exposed versus EGCG ? CS exposed NS non significant * p \ 0.05

Table 2 Effect of EGCG on heart mitochondrial TCA cycle enzyme activities in control and experimental groups Particulars

Isocitrate dehydrogenase

a Ketoglutarate dehydrogenase

Succinate dehydrogenase

Malate dehydrogenase

NADH dehydrogenase

Cytochrome-coxidase

Group I

748.63 ± 56.91

73.58 ± 6.95

245.48 ± 19.13

342.34 ± 24.38

139.38 ± 12.84

47.96 ± 2.15

Group II

537.08 ± 47.53*

42.99 ± 4.21*

163.74 ± 14.61*

243.17 ± 1.44*

86.49 ± 4.16*

22.64 ± 1.87*

Group III

751.34 ± 46.97NS

72.78 ± 5.88NS

249.53 ± 17.62NS

346.03 ± 28.29NS

137.87 ± 14.41NS

46.15 ± 1.78NS

Group IV

708.93 ± 35.98*

66.22 ± 4.23*

232.49 ± 12.35*

328.78 ± 23.04*

128.52 ± 11.83*

52.26 ± 1.89*

Units Activities are expressed as moles of NADH oxidized/h/mg of mitochondrial protein for ICDH; nmol of ferrocyanide formed/h/mg of mitochondrial protein for a-KGDH; nmol of succinate oxidized/min/mg of mitochondrial protein for SDH; nmoles of NADH oxidized/min/mg of mitochondrial protein for MDH; nmol of NADH oxidized/min/mg of mitochondrial protein for NADH-dehydrogenase; nmol of cytochrome/min/mg of mitochondrial protein for cytochrome-c-oxidase. Results are expressed as mean ± SD, for six animals in each group. Statistical significant variations are compared as follows: control versus CS exposed and EGCG alone treated; CS exposed versus EGCG ? CS exposed NS non significant * p \ 0.05

(Group II). In EGCG alone treated rats there was no significant change in LPO and GSH levels when compared to control rats. Antioxidant enzyme activities reflect the level of oxidative stress of the tissue examined. The antioxidant enzyme activities in heart mitochondria of control and experimental rats are shown in Table 1. CS-exposure caused a significant decrease in antioxidant enzyme activities such as SOD, CAT, GPx, GST, and GR when compared to control rats. EGCG treatment showed a significant increase in SOD, CAT, GPx, GST, and GR (p \ 0.05) activities in heart mitochondria when compared to CS alone exposed rats. In EGCG alone treated rats there were no alterations in any of the above antioxidant enzyme activities when compared to control rats. Effect of EGCG on TCA cycle enzyme activities The activities of the TCA cycle and respiratory enzymes in the control and experimental groups are presented in Table 2. The activities of the ICDH, a-KGDH, SDH, MDH, NADH dehydrogenase and cytochrome-c-oxidase

(p \ 0.05) were found to be significantly lower in the mitochondria of rats subjected to CS-exposure when compared with control (Group I). EGCG treatment resulted in a significant (p \ 0.05) increase in the above enzyme activities in Group IV animals when compared to Group II animals. No significant difference was seen between Group III and Group I (control animals). Effect of EGCG on CS-induced cardiac apoptosis Oxidative damage to mitochondria triggers apoptosis by the release of cytochrome c into the cytosol [37]. To ascertain whether myocardial damage is accompanied by the release of mitochondrial cytochrome c in CS-exposed rats, the levels of mitochondria free, cytosolic proteins were determined by immunoblot analysis (Fig. 3a). The cytochrome c content in the cytosol of CS-exposed rats was found to increase when compared to control rats (p \ 0.05) (Fig. 3b). Given that cytochrome c is primarily located in the mitochondria under normal conditions, this increase indicated that cytochrome c was released to the cytosol during CS exposure. Oral supplementation of EGCG to CS

123

6538 Fig. 3 Effect of EGCG on levels of proteins related to apoptotic pathways in cardiac tissues. a Level of cytosolic cytochrome c and the activated form of caspase-9 by Western blot analysis and b quantitation of signal intensity of bands normalised using b-actin. c Activation of pro-caspase-3 analyzed by immunoblotting. Activation of caspases-3 is evidenced by the formation of cleaved caspases (19 KD) (bar diagram shows mean ± SD of three independent experiments)

Mol Biol Rep (2013) 40:6533–6545

A

B

C

intoxicated rats reduces the cytosolic cytochrome c content (p \ 0.05), possibly by protecting mitochondrial membrane integrity. To determine the downstream effect of cytochrome c release, the levels of active caspase-3 were evaluated by immunoblot (Fig. 3c). Caspases are cytosolic proteins that exist normally as inactive precursors with higher molecular weights (55, 46 and 32 kDa). They are cleaved proteolytically into low molecular weights (20–13 kDa) when cell undergoes apoptosis [37]. The activation of caspase-3 by proteolytic processing of pro-caspase-3 into 17 and 12 kDa subunits serves as an early marker of apoptosis in various cell types. In our immunoblot analysis, we detected diminished band of inactive caspases (procaspases) and increased cleaved fragments in rat heart exposed to the CS. EGCG treatment, however, prevented the CS-induced fragmentation of caspase-3 on the other hand, EGCG alone did not show any effect on caspase-3 activation (Fig. 3c). To further understand the upstream signaling pathways associated with the activation of caspase-3, the levels of caspase-9 were evaluated by immunoblot analysis (Fig. 3a). CS-exposed groups showed significantly higher levels of active caspase-9 (p \ 0.05) than did the control

123

group (Fig. 3a). EGCG administration prevented the CSinduced activation of caspase-9 however, EGCG alone did not show any effect on caspase-9 activation (Fig. 3b). These changes indicate that the mitochondria-mediated apoptotic caspases cascade was activated in the CSexposed rat. Bcl-2, a family of proteins plays pivotal role in upstream signaling of cytochrome c release and to constitute a key intracellular check point in apoptotic signal transduction. In particular, among the entire Bcl-2 family, Bax (a proapoptotic member) and Bcl-2 (an antiapoptotic member) have been well examined and that the relative content of Bax/Bcl-2 provides a tight control in promoting the execution of apoptotic cascades [12, 37]. In concert with this idea, we assessed whether Bcl-2 family members play any regulatory role in the cytochrome c pathway of apoptosis in CS-exposed rats, by determining the levels of Bcl-2 and Bax. In our immuno blot analysis, Bcl-2 protein content decreased and Bax protein content increased in CSexposed rats than the control rats (Fig. 4a). Oral administration of EGCG to CS-exposed rats significantly decrease the Bax level and increases the Bcl-2 level toward that of

Mol Biol Rep (2013) 40:6533–6545 Fig. 4 Effects of EGCG on levels of Bcl-2 and Bax protein expression. a Western blot analysis. b Quantitation of the signal intensity of Bax and Bcl2 (bar diagram shows mean ± SD of three independent experiments)

6539

A

B

control rats (p \ 0.05). Following EGCG treatment Bcl-2 is upregulated in CS-exposed rats when compared with control however, Bax expression appeared to be downregulated. These results show that the antiapoptotic property of EGCG was achieved possibly through regulating the levels of pro- and antiapoptotic Bcl-2 family proteins. Effect of EGCG on myocardial ultra-structure Figure 5 shows the electron microscopic heart sections in control and experimental groups of rats. Control rats showed normal architecture of cardiac mitochondria (Fig. 5a). Figure 5c, d were the CS-exposed rats shows highly abnormal architecture of mitochondria, swelling of mitochondria, increased areas of vacuolation indicative of edema and mitochondrial cristae were also disrupted. The mitochondria of EGCG alone treated rats showed a normal structure (Fig. 5b). EGCG administrated rats with CSexposure (Fig. 5e) shows mild distortion in the mitochondrial structure, with out swelling and normal cristae. Electron microscopy was used to identify ultra structural details of typical apoptosis within a cardiac myocyte. Figure 6 shows the ultra structure of myocardium from the control and experimental groups of animals. Control rats showed cells with intact plasma membrane, nucleus and mitochondria (Fig. 6a). CS-exposed rats showed distinct ultra structural alterations with chromatin condensation, vacuolation of cytoplasm, formation of apoptotic bodies and mitochondrial swelling, which are the characteristics of

apoptosis (Fig. 6c, d). EGCG ? CS-exposed rats did not show cigarette smoking-induced alterations by the absence of apoptotic features (Fig. 6e). EGCG alone administered rats showed normal architecture with no significant changes (Fig. 6b). DNA fragmentation is considered to be the hallmark of apoptosis. In this study, we have evaluated DNA fragmentation (Fig. 7), an evident for apoptosis, CSexposed rats showed typical DNA fragmentation laddering pattern which is the characteristic of apoptosis. Administration of EGCG during CS-exposure reduced the formation of DNA fragments, which shows the anti apoptotic activity of EGCG (Fig. 7). Histopathology A histopathological analysis was done to understand the myocardial architecture and cardiac fibrosis of control and experimental groups with hematoxylin–eosin staining (Fig. 8a–d). Myocardium of healthy control rats showed normal architecture and orderly alignment of myocytes (Fig. 8a). Histopathological findings of the CS-exposed myocardium showed enlarged interstitium disorganization of myocardial fibers with interstitial fibrosis (area of infarction with splitting of cardiac muscle fibers) (Fig. 8b). EGCG treated ? CS-exposed myocardium showed normal cardiac muscle bundles with minimal interstitial fibrosis in tissues observed at 2009 magnification (Fig. 8c). Rats supplemented with EGCG (Group III) showed normal cardiac muscle bundles without any damage (Fig. 8d).

123

6540

Mol Biol Rep (2013) 40:6533–6545 Control

EGCG alone

A

Control

A

B

20,000 x

C

B

10,000 x

10,000 x

CS induced Mitochondrial damage

EGCG alone

CS induced Nuclear fragmentation

CS induced cristae damage

C

D

30,000 x

10,000 x

CS induced shrunken nucleus

D

15,000 x

20,000 x

10,000 x

CS induced + EGCG Treated

CS induced + EGCG Treated

E

E

10,000 x 10,000 x

Fig. 5 Transmission electron microscope images of heart mitochondria in control and experimental rats. a Control group showing normal architecture of the heart mitochondria. b Control ? EGCG treated rats showing normal architecture of the heart mitochondria. c and d CS-intoxicated heart mitochondria showing swelling of mitochondria and disruption of cristae with vacuolation. e EGCG ? CSintoxicated heart mitochondria showing mild separation of cristae without swelling and vacuolation

Fig. 6 Transmission electron microscope images of cardiac nuclear changes in control and experimental groups of rats. a Control group showing normal nucleus. b Control ? EGCG treated rats showing normal nucleus. c and d CS exposed heart showing chromatin condensation, apoptotic bodies and fragmented nucleus. e EGCG ? CS exposed heart showing absence of apoptotic features

Discussion Heart sections were stained with Sirius red for detecting the deposition of collagen to assess the impact of EGCG on cardiac fibrosis caused by CS-exposure. Significant accumulation of collagen (red colour-indicated by the arrow) and myofibril disarray were observed in the CS-exposed rats (Fig. 8f) when compared to normal control (Fig. 8e). Accumulated collagen levels and myofibril disarray were attenuated in EGCG-treated ? CS-exposed rats (Fig. 8g). Noticeable changes were not observed in the EGCG alone treated animals (Fig. 8h) when compared to normal control. Collectively, these results demonstrated that EGCG protected the rat heart from CS-induced fibrogenesis.

123

Significant reduction in ratio of heart weight to body weight (Fig. 1) following CS-exposure confirmed the previous report [4]. Cigarette smoking was shown to inhibit the activity of orexigenic peptides and stimulate the activity of anorexigenic peptides, which in turn significantly decreased food intake, body weight and fat mass [38]. Thus our finding of decreased weight gain caused by CS-exposure is consistent with the multitudinous effects of plasma nicotine on food intake and energy expenditure. The decreased heart weight might be due to extensive interstitial fibrosis and collagen deposition. Simultaneous

Mol Biol Rep (2013) 40:6533–6545

Fig. 7 Agarose gel electrophoretic pattern of cardiac DNA in control and experimental groups of rats. The results are representative of three independent experiments. Lane 1 Group I (control); lane 2 Group II (CS); lane 3 Group III (EGCG); lane 4 Group IV (CS ? EGCG)

administration of EGCG on CS-exposed rats brought up the ratio of heart weight to body weight, indicative of protection of myocardium against infiltration and it could also be due to the decrease in water content of the myocardium. We observed an increased lipid peroxidation and decreased GSH level in the mitochondrial fraction of heart in CS-exposed rats (Fig. 2). CS was shown to increase LPO in rat brain mitochondria [20]. An elevated level of lipid peroxidation in lymphocytes of chronic smokers has been correlated to mitochondrial respiratory chain dysfunction [10]. Elevation of lipid peroxides in CS-exposed rats could be attributed to the sustained release of reactive free radicals from the tar and gas phases of CS. GSH serves as the primary antioxidant defense against oxidative stress. Heart mitochondrial GSH was significantly lowered after exposure to CS. In agreement with this finding Ramesh and Begum [39] have demonstrated that CS-exposure deplete the GSH level. Supplementation of EGCG reduced the levels of LPO and increased GSH in heart mitochondria of CS-intoxicated rats. Scavenging of superoxide, hydroxyl and peroxyl radical, which is responsible for the initiation and propagation of LPO, may be one of the reasons for the LPO inhibitory activity of EGCG, and the other possible mechanism might be the rejuvenation of tocopherol content

6541

and the iron chelating activity [18, 40]. Fu et al. [41] showed that the EGCG enhanced the level of cytoplasmic and mitochondrial GSH, which may be due to increase in gene expression of the catalytic subunit of glutamate-cysteine ligase (GCL), which is the rate-limiting enzyme in the de novo synthesis of GSH. Antioxidant enzymes are considered to be the primary defense agent that protects biological macromolecules from oxidative damage. Aerobic cells contain various amounts of two main antioxidant enzymes, SOD and CAT. In this study, significantly decreased activities of SOD and CAT were observed in mitochondrial fraction of heart tissues of CS-exposed rats when compared to control rats (Table 1). The observed decrease in the activities of these enzymes might be due to their increased utilization for scavenging ROS and their inactivation by excessive CS oxidants. EGCG comprising the ortho-hydroxyl group in the B-ring and galloyl moiety in the C-ring could react directly with superoxide may reduce the formation of H2O2 and in addition it possess a direct scavenging effect on H2O2 and restores SOD and CAT activities [18]. The activities of GPx, GST and GR were declined in mitochondrial fraction of heart tissues of CS-exposed rats (Table 1). A decreased activity of these GSH dependent antioxidant enzymes reduces LPO, detoxifies toxic electrophiles and maintains intracellular concentration of GSH. CS-exposed has been demonstrated to reduce GPx, GST and GR activities in rat whole heart [39]. In mitochondria, the decreased GSH level and increased lipid peroxidation inhibit GSH-dependent enzymes such as GPx, GST and GR in CS-intoxicated rats. The decreased activities of these antioxidant enzymes by CS-exposure might be associated with the depletion of GSH level and an increase of LPO level. Simultaneous administration of EGCG to CSexposed rats increased the activities of these enzymes, indicating the protective nature of the EGCG against CSinduced cardiac injury. Enhancement of these enzyme activities in cardiac tissues by EGCG has been reported under different conditions [40, 42, 43]. Restoration of GSH level and decrease LPO may be responsible for the increase in the activities of GPx, GST and GR in EGCG administered CS-exposed rats. Cardiomyocytes are rich in mitochondria, which are the key organelles involved in myocardial injury. The activities of enzyme involved in the aerobic oxidation of pyruvate in mitochondria are ICDH, a-KGDH, SDH and MDH were significantly decreased in CS-exposed rats (Table 2). It has been shown that CS can decrease the activities of TCA cycle enzymes and reduce the energy production in brain mitochondria [20]. These dehydrogenases are located in the outer membrane of the mitochondria and affected by increased levels of free radicals and aldehydes produced on CSintoxication [44]. The decreased activities of respiratory

123

6542 Fig. 8 Histopathological observation of heart tissue stained with haematoxylin and eosin (H&E) (a–d) and Sirius red staining (e–h). a Control group, showing normal architecture and orderly alignment of myofibrils. b Cigarette smoke exposed rats showing area of infarction with splitting of cardiac muscle fibers and enlarged interstitium; arrows indicate myocardial interstitium. c EGCG treated cigarette smoke exposed rats showing a near normal myocardium. d EGCG supplemented rats showing normal cardiac muscle bundles. e Control rat showing orderly alignment of myofibrils without collagen deposition. f CSexposed rats shows significant accumulation of collagen (red colour-indicated by the arrow) and myofibril disarray. g EGCG treated CS exposed rat showing limited collagen deposition and myofibril disarray. EGCG alone treated animals showing no collagen deposition and myofibril disarray (2009; n = 3). (Color figure online)

marker enzymes such as NADH-dehydrogenase and cytochrome c oxidase observed in CS-exposed rats were in line with previous reports [7, 45]. The decreased activities of respiratory marker enzymes observed in CS-intoxicated rats might be due to increased serum carboxyhemoglobin levels [7, 8] and enhanced phospholipid degradation resulting in the non-availability of cardiolipin for their functional activity. CS was shown to promote auto antibodies to cardiolipin [45]. Treatment of EGCG resulted in restoring the activities of these enzymes to near normal in CS-intoxicated rats. In previous studies, we found that EGCG is protective against lipid peroxidation in whole heart tissues [5]. Thus, the capacity of EGCG to restore the mitochondrial membrane

123

Mol Biol Rep (2013) 40:6533–6545

A

B

C

D

E

F

G

H

components from oxidative insults would have proven essential in preserving these enzymes against lipid peroxidative damage and deterioration. Myocyte apoptosis contributes to progressive deterioration of cardiac function [11]. Caspases play critical roles in the apoptotic process and activation of caspase-3 is a pivotal downstream event in the execution of apoptosis. In the present study, we observed that CS caused cleavage of procaspase 3 to active caspase-3 (17 kDa, Fig. 3c) in the rat cardiac tissues. When the CS-exposed rats were treated with EGCG, activation of caspase-3 was prevented. Recent studies have demonstrated that mitochondria may play a key role in apoptosis by releasing cytochrome c and

Mol Biol Rep (2013) 40:6533–6545

activating caspase-9, which activates caspase-3 that is responsible for DNA-cleavage action and myocyte death [12, 37]. In this study, we also found that CS exposure apparently increased the caspase-9 (Fig. 3a). The principal component in CS such as nicotine was shown to increase caspase activity and increase cardiac apoptosis [46]. However, EGCG treatment decreased the activation of caspase-3 and caspase-9 which were induced by CS. Previous study shown that EGCG decreased caspase-3 activity and inhibits cardiac apoptosis through its antioxidant potential [17]. The level of cytochrome c in the cytosol, serves as a marker of mitochondrial damage. Data from our present study revealed that CS-exposure elicits the increased level of cytochrome c in the cytosol. CS-exposure is known to increase oxidative radicals in myocytes, susceptibility to the mitochondrial permeability transition (MPT) and increase myocardial infarct size [6]. Initiation of the MPT is thought to result from increased Ca2? loading and increased ROS in mitochondria [12] and results in the release of mitochondrial Ca2? and cytochrome c. These events can cause apoptotic myocyte death [37]. Therefore, oxidative stress and their associated depolarization of mitochondrial membranes coupled with MPT may be considered as possible causes for outer mitochondrial membrane permeability to release cytochrome c and their subsequent activation of apoptosis. Treatment with EGCG decreased the level of cytochrome c in the cytosol of the EGCG-treated CS-exposed group compared to CS alone exposed group. Interestingly, in the present observation we found that EGCG inhibits the release of cytochrome c from mitochondria into cytosol and inhibited the mitochondria-mediated apoptosis pathway. EGCG supplementation can improve antioxidant status, especially glutathione and also maintain critical thiol groups in a reduced state, which may be attributed to the inhibition of endogenous MPT activity. Hence, it may be concluded that the increase in mitochondrial redox status accompanying inhibition of MPT is considered as the potential antiapoptotic mechanism of EGCG by regulating the release of cytochrome c into the cytosol. Apoptosis is usually regulated by a complex interplay of a number of proapoptotic and antiapoptotic proteins. Following oxidative stress-induced activation, Bax, a proapoptotic protein translocates to mitochondria, by exposing its N-terminus, which allows the Bax/Bax-homo oligomerization, resulting in MPT pores and subsequent release of the mitochondrial residing apoptogenic factors, including cytochrome c [12, 37]. The antiapoptotic role of Bcl-2 has been established by showing that Bcl-2 forms Bcl-2/Bax heterodimers, which oppose the proapoptotic activity of Bax by preventing the process of Bax/Bax-homo oligomerization [12, 47]. Bcl-2 targets the voltage-dependent anionic channel and blocks the release of cytochrome c, thereby

6543

blocks apoptosis. So, the ratio of Bax and Bcl-2 (an index of cell resistance to apoptosis) determines whether a cell will undergo apoptosis or to survive in pathophysiology [37]. We found that CS-exposure to rats has decreased the level of Bcl-2, and significantly increased Bax protein (Fig. 4a, b), resulting in an overall decrease of Bcl-2/Bax ratio. Bax induces apoptosis by the formation of large pores by Bax oligomers alone or in association with voltage-dependent anion channel [37, 47]. Our study demonstrated that CSexposed suppressed cardiac expression of Bcl-2 protein, which might prevent permeability transition pore opening, cytochrome c release and might facilitate the mitochondriadependent apoptotic cascade. Earlier report showed that CSexposed triggered apoptosis in gastric cells through the inhibition of Bcl-2 and by the release of cytochrome c [12]. When the CS-exposed rats were treated with EGCG, there was decreased expression of Bax and increased expression of Bcl-2 protein. This resulted in a reversal of the Bcl-2/Bax ratio. Thus apoptosis-resistance index could be largely protected by EGCG treatment, implying the involvement of Bcl-2 family proteins in the anti-apoptotic action of EGCG. The present study showed that CS activated the mitochondrial dependent apoptotic signaling in the heart via, Bcl-2 family protein dysregulation, Bax upregulation, cytochrome c release, and downstream caspase-3 activation. EGCG, with potent antioxidant effect, attenuates CS-induced cardiomyocyte apoptosis through the inhibition of mitochondrial dependent apoptotic pathway. Apoptosis-associated DNA degradation in cells has been attributed to several endonucleases and exhibits the typical ‘‘ladder’’-like formation when subjected to electrophoresis. In the present study, chronic CS exposure induced apoptosis in myocardium, which was characterized by DNA fragmentation as evidenced by the laddering pattern, characteristics of apoptosis. Generation of oxygen free radicals from CS induces oxidative DNA damage that leads to apoptosis. However, treatment with EGCG could inhibit ladder formation. The transmission electron microscopy results showed that CS-exposure induces chromatin condensation, nuclei fragmentation and formation of apoptotic bodies which are the characteristics of apoptosis. Administration of EGCG inhibited smoking-induced apoptosis in cardiac tissues, as indicated by the absence of DNA fragmentation and absence of apoptotic features by electron microscopy. Sheng et al. [42] reported that EGCG, by reducing the intracellular oxidants, protect the genomic DNA and there by decreased apoptosis and cell damage. The present study confirms that EGCG treatment protects the cardiac morphological architecture by reducing mitochondrial oxidative stress. The aberrant cardiac mitochondrial change associated with CS-exposure was also visualized with electron microscope (Fig. 5a–e). The registered ultra structural

123

6544

alterations in myocardial mitochondria were in line with the previous work [48] which reported that 30 days of environmental tobacco smoke exposure in male Wistar rats was associated with disorganization of the myofibrils and mitochondrial swelling. They have also shown that the injury to myocardial mitochondria was elicited by carbon monoxide which enhanced the level of carboxyhaemoglobin with consequent hypoxia of the myocardium. EGCG treatment prevented these ultra structural alterations.

Conclusion The CS-exposed rats showed significant increase in mitochondrial LPO and significant decrease in GSH level, antioxidant enzymes and TCA cycle enzyme activities. CSexposure was shown to promote cardiac apoptosis by releasing the cytochrome c into cytosol, activation of caspase-3, caspase-9 and significant down regulation of the anti-apoptotic protein (Bcl-2) with simultaneous up regulation of the pro-apoptotic protein (Bax). The CS-induced apoptosis was further confirmed by ultra structural apoptotic features. EGCG administration decreased the level of LPO and significantly increased the activities of antioxidant. As evidenced by TCA cycle key enzyme activities, EGCG treatment protects the mitochondrial function. We further observed that treatment with EGCG inhibited apoptosis through the inhibition of cytochrome c release into cytosol, inhibition of activation caspase-3 and caspase9, down regulation of the Bax and significant up regulation of the anti-apoptotic protein in the CS-exposed rats. EGCG also prevented DNA fragmentation and ultra structural apoptotic alteration. These results suggest that EGCG reduces the oxidative stress in the myocardium and play a significant role in the inhibition of mitochondria dependent apoptotic pathways leading to cardio protection. Acknowledgments We gratefully acknowledge the assistance from Dr. P. V. Anandhbabu, Dr. Murali, Dr. P. Ashok Kumar, and Dr. S. Bharathiraja. We also acknowledge Prof. B. Kannabiran (Retired), Department of Biochemistry and Molecular Biology, Pondicherry University, Puducherry, India for his assistance while revising this manuscript.

References 1. Goette A, Lendeckel U, Kuchenbecker A, Bukowska A, Peters B, Klein HU, Huth C, Rocken C (2007) Cigarette smoking induces atrial fibrosis in humans via nicotine. Heart 93(9):1056–1063 2. Zhou X, Sheng Y, Yang R, Kong X (2010) Nicotine promotes cardiomyocyte apoptosis via oxidative stress and altered apoptosis-related gene expression. Cardiology 115(4):243–250 3. Barnoya J, Glantz SA (2005) Cardiovascular effects of second hand smoke: nearly as large as smoking. Circulation 111(20):2684–2698

123

Mol Biol Rep (2013) 40:6533–6545 4. Kuo WW, Wu CH, Lee SD, Lin JA, Chu CY, Hwang JM, Ueng KC, Chang MH, Yeh YL, Wang CJ, Liu JY, Huang CY (2005) Second-hand smoke-induced cardiac fibrosis is related to the Fas death receptor apoptotic pathway without mitochondria-dependent pathway involvement in rats. Environ Health Perspect 113(10):1349–1353 5. Gokulakrishnan A, Jayachandran DB, Thirunavukkarasu C (2011) Attenuation of the cardiac inflammatory changes and lipid anomalies by (-)-epigallocatechin-gallate in cigarette smoke exposed rats. Mol Cell Biochem 354(1–2):1–10 6. Yamada S, Zhang XQ, Kadono T, Matsuoka N, Rollins D, Badger T, Rodesch CK, Barry WH (2009) Direct toxic effects of aqueous extract of cigarette smoke on cardiac myocytes at clinically relevant concentrations. Toxicol Appl Pharmacol 236(1):71–77 7. Gvozdjakova A, Kucharska J, Gvozdjak J (1992) Effect of smoking on the oxidative processes of cardiomyocytes. Cardiology 81(2–3):81–84 8. Van Jaarsveld H, Kuyl J, Alberts DW (1992) Antioxidant vitamin supplementation of smoke exposed rats partially protects against myocardial ischemic reperfusion injury. Free Radic Res Commun 17(4):263–269 9. Aoshiba K, Tamaoki J, Nagai A (2001) Acute cigarette smoke exposure induces apoptosis of alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 281(6):L1392–L1401 10. Miro O, Alonso JR, Jarreta D, Casademont J, Urbano-Ma´rquez A, Cardellach F (1999) Smoking disturbs mitochondrial respiratory chain function and enhances lipid peroxidation on human circulating lymphocytes. Carcinogenesis 20(7):1331–1336 11. Kim NH, Kang PM (2010) Apoptosis in cardiovascular diseases: mechanism and clinical implications. Korean Circ J 40(7):299–305 12. Clerk A, Cole SM, Cullingford TE, Harrison JG, Jormakka M, Valks DM (2003) Regulation of cardiac myocyte cell death. Pharmacol Ther 97(3):223–261 13. Zhou X, Li C, Xu W, Chen J (2012) Trimetazidine protects against smoking-induced left ventricular remodeling via attenuating oxidative stress, apoptosis, and inflammation. PLoS ONE 7(7):e40424 14. Das A, Dey N, Ghosh A, Das S, Chattopadhyay DJ, Chatterjee IB (2012) Molecular and cellular mechanisms of cigarette smokeinduced myocardial injury: prevention by vitamin C. PLoS ONE 7(9):e44151 15. Ramesh T, Sureka C, Bhuvana S, Hazeena Begum V (2010) Sesbania grandiflora diminishes oxidative stress and ameliorates antioxidant capacity in liver and kidney of rats exposed to cigarette smoke. J Physiol Pharmacol 61(4):467–476 16. Banerjee S, Chattopadhyay R, Ghosh A, Koley H, Panda K, Roy S, Chattopadhyay D, Chatterjee IB (2008) Cellular and molecular mechanisms of cigarette smoke-induced lung damage and prevention by vitamin C. J Inflamm 5:21 17. Hirai M, Hotta Y, Ishikawa N, Wakida Y, Fukuzawa Y, Isobe F, Nakano A, Chiba T, Kawamura N (2007) Protective effects of EGCG or GCG, a green tea catechin epimer, against post ischemic myocardial dysfunction in guinea-pig hearts. Life Sci 80(11):1020–1032 18. Velayutham P, Babu A, Liu D (2008) Green tea catechins and cardiovascular health. Curr Med Chem 15(18):1840–1850 19. Sheng R, Gu ZL, Xie ML, Zhou WX, Guo CY (2007) EGCG inhibits cardiomyocyte apoptosis in pressure overload induced cardiac hypertrophy rats and protects cardiomyocyte from oxidative stress. Acta Pharmacol Sin 28(2):191–201 20. Anbarasi K, Vani G, Shyamala Devi CS (2005) Protective effect of bacoside A on cigarette smoking-induced brain mitochondrial dysfunction in rats. J Environ Pathol Toxicol Oncol 24(3):225–234

Mol Biol Rep (2013) 40:6533–6545 21. Jhonson D, Lardy H (1967) Isolation of liver and kidney mitochondria. In: Estabrook RW (ed) Methods in enzymology, vol 10. Academic Press, London, pp 94–96 22. Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissue by thiobarbituric acid reaction. Anal Biochem 95(2):351–358 23. Ellman GL (1959) Tissue sulphydryl groups. Arch Biochem Biophys 82(1):70–77 24. Misra HP, Fridovich I (1972) The role of superoxide anion in the auto oxidation of epinephrine and a simple assay of superoxide dismutase. J Biol Chem 247(10):3170–3175 25. Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47(2):389–394 26. Rotruck JT, Pope AL, Ganther HE, Hafeman DG, Hoekstra G (1973) Selenium biochemical role as a component of glutathione peroxidase. Science 179(4073):588–590 27. Habig WH, Jakoby WB (1981) Assays for differentiation of glutathione-s-transferases. Methods Enzymol 77:398–405 28. Carlberg I, Mannervik B (1975) Purification and characterization of the flavo enzyme glutathione reductase from rat liver. J Biol Chem 250(14):5475–5480 29. King J (1965) Isocitrate dehydrogenase. In: King JC, Van D (eds) Practical clinical enzymology. Nostrand Co., London, p 363 30. Reed LJ, Mukherjee RB (1969) a-Ketoglutarate dehydrogenase complex from Escherichia coli. In: Lewinstein JM (ed) Methods in enzymology. Academic Press, New York, pp 55–61 31. Slater EC, Borner WD Jr (1952) The effect of fluoride on the succinic oxidase system. Biochem J 52(2):185–196 32. Mehler AH, Kornberg A, Criscuoli S, Ochoa S (1948) The enzymatic mechanism of oxidation reductions between malate or isocitrate or pyruvate. J Biol Chem 174(3):961–977 33. Minakami S, Ringler RL, Singer TP (1962) Studies on the respiratory chain-linked dihydro diphospho pyridine nucleotide dehydrogenase. I. Assay of the enzyme in particulate and in soluble preparations. J Biol Chem 237:569–576 34. Pearl W, Caocarano J, Zweifach BW (1963) Microdetermination of cytochrome oxidase in rat tissues by the oxidation on N-phenyl-phenylenediamine or ascorbic acid. J Histochem Cytochem 11(1):102–107 35. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275 36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning—a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, p 1.74 37. Crow MT, Mani K, Nam YJ, Kitsis RN (2004) The mitochondrial death pathway and cardiac myocyte apoptosis. Circ Res 95(10):957–970

6545 38. Chen H, Vlahos R, Bozinovski S, Jones J, Anderson GP, Morris MJ (2005) Effect of short-term cigarette smoke exposure on body weight, appetite and brain neuropeptide Y in mice. Neuropsychopharmacology 30(4):713–719 39. Ramesh T, Begum VH (2008) Protective effect of Sesbania grandiflora against cigarette smoke-induced oxidative damage in rats. J Med Food 11(2):369–375 40. Devika PT, Stanley Mainzen Prince P (2008) (-)Epigallocatechin-gallate (EGCG) prevents mitochondrial damage in isoproterenol-induced cardiac toxicity in albino Wistar rats: a transmission electron microscopic and in vitro study. Pharmacol Res 57(5):351–357 41. Fu Y, Zheng S, Lu SC, Chen A (2008) Epigallocatechin-3-gallate inhibits growth of activated hepatic stellate cells by enhancing the capacity of glutathione synthesis. Mol Pharmacol 73(5):1465–1473 42. Sheng R, Gu ZL, Xie ML, Zhou WX, Guo CY (2009) EGCG inhibits proliferation of cardiac fibroblasts in rats with cardiac hypertrophy. Planta Med 75(2):113–120 43. Sheng R, Gu ZL, Xie ML, Zhou WX, Guo CY (2010) Epigallocatechin gallate protects H9c2 cardiomyoblasts against hydrogen dioxides-induced apoptosis and telomere attrition. Eur J Pharmacol 641(2–3):199–206 44. Sun L, Luo C, Long J, Wei D, Liu J (2006) Acrolein is a mitochondrial toxin: effects on respiratory function and enzyme activities in isolated rat liver mitochondria. Mitochondrion 6(3):136–142 45. Fickl H, Van Antwerpen VL, Richards GA, Van der Westhuyzen DR, Davies N, Van der Walt R, Van der Merwe CA, Anderson R (1996) Increased levels of autoantibodies to cardiolipin and oxidised low density lipoprotein are inversely associated with plasma vitamin C status in cigarette smokers. Atherosclerosis 124(1):75–81 46. Hu N, Guo R, Han X, Zhu B, Ren J (2011) Cardiac-specific over expression of metallothionein rescues nicotine-induced cardiac contractile dysfunction and interstitial fibrosis. Toxicol Lett 202(1):8–14 47. Adams JM (2003) Ways of dying: multiple pathways to apoptosis. Genes Dev 17(20):2481–2495 48. Zornoff LA, Matsubara LS, Matsubara BB, Okoshi MP, Okoshi K, Dal Pai-Silva M, Carvalho RF, Cicogna AC, Padovani CR, Novelli EL, Novo R, Campana AO, Paiva SA (2006) Beta-carotene supplementation attenuates cardiac remodeling induced by one-month tobacco-smoke exposure in rats. Toxicol Sci 90(1):259–266

123