Lycopene attenuates oxidative stress and heart ... - Pathophysiology

Pathophysiology 19 (2012) 121–130

Lycopene attenuates oxidative stress and heart lysosomal damage in isoproterenol induced cardiotoxicity in rats: A biochemical study Ahmed M. Mohamadin a,∗ , Ahmed A. Elberry b , Amr D. Mariee c,d , Gehan M. Morsy e , Fahad A. Al-Abbasi f a

Department of Chemistry for Health Sciences, Deanery of Academic Services, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia b Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia c Department of Biochemistry, College of Pharmacy, Al-Azhar University, Cairo, Egypt d Department of Pharmacology and Toxicology, College of Pharmacy, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia e Department of Biochemistry, Applied Science College, Taibah University, Al-Madinah Al-Munawarah, Saudi Arabia f Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia Received 10 October 2010; received in revised form 21 November 2010; accepted 14 February 2011

Abstract The present study was designed to investigate the cardioprotective potential of lycopene (LYC) on isoproterenol (ISO)-induced oxidative stress and heart lysosomal damage in rats. Male Sprague Dawley rats were pretreated with LYC (4 mg/kg, p.o.) once daily for 21 days. After the treatment period, ISO (85 mg/kg) was injected subcutaneously, once daily, to rats for 2 days. Hemodynamic parameters, cardiac marker enzymes, antioxidant, and oxidative stress parameters in serum and heart tissues were measured. ISO treated rats showed significant changes in heart rates, heart weights and serum lipid profiles. The activity of aspartate aminotranferase (AST), lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT) were increased significantly (p < 0.01) in the serum of ISO rats. The levels of lipid peroxides (thiobarbituric acid reactive substances, TBARS), protein carbonyl content (PCC) and neutrophil infiltration marker; myeloperoxidase (MPO) were significantly (p < 0.01) increased. In addition, the activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetylglucosaminidase, and cathepsin-d) in the serum and heart of ISO rats were increased significantly. Furthermore, a marked decrease in the levels of serum and cardiac reduced glutathione (GSH), vitamin C and cardiac enzymatic antioxidants; superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were observed. In vitro study confirmed the strong antioxidant effect of LYC on total antioxidant activity. In conclusion, the present study demonstrated that LYC supplementation to ISO rats significantly ameliorated lysosomal membrane damage as well as the alterations in cardiac enzymes, lipid profile and oxidative stress markers. These findings revealed the cardioprotective effects of LYC against ISO-induced oxidative stress and cardiotoxicity in rats. These observed effects are mediated via antioxidant power and free radical scavenging activity of LYC. © 2012 Elsevier Ireland Ltd. All rights reserved. Keywords: Lycopene; Isoproterenol; Lysosomal damage; Oxidative stress; Cardiotoxicity

1. Introduction Myocardial infarction (MI) is one of the common ischemic heart diseases, which is the leading cause of death in ∗ Corresponding author. Permanent address: Biochemistry Department, College of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt. Tel.: +966 50 846 8142. E-mail address: [email protected] (A.M. Mohamadin).

0928-4680/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pathophys.2012.04.005

developed countries. Experimental and clinical studies have shown that reactive oxygen species (ROS) are involved in the formation of lipid peroxides, damage of cell membrane and destruction of antioxidative defense system which thereby results in myocardial cell membrane destruction [1]. Consequences of MI include hyperlipidemia, peroxidation of membrane lipids and loss of plasma membrane integrity [2]. Isoproterenol (ISO), a synthetic catecholamine, has been reported to cause infarct like necrosis of the cardiac muscle

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in doses exceeding the physiological concentrations. Excessive production of free radicals resulting from oxidative metabolism of catecholamines is one of the mechanisms proposed to explain the ISO-induced injury to myocardial cells [3,4]. ISO-induced myocardial damage in rats is a widely used experimental model for evaluation of cardioprotective effect of various herbal drugs [1,4], because the pathophysiological changes following ISO administration in rats are comparable to those taking place during MI in humans [3]. Lysosomal enzymes [␤-d-glucuronidase (␤-Glu), ␤-d-Nacetylglucosaminidase (NAG) and cathepsin-d (Cat-d)] are important mediators of acute MI. Their release into the cytoplasm stimulates the formation of inflammatory mediators, such as oxygen radicals and prostaglandins [5]. Lysosomal destabilization may be prevented either by inhibition of cellular peroxidation or by prevention of iron catalyzed oxidative reactions [6]. Oxidant/antioxidant status is in balance under normal physiological conditions [7]. This balance is disrupted with high production of ROS and oxidative damage, as a result of the perturbing effects of ISO metabolism [4]. ROS or their derivatives may be able to extract a doubly allylic hydrogen atom from unsaturated lipids and initiate lipid peroxidation (LPO) [8]. Detection of protein carbonyl content (PCC) is a specific marker for tissue injury caused by oxygen radicals [9]. During the inflammatory process, neutrophils are stimulated and release myeloperoxidase (MPO), which catalyzes the formation of hypochlorous acid (HOCl). The latter initiates oxidative injury, whereby organic hydroperoxides are served as primary targets for HOCl [10]. To protect themselves from oxidative damage, cells have developed a sophisticated antioxidant enzyme defense system. In this system, superoxide dismutase (SOD) converts superoxide radical (O2 •− ) into hydrogen peroxide (H2 O2 ) and seems to be the first line of defense against ISO-induced ROS production, whereas glutathione peroxidase (GSH-Px) and catalase (CAT) convert H2 O2 into water [8]. In normal conditions, these enzymes work in coordination to convert two toxic species, namely O2 •− and H2 O2 , into the harmless products, namely water and molecular oxygen. If ROS production overwhelms the enzymatic defense, this would cause cytotoxicity, leading to apoptosis or necrosis of the cells [11]. In recent years, the prevention of cardiovascular diseases has been associated with the ingestion of fresh fruits, vegetables or plants rich in natural antioxidants. Nutrition is perhaps the most significant environmental factor that has been implicated in either the development or prevention of chronic degenerative diseases [12]. Nevertheless, foods that are particularly rich in natural antioxidant nutrients such as vitamins, minerals and carotenoids may have the health protecting power to alleviate some of these chronic symptoms and diseases. Lycopene (LYC) is a dietary carotenoid synthesized by plants and microorganisms. Different fruits, vegetables and their products contain different concentrations of LYC (Table 1) [13]. LYC is one of the most potent antioxidants among the dietary carotenoids due to its

Table 1 Lycopene content of various fruits and vegetables [13]. Food

Lycopene content (mg/100 g)

Tomato foods Tomatoes, raw Tomato sauce Tomato paste Tomato soup (condensed) Tomato juice Ketchup Apricots, fresh Watermelon, fresh Papaya, fresh Grapefruit, pink/red Guava, raw Vegetable juice

0.9–4.2 3.7–4.4 7.3–18.0 5.4–55.5 8.0–10.9 5.0–11.6 9.9–13.4 0.0005 2.3–7.2 2.0–5.3 0.2–3.4 5.3–5.5 7.3–9.7

many conjugated double bonds [14]. Moreover, LYC has the strongest singlet oxygen quenching ability compared to other carotenoids [15,16]. This ability is twice as high as that of ␤-carotene and 10 times higher than that of ␣-tocopherol [17]. Besides, strong interaction of LYC has been shown to occur with other ROS such as H2 O2 [18], which can generate the hydroxyl radical (• OH), known to induce membrane lipid peroxidation and DNA strand scission [19]. Bignotto et al. [20] reported that LYC has an anti-inflammatory action against carrageenan-induced paw edema through inhibition of cytokines generation and membrane lipid peroxidation (LPO). The present study was designed to investigate the possible cardioprotective effects of LYC against ISO-induced oxidative stress and heart lysosomal damage in rats.

2. Materials and methods 2.1. Chemicals Lycopene was obtained from Toronto Research Chemicals (Toronto, ON, Canada). Isoproterenol hydrochloride, Ellman’s reagent (5,5 -dithio-bis-2-nitrobenzoic acid, DNTB); thiobarbituric acid (TBA), trichloroacetic acid (TCA), glutathione (GSH), 2,6-dichlorophenol, 1-chloro-2, 4-dinitrobenzene (CDNB), 1,1,3,3-tetramethoxypropane and malondialdehyde (MDA) were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals used were of high analytical grade. 2.2. Animals Adult male Sprague-Dawley, weighing 210–220 g, were used in this study. They were obtained from the Animal Care Centre, College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. All animals were fed a standard rat chow and water ad libitum. Animals were kept in a temperature-controlled environment (20–22 ◦ C) with an alternating cycle of l2-h light and dark. The experimental


design and procedures were approved by the Institutional Ethical Committee for Animal Care and Use at the King Abdulaziz University, Jeddah, Saudi Arabia. 2.3. Experimental design A total of forty eight rats were used in the study. They were randomly divided into four groups, 12 animals in each group: Group1 (Control) was administered saline orally (3 ml/kg/day) using intra gastric tube for 21 days and on 20th and 21st day received 0.3 ml saline s.c., once daily. Group 2 (LYC) was treated orally with LYC suspended in corn oil (4 mg/kg/day) [21], for a period of 21 days. On day 20 and 21 animals were given 0.3 ml LYC, s.c., once daily. Group 3 (ISO) was administered saline orally (3 ml/kg/day) for 21 days along with ISO, 85 mg/kg/day, s.c., at days 20 and 21 [22]. Group 4 (LYC + ISO) was treated with LYC, 4 mg/kg/day orally for a period of 21 days along with ISO (85 mg/kg, s.c., once daily) on 20th and 21st day. Twelve hours after the second ISO-injection, animals were anesthetized with pentobarbital sodium (35 mg/kg, i.p.) and sacrificed by cervical decapitation. Blood was collected and serum was separated by centrifugation. The heart tissue was excised immediately from the animals, washed with ice-chilled physiological saline and stored for further biochemical estimations. A known weight (200 mg) of the heart tissue was homogenized in 5 ml of 0.1 M Tris–HCl buffer (pH 7.4) solution. The homogenate was centrifuged at 3000 rpm for 5 min. The supernatant was used for the assay of lysosomal hydrolases and for estimation of various biochemical parameters. 2.4. Survival study and general toxicity Mortality and general condition of the animals were observed daily throughout the experiment. Fluid accumulation in the abdominal cavity was determined at the end of the experiment after abdominal dissection and quantitated according to a graded scale of 0 to 3+: 0, none; 1+, mild; 2+, moderate and 3+, severe [23]. 2.5. Heart rate (HR) measurements All animals were anesthetized with ether, needle electrodes were inserted under the skin for the limb and HR (expressed in beats/min) was measured using an electrocardiograph (400MD2C, Bioscience, Sheerness, Kent, UK). 2.6. Biochemical assays 2.6.1. Assay of cardiac marker enzymes Serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase isoenzyme-MB (CK-MB) activities were determined according to standard methods using diagnostic kits from BioSystems S.A. (Barcelona, Spain). Assessment of serum cardiac troponin T (cTnT)

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was carried out using standard kit by chemiluminescence immunoassay (Roche Diagnostics, Switzerland). 2.6.2. Assay of lipid proﬁle The levels of total cholesterol (TC), HDL-cholesterol (HDL-C) and triglyceride (TG) were measured using commercially available assay kits (Abbott® , IL, USA) with an auto analyzer (Aeroset® , Abbott® , IL, USA). LDLcholesterol (LDL-C) was calculated using the Friedewald formula [24]. 2.6.3. Vitamin C assay Vitamin C in the serum was estimated by the method of Omaye et al. [25]. Serum (0.166 ml) was mixed thoroughly with 0.5 ml of 6% trichloroacetic acid and centrifuged for 20 min at 3500 × g. A volume of 0.25 ml of the supernatant was mixed with 0.25 ml of dinitrophenylhydrazine reagent. The tubes were allowed to stand at room temperature for 3 h, removed, and placed in ice-cold water. Then, 1.25 ml of 85% sulfuric acid was added to all the tubes and allowed to stand for 30 min at room temperature. The color developed was read at 530 nm and vitamin C values were expressed as mg/dl. 2.6.4. Reduced glutathione (GSH) assay Serum/cardiac GSH was determined according to the method of Ellman [26] and values were expressed as mg/dl and nmol/mg protein, respectively. 2.6.5. Lipid peroxidation (LPO) assay LPO products were determined by measuring thiobarbituric acid reactive substances (TBARS) content in tissue homogenates according to the method of Ohkawa et al. [27]. The MDA content was measured spectrophotometrically at 532 nm. The MDA content was calculated based on a standard curve using 1,1,3,3-tetraethoxypropane as a standard. Values were expressed as nmol/g protein. 2.6.6. Protein carbonyl content (PCC) assay Cardiac PCC was determined spectrophotometrically by a method based on the reaction of the carbonyl group with 2,4dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone [9] and values were expressed as nmol/mg protein. 2.6.7. Catalase (CAT) activity Cardiac CAT activity was determined according to the method described by Aebi [28] based on determination of the H2 O2 decomposition rate at 240 nm and values were expressed as U/mg protein. 2.6.8. Superoxide dismutase (SOD) activity Cardiac SOD activity was determined according to the method of Sun et al. [29]. The principle of the method is based on the inhibition of nitroblue-tetrazolium reduction by the

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xanthine–xanthine oxidase system as a superoxide generator. Values were expressed as U/mg protein. 2.6.9. Glutathione peroxidase (GSH-Px) activity Cardiac GSH-Px activity was assessed spectrophotometrically according to the method of Paglia and Valentine [30] and expressed as U/mg protein. 2.6.10. Myeloperoxidase (MPO) activity Cardiac MPO activity was determined by the method of Mullane et al. [31]. The principle of the assay is based on using 4-aminoantipyrine/phenol solution as the substrate for MPO-mediated oxidation by H2 O2 and recording the changes in absorbance at 510 nm. Values were expressed as mU/g protein. 2.6.11. Determination of protein content The protein content of cardiac tissue homogenates was determined by the Lowry protein assay using bovine serum albumin as the standard [32]. 2.6.12. Assay of lysosomal enzymes The activity of ␤-glucuronidase (␤-Glu) was estimated by the method of Kawai and Anno [33]. The substrate for the enzyme reaction was p-nitrophenyl ␤d-glucuronide and the enzyme activity was assessed in terms of ␮mol of p-nitrophenol liberated/h/mg protein. ␤-Nacetylglucosaminidase (NAG) activity was assessed by the method of Moore and Morris [34] using 4-nitrophenyl-Nacetyl glucosaminide as the substrate. The enzyme activity was expressed as ␮mol of p-nitrophenol formed/h/mg protein. Cathepsin-d (Cat-d) was estimated by the method of Etherington [35]. The incubation mixture contained the tissue homogenate or plasma and buffered substrate (1.5% hemoglobin in sodium acetate buffer). The enzyme activity was expressed as ␮mol of tyrosine liberated/h/mg protein. 2.6.13. In vitro total antioxidant activity of LYC In vitro total antioxidant potential of LYC was determined by the 2,2 -azinobis-(3-ethyl-benzothiazoline6-sulfonic acid) radical assay, as described by Miller et al. [36]. The reaction mixture contained 2,2 -azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS, 0.002 M), LYC (10–50 ␮M) and buffer in a total volume of 3.5 ml. The absorbance was measured at 734 nm using Systronics UV–visible spectrophotometer. 2.7. Statistical analysis Survival and effusion intensity score were analyzed using χ2 test. All other data were expressed as means ± SEM. Assessment of these results was performed using one-way ANOVA procedure followed by Tukey–Kramer multiple comparisons tests using Software GraphPad InStat, Version 4

(GraphPad Software Inc., La Jolla, CA, USA). Statistical significance was determined as p value below 0.05.

3. Results At baseline, heart weights as well as HR were similar in all groups. In ISO group, two rats died (16.6%) and HR increased significantly (p < 0.01) compared to the control group. In LYC + ISO group the HR did not increase significantly compared to control group. Heart weights in ISO group rats were significantly larger than those of control and LYC + ISO groups. Rats in LYC + ISO group showed nonsignificant differences in absolute and relative heart weights as compared with the control group (Table 2). Rats in the ISO group showed red exudates around the eyes and appeared to be weaker and lethargic as compared to LYC + ISO rats. Strikingly, these animals developed ascites, as determined by a grossly distended abdomen and later confirmed during necropsy. The hallmark gross pathologic changes in the ISO rats were excessive amounts of pericardial, pleural and peritoneal fluids. Effusion intensity score was moderate in 28.5% and severe in 71.4% in ISO rats, compared with five rats only (50%) in the LYC + ISO group, where the intensity score was severe (Table 3). Fig. 1 shows the activities of AST, LDH, CK-MB and cTnT in the sera of control and ISO rats. The activities of these cardiac marker enzymes were increased significantly (p < 0.01) in ISO rats when compared to control rats. Pretreatment with LYC significantly (p < 0.01) decreased the activities of these enzymes in LYC + ISO rats when compared with ISO rats. The data in Table 4 indicates the effect of treatments on serum lipid profiles in control and ISO rats. ISO rats had significantly higher levels of T-Ch, TG and LDL-C (p < 0.01), while having significantly lower HDL-C (p < 0.01) levels when compared with the control rats. Pretreatment with LYC showed decrease in lipid profile levels (p < 0.05) with a slight increase in HDL-C when compared with the ISO rats. As shown in Fig. 2, GSH, TBARS and PCC levels of the heart homogenates were similar between the control and LYC rats. In the ISO rats, TBARS and PCC levels increased significantly compared with the control group. These changes were accompanied with a significant decrease (p < 0.05) in GSH level compared to control group. Pretreatment with LYC significantly (p < 0.05) decreased the levels of TBARS and PCC and significantly (p < 0.05) increased the levels of heart GSH compared with ISO rats. Fig. 3 shows the effect of LYC on serum GSH and vitamin C in normal and ISO-induced rats. Rats treated with ISO showed a significant decrease in the concentration of GSH and vitamin C in comparison with control rats. Administration of LYC to ISO rats significantly (p < 0.01) increased the concentrations of these non-enzymatic antioxidants when compared with ISO rats.


125

Table 2 Effect of lycopene (LYC) administration on isoproterenol (ISO)-induced alterations in heart rate and heart weight of experimental groups. Groups

Heart rate (bpm) Baseline

Control LYC ISO LYC + ISO

371 365 362 373

± ± ± ±

7.1 8.3 7.4 8.5

Heart weight (g) After ISO injection 356 370 472 385

± ± ± ±

Absolute heart weight

6.8 9.3 11.2a,c 10.2b

0.69 0.70 0.82 0.72

± ± ± ±

Right ventricular weight

0.03 0.01 0.02a,c 0.05

0.28 0.29 0.36 0.31

± ± ± ±

Left ventricular weight

0.01 0.02 0.03 0.02

0.37 0.36 0.48 0.38

± ± ± ±

0.03 0.05 0.04 0.02

Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group. b p < 0.01 compared with the ISO group. c p < 0.01 compared with the LYC + ISO group.

Table 3 Effect of lycopene (LYC) on isoproterenol-induced pleural, pericardial and peritoneal effusion intensity score in surviving rats. Groups

No. of animals

Effusion intensity score 0


12 12 10 12

+1

+2

+3

No.

%

No.

%

No.

%

No.

%

12 12 0 0

100 100 0 0

0 0 0 2

0 0 0 16.6

0 0 2 6

0 0 16.6 50

0 0 8 4

0 0 66.6a 33.3b

Score: (0) non, (1) mild, (2) moderate, (3) severe. a p < 0.05 vs. corresponding control group. b p < 0.05 vs. corresponding ISO group.

75

250

(A)

(C)

a,c

a,c

50

CK-MB (IU/L)

AST (IU/L)

200

b

25

150

b 100 50

0

Control

LYC

ISO

0

LYC+ISO

Control

500

b

200

cTnT (ng/ml)

300

LYC+ISO

(D)

a,c

400

ISO a,c

3

(B) LDH (IU/L)

LYC

2

b

1

100 0

0

Control

LYC

ISO

LYC+ISO

Control

LYC

ISO

LYC+ISO

Fig. 1. Effect of lycopene (LYC) on the activity of cardiac marker enzymes in the serum of control and isoproterenol (ISO)-induced oxidative stress and cardiotoxicity in rats. Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group, b p < 0.01 compared with the ISO group, c p < 0.01 compared with the LYC + ISO group.

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Table 4 Effect of lycopene (LYC) on isoproterenol (ISO)-induced alterations in lipid profile in experimental groups including total cholesterol (T-Ch), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C). Groups

Lipids profile (mg/dl) T-Ch


97.8 96.3 130.7 108.2

TG ± ± ± ±

2.3 3.6 5.4a,c 5.2b

41.2 38.9 66.2 47.3

HDL-C ± ± ± ±

1.2 1.3 2.7a,c 1.6b

24.8 25.6 19.8 22.0

± ± ± ±

LDL-C 0.63 0.57 0.48a,c 0.37b

64.7 62.9 97.6 76.7

± ± ± ±

1.3 1.8 3.3a,c 2.6b

Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group. b p < 0.01 compared with the ISO group. c p < 0.01 compared with the LYC + ISO group.

GSH (µmol/g tissue)

15

(A)

12

40

a,c

6

b

24

a,c

16 8

b

9

(A)

32

GSH (mg/dl)

Fig. 4 represents the effect of LYC on the activities of SOD, CAT and GSH-Px in the heart of control and ISO rats. The ISO rats showed a significant (p < 0.01) decrease in the

0

Control

LYC

ISO

LYC+ISO

3 3

(B)

Control

TBARS (nmol/g tissue)

10.0

LYC

ISO

LYC+ISO

a,c

(B)

7.5

b

b

2

a,c

1

5.0 0

Control

2.5

0.0

Control 8

PCC (nmol/mg tissue)

Vitamin C (mg/dl)

0

LYC

ISO

LYC+ISO

(C)

6

a,c 4

b

2 0

Control

LYC

ISO

LYC+ISO

Fig. 2. Effect of lycopene (LYC) on the level of cardiac oxidative stress markers (A) GSH, (B) TBARS and (C) PCC in control and isoproterenol(ISO) induced oxidative stress and cardiotoxicity in rats. Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group, b p < 0.01 compared with the ISO group, c p < 0.01 compared with the LYC + ISO group.

LYC

ISO

LYC+ISO

Fig. 3. Effect of lycopene (LYC) on the level of serum antioxidants (A) GSH and (B) vitamin C in control and isoproterenol (ISO)-induced oxidative stress and cardiotoxicity in rats. Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group, b p < 0.01 compared with the ISO group, c p < 0.01 compared with the LYC + ISO group.

activities of these enzymes in the heart compared with the control rats. Pretreatment with LYC enhanced the activities of these enzymes significantly (p < 0.01) compared with ISO rats. Table 5 illustrates the effect of LYC on serum and heart lysosomal enzymes; ␤-Glu, NAG and Cat-d in control and ISO rats. ISO rats showed a significant increase in the activities of these lysosomal enzymes in comparison with control rats. The activities of these enzymes were significantly (p < 0.01) decreased in LYC + ISO rats when compared with ISO rats. Fig. 5 shows the activity of MPO enzyme in hearts of control and ISO rats. MPO activity was significantly (p < 0.01) higher in the ISO rats when compared with control rats.


127

Table 5 Effect of lycopene (LYC) on isoproterenol (ISO)-induced alterations in the activities of serum and heart lysosomal hydrolases in experimental groups. ␤-d-Glucouronidase

Groups

Serum Control LYC ISO LYC + ISO

11.7 12.4 24.3 15.7

± ± ± ±

Heart 1.0 1.1 1.4a,c 1.3b

25.6 24.3 41.5 29.9

± ± ± ±

1.3 1.7 2.6a,c 1.5b

␤-N-acetyl glucosaminidase

Cathepsin-d

Serum

Serum

27.1 27.3 38.7 28.3

± ± ± ±

Heart 1.2 1.1 1.6a,c 1.4b

44.8 41.5 57.4 47.3

± ± ± ±

2.6 1.8 2.3a,c 3.1b

18.2 18.6 30.4 21.3

± ± ± ±

Heart 1.1 1.3 1.7a,c 1.4b

26.1 24.9 37.1 27.6

± ± ± ±

2.1 2.3 2.7a,c 2.2b

Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group. b p < 0.01 compared with the ISO group. c p < 0.01 compared with the LYC + ISO group. 8

(A)

40

b

30

a,c 20 10

MPO (mU/g protein)

SOD (U/mg protein)

50

a,c

6

4

b 2

0

Control

LYC

ISO

LYC+ISO

0

Control (B)

9

b

6

ISO

LYC+ISO

a,c 3

0

Control 6

GSH-Px (U/mg protein)

LYC

Fig. 5. Effect of lycopene (LYC) on cardiac myeloperoxidase (MPO) activity in control and isoproterenol-(ISO) induced oxidative stress and cardiotoxicity in rats. Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group, b p < 0.01 compared with the ISO group, c p < 0.01 compared with the LYC + ISO group.

LYC

ISO

LYC+ISO

(C) b

4

LYC + ISO rats showed significantly decreased the activity of MPO compared with ISO rats. Fig. 6 shows the percentage scavenging effect of various concentrations of LYC on ABTS+ radical (total antioxidant activity). This effect was dose-dependent and the percentages scavenging of LYC at the concentrations of 10, 20, 30, 40 and 50 ␮M were 8.3%, 17.3%, 28.8%, 46.3% and 67.5%, respectively.

a,c

2

75

0

Control

LYC

ISO

LYC+ISO

Fig. 4. Effect of lycopene (LYC) on the activities of heart enzymatic antioxidants (A) SOD, (B) CAT and (C) GSH-Px in control and isoproterenol (ISO)-induced oxidative stress and cardiotoxicity in rats. Values are the mean ± SEM for 12 animals in each group. a p < 0.01 compared with the control group, b p < 0.01 compared with the ISO group, c p < 0.01 compared with the LYC + ISO group.

% Scavenging of ABTS+ radical

Catalase (U/mg protein)

12

50

25

0 0

10

20

30

40

50

60

Lycopene concentration (µmoles) Fig. 6. In vitro scavenging effect of lycopene on 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radical (total antioxidant assay).

128


4. Discussion The present study indicated that LYC significantly protected against ISO-induced oxidative stress and cardiotoxicity in rats. The mechanisms underlying the ISO-induced cardiotoxicity have not been fully understood, however, several investigators have shown that ROS are closely related to the oxidative stress and cardiotoxicity induced by ISO [4,37]. In the present study, it has been shown that administration of ISO to rats caused an elevation in serum cardiac enzymes; AST, LDH, CK-MB and cTnT, which was correlated with increase in heart lysosomal enzymes’ activities. Also, ISOinduced cardiotoxicity was accompanied by increases in TBARS and PCC with reduction of the levels of nonenzymatic antioxidants (GSH and vitamin C) and activities of enzymatic antioxidants (SOD, CAT and GSH-Px) in the heart tissues. LYC + ISO group showed lower levels of T-Ch, TG and LDL-C and increased level of HDL-C in serum compared with ISO group, indicating improvement of the lipid profiles. Our results are in agreement with previously published data by Zeng et al. [38], who indicated that LYC has improved lipid profile parameters in hypercholesterolemic rats. Similarly, Ried and Fakler [39] showed that LYC treatment for 2 weeks in patients with dislipidemia significantly decreased LDL-C and blood pressure. Previous studies showed that ISO caused increase in LPO and PCC in myocardial tissue as important indices of oxidant injury [37,40,41]. In the present investigation LPO and PCC levels were increased significantly in ISO group. LYC has antagonized the enhancement of LPO and PCC levels to a considerable extent, thereby confirming its antioxidant role in ISO-induced oxidative stress. These changes in the oxidative stress parameters are in accordance with several published reports [1,22,37]. The mechanism of action for LYC toward the reactive species can be predicted through three possible mechanisms: (i) adduct formation, (ii) electron transfer to the radical and (iii) allylic hydrogen abstraction [42,43]. Lycopene + R• → R-Lycopene• (adduct formation)

(1)

Lycopene + R• → Lycopene• + R(electron transfer) (2) Lycopene + R• → Lycopene• + RH(allylic H abstraction)

(3)

Two of the most important intracellular non-enzymatic antioxidant systems are GSH and vitamin C. Depletion in the heart GSH level has been observed in rats in response to oxidative stress caused by ISO [37]. In our studies, GSH and vitamin C were decreased by 45% and 57%, respectively following ISO administration. The lowered concentrations of GSH and vitamins C observed in ISO group rats might be due to neutralizing the increased production of free radicals. Pretreatment with LYC enhanced the levels of these antioxidants in the LYC + ISO group rats. The increased concentration of these antioxidants may protect the heart

against ISO-mediated free radicals. These results are in line with the previously published data of Yilmaz et al. [44], which indicated that LYC increased GSH and vitamin C concentrations in adriamycin-induced cardiotoxicity in rats. All of these data are in concordance with our previously mentioned results, in which LYC significantly ameliorated oxidative myocardial damage induced by catecholamine in rats [45]. ISO treatment caused a significant decrease in the activities of cardiac SOD, CAT and GSH-Px. The decreased activities of these enzymes were increased to nearly normal levels by LYC pretreatment. The exact mechanism of LYC on the enzyme activities is not yet known. Our results justify protective effect of LYC in terms of its free radical scavenging and antioxidant activity. Based on the similar proposed mechanism in other studies, LYC administration may prevent ISO-induced cardiac damage in rat by its free radical trapping activity [46]. Likewise, it was reported that LYC treatment protected the tissues from ischemia-reperfusion injury [20] and oxidative damages [47,48]. These reports are consistent with the results of the current study. Our study showed a significant increase in the activities of lysosomal enzymes in serum and heart tissue of ISO rats. Increased LPO in ISO-administered rats was observed in a previous study [37]. The membrane deterioration of lysosomes by ISO-induced lipid peroxide could have resulted in the leakage of enzymes. The phospholipid-rich lysosomal membrane is a potential site of free radical attack with subsequent loss of membrane stability [6,12]. LYC has an antioxidant effect [45] by inhibiting ISO-induced increase in LPO, ultimately resulting in the decreased lysosomal membrane damage and enhancing its stability. The sources of oxidants which may contribute to ISOinduced myocardial injury include mitochondrial electron transport chain and neutrophils which leak superoxide radical [49]. Neutrophils migrate to the tissue during tissue injury and have a role in oxidant injury mechanisms through the action of NADPH oxidase or MPO system. Our study demonstrated that ISO caused high MPO activity, resulting in increased oxidative stress. The prior administration of LYC was found to significantly lower ISO-induced elevation in the activity of MPO enzyme. Our results are in agreement with the work of Reifen et al. [49] who reported that LYC supplementation attenuates the inflammatory status in experimental colitis. Recently, an in vitro study indicated that LYC has antioxidative activity via scavenging hypochlorous acid [50]. In vitro studies also confirmed the free-radical-scavenging activity of LYC. Generation of the ABTS+ radical cation forms the basis of one of the spectrophotometric methods that have been applied to determine the total antioxidant activities of solutions of pure substances [36]. In the present study, LYC scavenged ABTS+ in a dose dependent manner. The highest percentage of scavenging effect of LYC on ABTS+ was achieved at a concentration of 50 ␮mol and was found to be 73.6%. Our result is consistent with Hsiao et al. [51] who reported that LYC has a free radical scavenging properties in


vitro. The decreased levels of these free radicals resulted in reduced cardiac damage in ISO-rats. The ability of LYC to act as an antioxidant and scavenger of free radicals is considered by most investigators as the most likely mechanism which accounts for its hypothesized beneficial effects on human health [43]. As a result of having an extensive chromophore system of conjugated carbon–carbon double bonds, LYC can accept energy from various electronically excited species. This is due to its ability to quench singlet oxygen (1 O2 ), formed by energy transfer from a meta-stable excited photosensitizer [15]. LYC may also interact with ROS such as hydrogen peroxide and nitrogen dioxide [19]. In conclusion, the present study demonstrated that LYC supplementation to ISO rats significantly ameliorated lysosomal membrane damage as well as the alterations in cardiac enzymes, lipid profile and oxidative stress markers. These findings revealed the cardioprotective effects of LYC against ISO-induced oxidative stress and cardiotoxicity in rats. These observed effects are mediated via antioxidant power and free radical scavenging activity of LYC.

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