PROTECTIVE EFFECT OF MELATONIN AND SOME DIETARY SUBSTANCES ON THE INDUCED HYPERCHOLESTEROLEMIA IN ADULT MALE RATS

Assiut Vet. Med. J. Vol. 52 No. 110 July 2006

Dept. of Physiology, Faculty of Medicine, Assiut University.

PROTECTIVE EFFECT OF MELATONIN AND SOME DIETARY SUBSTANCES ON THE INDUCED

HYPERCHOLESTEROLEMIA

IN ADULT MALE RATS (With 3 Tables and 6 Figures)

By M.Y. HAMED; IBTISAM M.H. EL. MILEEGY; FATMA A.M. BADARY* and SALWA I.A. WASFY *Pathology Departments, Faculty of Medicine, Assiut University.

(Received at 28/5/2006)

التأثير الواقي للميلاتونين وبعض المواد الغذائية من زيادة الکولسترول

المحدثة في ذکور الفئران البالغة محمد يسري حامد،ابتسام محمد حسن المليجي،فاطمة احمد محمود بداري،

سلوى اسماعيل وصفی تهدف هذه الدراسة إلى تقييم التأثير الواقي المحتمل لزيت السمک وزيت الزيتون والميلاتونين من زيادة الکولسترول المحدثة في ذکور الفئران البالغة. استخدم في هذه الدراسة .5 فارا قسمت إلى 5 مجاميع، ۱۰ فئران في کل مجموعة. ولقد تم تغذية حيوانات المجموعة الأولي

على الغذاء المعتاد والمجموعة الثانية تغذت على الغذاء المعتاد المضاف إليه الکولسترول

بنسبة 1% ( المجموعة المغذاة بالکولسترول). المجموعات من

3-5 تم تغذيتها بغذاء المجموعة الثانية والتي تم معالجتها في نفس الوقت بزيت السمک في المجموعة الثالثة

مجموعة زيت السمک) وزيت الزيتون في المجموعة الرابعة (مجموعة زيت الزيتون) والميلاتونين في المجموعة الخامسة (مجموعة الميلاتونين). وتم أخذ عينات دم من جميع الحيوانات في نهاية التجربة التي استمرت 10 أسابيع. ثم تم الحصول على شريان الأورطى من جميع الحيوانات بعد الذبح وفحصت هستولوجيا التقييم وجود تصلب الشرايين، وتم قياس مستوى الکولسترول الکلى بالبلازما ومستوى الکولسترول عالي الکثافة والکولسترول منخفض الکثافة والتراي جليسريد. وتم أيضا قياس إنزيم السوبر أوکسيد ديسميوتيز والثيول الکلى وأکسيد النتريک وفوق أکسيد الدهون. أظهرت النتائج أن التغذية بالکولسترول تحدث زيادة ذات دلالة إحصائية في مستوى الکولسترول الکلى ومستوى الکولسترول منخفض الکثافة والترای جلیسرید و فوق أکسيد الدهون وانخفاض ذو دلالة إحصائية في مستوى الکولسترول مرتفع الکثافة و السوبر أوکسيد ديسميوتاز وأکسيد النيتريک والثيول الکلى. وقد أدت المعالجة بزيت السمک وزيت الزيتون والميلاتونين مع التغذية بالکولسترول إلى نقص ذو دلالة إحصائية في مستوى الکولسترول الکلى والکولسترول منخفض الکثافة والترای

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جليسريد وفوق أکسيد الدهون وزيادة ذات دلالة إحصائية في مستوى السوبر أو کسيد ديسميوتيز وأکسيد النيتريک والثيول الکلى وزيادة غير معنوية في مستوى الکولسترول عالي

الهستولوجي نشريان الأورطي لحيوانات مجموعتي زيت السمک

الکثافة . وقد أظهر الفحص و زيت الزيتون بداية حدوث تصلب شریاني في المراحل الأولى) مقارنة بالمجموعة الضابطة التي کان تصلب الشرايين بها مکتمل المراحل. أما مجموعة الميلاتونين فأظهرت مرحلة مبکرة جدا من التصلب الشرياني مقارنة بمجموعتي زيت السمک وزيت الزيتون ويمکن استنتاج أن زيادة الکولسترول تؤدي إلى زيادة الترای جليسريد والکولسترول منخفض الکثافة وفوق أکسيد الدهون والإجهاد التأکسدي وتؤدي إلى نقص في الکولسترول عالي الکثافة

الأکبر في الترای

ونضوب الإنزيمات المضادة للأکسدة وقد سبب زيت السمک النقص جليسريد والزيادة الأکبر في اکسيد النيتريک. أما زيت الزيتون فکان الأکثر تأثيرا في زيادة

فوق أکسيد الدهون وکان الميلاتونين الأفضل تأثيرا في خفض

الثيول الکلي وفي خفض

الکثافة وبالتالي تحسن تصلب الشرايين. وهکذا

الکولسترول الکلى و الکولسترول منخفض يوصى باستخدام زيت السمک وزيت الزيتون والميلاتونين أمنع حدوث تصلب الشرايين الذي يؤدي إلى نوبات القلب والسکتة الدماغية وأشکال أخرى من إصابات الجهاز الدوري.

SUMMARY

This study aims to evaluate the possible protective effects of fish oil, olive oil and melatonin on the induced hypercholesterolemia in adult male rats. 50 rats were used in this study and were divided into 5 groups 10 rats each. Rats of group 1 were fed on a standard diet and those of group 2 were fed on a standard diet enriched with 1% cholesterol (cholesterol fed group). Groups 3-5 were fed as in group 2 and treated at the same time with fish oil in group 3 (fish oil group), with olive oil in group 4 (olive oil group) and with melatonin in group 5 (melatonin group). Blood samples were taken from all animals at the end of the experiment after 10 weeks. The aorta of all animals were obtained after slaughtering and examined histologically to assess the presence of atherosclerosis. Parameters of the lipogram (total plasma cholesterol (TPC), high density lipoprotein (HDL), low density lipoprotein (LDL) and triglycerides (TG)], superoxide dismutase (SOD), total thiol, nitric oxide (NO) and lipid peroxide (LP) were measured. Feeding cholesterol significantly increased TPC, LDL, TG and LP and significantly decreased HDL, SOD, NO and total thiol. There was a significant decrease in TPC, LDL, TG and LP by using fish oil, olive oil and melatonin while, the level of SOD, NO and total thiol were significantly increased and non significant increase in the level of HDL. Histological examination of the aorta from rats of the fish oil and olive oil groups showed fatty streaks, which are early atheromatous lesions, compared with typical atherosclerotic fibrous plaques in cholesterol fed group. In

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melatonin group very early atheromatous lesions were found and they were less pronounced than in fish oil and olive oil group. It was concluded that hypercholesterolemia leads to an increase in TG, LDL, LP and oxidative stress and a decrease in HDL and a depletion of antioxidant enzymes. Fish oil caused the greatest reduction in TG and the greatest increase in NO. Olive oil was the most effective in increasing total thiol and in reducing LP, and melatonin was the best factor reducing TPC, LDL and consequently atherogenesis. Therefore, fish oil, olive oil and melatonin supplementation is recommended to prevent arteriosclerosis that leads to heart attacks, strokes and other forms of cardiovascular damage.

Key words: Fish oil, olive oil, melatonin, hypercholesterolemia, rat.

INTRODUCTION

Cholesterol, a very important lipid, has many important functions in the body. It serves as a precursor for plasma membranes, bile salts, steroid hormones and other specialized molecules. On the other hand, high plasma concentrations of cholesterol enhance the development of atherosclerosis, the arterial thickening that leads to heart attacks, strokes and other forms of cardiovascular damage (Vander et al., 1998).

Fish oil as a rich source of omega-3 polyunsaturated fatty acids (PUSFA) has been shown to be a hypolipidemic, anti-inflammatory and anti-platelet agent. So it is a cardioprotectant reducing risk factors for coronary heart disease (Collier et al., 1993, Yilmaz et al., 2002 and Higuchi et al., 2006).

Olive oil with its high oleic acid content and abundant polyphenols guards against atherogenesis. Olive oil increases antioxidant capacity in the liver, heart, aorta, platelets and brain. In addition, olive oil has got nitric oxide (NO) releasing properties (Visioli and Galli, 2001, Puiggros et al., 2002, Faine et al., 2004 and Gonzalez-Santiago et al., 2005).

Melatonin is a potent antioxidant that plays a critical role in free radical scavenging (Reiter et al., 1994 and Ahmed et al., 2005). Melatonin has a hypocholesterolemic effect and thus it has protective effects on the cardiovascular system by reducing risk of atherosclerosis (Bubenik et al., 1998, Sener et al., 2004 and Nishida, 2005).

In this work, evaluation of the possible protective effects of fish oil, olive oil and melatonin on the induced hypercholesterolemia in adult male rats was studied.

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MATERIALS and METHODS This study was carried out on 50 Sprague-Dawley adult male rats weighing between 200-250 grams. The animals were obtained from, and kept in the Animal House Facility of Assiut Faculty of Medicine. Animals were divided into 5 groups 10 rats each. Rats of group 1 (standard diet group) were fed on a standard commercial pellet diet for 10 weeks, animals of group 2 (cholesterol fed group) were fed on the standard diet enriched with 1% cholesterol (Sigma chemical company USA) dissolved in 0.5% cholic acid for 10 weeks. Groups 3-5 were fed as in group 2 and treated at the same time with fish oil (Menhaden Oil, Sigma) at a dose of 0.5 ml / day administered orally in group 3 (fish oil group), with extravergin olive oil (Wadi food, Egypt) at a dose of 0.5 ml / day administered orally in group 4 (olive oil group) and with melatonin (Sigma) injected subcutaneously at a dose of 75 ug / day 3-2 hours before sunset in group 5 (melatonin group).

Blood samples were taken at the end of the experiment. 3 ml blood sample was collected from each rat after over night fasting in a clean sterile centrifuge tube with anticoagulant (EDTA) by puncture of the retro-orbital sinus. Plasma was separated by centrifugation and divided into small aliquots and frozen at -20° C until processed.

Lipogram parameters [total plasma cholesterol (TPC) high density lipoprotein (HDL), low density lipoprotein (LDL) and triglycerides (TG) were measured by using fluorimetric kits (Boehringer-Mannhim, Germany). TPC was determined by the method of Flegg (1973). HDL was determined according to the method described by Finely (1978). LDL was determined by the method of Friedewald et al. (1972). The method for determination of TG was described by Fredrickson et al. (1967). Super oxide dismutase (SOD) was estimated according to Misra and Fridovich (1972) using spectrophotometer. Total thiol was determined colorimetrically after Ellman (1959). Nitric oxide (NO) was measured according to Ding et al. (1988) using spectrophotometer. Lipid peroxide (LP) was determined as thiobarbituric acid reactive substances colorimetrically according to the method of Satoh (1978).

The rats were killed by slaughtering and the aorta and its major branches from each animal were obtained, washed in saline and fixed in 10% formalin. Sections were prepared and stained with H&E stain (Carleton and Drury, 1957) for histological examination.

Data were expressed as mean = standard error (S.E.). t-test was used to compare between groups to determine significance.

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RESULTS

As regard the effect of feeding cholesterol on the measured blood parameters, Table (1) showed that TPC levels, LDL, TG and LP levels were significantly increased in the cholesterol fed group (G2) in comparison with the standard diet group (G1). While, HDL, SOD, NO and total thiol levels were significantly decreased in G2 in comparison with G1.

Table (2) and Figure (1) showed that plasma cholesterol level was 593.1 + 20.90 mg % in the cholesterol fed group and significantly (P<0.001) decreased to 411.3 + 8.79, 391.6 $11.75 and 368.0 – 10.54 mg% in the fish oil, olive oil and melatonin groups respectively.

The high density lipoprotein level in the cholesterol fed group was 36.09 + 0.63 mg% and non significantly increased to 37.66 + 0.69, 38.16 = 0.62 and 36.53 $ 0:76 mg% in the fish oil, olive oil and melatonin groups respectively.

In the cholesterol fed group the low density lipoprotein was 393.8 10.21.and significantly (P<0.001) decreased to 275.3 $ 7.72, 255.7 $ 9.01 and 226.5 5.03 mg% in the fish oil, olive oil and melatonin groups respectively as shown in Table (2) and Figure (1).

Triglycerides level in the cholesterol fed group was 177.9 = 5.43 and significantly (p< 0.001) decreased to 94.7 = 2.03, 120.9 5.44 and 102.9 $ 2.74mg% in the fish oil, olive oil and melatonin groups respectively.

Table (3) and Figure (2) showed that superoxide dismutase level was 19.00 + 0. 47 and became 24.0 + 0.63, 21.70 = 0.55 and 24.60 = 0.70 unit/ml in the fish oil, olive oil and melatonin groups respectively. There was a significant increase in the fish oil and melatonin groups (P<0.001) and in the olive oil group (P<0.01). Nitric oxide level in the cholesterol fed group was 28.80 = 0.46 umol/L and significantly (P< 0.001) increased to 36.20 + 0.74, 33.50 + 0.65 and 32.30 = 0.42 umol/L in the fish oil, olive oil and melatonin groups respectively. Lipid peroxide level in the cholesterol fed group was 2.37 + 0.10 and decreased to 0.69 = 0.02, 0.61 = 0.01 and 0.96 = 0.02 nmol/ml in the three groups respectively. This decrease was significant (P<0.001) as shown in Table (3) and Figure (2)

In the cholesterol fed group the total thiol level was 161.3 £ 5.10 umol/L and significantly (P < 0.001) increased to 268.0 + 7.42, 321.4, + 7.58 and 292.9 5.53 umol/L in the fish oil, olive oil and melatonin groups respectively.

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Histological examination of the aorta from groupl under a light microscope revealed normal histological features of intima, media and adventitia (Fig. 3) while, in group2, it showed typical atherosclerotic fibrous plaques. An atheromatous plaque consists of lipid rich necrotic core filled with cellular debris and cholesterol clefts covered by a well developed fibrous cap (Fig. 4). Examination of the aortic wall from fish oil and olive oil groups revealed presence of early atheromatous lesions (fatty streaks). The lesions were composed of loosly textured connective tissue with varying amounts of vaculated lipid laden macrophages demonstrated to some extent in all animals of these groups (Fig. 5). In melatonin group, the atheromatous lesion was less pronounced than in olive oil and fish oil groups. The lesion consists of lipid laden macrophages present in between muscle bundles. There is also splitting of elastic lamina due to the presence of foam cells (Fig. 6).

Table 1: Effect of feeding cholesterol on the measured biochemical

parameters in adult male rats.

Parameters Cholesterol (mg/dl) HDL (mg/dl) LDL (mg/dl)

Triglycerides (mg/dl) Superoxide Dismutase (unit/ml) Nitric oxide (umol/L) Lipid peroxide (nmol/L) Total thiol (umol/L)

**P<0.01

Standard diet group

104.5+2.99 39.14+0.71 53.021.85 83.5+1.74 27.9+0.64 39.1+0.50 0.40+0.01 388.6+13.89

Cholesterol fed group

593.1+20.90***

36.09+0.63** 393.8=10.21*** 177.915.43*** 19.00+0.47*** 28.80+0.46*** 2.37+0.10*** 161.335.10***

***P<0.001

Table 2: Effect of fish oil, olive oil and melatonin on plasma levels of

total cholesterol, high density lipoprotein, low density lipoprotein and triglycerides in cholesterol fed rats.

Group

Total Plasma

Cholesterol(mg/dl) Cholesterol fed group 593.1+20.90 Fish oil group

411.348.79*** Olive oil group 391.6+11.75*** Melatonin group 368.0+10.54***

NS: Non significant

High density lipoprotein(mg/dl)

36.09+0.63 37.66+0.69 NS 38.16+0.62 NS 36.53+0.76 NS

Low density lipoprotein(mg/dl)

393.8 +10.21 275.337.72*** 255.729.01*** 226.5=5.03***

Triglycerides

(mg/dl) 177.0=5.43 94.7+2.03*** 120.9+5.44*** 102.932.74***

*** P<0.001

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Table 3: Effect of fish oil, olive oil and melatonin on plasma levels of

super oxide dismutase, nitric oxide, lipid peroxide and total thiol in cholesterol fed rats.

Group

Cholesterol fed group Fish oil group Olive oil group Melatonin group

** P<0.01

Superoxide Nitric oxide dismutase u mol/L 1 umol/L

19.00:0.47

28.80+0.46 24.0+0.63*** 36.2+0.74*** 21.70=0.55** 33.50+0.65*** 24.60£0.70*** 32.30+0.42***

Lipid peroxide 1 umol/L

2.37+0.11 0.69+0.02*** 0.61+0.01*** 0.96-0.02***

Total thiol

u mol/L 161.3–5.10 268.0=7.42*** 321.437.58*** 292.9=5.53***

*** P<0.001

High Density lipoprotein (HDL)

Total Plasma Cholesterol TPCL

VOY

Cholesterol

Fall Central

Olive On

Melatonin

Cholesterol

Fed Control

Hch Oil

Olive On

Melatonin

Low Density Lipoprotein ILDL

Triglycerides Het

•

450

200

180

400

180

350

140

300

120

250

100

200

SO

150

100+

50

Cholesterol

Fed Control

Fish Oil

HO DAYO

Melatonin

Cholesterol

Fed Control

Fish Ou

Olive On

Melatonin

Fig. 1: Effect of fish oil, olive oil and melatonin on plasma levels of

total cholesterol, high density lipoprotein, low density lipoprotein and triglycerides in cholesterol fed rats.

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Nitric Oxide

Lipd peroxide HP

Cholesterol

Ted Conteel

Fish Oil

Olive Oil

Melatonin

POY

Cholesterol

Fed Control

Olive On

Melatonin

Total Thiet

Superoxide Dismutase +900+

250

----

-

Cholesterol

Fed Control

Fish Oil

Olive Oil

Melatonin

Cholesterol

Ved Control

Fish Oil

Olive OH

Melatonin

Fig. 2: Effect of fish oil, olive oil and melatonin on plasma levels of

superoxide dismutase, nitric oxide, lipid peroxide and total thiol in cholesterol fed rats.

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Fig. 3: Transverse section of the aortic wall of male rats from standard

diet group showing normal intima, media and adventitia (H&EX 40).

PERT

Fig. 4: Transverse section of the aortic wall of male rats from cholesterol

fed group showing typical fibrous plaque (H&E X 100).

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Fig. 5: Transverse section of the aortic wall of male rats from fish oil

group illustrating the presence of fatty streak (early atheromatous lesion) (H&E X100).

Fig. 6: Transverse section of the aortic wall of male rats from melatonin

group showing lipid-laden macrophages between muscle bundles of media (H&E X40).

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DISCUSSION

In this work the possible protective effect of fish oil, olive oil and melatonin against hypercholesterolemia was studied. Feeding cholesterol significantly increased (P<0.001) TPC, LDL and TG and significantly decreased (P<0.01) HDL. The marked hypercholesterolemia occurring in this work goes with the results of Nakayama et al. (1983) and Leth Espensen et al. (1988) in rabbits, Kunitomo et al. (1981) in rats and Sener et al. (2004) in mice. Biomarkers of oxidative stress were affected by feeding cholesterol. SOD, NO and total thiol were significantly decreased (P<0.001) and LP was significantly increased (P<0.001) in the cholesterol fed group (G2) in comparison with the standard diet group (G1). Other studies reported that hypercholesterolemia increase lipid peroxidation and oxidative stress and causes depletion of antioxidant enzymes (Bednarek- Tupikowska et al., 2000 and Gonzalez-Santiago, 2005). Histological examination of the aorta from cholesterol fed group showed typical atherosclerotic fibrous plaques. While, Sener et al. (2004) reported that no fatty strakes or plaques developed in the aorta of mice following high cholesterol diet containing 1.5% cholesterol and 0.5% cholic acid for 4 months but in some sections derangment of the endothelial layer was detected.

Fish oil significantly decreased (P< 0.001) TPC, LDL and TG and non-significantly increased HDL in comparison with the cholesterol fed group. Many studies reported similar effect of fish oil on TPC (Kris Etherton et al., 1999 and Yilmaz et al., 2002), HDL (Harris et al., 1997) and TG (Flaten et al., 1990). In addition, Baydas et al. (2002) found that plasma lipid levels in rats treated with fish oil were significantly lower than those of the control. However, Franzen et al. (1993) found that TPC and LDL did not change significantly by fish oil. As regard the effect of fish oil on biomarkers of oxidative stress, it was found that fish oil significantly increased (P<0.001) SOD, NO and total thiol and significantly decreased lipid peroxidation. Harris et al. (1997) reported similar effect on NO and Vecera et al. (2003) on total thiol. While contradictory effect on LP was reported by Baydas et al. (2002) who found non significant increase in LP with fish oil. Histological examination of the aorta from fish oil group showed fatty streaks which are early atherosclerotic lesions compared with typical atherosclerotic fibrous plaques detected in G2. In agreement with our results, studies in swine fed high cholesterol diets with and without cod liver oil showed that there was less coronary atherosclerosis in the cod liver group

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(Weiner et al., 1986). Also, dietary polyunsaturated fat protected African green monkey from coronary artery atherosclerosis compared with saturated fat (Rudel et al., 1995).

Olive oil significantly decreased (P< 0.001) TPC, LDL and TG and non significantly increased HDL in comparison with the cholesterol fed group. Olive oil induced better cholesterol reducing results than fish oil. This is in accordance with the results of Kris-Etherton et al. (1999) but contradict that of mortensen et al. (1998). In addition, Bayindir et al. (2002) found that dietary treatment with olive oil improves the lipid profile by lowering TPC in rabbits. While, Gonzalez-Santiago (2005) found that hydroxytyrosol, a phenolic antioxidant present in olive oil reduces TPC by 50% in hyperlipemic rabbits (treating effect) but has no protective effect. In agreement with our results, LDL lowering effect of olive oil was reported by Kiritsakis (1998). Triglycerides lowering effect of olive oil was also reported by Faine et al. (2004) in rats and Ahuja et al. (2006) in human. The non significant increase in HDL by olive oil in this work contradicts the significant increase reported by Mortensen et al. (1992) in rabbits, Faine et al. (2004) in rats and Ahuja et al. (2006) in human and contradict the significant decrease reported by Ima et al. (1979) in rats. This different effect may be due to difference in the dose, duration or species. Concerning the effect on biomarkers of oxidative stress, olive oil significantly increased SOD, NO and total thiol (P<0.01, P<0.001 and P<0.001, respectively) and significantly decreased (P<0.001) LP. Accordant results, were increased myocardial SOD by olive oil in rats (Faine et al., 2004) and a potent antioxidant and anti inflammatory effect reported by El-Sweidy et al. (2005). Histological examination of the aorta from olive oil group showed early atherosclerotic lesions (fatty streaks) compared with typical atherosclerotic fibrous plaques in G2. In agreement with our results, microscopical examination of the aorta of rabbits fed olive oil showed a lower extent of degeneration in tunica intima with better organized endothelium and normal internal elastic membrane compared to corn oil fed and butter-fed rabbits (Bayindir et al., 2002) indicating that high dietary intake of olive oil may be more effective in the protection of endothelial integrity as evidenced by the lower incidence of atherosclerotic disease in the Mediterranean countries where olive oil is consumed in substantial amounts.

Melatonin significantly decreased (P<0.001) TPC, LDL and TG and non significantly increased HDL in comparison with the cholesterol fed group. Melatonin was more beneficial in lowering TPC than olive oil

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and fish oil. The hypocholesterolemic effect of melatonin was reported also by Sewerynek (2002) and Sener et al. (2004). The hypocholesterolemic effect of melatonin may work through augmentation of the endogenous cholesterol clearance mechanisms. Melatonin suppressed the formation of cholesterol by 38% and reduce LDL accumulation by 42% (Sewerynek, 2002) and reversed doxorubicin (induce acute cardiac toxicity in rats) induced increase in LDL towards the normal values (Ahmed et al. 2005). As regard the effect of melatonin on biomarkers of oxidative stress, melatonin significantly decreased (P<0.001) LP. Melatonin and fish oil were about equal in raising levels of SOD more than olive oil, and melatonin restored the level of NO less than fish oil and olive oil and has median effect between olive oil and fish oil on total thiol. Stimulating effect of melatonin on antioxidant enzymes was also reported by Ahmed et al. (2005) and Nishida (2005). Melatonin inhibit the elevation of LP but less than olive oil and fish oil. Inhibitory effect of melatonin on plasma LP was found also by Hoyos et al. (2000) and Baydas et al. (2002). Melatonin inhibited also lipid peroxidation in the heart (Ahmed et al., 2005), in the brain of methionine treated rats (Bouzouf et al., 2005) and high doses of melatonin inhibit lipid peroxidation in vitro (Tailleux et al., 2005).

Histological examination of the aorta from rats injected with melatonin showed that the atheromatous lesions were less pronounced than olive oil and fish oil groups. The lesions were very early in which the lipid laden macrophages were present in between muscle bundles which means that melatonin decreased the development of atherosclerosis. The results of Pita et al. (2002) agree with our results while in the study of Sener et al. (2004), there were no difference in the aortic histological findings of mice fed on high cholesterol diet with and without melatonin treatment (10mg/L in drinking water for 4 months).

Fish oil was the most effective in reducing TG level and in improving vascular endothelial function as evidenced in a rise of NO level in plasma. Olive oil was the most effective in restoring level of total thiol and in inhibiting LP and melatonin was the best factor reducing TPC, LDL and consequently atherogenesis. It was also the most effective in restoring SOD levels. Fish oil was less effective than olive oil in correcting lipid peroxidation because the highly unsaturated fatty acids of fish oil are the most susceptible to peroxidation (Satio and Nakatsugawa, 1994), while monounsaturated fatty acids in olive oil resist lipid peroxidation and attack by free radicals on account of having a single double bond (Bhadra et al., 1993).

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Fish oil supplementation beneficially affect persons with cardiovascular disease by at least three mechanisms. It reduces plasma triglycerides by about 30% (Harris et al., 1997) and reduces blood pressure significantly (Morris et al., 1993). Fish oil also has antithrombotic properties, it reduces platelet aggregation by decreasing thromboxane production (Goodnight et al., 1981). Also, olive oil has been associated with a lower incidence of coronary heart disease and cancer. Olive oil contains a high proportion of monounsaturated oleic acid and high quantities of phenol compounds, hydroxytyrosol and oleuropein with potent biologic activities that may partially account for the cardio protective effects of the Mediterranean diet (Visioli and Galli, 2001). Oleic acid in olive oil is the preferred substrate for acyl-CoA cholesterol acyltransverase (ACAT), thus favouring the formation of cholesterol esters and promoting LDL receptor synthesis. Increased LDL receptor activity results in a higher rate of LDL uptake and clearance from the plasma (Dietschy, 1997).

Melatonin has potent antioxidant properties, so may prevent the development of atherosclerosis, cancer and other consequences of agi (Reiter et al., 1994). In a human study, nocturnal secretion of melatonin was decreased in patients with coronary atherosclerosis (Brugger et al. 1995). Melatonin significantly suppressed the vasospastic effect of oxidized LDL (which has been reported to be the most important risk factor for atherosclerosis) probably because it scavenges hydroxyl radicals arising from oxidized LDL (Okatani et al., 2000).

It can be concluded that hypercholesterolemia lead to an increase in TG, LDL, lipid peroxidation and oxidative stress and a decrease in HDL and depletion of antioxidant enzymes, Fish oil supplementation caused marked reduction in TG and improvement of vascular endothelial function as evidenced in a rise of NO more than with olive oil and melatonin. While olive oil supplementation was the most effective in preventing the marked reduction of total thiol and in reducing LP and melatonin was the best factor reducing TPC, LDL and consequently atherogenesis. So, fish oil, olive oil and melatonin supplementation is recommended for prevention of atherosclerosis that lead to heart attacks, strokes and other froms of cardiorascular damage.

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