Synergistic protective role of mirazid (Commiphora molmol) and ascorbic acid against tilmicosin-induced cardiotoxicity in mice.

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Synergistic protective role of Mirazid® (Commiphora molmol) and ascorbic acid against

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tilmicosin-induced cardiotoxicity in mice

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Mohamed M. Abdel-Daim1*, Emad W. Ghazy2 and Mostafa Fayez 1

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41522, Egypt.

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University, Kafr El-Sheikh, 33516, Egypt.

Pharmacology Department, Faculty of Veterinary Medicine, Suez Canal University, Ismailia,

Department of Clinical Pathology, Faculty of Veterinary Medicine, Kafrelsheikh

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Running title: Myrrh and Vitamin C against tilmicosin cardiotoxicity

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* Corresponding author. Address: Pharmacology Department, Faculty of Veterinary Medicine,

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Suez Canal University, Ismailia, 41522, Egypt.

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Tel/Fax.: +20643207052

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E-mail address: [email protected], [email protected] (Abdel-Daim M.)

Mob. +201117761570

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ABSTRACT

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Tilmicosin (TIL) is a long-acting macrolide antibiotic approved for the treatment of cattle with

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Bovine Respiratory Disease. However, it has been reported to induce cardiac toxicity when taken

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in an overdose. Our experimental objective was to evaluate the protective effects of Commiphora

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molmol; Mirazid® (MRZ) and/or Ascorbic acid (AA) against TIL-induced cardiotoxicity in

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mice. MRZ and AA were orally administered using stomach gavage each either alone or in

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combination for 5 consecutive days followed by single Til overdose. Til overdosage induced a

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significant increase in serum cardiac damage biomarkers; AST, LDH, CK, CK-MB and cTnT as

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well as cardiac lipid peroxidation but caused an inhibition in cardiac antioxidant biomarkers;

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GSH, SOD, CAT and TAC. Each of MRZ and AA tends to normalize the elevated serum levels

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of cardiac injury biomarkers. Furthermore, it reduced TIL-induced lipid peroxidation and

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oxidative stress parameters. Both MRZ and AA induced a synergistic cardioprotective effect

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when given together. It could be concluded that myrrh and/or vitamin C administration are able

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to minimize the toxic effects of TIL by its free radical-scavenging and potent antioxidant

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activities.

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Keywords: macrolide; oxidative stress; myrrh; vitamin C; cardioprotective

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1.

Introduction Tilmicosin (TIL) is a semi-synthetic long acting macrolide antibiotic, widely used in

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veterinary practice for the treatment and prevention of pneumonia in cattle, sheep and pigs,

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associated with Pasteurella multocida, P. haemolytica, Actinobacillus pleuropneumoniae,

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Mycoplasma spp, Staphylococcus spp, Streptococcus spp (Modric et al. 1998). Moreover, it is

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used for prevention and treatment of mastitis in ruminants (Dingwell et al. 2002). In addition, it

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is used in poultry industry for treatment of respiratory infection caused by susceptible organisms

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(Abu-Basha et al. 2007). It is a 16-membered macrolide, prepared by chemical modification of

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tylosin-related compound and inhibits microbial protein synthesis through binding with the 50S

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ribosomal subunit. The efficacy of TIL is attributed to its large distribution volume, long

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elimination half-life as well as low inhibitory concentrations, and other pharmacodynamic

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characters (Scorneaux and Shryock 1999; Ziv et al. 1995). However, the drug may have cardiac

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toxic effects in animals, whereby in general; overdose causes positive chronotropic and negative

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inotropic cardiovascular action with increased HR and loss of ventricular function (Jordan et al.

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1993), particularly in young animals. This effect is mainly depending upon animal species, the

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dose, and the route of administration (Altunok et al. 2002; Yazar et al. 2002) . An accidental

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human administration of the TIL resulted in chest pain, alterations in electrocardiogram (ECG)

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and cardiac injury marker levels, including an increase in creatine kinase (CK) and CK-MB (Von

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Essen et al. 2003). Previous studies definitely indicate that, TIL caused elevation of cardiac

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damage biomarkers and oxidative stress by decreasing antioxidant enzymes or by reducing

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synthesis of these enzymes in the cardiac tissue (Kart et al. 2007; Yazar et al. 2002). The cells

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combat oxidative stress by either directly scavenging oxygen radicals via endogenous enzymatic

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and non-enzymatic antioxidants or removing the damaged nucleotides and lipid peroxidation

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products (Abdel-Daim et al. 2013; Al-Sayed et al. 2014; Azab et al. 2013; Madkour and Abdel-

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Daim 2013).

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Commiphora molmol (myrrh) is known commercially in the Egyptian pharmacies as

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Mirazid® (MRZ). It consists of approximately 20–40% alcohol soluble resin, 30–60% water-

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soluble gum, and 3–8% volatile oil (al-Harbi et al. 1997). It also contains terpenes,

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sesquiterpenes, cuminic aldehyde, and eugenol (al-Harbi et al. 1997). It has been reported that

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myrrh has many pharmacological activities, including: antimicrobial, antiparasitic, analgesic, 3


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anti-inflammatory, local anesthetic, anti-ulcer, anticancer and immunomodulatory (al-Harbi et al.

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1997; Ashry et al. 2010). Myrrh protected liver against diazinon-induced hepatic and cardiac

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damage (Abdel-Daim and Halawa 2014). Myrrh active constituent; guggulsterone induced

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cardioprotective effects against doxorubicin cardiomyocyte injury (Wang et al. 2012). Its

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antioxidant properties might be through its free radical-scavenging activities (Ashry et al. 2010;

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El-Ashmawy et al. 2006). Myrrh reduced lead-induced oxidative damage in mice hepatic tissue

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by reducing lipid peroxidation and enhancing glutathione s-transferase activity (Ashry et al.

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2010; El-Ashmawy et al. 2006). It protected liver against diethylnitrosamine-induced injury and

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carcinogenesis (El-Shahat et al. 2012). Furthermore, the free radical-scavenging effect of myrrh

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essential oils provides a protection against lipid peroxidation induced by cosmetic preparations

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and lipophilic pharmaceuticals. Ascorbic acid; vitamin C is one of the most commonly used vitamins and probably the

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most important antioxidant in extracellular fluids. It is an important component in the diet of

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animals and human as well (Lindblad et al. 2013). It is highly water soluble and acts as an

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effective reducing agent. It is one of the most effective antioxidant in inhibiting lipid

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peroxidation initiated by peroxyl radicals, and considered as an effective free radical scavenger

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(El-Demerdash et al. 2005; Kojo 2004). Moreover, it may regenerate other antioxidants such as

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vitamin E (Carr and Frei 1999). To our knowledge, the role of myrrh and vitamin C against TIL-

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induced serum biochemical alteration as well as lipid peroxidation and antioxidant status in mice

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has not been studied yet. Therefore, the present study was designed to investigate the alterations

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in serum biochemical parameters related heart damage as well as cardiac lipid peroxidation and

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oxidative stress induced by TIL in mice. Moreover, the role of myrrh or/and vitamin C

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supplementation in alleviating these TIL-induced hazard effects could be evaluated.

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2.

Materials and Methods

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2.1.

Chemicals

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Tilmicosin (Tilmicosin® vial, 33 mg/ml) in clinical formulation was purchased from

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Arabcomid, Egypt. Mirazid; MRZ (Pharco Pharmaceuticals, Alexandria, Egypt) was purchased

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from a local pharmacy and prepared freshly before the treatment. Ascorbic acid was obtained

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from Adwia Pharmaceuticals, Cairo, Egypt. All kits were purchased from Biodiagnostics, Egypt 4


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except, lactate dehydrogenase (LDH), obtained from Randox Laboratories Ltd, U.K., creatine

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kinase (CK), creatine kinase-MB (CK-MB) were purchased from using Stanbio™ (Texas, USA),

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and ELIZA cardiac troponin T (cTnT) kit from Roche Diagnostics, Mannheim, Germany. All

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other chemicals used in the experiment were of analytical grade.

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2.2.

Animals and experimental design Fifty six male albino mice, weighing 25±3 g, were purchased from The Egyptian

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Organization for Biological Products and Vaccines. Mice were kept in ventilated room under

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controlled laboratory conditions of normal light –dark cycle (12 h light/dark) and temperature

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(25 ± 2 °C). Food and water were provided ad libitum. The animals were treated in accordance

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with the Guidelines for Animal Experimentation (of the Ethics Review Committee) of Faculty of

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Veterinary Medicine, Suez Canal University. After 1week acclimation period, mice were

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randomly assigned to seven different groups; eight animals each. The 1st served as a control

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group, and was given saline. The 2nd and 3rd groups were orally administered myrrh and ascorbic

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acid using gastric gavage at a dose of 100 and 20 mg/kg body weight, for five days respectively

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(EL-Naggar 2011; Ozdem et al. 2011). The 4th group received a single dose of TIL (75 mg/kg,

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subcutaneously) (Kart et al. 2007). The 5th and 6th groups were given myrrh and ascorbic acid at

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the same dose regimen used for the 2nd and 3rd groups before TIL administration at the same dose

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and regimen used for the 4th group. The 7th group was given both myrrh and ascorbic acid one

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hour before TIL administration (Table 1).

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2.3.

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Serum collection and tissue preparation At the end of experiment (24 hours after TIL injection), blood samples were collected via

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direct heart puncture under light ether anaesthesia. Blood samples were left to clot at room

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temperature and centrifuged at 3000 rpm for 15 minutes. Sera were then, separated and stored at

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-20°C as aliquots for further biochemical analysis.

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After blood collection, mice were sacrificed by deep ether anaesthesia. Heart was rapidly

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excised from each animal, trimmed of connecting tissue, and washed free of blood with 0.9%

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NaCl solution and distalled water. It was blotted over a piece of filter paper and perfused with a

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50 mM (sodium phosphate buffer saline (100 mM Na2HPO4 / NaH2PO4) (PH 7.4) in an ice-

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containing medium, containing 0.1 mM ethylene di amine tetra acetic acid (EDTA) to remove

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any red blood cells and clots. Then, heart tissues were homogenized in 5 – 10 ml cold buffer per 5


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gram tissue and centrifuged at 5000 rpm for 30 minutes. The resulting supernatant was

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transferred into Eppendorf tubes, and preserved at -80°C in a deep freezer until used for various

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biochemical assays.

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2.4.

Serum biochemical analysis Sera stored at -20°C were used for estimation of serum cardiac injury marker enzymes;

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aspartate aminotransferase (AST), LDH, CK, CK-MB, and cTnT. AST was evaluated according

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to Reitman and Frankel (1957), LDH activity was determined enzymatically according to the

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manufacturer's protocol (Buhl and Jackson 1978). CK activity was estimated according to the

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method developed by Szasz et al. (1979) using Stanbio™ CK-NAC (UV-Rate) kit (Texas, USA).

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Serum CK-MB activity was assessed by an immunoinhibition method developed by Wurzburg et

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al. (1976) using Stanbio™ Creatine KinaseMB kit (Texas, USA). Serum cTnT level was

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evaluated using ELIZA Kit according to the manufacturer protocol Mair et al. (1991).

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2.5.

Evaluation of tissue lipid peroxidation and antioxidant enzymes Lipid peroxidation was evaluated by measurement of MDA content in cardiac tissues

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according to Mihara and Uchiyama (1978). The non-enzymatic antioxidant biomarker; reduced

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glutathione (GSH) was assessed according to Beutler et al. (1963). The enzymatic antioxidant

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biomarkers; superoxide dismutase (SOD) was evaluated according to Nishikimi et al. (1972) and

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catalase (CAT) according to Aebi, (1984). In addition, total antioxidant capacity (TAC) was

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determined according to Koracevic et al. (2001).

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2.6.

Histopathological examination Cardiac tissues were taken immediately from mice, fixed in 10% buffered formalin,

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dehydrated in ethanol (50–100 %), cleared in xylene, and embedded in paraffin. Sections (4–5

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µm thick) were prepared, and then stained with hematoxilin and eosin (H–E). The sections were

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examined for the pathological findings of cardiac changes.

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2.7.

Statistical analysis

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Data are presented as mean ± S.E.. Statistical significance of the data was analyzed using

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SPSS programme (Statistical package for Social Science) version 16. For comparison, One-Way

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analysis of variance (ANOVA) test and post-comparison was carried out with Tukey's Range

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Test for post-hoc analysis. Statistical significance was acceptable to a level of P ≤ 0.05.

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3.

Results

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3.1.

Serum biochemical analysis The effects of TIL intoxication as well as the preventive effects of myrrh and/or AA on

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serum biochemical analyses are shown in figures 1& 2. Significant increases (P≤0.05) in serum

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cardiac injury marker (AST, LDH, CK, CK-MB and cTnT) were recorded in TIL intoxicated

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mice as compared to the untreated control group (about 250.40%, 242.85%, 392.97%, 833. 38%

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and 261.58%, respectively). Pre-treatment with MRZ at doses of 100 mg/kg for 5 consecutive days significantly

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(P≤0.05) reduced the serum cardiac injury biomarkers; LDH, CK and CK-MB ( about 55.68,

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60.29%, 46.07%, 59.14% and 79.98%, respectively), while AA pre-treatment at a dose of 20

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mg/kg significantly (P≤0.05) reduced these biomarkers ( about 51.56%, 58.85%, 43.78%,55.65%

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and 72.92, respectively) compared with TIL-overdosed group. Moreover, both MRZ and AA

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were given in combination and significantly (P≤0.05) reduced the same parameters to a greater

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extent than that induced by each of them when given alone ( about40.82%, 47.29%,

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26.27%,18.96% and 43.74%, respectively) There were no significant changes in serum biomarkers in mice received either MRZ or

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AA alone (2nd and 3rd groups, respectively) if compared to the normal control (1st group),

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indicating the safety of MRZ and AA at the selected doses used in this study.

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3.2.

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Cardiac lipid peroxidation and antioxidant biomarkers The effects of TIL intoxication as well as preventive effects of MRZ and/or AA on

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cardiac tissue homogenate lipid peroxidation and antioxidant parameters are shown in figures

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3&4. A significant increase (P≤0.05) in cardiac MDA content (242.97%) was observed

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compared to the control group. On the other hand, cardiac GSH, SOD, CAT and TAC were

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significantly (P≤0.05) decreased (48.68%, 44.40%, 31.07% and 74.12%, respectively).

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Concerning TIL-MRZ group, cardiac MDA was decreased (57.87%) while GSH, SOD, CAT, 7


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and TAC were increased (131.37%, 158.08%, 158.95%, and 128.26%, respectively) compared to

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TIL-intoxicated group. Regarding to TIL-AA group, cardiac MDA was decreased (51.67%),

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while GHS, SOD, CAT, and TAC were increased (about 157.86%, 168.05%, 165.97% and

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131.03%, respectively). In addition, both MRZ and AA were given in combination, significantly

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(P≤0.05) reduced cardiac MDA level (36.76% ) and increased GHS, SOD, CAT, and TAC

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more than when each of them used alone ( about 206.50%, 231.30%, 310.70 and 139.62%,

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respectively).

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3.3.

Histopathological findings The Til-overdosed group revealed cardiac degeneration and necrosis. The cardiac bundles

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were vacuolated and sometimes interspersed with hemorrhage (Figure 5.b.). Regeneration of

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cardiac bundles was more observed when treated with vitamin C (Figure 5.d.) than when treated

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with MRZ alone (Figure 5.c.). The group pretreated with both vitamin C and MRZ in

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combination revealed complete regeneration and recovery (Figure 5.e).

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4.

Discussion Drug-induced cardiotoxicity was considered as a major limitation in standard and high-

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dose macrolide antibiotic, a phenomenon which is particularity applicable for TIL treatment

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(Jordan et al. 1993; Main et al. 1996). Oxidative stress plays a major role in TIL-induced

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cardiotoxicity during the normal clinical treatment regimens, which results in dose escalation

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limitation and hindrance of the clinical consequences (Kart et al. 2007; Yazar et al. 2002). As a

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result of TIL-induced oxidative stress, it could induce its cardiac side effect when either given

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alone or given in combination with other cardiotoxic drugs (Main et al. 1996). Although several

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reports about TIL toxicity have been published, a little studies has been performed about the use

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of natural products for prevention of such toxicity and different mechanisms of their preventive

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action (Kart et al. 2007; Yazar et al. 2002).

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In the present study, cardiotoxicity caused by TIL may be attributed to the oxidative

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stress resulted from free radical production. TIL intoxication increased serum cardiac injury

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marker enzymes; AST, LDH, CK, CK-MB and cTnT. Moreover, increased lipid peroxidation 8


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through elevated cardiac MDA level, decreased cardiac non-enzymatic; GSH as well as

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enzymatic; SOD and CAT antioxidant level. Moreover, TAC was reduced in the TIL-intoxicated

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group compared to the normal control group. All these effects are involved in the cascade of events leading to TIL-mediated cardiac

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toxicity and oxidative stress. This indicates that cardiac damage induced by TIL is the result of

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oxidative stress that arises as a result of excessive ROS generation, which have been reported to

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attack various biological molecules, including lipids and causing lipid peroxidation. The

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antioxidant enzyme activities, including these involved in glutathione metabolism were also

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altered in TIL treated group indicating the involvement of oxidative damage in TIL-mediated

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cardiac injury. These results are consistent with the literature (Kart et al. 2007; Yazar et al. 2002)

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and point towards the role of ROS in TIL mediated injury and toxicity.

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The cardiovascular toxicities of TIL were reported in many animal species (Jordan et al.

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1993; Kart et al. 2007). The clinical evidence of this toxicity is generally a manifestation of the

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positive chronotropic and negative inotropic cardiovascular events (Kart et al. 2007).

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Toxicological data indicate that lethal doses of TIL are accompanied by an altered myocardial

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contractility and an increase in heart rate (Main et al. 1996). In conscious dogs, intravenous

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injection of TIL (2.5 mg/kg body weight) resulted in a sinus tachycardia, a reduction in arterial

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pulse and a pronounced myocardial depression (negative inotropy) (Main et al. 1996). The

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mechanisms of cardiotoxicity induced by macrolides, including TIL have not been clearly

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suggested in the literature. One report indicated that, apart from direct toxicity to the cardiac

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cells, TIL promotes the release of adrenaline, contributing to cardiovascular overload (Tamargo

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et al. 1982). Another suggestion is that TIL’s mechanism of toxicity may be mediated through

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the rapid depletion of intracellular calcium through interference with sarcolemmal calcium

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channels and might be leading to negative inotropic actions (Main et al. 1996). Tamargo et al

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reported that the macrolide antibiotics; josamycin and erythromycin inhibited transmembrane

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calcium flux (Tamargo et al. 1982). Calcium-channel blockers have been reported to have a

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negative inotropic effect through direct calcium ions antagonism (Boddeke et al. 1988). The

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possibility of intracellular calcium flux alteration by TIL, has not been confirmed (Main et al.

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1996).

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Additionally, TIL toxic and potentially fatal doses might be depend upon the route of

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administration and the animal species. Severe toxicity was reported with administering doses as

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low as 5 mg/kg, IV, in cattle (death), 10 mg/kg, IM, in swine (convulsions) and 30 mg/kg, IM,

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in a rhesus monkey (death) (Jordan et al. 1993). Factors affecting TIL-induced cardiotoxicity

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include drug dosage, significant preexisting circulatory dysfunction or systemic illness and

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conditions that directly or indirectly interfere with the normal physiologic cardiovascular

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responses (Jordan et al. 1993). Multiple ventricular septal defects were noticed at necropsy of

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lamb suddenly died after TIL administration (Christodoulopoulos 2009). Unintentional human

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injection of TIL leading to cardiac manifestations and laboratory evidence of myocardial injury

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(Von Essen et al. 2003). Kart et al. 2007 and Yazar et al 2002 studied Til acute toxicity in mice

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and stated that, TIL-induced cardiotoxic consequences; increasing serum cardiac injury markers;

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total sialic acid, CK and CK-MB as well as altering serum and cardiac tissue MDA, GSH and

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SOD levels. Pre-administration of L-carnitine as an antioxidant reduced these cardiac injury

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markers and reduced both serum and cardiac tissue lipid peroxidation and oxidative stress

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parameters (Kart et al. 2007; Yazar et al. 2002).

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In the last few years, many countries have shown a renewed interest from consumers and

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researchers in using natural products for treatment of human and animal diseases. These

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materials are very cheap, readily available, and devoid of many side effects of synthetic

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chemicals (Abdel-Daim 2014a, b; Abdou and Abdel-Daim 2014). Therefore, MRZ, AA were

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evaluated in our study for their protection against TIL-induced cardiotoxicity. In the current

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study, the pre-administration of MRZ, AA or their combination reduced the serum cardiac injury

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biomarkers. Moreover, they reduced the lipid peroxidation in cardiac tissues. In addition, there

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were elevations of cardiac antioxidant enzymes and glutathione levels due to their

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administration. Furthermore, the cardioprotective effects of MRZ and/or AA were confirmed

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using histopathological examination of the cardiac muscles, and both agents pretreatment

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revealed the maximum protective effects against Til-induced cardiac degeneration and necrosis.

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The antioxidant and protective effects of MRZ are owed to their content of antioxidant active

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constituents such as cuminic aldehyde, eugenol, and sesquiterpenes (al-Harbi et al. 1997). Many

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previous literatures showed the cardioprotective effects of myrrh against drugs, chemicals and

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xenobiotics-induced cardiopathy (Ashry et al. 2010; El-Ashmawy et al. 2006; El-Shahat et al.

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2012; Wang et al. 2012). Pre-treatment with AA might play a role in reducing the toxic effect of 10


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TIL and its antioxidant properties seem to mediate such a protective effect, indicated by the

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reduction of cardiac MDA as well as the elevation of GSH, SOD, CAT and TAC levels (El-

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Demerdash et al. 2005; Kojo 2004). Mirazid and/or AA protective effect against TIL-induced oxidative stress in our study

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could be either by direct inhibition of lipid peroxidation and scavenging free radicals or indirect

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through the enhancement of the activity the enzymatic free radicals scavengers; CAT and SOD

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in the cells. Therefore, MRZ and/or AA could be used to prevent and treat cardiac diseases,

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especially those induced by oxidative damage and both agents might have a synergistic

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ameliorative effect against TIL-induced cardiotoxicity in mice.

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5.

Conclusions

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Oxidative stress plays a major role in TIL-induced cardiotoxicity during the normal

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clinical treatment regimens, which results in dose escalation limitation and hindrance of the

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clinical consequences. Antioxidants have proven to be effective in preventing macrolide-induced

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toxicity in some previous studies. MRZ and AA are potent natural antioxidants, which are

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reported to have cardioprotective effect and to enhance the effect of many known

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chemotherapeutic agents in addition to reducing their toxicities as well.

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In the present study, clearly TIL administration resulted in varying degree of lipid

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peroxidation, inhibition in the antioxidant enzymes' activities and alterations of serum

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biochemical parameters of mice. MRZ and/ or AA treatment prior to TIL provided near complete

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protection when given in combination in terms of serum biochemical changes and cardiac

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antioxidant biomarker activity and oxidative stress.

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Acknowledgements This research received no grant from any funding agency. The authors would like to

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thank both Dr. Mohie Haridy; the assistant professor of Pathology, Faculty of Veterinary

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medicine South Valley University, Qena, Egypt, and Dr. Abdelazim Ibrahim; the assistant

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professor of Pathology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt,

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for their help in histopathological examination.

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Conflict of interest statement

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The authors declare that there are no conflict of interest.

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References

304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342

Abdel-Daim, M., and Halawa, S. 2014. Synergistic hepatocardioprotective and antioxidant effects of myrrh and ascorbic acid against diazinon-induced toxicity in rabbits International Research Journal of Humanities, Engineering & Pharmaceutical Sciences 1(7): 1-7. Abdel-Daim, M.M. 2014a. Pharmacodynamic interaction of Spirulina platensis with erythromycin in Egyptian Baladi bucks (Capra hircus). Small Ruminant Research 120(2–3): 234-241. doi: http://dx.doi.org/10.1016/j.smallrumres.2014.05.013. Abdel-Daim, M.M. 2014b. Synergistic protective role of ceftriaxone and ascorbic acid against subacute diazinon-induced nephrotoxicity in rats. Cytotechnology In Press. doi: 10.1007/s10616-014-9779-z. Abdel-Daim, M.M., Abuzead, S.M., and Halawa, S.M. 2013. Protective role of Spirulina platensis against acute deltamethrin-induced toxicity in rats. PLoS One 8(9): e72991. doi: 10.1371/journal.pone.0072991 PONE-D-13-21292 [pii]. Abdou, R.H., and Abdel-Daim, M.M. 2014. Alpha-lipoic acid improves acute deltamethrin-induced toxicity in rats. Canadian Journal of Physiology and Pharmacology 92(9): 773-779. doi: 10.1139/cjpp2014-0280. Abu-Basha, E.A., Idkaidek, N.M., and Al-Shunnaq, A.F. 2007. Pharmacokinetics of tilmicosin (Provitil powder and Pulmotil liquid AC) oral formulations in chickens. Vet Res Commun 31(4): 477-485. doi: 10.1007/s11259-006-3543-6. Aebi, H. 1984. Catalase in vitro. Methods Enzymol 105: 121-126. al-Harbi, M.M., Qureshi, S., Raza, M., Ahmed, M.M., Afzal, M., and Shah, A.H. 1997. Gastric antiulcer and cytoprotective effect of Commiphora molmol in rats. J Ethnopharmacol 55(2): 141-150. doi: S03788741(96)01488-2 [pii]. Al-Sayed, E., Martiskainen, O., Seif el-Din, S.H., Sabra, A.-N.A., Hammam, O.A., El-Lakkany, N.M., et al. 2014. Hepatoprotective and Antioxidant Effect of Bauhinia Hookeri Extract against Carbon Tetrachloride-Induced Hepatotoxicity in Mice and Characterization of Its Bioactive Compounds by HPLCPDA-ESI-MS/MS. BioMed Research International 2014: 9. doi: 10.1155/2014/245171. Altunok, V., Yazar, E., Elmas, M., Tras, B., Bas, A.L., and Col, R. 2002. Investigation of haematological and biochemical side effects of tilmicosin in healthy New Zealand rabbits. J Vet Med B Infect Dis Vet Public Health 49(2): 68-70. Ashry, K.M., El-Sayed, Y.S., Khamiss, R.M., and El-Ashmawy, I.M. 2010. Oxidative stress and immunotoxic effects of lead and their amelioration with myrrh (Commiphora molmol) emulsion. Food Chem Toxicol 48(1): 236-241. doi: S0278-6915(09)00466-9 [pii] 10.1016/j.fct.2009.10.006. Azab, S., Abdel-Daim, M., and Eldahshan, O. 2013. Phytochemical, cytotoxic, hepatoprotective and antioxidant properties of Delonix regia leaves extract. Med Chem Res 22(9): 4269-4277. doi: 10.1007/s00044-012-0420-4. Beutler, E., Duron, O., and Kelly, B.M. 1963. Improved method for the determination of blood glutathione. J Lab Clin Med 61: 882-888. Boddeke, H.W., Wilffert, B., Heynis, J.B., and van Zwieten, P.A. 1988. Investigation of the mechanism of negative inotropic activity of some calcium antagonists. J Cardiovasc Pharmacol 11(3): 321-325.

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Buhl, S.N., and Jackson, K.Y. 1978. Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30, and 37 degrees C. Clin Chem 24(5): 828-831. Carr, A.C., and Frei, B. 1999. Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. Am J Clin Nutr 69(6): 1086-1107. Christodoulopoulos, G. 2009. Adverse outcome of using tilmicosin in a lamb with multiple ventricular septal defects. Can Vet J 50(1): 61-63. Dingwell, R.T., Duffield, T.F., Leslie, K.E., Keefe, G.P., DesCoteaux, L., Kelton, D.F., et al. 2002. The efficacy of intramammary tilmicosin at drying-off, and other risk factors for the prevention of new intramammary infections during the dry period. J Dairy Sci 85(12): 3250-3259. doi: S00220302(02)74413-5 [pii] 10.3168/jds.S0022-0302(02)74413-5. El-Ashmawy, I.M., Ashry, K.M., El-Nahas, A.F., and Salama, O.M. 2006. Protection by turmeric and myrrh against liver oxidative damage and genotoxicity induced by lead acetate in mice. Basic Clin Pharmacol Toxicol 98(1): 32-37. doi: PTOpto_228 [pii] 10.1111/j.1742-7843.2006.pto_228.x. El-Demerdash, F.M., Yousef, M.I., and Zoheir, M.A. 2005. Stannous chloride induces alterations in enzyme activities, lipid peroxidation and histopathology in male rabbit: antioxidant role of vitamin C. Food Chem Toxicol 43(12): 1743-1752. doi: S0278-6915(05)00161-4 [pii] 10.1016/j.fct.2005.05.017. EL-Naggar, S. 2011. Lack of the Beneficial Effects of Mirazid (Commiphora molmol) When administered with Chemotherapeutic Agents on Ehrlich Ascetic Carcinoma Bearing Mice. Advances in Biological Research 5(4): 193-199. El-Shahat, M., El-Abd, S., Alkafafy, M., and El-Khatib, G. 2012. Potential chemoprevention of diethylnitrosamine-induced hepatocarcinogenesis in rats: myrrh (Commiphora molmol) vs. turmeric (Curcuma longa). Acta Histochem 114(5): 421-428. doi: S0065-1281(11)00126-7 [pii] 10.1016/j.acthis.2011.08.002. Jordan, W.H., Byrd, R.A., Cochrane, R.L., Hanasono, G.K., Hoyt, J.A., Main, B.W., et al. 1993. A review of the toxicology of the antibiotic MICOTIL 300. Vet Hum Toxicol 35(2): 151-158. Kart, A., Yapar, K., Karapehlivan, M., and Citil, M. 2007. The possible protective effect of L-carnitine on tilmicosin-induced cardiotoxicity in mice. J Vet Med A Physiol Pathol Clin Med 54(3): 144-146. doi: JVA897 [pii] 10.1111/j.1439-0442.2007.00897.x. Kojo, S. 2004. Vitamin C: basic metabolism and its function as an index of oxidative stress. Curr Med Chem 11(8): 1041-1064. Koracevic, D., Koracevic, G., Djordjevic, V., Andrejevic, S., and Cosic, V. 2001. Method for the measurement of antioxidant activity in human fluids. J Clin Pathol 54(5): 356-361. Lindblad, M., Tveden-Nyborg, P., and Lykkesfeldt, J. 2013. Regulation of vitamin C homeostasis during deficiency. Nutrients 5(8): 2860-2879. doi: nu5082860 [pii] 10.3390/nu5082860. Madkour, F.F., and Abdel-Daim, M.M. 2013. Hepatoprotective and Antioxidant Activity of Dunaliella salina in Paracetamol-induced Acute Toxicity in Rats. Indian J Pharm Sci 75(6): 642-648. Main, B.W., Means, J.R., Rinkema, L.E., Smith, W.C., and Sarazan, R.D. 1996. Cardiovascular effects of the macrolide antibiotic tilmicosin, administered alone and in combination with propranolol or dobutamine, in conscious unrestrained dogs. J Vet Pharmacol Ther 19(3): 225-232. Mair, J., Artner-Dworzak, E., Lechleitner, P., Smidt, J., Wagner, I., Dienstl, F., et al. 1991. Cardiac troponin T in diagnosis of acute myocardial infarction. Clin Chem 37(6): 845-852.

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390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426

Mihara, M., and Uchiyama, M. 1978. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86(1): 271-278. doi: 0003-2697(78)90342-1 [pii]. Modric, S., Webb, A.I., and Derendorf, H. 1998. Pharmacokinetics and pharmacodynamics of tilmicosin in sheep and cattle. J Vet Pharmacol Ther 21(6): 444-452. Nishikimi, M., Appaji, N., and Yagi, K. 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Commun 46(2): 849-854. Ozdem, S., Nacitarhan, C., Gulay, M.S., Hatipoglu, F.S., and Ozdem, S.S. 2011. The effect of ascorbic acid supplementation on endosulfan toxicity in rabbits. Toxicol Ind Health 27(5): 437-446. doi: 0748233710388450 [pii] 10.1177/0748233710388450. Reitman, S., and Frankel, S. 1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 28(1): 56-63. Scorneaux, B., and Shryock, T.R. 1999. Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in bovine mammary, blood, and lung cells. J Dairy Sci 82(6): 1202-1212. doi: S00220302(99)75343-9 [pii] 10.3168/jds.S0022-0302(99)75343-9. Szasz, G., Waldenstrom, J., and Gruber, W. 1979. Creatine kinase in serum: 6. Inhibition by endogenous polyvalent cations, and effect of chelators on the activity and stability of some assay components. Clin Chem 25(3): 446-452. Tamargo, J., De Miguel, B., and Tejerina, M.T. 1982. A comparison of josamycin with macrolides and related antibiotics on isolated rat atria. Eur J Pharmacol 80(4): 285-293. Von Essen, S., Spencer, J., Hass, B., List, P., and Seifert, S.A. 2003. Unintentional human exposure to tilmicosin (Micotil 300). J Toxicol Clin Toxicol 41(3): 229-233. Wang, W.C., Uen, Y.H., Chang, M.L., Cheah, K.P., Li, J.S., Yu, W.Y., et al. 2012. Protective effect of guggulsterone against cardiomyocyte injury induced by doxorubicin in vitro. BMC Complement Altern Med 12: 138. doi: 1472-6882-12-138 [pii] 10.1186/1472-6882-12-138. Wurzburg, U., Hennrich, N., Lang, H., Prellwitz, W., Neumeier, D., and Knedel, M. 1976. [Determination of creatine kinase-MB in serum using inhibiting antibodies (author's transl)]. Klin Wochenschr 54(8): 357-360. Yazar, E., Altunok, V., Elmas, M., Tras, B., Bas, A.L., and Ozdemir, V. 2002. The effect of tilmicosin on cardiac superoxide dismutase and glutathione peroxidase activities. J Vet Med B Infect Dis Vet Public Health 49(4): 209-210. Ziv, G., Shem-Tov, M., Glickman, A., Winkler, M., and Saran, A. 1995. Tilmicosin antibacterial activity and pharmacokinetics in cows. J Vet Pharmacol Ther 18(5): 340-345.

427

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428

List of tables

429

Table 1: Summary of different mice groups and their treatment Group

TIL

MRZ

AA

Control

-

-

-

MRZ

-

+

-

AA

-

-

+

TIL

+

-

-

TIL-MRZ

+

+

-

TIL-AA

+

-

+

TIL-MRZ-AA

+

+

+

430

Tilmicosin; single SC 75 mg/kg body weight, (TIL), Mirazid; Commiphora molmol (myrrh) oral

431

100 mg/kg body weight, daily for 5 days (MRZ), Ascorbic acid; vitamin C oral 20 mg/Kg body

432

weight daily for 5 days (AA)

433

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434

Figures Legends

435

Figure 1: Serum aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)

436

levels in control and different treated mice groups.

437

Legend: a) Serum aspartate aminotransferase (AST) levels in control and different treated mice

438

groups, b) Serum lactate dehydrogenase (LDH) levels in control and different treated mice

439

groups,

440

Data are expressed as means ± SE (n=8), Tilmicosin; single SC 75 mg/kg body weight, (TIL),

441

Mirazid; Commiphora molmol (myrrh) oral 100 mg/kg body weight, daily for 5 days (MRZ),

442

Ascorbic acid; vitamin C oral 20 mg/Kg body weight daily for 5 days (AA),

443

* Significantly different from the control group (P≤0.05)

444

# Significantly different from the tilmicosin-intoxicated group (P≤0.05)

445 446

Figure 2: Serum creatine kinase (CK), creatine kinase-mb isoenzyme (CK-MB) and

447

cardiac troponin T (cTnT) in control and different treated mice groups.

448

Legend: a) Serum creatine kinase (CK) levels in control and different treated mice groups, b)

449

Serum creatine kinase-mb isoenzyme (CK-MB) levels in control and different treated mice

450

groups, c) Serum cardiac troponin T (cTnT) levels in control and different treated mice groups,

451


452


453


454


455


456 457

Figure 3: Cardiac malondialdehyde (MDA) and total antioxidant capacity (TAC) levels in

458

control and different treated mice groups.

459

Legend: a) Cardiac malondialdehyde (MDA) level in control and different treated mice groups,

460

b) Cardiac total antioxidant capacity (TAC) level in control and different treated mice groups.

461


462


463

Ascorbic acid; vitamin C oral 20 mg/Kg body weight daily for 5 days (AA), 16


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464


465


466 467

Figure 4: Cardiac reduced glutathione (GSH) levels, superoxide dismutase (SOD) and

468

catalase (CAT) activities in the control and different treated mice groups.

469

Legend: a) Cardiac reduced glutathione (GSH) level in control and different treated mice

470

groups, b) Cardiac superoxide dismutase (SOD) activity in control and different treated mice

471

groups, c) Cardiac catalase (CAT) activity in control and different treated mice groups.

472


473


474


475


476


477 478

Figure 5 Histopathological finding of the control and different treated mice groups.

479

Legend: a) Cardiac muscles from control group showing normal architecture. H&E 400x

480

b) Cardiac muscles of tilmicosin group showed hemorrhage, degeneration and necrosis. H&E

481

400x

482

c) Cardiac muscles of MRZ pretreated group revealed coagulative necrotic changes of deep

483

pyknotic nucleus and acidophilic cytoplasm, H&E 400x

484

d) Cardiac muscles of vitamin C pretreated group revealed regeneration with eosinophilic fibers

485

and striation. H&E 400x

486

e) Cardiac muscles of the group pretreated with MRZ and vitamin C in combination, revealed

487

almost normal myocardial architecture, complete regeneration with eosinophilic fibers and

488

striation H&E 400x

17


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Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by University of Queensland on 11/24/14 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record. Page 21 of 22

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Synergistic protective role of ceftriaxone and ascorbic acid against subacute diazinon-induced nephrotoxicity in rats.

Synergistic protective effects of ceftriaxone and ascorbic acid against subacute deltamethrin-induced nephrotoxicity in rats.

Protective role of ascorbic acid against asbestos induced toxicity in rat lung: in vitro study.

The Protective Role of Resveratrol against Arsenic Trioxide-Induced Cardiotoxicity.

Protective action of ascorbic acid and sulfur compounds against acetaldehyde toxicity: implications in alcoholism and smoking.

In Vivo Protective Effects of Diosgenin against Doxorubicin-Induced Cardiotoxicity.

Differential cytotoxic activity of the petroleum ether extract and its furanosesquiterpenoid constituents from Commiphora molmol resin.

Analgesic, anti-inflammatory and anti-hyperlipidemic activities of Commiphora molmol extract (Myrrh).

Protective effect of silymarin against chemical-induced cardiotoxicity.

Protective effect of Syzygium cumini against pesticide-induced cardiotoxicity.

Role of ascorbic acid in photosynthesis.

Synergistic protection of N-acetylcysteine and ascorbic acid 2-phosphate on human mesenchymal stem cells against mitoptosis, necroptosis and apoptosis.

Protective effects of ascorbic acid against the genetic and epigenetic alterations induced by 3,5-dimethylaminophenol in AA8 cells.

Protective role of Cupressuflavone from Cupressus macrocarpa against carbon tetrachloride-induced hepato- and nephrotoxicity in mice.

Protective role of S-adenosyl-l-methionine against acetaminophen induced mortality and hepatotoxicity in mice.

db mice.

Hepatotoxicity of pentavalent antimonial drug: possible role of residual Sb(III) and protective effect of ascorbic acid.

Studies on the role of ascorbic acid in olfactory tissue.

The role of ascorbic acid in mesenchymal differentiation.

Protective effects of deep sea water against doxorubicin‑induced cardiotoxicity in H9c2 cardiac muscle cells.

Essential role of Nrf2 in the protective effect of lipoic acid against lipoapoptosis in hepatocytes.

The protective effect of thiamine pyrophosphate, but not thiamine, against cardiotoxicity induced with cisplatin in rats.

Protective effects of maslinic acid against alcohol-induced acute liver injury in mice.

Glutathione deficiency increases hepatic ascorbic acid synthesis in adult mice.