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
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Keywords : Apium graveolens,Doxorubicin in Male Rabbits
Medical Journal of Babylon  10:1 , 2014 doi:1812-156X-10-1
Published :2013

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.

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 animal

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].


Results

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. Nevertheless, it is evident from the results of the present study in DOX with A. graveolens group as campared to DOX group that the A. graveolens plant could prevent myelosuppression induced by DOX, as seen by a significant increase in RBC counts and Hb concentrations, which is suggestive of positive erythopoisis; as well as significant increase in WBC counts which suggests induction of the immune system defensive mechanism [63] (Table 1). The protective effect of A. graveolens could be mediated through several mechanisms since the plant itself is a complex mixture of many chemicals. Obviously, A. graveolens is rich in several bioactive components with antioxidant and anti-inflammatory properties such as flavanoids (apigenin, quetiricin), phenolic acids, phthalides, coumarins, furanocoumarins and sterols [34]. Flavonoids and other polyphenols have powerful antioxidant activities, being able to scavenge a wide range of reactive oxygen and nitrogen species and chelate transition metal ions, often decreasing the pro-oxidant activity of metal ions [64]. They might also function to inhibit the activity of pro-oxidant enzymes, such as cyclooxygenase, lipoxygenase, xanthine oxidase, inducible nitric oxide, and NADPH oxidases and induction of antioxidant enzymes, such as superoxide dismutase and glutathione S-transferase (65), thus protecting blood tissues from DOX toxic actions. A. graveolens also contains a variety of various minerals and nutrients such as tryptophan, folate,nutritional fiber, molybdenum, manganese, phosphorus, potassium, calcium, magnesium and iron [66, 67]. Among vitamins, complex of vitamins B and C as well as ?-carotene are worth mentioning [68]. These phytocon-stituents have antioxidant and anti-inflammatory activitie [20, 66]. Yet, these findings agree wih Khuon [69] who observed that the oral administration of aqueous extract of A. graveolens has resulted in increasing of RBC, WBC and Hb levels significantly in female rats subjected to the hematotoxicity induced by CCl4. However, this study match with Abd El-Ghany et al. [67] who observed that A. graveolens plant has resulted in a significant increase in RBC and WBC counts in animals groups treated with nephrotoxic dose of gentamicin antibiotic. Also, Parabathina et al. [55] showed that the flavonoids rutin, quercetin and vitamin E could impede the down regulation of erythropoiesis and leukopoiesis induced by DOX. Result in Table (2) showed that there was significant decrease in neutrophile percentage in animals received DOX as compared with the control group and with its percentage before treatment (P<0.05) whereas the percentages of monocytes, lymphocytes, eosinophiles and basophiles remained unaffected in all groups of treatment (P>0.05). The present findings did not match with Parabathina et al. [55] who found that DOX was resulted in significant decrease in monocytes, lymphocytes, eosinophiles and basophiles counts while maintained normal neutrophiles levels in treated rabbits. Still, this controversy may be attributed to the differences in the expermental design and duration of tratment. A decreased percentage of neutrophile indicates suppression of neutrophiles production from bone marrow as a result of cumalative DOX doses [70]. Therefore, an increased percentage of neutrophiles in A. graveolens treated animals, as observed in the present study, suggests that A. graveolens is capable of impeding the down regulation of neutrophils induced by DOX and might be having a good potentiality to boost immune system [63]. A. graveolens contains a highly large amount of vitamin C which act to prevent the free radical formation and protect neutrophils and differential leucocytes from the ROS overproduction and thus may be responsible for heamatoprotective activities of A. graveolens. Regarding the effects of A. graveolens on monocytes and lymphocytes percentages, Khuon [69] studied the effect of the same plant against the hematotoxicity induced by CCl4 and showed similar findings. Up To our knowledge, there is no available study to compare with it for the A. graveolens effect on eosinophiles and basophiles percentages. A. graveolens with DOX cause significant decrease in serum MDA as compared with DOX group (P<0.05). Additionally, the result revealed that DOX decreased serum GSH level significantly (P<0.05), whereas A. graveolens alone or with DOX increased serum GSH level significantly (P<0.05) as compared with DOX group, with the control group or with its level before treatment. The increased levels of MDA might indicated excessive formation of the free radicals by DOX and activation of the lipid peroxidative process resulting in oxidative damage to the heart and liver tissues in rabbits subjected to DOX stress [12, 60]. Moreover, The depletion of GSH recorded in the present study signifies that free radicals and oxidative stress are increasing due to a toxic cumulative doses of DOX (16 mg/kg), and this might be attributed to a direct action of DOX on the GSH synthesis pathways, and/or an indirect function of the overwhelming response of DOX-stimulated generation of ROS [71]. Indeed, the observed decline in the level of GSH seems to be a prime factor that permits lipid peroxidation [72] and excessive lipid peroxidation causes increased GSH consumption [73]. Even so, the administration of A. graveolen significantly prevented the DOX induced elevation in serum MDA levels suggesting its protective activity. This could explain that the protective effects of A. graveolens may be attributed to its antioxidant and lipid-lowering properties that could directly eliminated free radicals and helped in the support of healthy cholesterol levels, thereby protecting the cardiac and hepatic cells from lipid peroxidation resulting in membrane stabilization [74, 75]. Further, the elevated levels of GSH could effectively provide thiol group for the possible GSH mediated detoxification reactions of glutathione S-transferase and glutathione peroxidase [76]. Also, A. graveolens contains large amounts of vitamin C which is a known immune system booster and reduces the free radicals in the body [77, 78]. On the other hand, metal chelating activity was claimed as one of the antioxidant activity mechanisms, since it reduced the concentration of the catalyzing transition metals in lipid peroxidation [79]. Nontheless, the use of A. graveolens could demonstrate effective capacity for iron binding, suggesting that its action as antioxidant may be related in part to its iron-binding capacity [80]. These findings were in agreement with findings reported by previous researches in which A. graveolens has resulted in decreased intensity of lipid peroxidation and enhanced GSH concenteration in animals treated with DOX, CCl4 or Di-2- ethylhexyl phthalate [13, 74, 81]. With respect to serum CAT, the present study showed non-significant changes in activity of this enzyme in all groups of treatment (P>0.05) (Table 3). Similar results were obtained by Kolarovic et al. [13] who suggested that cumulative DOX dose (6 mg/kg), A. graveolens leaves juice, and their combinatoin showed no significant influence on serum CAT activity in experimental animals. While Alshabanah et al. [59] and Mohan et al., [82] found that serum CAT activity notably lowered after DOX treatment, Raskovic et al. (16) concluded that CAT activity increased significantly in serum after DOX treatment. Conversly, Abd El-Ghany et al. [67] found that A. graveolens consumption had resulted in a significant elevation in serum CAT activity. This contraversy may be due to that CAT and other antioxidant enzymes other than GPx have been shown to be relatively less sensitive to oxidative stress [83] or it may be due to the differences in species variation, dose or duration of treatment. Data in Table (4) demonstrated that A. graveolens caused non-significant decrease in serum levels of ALT, AST and LDH enzymes (P>0.05), in contrast with DOX which cause significant increase in activities of ALT, AST and LDH in serum as compared with the control group or with its level before treatment (P<0.05). The combination of A.graveolens with DOX caused significant decrease in serum ALT, AST and LDH as compared with DOX group (P<0.05). DOX in the form of DOX semiquinone has been suggested to play a major role in its hepatotoxic action. Semiquinones are unstable under aerobic conditions thereby generating ROS by reacting with molecular oxygen [82]. Hepatocytes are the likely targets of ROS attack in the failing liver. It is conceivable that free radicals cause damage at their formation. Serum transaminases have long been considered as sensitive indicators of liver injury (84). DOX induced oxidative damage to hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells [82, 98]. This leakage causes a decrease in the levels of ALT and AST in hepatic cells but increase in levels of ALT and AST in serum (82). ALT is a more liver specific enzyme. Increase in AST activity is also roughly proportional to the extent of cardiac damage [16]. This indicate that DOX could not showed adverse effects at the hepatic level only, but also the possibility of significant damage of the heart function [11, 85]. Interestingly, A. graveolens prevents the increase in ALT and AST during its administration in combination with a cumulative dose of DOX and restored them towards normal side (Tables 4). These results suggested that A. graveolens may have protective effect against DOX-induced cardio and hepatotoxicity. This protective effect might be due to its ability for stabilizing of the cell membrane as well as repairing the hepatic and cardiac tissue damage by A. graveolens with the consequent decrease in the leakage of these enzymes. A. graveolens probably does so by the free radical scavenging which has been established in the in vitro studies indicating its antioxidant activity [74]. These results are in agreement with previous findings, which have also reported significant prevention into the rise of serum, ALT and AST levels by A. graveolens plant in animals treated with various hepatotoxin supporting the anti-hepatotoxic action of A. graveolens by virtue of membrane stabilizing activity [77, 26]. Similarlly, the dramatic augmentation of the LDH activity (Table 4) could suggest the great permeability of the hepatic cell membrane. Further, LDH is not specific for hepatic injury individually; the rapid cell swelling of sub-sarcolemmal bulbs and injured myocardium could facilitate the loss of intracellular enzymes in DOX group [12, 86]. It was previously known that the heart is very sensitive to ROS induced damage because of its highly oxidative metabolism and fewer antioxidant defenses compared to other organs [87]. This might be the possible mechanism for marked elevation of LDH concentration in serum after multi-dose administration of DOX. Nevertheless, LDH activity significantly decreased after A. graveolens treatment (Table 4) suggesting amioleration the toxicity of DOX not only at the level of liver but also at the level of other organs that house LDH intracellulary including heart. This result is in agreement with Prisacaru and Burlacu [88] who confirm the antitoxic potentials of A. graveolens volatile oil and other extractive solutions against acrylamide intoxication . Non-significant (P>0.05) changes in serum albumin activity in all groups of treatment are shown in (Table 5). Albumin is the most abundant blood plasma protein and is produced in the liver and forms a large proportion of all plasma protein [89]. It might take a longer time for DOX to affect liver synthetic function (as albumin synthesis). Similar results were obtained by Firat et al. [90]. However, serum albumin level was not significantly affected after A. graveolens treatment (Table 5). The same findings have been observed by Abd El-Ghany et al. [67]. However, Prisacaru and Burlacu [88] suggested a possible correlation between antitoxic effect of phthalides from A. graveolens plant and the hyperalbuminemia of the acrylamide intoxication. This controversy might due to differences in the dose, duration of A. graveolens supplementation or different species variation. In fact, analysis of data in (Table 5) also showed that there were non-significant changes in serum total bilirubin concentration in all groups before treatment. Conversely, there was significant increase in bilirubin concentration in animals received DOX as compared with the control group and with its concentration before treatment (P<0.05). Since the liver is responsible for clearing the blood of bilirubin, so increasing the total serum bilirubin level indicated a reduction in the excretory capability of the liver as a consequence of liver injury [91]. This finding is in accordance to the results of Anandakumar et al. [92] and Alshabanah et al. [59] who suggested that increase in bilirubin is a clear marker of DOX induced hepatic dysfunction. Furthermore, there was non-significant changes in serum level of total bilirubin in animals received A. graveolens alone as compared with the control group (P>0.05), but there was a significant differences in its level in animals which received A. graveolens in combination with DOX as compared with the control group (P<0.05). This result may be due to the enhancement of bile acid excretion that may induced by A. graveolens and its active constituents indicating the improvement effects of th plant on liver function [77, 81]. Histopathological Findings Histological Changes Induced by DOX and A. graveolens in Hepatic Tissue Light microscopic observation revealed that the control and A. graveolens groups showing normal hepatocytes as illustrated in Figure (1) and Figure (2) respectively. Conversely, the hepatic sections from rabbits administered DOX exhibited a significant distribution of feathery degeneration of hepatocytes as summarized in Figure (3), moderate inflammatory cell infiltratation around portal tract, marked piecemeal necrosis, interlobular inflammatory cell infiltrate, and bridging necrosis, as recapitulated in Figure (4). However, the histopathological examination of hepatic tissues of DOX with A. graveolens group showed a mild degenerative changes as briefed in Figure (5). B. Histological Changes Induced by DOX and A. graveolens in Cardiac Tissue The histopathological examination of cardiac tissues of the control and A. graveolens groups exhibited normal cardiac myocytes as briefed in Figure (6) Figure (7) respectively. In contrast, light microscopic observation revealed that the heart sections from rabbits received DOX showed a significant distribution of myocarditis with focal chronic inflammatory cells infiltration as summarized in Figure (8) and marked vascular congestion in between cardiac myocytes as recapitulated in Figure (9). Though, histological sections of cardiac tissue of DOX with A. graveolens group revealed intact myocardiam as illustrated in Figure (10). The different patterns of cell death and inflammation noticed in the present study might be due to DOX induced heptocellular injury and intrahepatic cholestasis resulting from cumlative drug administration. These findings were in agreement with Rasha and Abdella [93] and Ibrahim et al. [94] who observed portal and interlobular inflammation in hepatic sections isolated from DOX treated animals. Also, Abbas [95] and Mohan et al. [82] conclude that animals subjected to DOX stress showed histological alterations in many organelles including feathery hepatocyte degeneration, piecemeal necrosis and 4 necrosis. Besides, myocarditis is an inflammation of the myocardium associated with cardiac dysfunction, it is frequently characterised by the evolution in dilated cardiomyopathy [96] and most often results from infectious agents, hypersensitivity responses, or immune-related injury [97]. Actually, DOX associated myocarditis developed in a dose-dependent fashion, and myocyte damage on biopsy has a linear relation with cumulative dose [98]. Thus, the histological cardiac lesions observed in the current study might result as a response of cellular injury and electrolyte abnormalities mediated by DOX cumulative administration. Increased free radicals and reduced endogenous antioxidants, with subsequent increase in oxidative stress lead to loss of myofibrils and vacuolization of myocardial cells [99]. Moreover, there is a strong association between oxidative stress and the cardiac inflammatory response, including cytokine release after DOX administration [93, 100]. In that regard, DOX is known to induce NFK-B activation and COX-2 expression in cardiomyocytes [101]. Also, it was reported that DOX induced changes in the properties of membrane bound ATPases of cardiac cells affect cardiac function and may be a relevant mechanism leading to lethal myocardial cellular injury [102]. In addition to that, DOX mediated vascular congestion may contribute to this pathogenesis [103]. The resulting peripheral release of vasoactive and pro-inflammatory neurohormones from the stretched endothelium and from the perivascular congested tissue may promote the development and progression of heart failure and thus compromising organ function [104]. These histopathological changes were found to be in a good agreement with the work of other authors such as [71, 93, 102]. Interestingly, combined supplementations of A. graveolens plant along with DOX antibiotic in this study resulted in significant reduction in histological changes that were induced by DOX in liver and heart tissues as summarized in Figures (4-5) and (4-10) respectively. This might explain the ability of A. graveolens to attenuate the DOX-related toxic effects via more than one mechanism including the powerful scavinging of ROS, the inhibition of lipid peroxidation, the decrease of the inflammatory response and the stimulation of the synthesis of cellular antioxidants that protect hepatic and cardiac cells from DOX induced liver and heart injuries. Similar results were observed by Sultana et al. [66] and Jain et al. [81] who studied the effect of A. graveolens on isolated hepatic sections. However, there is no available study to compare with for the effect of A. graveolens on isolated cardiac sections.

Discussions

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. Nevertheless, it is evident from the results of the present study in DOX with A. graveolens group as campared to DOX group that the A. graveolens plant could prevent myelosuppression induced by DOX, as seen by a significant increase in RBC counts and Hb concentrations, which is suggestive of positive erythopoisis; as well as significant increase in WBC counts which suggests induction of the immune system defensive mechanism [63] (Table 1). The protective effect of A. graveolens could be mediated through several mechanisms since the plant itself is a complex mixture of many chemicals. Obviously, A. graveolens is rich in several bioactive components with antioxidant and anti-inflammatory properties such as flavanoids (apigenin, quetiricin), phenolic acids, phthalides, coumarins, furanocoumarins and sterols [34]. Flavonoids and other polyphenols have powerful antioxidant activities, being able to scavenge a wide range of reactive oxygen and nitrogen species and chelate transition metal ions, often decreasing the pro-oxidant activity of metal ions [64]. They might also function to inhibit the activity of pro-oxidant enzymes, such as cyclooxygenase, lipoxygenase, xanthine oxidase, inducible nitric oxide, and NADPH oxidases and induction of antioxidant enzymes, such as superoxide dismutase and glutathione S-transferase (65), thus protecting blood tissues from DOX toxic actions. A. graveolens also contains a variety of various minerals and nutrients such as tryptophan, folate,nutritional fiber, molybdenum, manganese, phosphorus, potassium, calcium, magnesium and iron [66, 67]. Among vitamins, complex of vitamins B and C as well as ?-carotene are worth mentioning [68]. These phytocon-stituents have antioxidant and anti-inflammatory activitie [20, 66]. Yet, these findings agree wih Khuon [69] who observed that the oral administration of aqueous extract of A. graveolens has resulted in increasing of RBC, WBC and Hb levels significantly in female rats subjected to the hematotoxicity induced by CCl4. However, this study match with Abd El-Ghany et al. [67] who observed that A. graveolens plant has resulted in a significant increase in RBC and WBC counts in animals groups treated with nephrotoxic dose of gentamicin antibiotic. Also, Parabathina et al. [55] showed that the flavonoids rutin, quercetin and vitamin E could impede the down regulation of erythropoiesis and leukopoiesis induced by DOX. Result in Table (2) showed that there was significant decrease in neutrophile percentage in animals received DOX as compared with the control group and with its percentage before treatment (P<0.05) whereas the percentages of monocytes, lymphocytes, eosinophiles and basophiles remained unaffected in all groups of treatment (P>0.05). The present findings did not match with Parabathina et al. [55] who found that DOX was resulted in significant decrease in monocytes, lymphocytes, eosinophiles and basophiles counts while maintained normal neutrophiles levels in treated rabbits. Still, this controversy may be attributed to the differences in the expermental design and duration of tratment. A decreased percentage of neutrophile indicates suppression of neutrophiles production from bone marrow as a result of cumalative DOX doses [70]. Therefore, an increased percentage of neutrophiles in A. graveolens treated animals, as observed in the present study, suggests that A. graveolens is capable of impeding the down regulation of neutrophils induced by DOX and might be having a good potentiality to boost immune system [63]. A. graveolens contains a highly large amount of vitamin C which act to prevent the free radical formation and protect neutrophils and differential leucocytes from the ROS overproduction and thus may be responsible for heamatoprotective activities of A. graveolens. Regarding the effects of A. graveolens on monocytes and lymphocytes percentages, Khuon [69] studied the effect of the same plant against the hematotoxicity induced by CCl4 and showed similar findings. Up To our knowledge, there is no available study to compare with it for the A. graveolens effect on eosinophiles and basophiles percentages. A. graveolens with DOX cause significant decrease in serum MDA as compared with DOX group (P<0.05). Additionally, the result revealed that DOX decreased serum GSH level significantly (P<0.05), whereas A. graveolens alone or with DOX increased serum GSH level significantly (P<0.05) as compared with DOX group, with the control group or with its level before treatment. The increased levels of MDA might indicated excessive formation of the free radicals by DOX and activation of the lipid peroxidative process resulting in oxidative damage to the heart and liver tissues in rabbits subjected to DOX stress [12, 60]. Moreover, The depletion of GSH recorded in the present study signifies that free radicals and oxidative stress are increasing due to a toxic cumulative doses of DOX (16 mg/kg), and this might be attributed to a direct action of DOX on the GSH synthesis pathways, and/or an indirect function of the overwhelming response of DOX-stimulated generation of ROS [71]. Indeed, the observed decline in the level of GSH seems to be a prime factor that permits lipid peroxidation [72] and excessive lipid peroxidation causes increased GSH consumption [73]. Even so, the administration of A. graveolen significantly prevented the DOX induced elevation in serum MDA levels suggesting its protective activity. This could explain that the protective effects of A. graveolens may be attributed to its antioxidant and lipid-lowering properties that could directly eliminated free radicals and helped in the support of healthy cholesterol levels, thereby protecting the cardiac and hepatic cells from lipid peroxidation resulting in membrane stabilization [74, 75]. Further, the elevated levels of GSH could effectively provide thiol group for the possible GSH mediated detoxification reactions of glutathione S-transferase and glutathione peroxidase [76]. Also, A. graveolens contains large amounts of vitamin C which is a known immune system booster and reduces the free radicals in the body [77, 78]. On the other hand, metal chelating activity was claimed as one of the antioxidant activity mechanisms, since it reduced the concentration of the catalyzing transition metals in lipid peroxidation [79]. Nontheless, the use of A. graveolens could demonstrate effective capacity for iron binding, suggesting that its action as antioxidant may be related in part to its iron-binding capacity [80]. These findings were in agreement with findings reported by previous researches in which A. graveolens has resulted in decreased intensity of lipid peroxidation and enhanced GSH concenteration in animals treated with DOX, CCl4 or Di-2- ethylhexyl phthalate [13, 74, 81]. With respect to serum CAT, the present study showed non-significant changes in activity of this enzyme in all groups of treatment (P>0.05) (Table 3). Similar results were obtained by Kolarovic et al. [13] who suggested that cumulative DOX dose (6 mg/kg), A. graveolens leaves juice, and their combinatoin showed no significant influence on serum CAT activity in experimental animals. While Alshabanah et al. [59] and Mohan et al., [82] found that serum CAT activity notably lowered after DOX treatment, Raskovic et al. (16) concluded that CAT activity increased significantly in serum after DOX treatment. Conversly, Abd El-Ghany et al. [67] found that A. graveolens consumption had resulted in a significant elevation in serum CAT activity. This contraversy may be due to that CAT and other antioxidant enzymes other than GPx have been shown to be relatively less sensitive to oxidative stress [83] or it may be due to the differences in species variation, dose or duration of treatment. Data in Table (4) demonstrated that A. graveolens caused non-significant decrease in serum levels of ALT, AST and LDH enzymes (P>0.05), in contrast with DOX which cause significant increase in activities of ALT, AST and LDH in serum as compared with the control group or with its level before treatment (P<0.05). The combination of A.graveolens with DOX caused significant decrease in serum ALT, AST and LDH as compared with DOX group (P<0.05). DOX in the form of DOX semiquinone has been suggested to play a major role in its hepatotoxic action. Semiquinones are unstable under aerobic conditions thereby generating ROS by reacting with molecular oxygen [82]. Hepatocytes are the likely targets of ROS attack in the failing liver. It is conceivable that free radicals cause damage at their formation. Serum transaminases have long been considered as sensitive indicators of liver injury (84). DOX induced oxidative damage to hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells [82, 98]. This leakage causes a decrease in the levels of ALT and AST in hepatic cells but increase in levels of ALT and AST in serum (82). ALT is a more liver specific enzyme. Increase in AST activity is also roughly proportional to the extent of cardiac damage [16]. This indicate that DOX could not showed adverse effects at the hepatic level only, but also the possibility of significant damage of the heart function [11, 85]. Interestingly, A. graveolens prevents the increase in ALT and AST during its administration in combination with a cumulative dose of DOX and restored them towards normal side (Tables 4). These results suggested that A. graveolens may have protective effect against DOX-induced cardio and hepatotoxicity. This protective effect might be due to its ability for stabilizing of the cell membrane as well as repairing the hepatic and cardiac tissue damage by A. graveolens with the consequent decrease in the leakage of these enzymes. A. graveolens probably does so by the free radical scavenging which has been established in the in vitro studies indicating its antioxidant activity [74]. These results are in agreement with previous findings, which have also reported significant prevention into the rise of serum, ALT and AST levels by A. graveolens plant in animals treated with various hepatotoxin supporting the anti-hepatotoxic action of A. graveolens by virtue of membrane stabilizing activity [77, 26]. Similarlly, the dramatic augmentation of the LDH activity (Table 4) could suggest the great permeability of the hepatic cell membrane. Further, LDH is not specific for hepatic injury individually; the rapid cell swelling of sub-sarcolemmal bulbs and injured myocardium could facilitate the loss of intracellular enzymes in DOX group [12, 86]. It was previously known that the heart is very sensitive to ROS induced damage because of its highly oxidative metabolism and fewer antioxidant defenses compared to other organs [87]. This might be the possible mechanism for marked elevation of LDH concentration in serum after multi-dose administration of DOX. Nevertheless, LDH activity significantly decreased after A. graveolens treatment (Table 4) suggesting amioleration the toxicity of DOX not only at the level of liver but also at the level of other organs that house LDH intracellulary including heart. This result is in agreement with Prisacaru and Burlacu [88] who confirm the antitoxic potentials of A. graveolens volatile oil and other extractive solutions against acrylamide intoxication . Non-significant (P>0.05) changes in serum albumin activity in all groups of treatment are shown in (Table 5). Albumin is the most abundant blood plasma protein and is produced in the liver and forms a large proportion of all plasma protein [89]. It might take a longer time for DOX to affect liver synthetic function (as albumin synthesis). Similar results were obtained by Firat et al. [90]. However, serum albumin level was not significantly affected after A. graveolens treatment (Table 5). The same findings have been observed by Abd El-Ghany et al. [67]. However, Prisacaru and Burlacu [88] suggested a possible correlation between antitoxic effect of phthalides from A. graveolens plant and the hyperalbuminemia of the acrylamide intoxication. This controversy might due to differences in the dose, duration of A. graveolens supplementation or different species variation. In fact, analysis of data in (Table 5) also showed that there were non-significant changes in serum total bilirubin concentration in all groups before treatment. Conversely, there was significant increase in bilirubin concentration in animals received DOX as compared with the control group and with its concentration before treatment (P<0.05). Since the liver is responsible for clearing the blood of bilirubin, so increasing the total serum bilirubin level indicated a reduction in the excretory capability of the liver as a consequence of liver injury [91]. This finding is in accordance to the results of Anandakumar et al. [92] and Alshabanah et al. [59] who suggested that increase in bilirubin is a clear marker of DOX induced hepatic dysfunction. Furthermore, there was non-significant changes in serum level of total bilirubin in animals received A. graveolens alone as compared with the control group (P>0.05), but there was a significant differences in its level in animals which received A. graveolens in combination with DOX as compared with the control group (P<0.05). This result may be due to the enhancement of bile acid excretion that may induced by A. graveolens and its active constituents indicating the improvement effects of th plant on liver function [77, 81]. Histopathological Findings Histological Changes Induced by DOX and A. graveolens in Hepatic Tissue Light microscopic observation revealed that the control and A. graveolens groups showing normal hepatocytes as illustrated in Figure (1) and Figure (2) respectively. Conversely, the hepatic sections from rabbits administered DOX exhibited a significant distribution of feathery degeneration of hepatocytes as summarized in Figure (3), moderate inflammatory cell infiltratation around portal tract, marked piecemeal necrosis, interlobular inflammatory cell infiltrate, and bridging necrosis, as recapitulated in Figure (4). However, the histopathological examination of hepatic tissues of DOX with A. graveolens group showed a mild degenerative changes as briefed in Figure (5). B. Histological Changes Induced by DOX and A. graveolens in Cardiac Tissue The histopathological examination of cardiac tissues of the control and A. graveolens groups exhibited normal cardiac myocytes as briefed in Figure (6) Figure (7) respectively. In contrast, light microscopic observation revealed that the heart sections from rabbits received DOX showed a significant distribution of myocarditis with focal chronic inflammatory cells infiltration as summarized in Figure (8) and marked vascular congestion in between cardiac myocytes as recapitulated in Figure (9). Though, histological sections of cardiac tissue of DOX with A. graveolens group revealed intact myocardiam as illustrated in Figure (10). The different patterns of cell death and inflammation noticed in the present study might be due to DOX induced heptocellular injury and intrahepatic cholestasis resulting from cumlative drug administration. These findings were in agreement with Rasha and Abdella [93] and Ibrahim et al. [94] who observed portal and interlobular inflammation in hepatic sections isolated from DOX treated animals. Also, Abbas [95] and Mohan et al. [82] conclude that animals subjected to DOX stress showed histological alterations in many organelles including feathery hepatocyte degeneration, piecemeal necrosis and 4 necrosis. Besides, myocarditis is an inflammation of the myocardium associated with cardiac dysfunction, it is frequently characterised by the evolution in dilated cardiomyopathy [96] and most often results from infectious agents, hypersensitivity responses, or immune-related injury [97]. Actually, DOX associated myocarditis developed in a dose-dependent fashion, and myocyte damage on biopsy has a linear relation with cumulative dose [98]. Thus, the histological cardiac lesions observed in the current study might result as a response of cellular injury and electrolyte abnormalities mediated by DOX cumulative administration. Increased free radicals and reduced endogenous antioxidants, with subsequent increase in oxidative stress lead to loss of myofibrils and vacuolization of myocardial cells [99]. Moreover, there is a strong association between oxidative stress and the cardiac inflammatory response, including cytokine release after DOX administration [93, 100]. In that regard, DOX is known to induce NFK-B activation and COX-2 expression in cardiomyocytes [101]. Also, it was reported that DOX induced changes in the properties of membrane bound ATPases of cardiac cells affect cardiac function and may be a relevant mechanism leading to lethal myocardial cellular injury [102]. In addition to that, DOX mediated vascular congestion may contribute to this pathogenesis [103]. The resulting peripheral release of vasoactive and pro-inflammatory neurohormones from the stretched endothelium and from the perivascular congested tissue may promote the development and progression of heart failure and thus compromising organ function [104]. These histopathological changes were found to be in a good agreement with the work of other authors such as [71, 93, 102]. Interestingly, combined supplementations of A. graveolens plant along with DOX antibiotic in this study resulted in significant reduction in histological changes that were induced by DOX in liver and heart tissues as summarized in Figures (4-5) and (4-10) respectively. This might explain the ability of A. graveolens to attenuate the DOX-related toxic effects via more than one mechanism including the powerful scavinging of ROS, the inhibition of lipid peroxidation, the decrease of the inflammatory response and the stimulation of the synthesis of cellular antioxidants that protect hepatic and cardiac cells from DOX induced liver and heart injuries. Similar results were observed by Sultana et al. [66] and Jain et al. [81] who studied the effect of A. graveolens on isolated hepatic sections. However, there is no available study to compare with for the effect of A. graveolens on isolated cardiac sections.

Conclusions

It seems that the cardiotoxic, hepatotoxic and hematototoxic effects of DOX might be attributed to the combination of free radical oxidative stress, and lipid peroxidation. A. graveolens coadministration significantly protected against DOX -induced toxicities on heart, liver, and blood component; thus suggesting its administration as a supportive care agent during anticancer treatment. The protective mechanism of A. graveolens may involve direct and indirect antioxidant activity against DOX-induced cardiac, hepatic and hematological damage.

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