Effects of Two Different Doses of Zinc Sulfate on Serum Troponin I 3 Enzyme Level and Cardiac Malondialdehyde Contents in Mitoxantrone- Induced Cardiotoxicity in Rats

115 Effects of Two Different Doses of Zinc Sulfate on Serum Troponin I 3 Enzyme Level and Cardiac Malondialdehyde


Introduction
Mitoxantrone (MTXN) is one of the most generally used antineoplastic drug used in treatment of acute leukemia ,breast and prostate cancer, lymphoma, and also in the treatment of multiple sclerosis (MS) because of its immunosuppressive features (1,2) . The MTXNprompted cardiotoxicity is irreversible, and depend on dose. The risk of MTXN-induced cardiac adverse effects can be seriously increased with cumulative dose > 140 mg/m 2 (2) . Clinically, the cardiotoxicity produced by MTXN can cause a reduction in congestive heart failure (CHF) and left ventricular stroke volume (3) ; furthermore, authors stated that the cardio-protective properties of several pharmacologic drugs and a plant have been confirmed by researchers; where, pyridoxine, statins and artichoke have been revealed to be potentially protective against various cardiotoxic agents (4,5,6) .
Zinc (Zn), is one of the main trace elements in the body. It is involved in homeostasis role in oxidative stress (OS). It is required for the metabolic activity of the body's enzymes that involved carbohydrates, fats, proteins the metabolism; and it has an important role in states of cardiovascular diseases (CVDs); and it may have cardio-protective effects in heart disease (7) .The aim of the study is to investigate the effects of two doses of zinc sulphate on MTXN -induced cardiotoxicity.

Preparation of drugs solution
Mitoxantrone (MTXN) vial (20mg/10ml) was diluted with 10ml D.W to obtain 1mg /ml to be IP injected at a dose 2.5 mg/kg. Each capsule of zinc sulfate (220mg) was reconstituted in 22ml of D.W to obtain 10mg/ml to be administered by oral gavage at doses 15mg/kg, and 30mg/kg (8,9 ) .

Animals
Forty-eight (48) healthy adult Wistar Albino rats of both sexes (24 male and 24 female), three months old, weighing range 150-240 gm were utilized in this research; they were gotten from in the animal House of College of Science, University of Dhi Qar. Rats were maintained in the College of Science, Wasit University under normal conditions of temperature, humidity and under a 12 h light/dark cycle .Rats were supplied commercial pellets and tap water throughout the experiment period. The animals had no manifestation of any illness upon examination. They were left for two weeks without interference for acclimatization. The study was accepted by the Graduate Studies and the Scientific Committees of the College of Pharmacy, University of Baghdad.

Experimental protocol
Healthy rats were randomly allocated into six groups, each containing 8 rats' (4 males and 4 females) as follows: Group I: Rats received 0.5 ml/day of distilled water (DW) intraperitoneally (IP) for 20 days. This group served as a negative control. Group II: Rats orally-administered zinc sulfate at a dose of (15mg/kg/day) alone for 20 days. Group III: Rats orally-administered zinc sulfate at a dose of (30mg/kg/day) alone for 20 days (8) Group IV: Rats intraperitoneally (IP) injected with a mitoxantrone (MTXN) at a dose (2.5 mg/kg) on day 0, 10, 20 to reach total cumulative dose of 7.5 mg/kg on day 20 ( 9 ) . This group served as positive control. Group V-Rats orally-administered zinc sulfate at a dose (15mg/kg/day) for 20 days, with an IP injection of mitoxantrone (MTXN) at a dose (2.5 mg/kg) was administered at the day 0, 10, 20 to reach total cumulative dose of 7.5 mg/kg on day 20. Group VI-Rats orally-administered zinc sulfate at a dose (30 mg/kg/day) for 20 days, and an IP injection of mitoxantrone (MTXN) at a dose (2.5 mg/kg) was administered at the day 0, 10, 20 to reach total cumulative dose of 7.5 mg/kg on day 20.
Forty-eight (48) hour after the end of the treatment duration (i.e. at day 22 nd ), each animal was euthanized by diethyl ether and ketamine. Then, after cervical dislocation, two and a half (2.5) ml blood was obtained from each rat by cardiac puncture and then was transferred into a gel tube and left to clot for 30 minutes before centrifugation for 15 minutes at 3000 round per minute (rpm) to obtain serum, which was collected using rubber micropipette, divided into small aliquots in labeled Eppendorf tubes, and kept frozen at -50 ºC until analyzed. The serum was utilized for the estimation of troponin I 3; where, the estimation is based on Sandwich-Enzyme-linked immunosorbent assay (ELISA) (10) . Serum level of troponin I 3 was expressed as pg/mL. Furthermore, cardiac tissues were obtained to prepare heart tissue homogenates for the determination of MDA contents with the use of phosphate buffer saline (PBS pH 7.4) which also based on ELIZA (11) . The contents of MDA in heart tissue homogenate were expressed as ng/mL.

Statistical Analysis
Statistical analysis was done by Statistical Package for Social Sciences (SPSS) version 24. Results were calculated as mean ± standard error of means (SEM). Comparison among groups was done by using a one-way Analysis of variance (ANOVA). The statistically significant differences were considered when P<0.05. Table 1 and figure 1showed that there were non-significant differences (P>0.05) in serum level of troponin I 3 in group of rats orally-administered zinc sulfate (15mg/kg/day alone for 20 days) (Group II) compared to the corresponding enzyme level in serum of negative control rats (Group I).

Mitoxantrone
(MTXN) is an antineoplastic agent used to treat several types of cancers and on multiple sclerosis (MS); such drug showed a high incidence of cardiotoxicity, which was reported to depend on several factors; where, age and cumulative dose being two important factors (12) .
In the current study, IP injection of MTXN can cause destruction of the myocardial cell membrane, which becomes more permeable; thus, serum troponin I 3 level were significantly elevated (P<0.05) compared to the corresponding levels in negative control rats' group (table 1 and figure 1). This comes in tune with a study of others (13) .
Biomarkers are molecules which occur naturally and can be used as an indicator of a particular disease state or some other physiological state of an organism. Troponins were medium-sized proteins which have an important role in regulating the cardiac muscle contractile elements (myosin and actin) (14) . Furthermore, troponins are well-established biomarkers for the discovery of heart damage in both human and animals; where, levels may increase after 2-3 hours; and serum levels of cTnI and cTnT components of the troponin complex of muscle cells, are of importance in the diagnosis of myocardial damage in several conditions, including acute myocarditis (15,16,17) .
The serum levels of troponin I 3 to detect myocardial destruction induced by MTXN was not previously estimated; but the study performed by Herman EH, et al at 2001 (18) and by the determination of troponin T levels showed that there were high levels of the biomarker in rats with chronic doxorubicin-and MTXN-induced cardiotoxicity . Thus, to our knowledge the current research is the first to estimate the serum level of troponin I 3 enzyme in MTXN-induced cardiotoxicity; thus, we did not have a chance to compare results obtained from this study with others concerning this respect.
The results of the present study also showed that oral administration of zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of MTXN] (Group V), resulted in a non-significant (P>0.05) difference in serum level of troponin I 3 compared to the corresponding serum level in group of rats IP injected with total cumulative dose of 7.5 mg/kg of MTXN (Group IV). Moreover, there were significant reduction (P<0.05) in serum level of troponin I 3 of rats orally-administered zinc sulfate [(30 mg/kg/day) with total cumulative dose (7.5mg/kg) of MTXN] (Group VI) compared to the corresponding levels in serum of rats IP injected with total cumulative dose of 7.5mg/kg of MTXN (Group IV) (table 1 and figure 1).
Zinc is a vital element in preserving the normal structure and physiology of cells. It appears to have protective effects in heart diseases (19) . Furthermore, authors reported ischemia and infarction may lead to release of zinc from proteins and cause myocardial damage. In such states, replacing with zinc has been shown to recover cardiac function and prevent further destruction (20) . Moreover, authors reported that rodents administered zinc salts during reperfusion has been stated to decrease arrhythmias and improve myocardial function (19) .
Additionally, to assess if MTXN is able to initiate or enhance lipid peroxidation, the most known secondary product of lipid peroxidation, MDA, was determined; where, it is one of the best indicators of OS (21) . Results of this study exhibited that the cardiac tissue MDA content was significantly elevated (P<0.05) in MTXN-treated rats (Group IV, positive control) compared to the corresponding cardiac contents in negative control (Group I) rats (table 2 and figure 2); these results are in line with the work of others. The increment in the product of lipid peroxidation (MDA) may be considered as a causative factor in MTXNinduced acute cardiotoxicity (22,23) Furthermore, results of this study showed that that oral administration of zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of MTXN] (Group V), resulted in a non-significant (P>0.05) difference in MDA contents in heart tissue homogenate compared to the corresponding contents in heart tissue in group of rats IP injected with total cumulative dose of 7.5 mg/kg of MTXN (Group IV). Moreover, there were significant reduction (P<0.05) in MDA contents in heart tissue homogenate of rats orallyadministered zinc sulfate [(30 mg/kg/day) with total cumulative dose (7.5mg/kg) of MTXN] (Group VI) compared to the corresponding MDA contents in heart tissue homogenate of rats IP injected with total cumulative dose of 7.5mg/kg of MTXN (Group IV).
Authors revealed that Zn, a necessary metal that is integral to the activity of many metalloenzymes required for cellular functions; and it has critical, anti-atherogenic, antiinflammatory and antioxidant actions to protect against various oxidative stresses Moreover, the authors describe that there is a strong basis to consider the strategy of Zn supplementation to cardioprotect the heart against MI (24) . Furthermore, Asri-Rezaei Siamak et al (2017) observed that, zinc sulfate administration could protect against lipid peroxidation, due to anti-oxidative effect that showed by a reduction in MDA contents in rats. Furthermore, others reported that zinc-induced a very efficient antioxidant in scavenging various free radicals [metallothionein (MT)] synthesis in the rat heart which in turn may alleviate diabetic cardiotoxicity (8) . In addition, at 2015 Hala Attia et al reported that addition of Zn to glibenclamide can significantly reduce MDA in cardiac tissues compared to glibenclamide alone; this can support the powerful antioxidant effect of Zn and potential cardio-protection from oxidative damage (25).

Conclusion
Zinc sulfate at a dose of (30 mg/kg/day) (in Group VI rats) diminished the cardiotoxicity induced by mitoxantrone as indicated by the reduction of serum troponin I 3 enzyme level but non-significant difference in lipid peroxidation marker ( malondialdehyde) compared to (15 mg/kg/day) (in Group V rats).