Medical Journal of Babylon-Vol. 10- no. 1 -2013 مجلة بابل الطبية- المجلد العاشر




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Medical Journal of Babylon-Vol. 10- No. 1 -2013 مجلة بابل الطبية- المجلد العاشر- العدد الاول- 2013



Effect of Apium graveolens Leaves and Stalks in Reducing

the Side Effects of Doxorubicin in Male Rabbits
Haider Ridha Salman1,2 Batool Amein Al-Khafaji Nisreen J. Mohammed1

1Dept. of Pharmacology and Toxicology, College of Medicine, University of Babylon, Hilla, Iraq.

2pharmacist_iraq@yahoo.com


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Abstract

The aim of the present study is to evaluate the potential protective effect of Apium graveolens (A. graveolens) against cumulative DOX-induced toxic effects to the heart, liver and blood components in male rabbits. DOX was administered intraperitoneally (i.p.) to the rabbits at a dose of (4mg/kg) four times within 14 days (cumulative dose 16 mg/kg). A. graveolens was given orally (7.5 g/kg/day) for 14 days. The results indicated that the concurrent use of A. graveolens with DOX significantly (p<0.05) protected against DOX-induced toxicities as evidenced by significant increase in RBC, WBC, Hb, and neutrophiles levels (p<0.05). No significant changes in monocytes, lymphocytes, eosinophiles and basophiles percentages were noticed in all groups of treatment. Also, Coadministration of A. graveolens significantly ameliorated the DOX-induced elevation in serum levels of malondialdehyde (MDA), lactate dehydrogenase (LDH), Alanine Transaminase (ALT), Aspartate Transaminase (AST) and bilirubin as well as inhibited DOX-provoked serum glutathione (GSH) depletion (p<0.05). No significant alterations in serum albumin level and catalase (CAT) activity were observed in all groups of treatment. Furthermore, the histopathological sections showed that the DOX caused significant structural changes in hepatic and cardiac tissues like necrosis and inflammation which were attenuated with combined use of A. graveolens. The study conclude that A. graveolens has the potential in protecting against DOX -induced cardiac, hepatic and hematological damage through a mechanism related to direct and indirect antioxidant property.



الخلاصة

تهدف هذه الدراسة الى تقصي تأثير نبات الكرفس على التأثيرات السمية التراكمية المستحثة لعقار الدوكسوروبيسين في القلب والكبد وعناصر الدم لدى ذكور الارانب. لقد تم استخدام عقار الدوكسوروبيسين بجرعة (4 ملغ/كغم) داخل البريتون اربع مرات في اربعة عشر يوما ( الجرعة التراكمية 16 ملغ/كغم), اما نبات الكرفس فقد تم اعطائه عن طريق الفم بجرعة (7,5 غم/كغم) في اليوم لمدة اربعة عشر يوما. دلت النتائج على ان الاستخدام المتزامن لنبات الكرفس مع عقار الدوكسوروبيسين ادى الى تقليل التاثيرات السمية المستحثة بواسطة هذا العقار وقد استدل على ذلك من خلال الزيادة المعنوية في اعداد كريات الدم الحمر وكريات الدم البيض وكريات الدم العدلة وكذلك الزيادة المعنوية في مستوى تركيز الهيموغلوبين في الدم. ولم تحدث اي تغيير في نسب كريات الدم الاحادية واللمفاوية والحمضة والقعدة في اي مجموعة من المجموعات في هذه الدراسة. وكذلك, فقد وجد ان استخدام الكرفس مع الدوكسوروبيسين ادى الى تقليل الزيادة الحاصلة في مستويات اكسدة الدهون (MDA) والإنزيمات (LDH وAST و (ALTوالبليروبين في مصل الدم بشكل معنوي كما ثبط من الانخفاض الحاصل في فعالية الكلوتاثايون في مصل الدم بشكل معنوي ولم يحدث اي تغيير في مستوى الالبومين او فعالية انزيم (CAT) في مصل الدم في اي مجموعة من المجاميع في هذه الدراسة. علاوة على ذلك, فان المقاطع النسيجية اظهرت ان عقار الدوكسوروبيسين سبب تغييرات تركيبية واضحة في نسيجي القلب والكبد للارنب كالتنخر والالتهاب والتي تحسنت بشكل واضح مع استخدام نبات الكرفس. تستنتج الدراسة أن نبات الكرفس يمتلك القابلية في توفير الحماية ضد الاضرار المسببة بواسطة الدوكسوروبيسين في القلب والكبد والدم , بطريقة تعود بصورة مباشرة و غير مباشرة الى الخاصية المضادة للاكسدة.

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Introduction

Doxorubicin (DOX) is an anthracycline (ANT) antibiotic that possesses a potent and broad spectrum antitumour activity against a variety of human solid tumors and hematological malignancies [1, 2]. It is commonly used against ovarian, breast, lung, uterine and cervical cancers, soft tissue sarcomas as well against several other cancer types [3, 4]. It is known in the medical world as Adriamycin [5]. The preferential target of DOX is the DNA of dividing cells; the drug intercalates within DNA strands causing cell cycle blockage in the G2 phase [6] and inhibition of the activity of some nuclear proteins, such as DNA and RNA-polimerase and DNA-topoisomerase II [7, 8]. Unfortunately, DOX use has been limited largely due to its diverse toxicities, including cardiac, hepatic and hematological damages [9, 10]. The mechanism of the organ toxicity is unclear. It is believed that oxidative stress and the formation of free radicals, which also involves a reaction of DOX with iron, play a crucial role in the mechanism of DOX induced toxicities [11-14].

Nevertheless, many researchers have tried to find ways to reduce the adverse effects associated with DOX therapy. There are promising preclinical results in protecting against DOX induced toxicity through application of medical herbs and different natural products, such as as antioxidants from natural sources may be useful in the protection of DOX induced toxicity [4, 15, 16]. Apium graveolens (A. graveolens) is a herbal member of the Apiaceae family commonly knonwn as celery. It is an annual or biennial plant native to Mediterranean regions [17, 18]. A review of the literature indicates that A. graveolens has been cultivated for the last 3.000 years [19- 21]. Its root, fresh leaves, and seeds are used as food, spice, and at various times consumed as a medicine [21, 22]. Studies have documented that A. graveolens possess broad range of biological activities including antioxidant [23, 24] antimicrobial [25, 109], anticancer [20, 26, 36, 112], hypolipidemic [27- 29], hypoglyceamic [77, 106], hepatoprotective, [20, 26, 77] , anti-inflamatory and analgesic effects [106, 107, 108, 110]. Actually, A. graveolens is used in the treatment of bronchitis, asthma, liver and spleen diseases and valuable in weight loss diets [30]. It can lower blood pressure and regulate heart function [31]. Potassium and sodium ions in A. graveolens juice helps to regulate body fluid and stimulate urine production [105]. Further, A. graveolens have a significant attenuating effect in reducing calcium deposits from renal tissues [41]. In addition to carminative, diuretic and uricosuric activities, A. graveolens exhibit also spasmolytic and sedative properties, which opens new possibilities of using A. graveolens in modern phytotherapy [111, 32].

Multiple benefits of A. graveolens are propaply caused by its rich compositions of several bioactive substances [13]. In fact, this vegetable has rich minerals and vitamins and contains nutritional fiber, essential oils and other secondary metabolites numbered to polyphenols among which flavonoids, phenolic acids, phthalides, coumarins, furanocoumarins and sterols can be distinguished [33, 34]. A. graveolens leaves and a purified component from this, Apiin, inhibits inducible nitric oxide and nitric oxide production in vitro [35]. Limonene of this plant protects against cell mutations [20, 26] and phthalides constituents have shown to inhibit benzopyrene-induced forestomach cancer in mice [36]. Luteolin, apigenin and quercentin flavonoids could be found in many plants including A. graveolens [37]. Previous research has shown dramatic protection of flavonoids against DOX induced bone marrow peroxidation [13, 38]. Besides, flavonoids protect against side effects on blood, hepatic and cardiac tissues in animals treated with anticancer drugs [39, 40].

Materials and Methods

Drug

DOX vial (50mg/25mL) was obtained from (Ebewe, Australia). The drug was administered in a dose of (4mg/kg i.p.) four times in 14 days to obtain treatments with cumulative doses of 16 mg/kg according to Kolarovic et al. [13] with few modification.



Plant

Fresh plant materials of A. graveolens were purchased from a local market in Hilla city /Iraq. A. graveolens was administered in a single oral dose of (7.5 g/kg/day) for 14 days, added to the animals food according to Al Jawad et al. [41] with few modification. Fresh A. graveolens leaves and stalks was used in this study.



Animals

Twenty four male adult rabbits were enrolled in this study. The animals were obtained from the Animal House of College of Science in Kufa University. Their weight was in range of (1500 to 2000 g). The rabbits were housed in Animal House of Babylon Medical College, under controlled temperature around 25 °C and 12 hours light-dark cycles in cages (3 rabbits in each cage). They were fed a standard diet and allowed free access to tap water [42].



Experimental design

After 2 weeks of adaptation, the rabbits were divided randomly into four groups, each group consist of six animals; group I was the control group and received 5mL/kg of 0.9% NaCl solution intraperitoneally (i.p.) four times in 14 days; group II received DOX (4mg/kg i.p.) four times in 14 days; group III given an oral dose of (75 g/kg/day) of A. graveolens in addition to four equal injections of 0.9% NaCl (5 ml/kg i.p.) in 14 days and group IV given an oral dose of (75 g/kg/day) of A. graveolens and received DOX (4mg/kg i.p.) four times in 14 days. On day 17, blood samples were collected from each rabbit, then all animals were sacrificed by overdose of anesthesia, livers and hearts were removed and homogenized and experimental parameters were measured.



Preparation of Serum samples

Blood samples were taken from each animal before starting drug treatment by direct heart puncture and on the day 17 of treatment (3 days after the last dose of DOX) by the same manner. Four ml of blood were collected from each rabbit by intracardiac puncture, one ml of blood was put in EDTA (1.2mg) tubes for hematological analysis including: Hb, RBC and total and differential WBC levels. Three ml of fresh blood was placed in test tube and left for 30 minutes at 37 °C in incubator to allow clotting. The serum was prepared by centrifugation at 3000 rpm for 10 minutes to determine levels of GSH, MDA, CAT, ALT, AST, LDH, bilirubin and albumin.



Estimation of Haematological Parameters

RBC counts were carried out using the improved Neubauer haemacytometer as described by Lewis et al. [43]. The counting chamber (neubaur hemocytometer ) was filled by holding the pipette at an angle 45 degree and touching the space between the coverslip and the chamber by the point of the of the pipette , an appropriate drop of the mixture is allowed to run under the coverslip by capillary action . The chamber is examined under 40X objective lens of the microscope to count in RBC counting area of the chamber ( in the four corners and center tertiary squares of the RBC) ,and the depth of the field is 0. 1 mm [43]. Estimation of total WBC count has been carried out using Neubaur improved chamber slide and 2% acetic acid solution as a diluents in about 1:20 ratio of dilution to dissolve RBC membrane according to [43]. Hb concentration was assessed by Sahli method which is based on converting Hb to acid hematin and then visually matching its color against a solid glass standard [44,45]. Differential leucocytes counts had been calculated in Leishman’s stained blood films. A total of 100 leucocytes per each slide are examined to count neutrophils, lymphocytes, monocytes, eosinophiles and basophiles percentages [46].



Estimation of Biochemical Parameters

MDA the end product of lipid peroxidation was analyzed according to Burtis and Ashwood [47]. The principle depends on spectrophotometric measurement of the purple color generated by the reaction of thiobarbituric acid (TBA) with MDA. GSH was evaluated as described by the method of Burtis and Ashwood [47] which is based on on the reduction of the Ellman’s reagent by the SH group (GSH) to form 5,5-dithio-2-nitrobenzoic acid in phosphate buffer. Serum catalase (CAT) activity was determined spectrophotometrically by the decrease in absorbance due to H2O2 consumption according to the principle of Aebi [48]. Biochemical analysis of the serum enzymes for ALT and AST was performed by the colorimetric method of Reitman and Frankel [49]. LDH was assayed according to the method of Howell and Coll [50] which is dependent on the conversion of pyruvate to lactate. Albumin level in serum was measured according to Doumas [51]. Albumin in the presence of bromcresol green at a slightly acidic pH produce a color change of the indicator. The intensity of the color formed is proportional to the albumin concentration in the sample. Total serum bilirubin was determined by sulfanilic acid method according to Walter and Gerarde [52]. The principle of this method depends on colorimetric measurement of azobilirubin that is formed from the reaction between bilirubin and diazotized sulfanilic acid.



Histopathological examination

The liver and heart tissues were excised and immediately fixed in 10% buffered formalin at the end of the experiment. The tissue specimens were embedded in paraffin after being dehydrated in alcohol and subsequently cleared with xylene [53]. Five-micrometer thick serial histological sections were obtained from the paraffin embedded blocks, dewaxed in xylene for 6 minutes and hydrated by decreasing concentrations of ethanol followed by distilled water for 2 minutes. Then, sections were stained with hematoxylin and eosin (H&E) and subsequently examined under light microscope to evaluate pathological changes and photomicrographs were taken.



Statistical Analysis

All values were expressed as Mean ± Standard error (SE). The data were analyzed by using of computer SPSS versin 17 and taking P-value less than 0.05 as the lowest limit of significant. Analysis Of Variance (ANOVA) test was used to examine the differences between different groups [54].


Result and Discussion

Table (1) showed that the RBC count, WBC count and Hb concentration were within the normal range in all groups before treatment. Howevr, the animals of DOX group showed a significant decrease in RBC count as compared with control group (P<0.05), while the animals of A.graveolens group and the animals of DOX with A. graveolens showed a significant increase in RBC count as compared with control group (P<0.05). Data also revealed a significant decrease in WBC count and Hb concentration in animals received DOX as compared with the control group and with their count before treatment (P<0.05). Besides, there was non-significant changes in these parameters in animals received A. graveolens alone or with DOX as compared with the control group (P>0.05), but there was a significant differences in both groups as compared with the group of DOX (P<0.05).



Actually, the depression in RBC, WBC and Hb levels recorded in the present work (Table 1) could be attributed to disturbed hematopoiesis and suppression of marrow cells mediated by DOX toxicity. It is well established that DOX was myelosupressent and thereby affecting erythropoiesis and leucopoiesis [55]. Bone marrow toxicity is primarily on the rapidly dividing early progenitor cells and it takes approximately 2 weeks before the peripheral blood count decrease becomes fully manifested [56, 57]. In addition, blood cells are under DOX mediated oxidative stress. This oxidative stress might resulted in free radical formation and membrane lipid peroxidation. Two different ways of free radical formation by DOX have been described, the first implicates the formation of a semiquinone free radical which yields O2−• radicals and the second way produces DOX-iron complex that can reduce O2 to H2O2 and other active species [58]. The decrease in Hb level may thus be associated with decrease in the total content of body iron as a result of forming DOX-iron complex. This could be another reason for decreasing hematological values [59, 60]. These finding were in agremeent with Al-Harbi et al. [61] and Parabathina et al. [55] who have concluded that DOX caused significant reduction in RBC, WBC and Hb levels in serum of expermental animals administred DOX antibiotic. Similarly, Piura and Rabinovich [62] showed that treatment of patients with advanced uterine sarcoma with a combination of DOX and ifosfamide had hematological toxicity represented in leukopenia in (80%), neutropenia in (80%), and anemia in (20%) of the patients.

Table 1 A comparison of RBC count, WBC count and Hb concentration after two weeks treatment of normal rabbit with DOX, A. graveolens and their combination.


Group

RBC count (x1012)/ L ± SE

Before treatment

After treatment

Control

5.1 ± 0.74

5.22 ± 0.77

DOX

5.0 ± 0.55

3.52 ± 0.67*

A. graveolens

5.0 ± 0.73

6.02 ± 0.72*

DOX with A.graveolens

4.8 ± 0.58

6.60 ± 0.65*

Group

WBC count (x109)/L ± SE

Before treatment

After treatment

Control

4.9 ± 1.51

5.0 ±1.22

DOX

5.4 ± 1.14

3.3 ±1.31*

A. graveolens

5.4 ± 1.14

5.4 ± 0.36

DOX with A.graveolens

5.1 ± 1.08

4.8 ± 1.31

Group

Hb (mg/dL) ± SE

Before treatment

After treatment

Control

10.32±0.42

10.64±0.62

DOX

10.53±0.40

6.64 ± 0.54*

A. graveolens

10.40±0.58

11.04± 0.45

DOX with A.graveolens

10.71±0.45

9.81±0.35
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