Effect of N-Acetyl Cysteine on Doxorubicin Induced Cardiotoxicity in Adult Male Albino Rat: Histological Study
Received Date: Mar 02, 2019 / Accepted Date: Mar 13, 2019 / Published Date: Mar 19, 2019
Introduction: Cardiotoxicity is one of the most important causes that limit the use of Doxorubicin (DOX) in treatment of the cancer. N-acetylcysteine (NAC) is an antioxidant substance that protects different cellular organelles from free radicals.
The aim of the work: To shed the spot on the role of NAC on cardiotoxicity induced by DOX.
Materials and methods: Forty adult male albino rats were divided into three groups. Group I: it was formed of 20 animals served as a control group. Further, it was divided into two subgroups; Subgroup Ia: formed of 10 animals received physiologic saline and Subgroup Ib: formed of 10 animals received NAC. Group II: formed of 10 animals that received DOX dissolved in normal saline. Group III: formed of 10 animals that received DOX similar togroup II and NAC similar to subgroup Ib. At the end of the second week, all animals were sacrificed; the heart specimens were dissected out and processed to light and electron microscopic examination. Morphometric and statistical analysis was also done.
Results: Light microscopic examination of group II showed deeply stained cardiac muscle fibers and congested coronary vessels. Distorted cardiac muscle fibers and deeply stained nuclei were also observed. Moreover, cellular infiltration was also observed among cardiac muscle fibers. Apparent increase in greenish collagen fibers was seen between cardiac muscle fibers by Masson's trichrome. Electron microscopic examination of group II showed the cardiac muscle with thinning out of some myofibrils, vacuolations of the sarcoplasm and irregular wavy nuclear envelop. Telocytes appeared between cardiac muscle fibers. Group III showed improvement of the cardiac muscle by light and electron microscope with minimal vacuolation in the cardiac muscle. Morphometric and statistical analysis confirmed the histological results.
Conclusion: The present study demonstrated that the administration of N-acetyl cysteine could protect against cardiaotoxicity induced by doxorubicin.
Keywords: Cardiac muscle; Doxorubicin; N-acetyl cysteine; Telocytes; Rats
Doxorubicin (DOX) is a common antineoplastic anthracycline antibiotic and is used for treatment of many types of cancer . However, the risk of cardiac, renal, pulmonary, testicular, and hematological toxicities largely limits its effective and widespread use in clinical oncology . The risk of developing cardiac impairment increases concomitantly with an increase in the cumulative dose . In a recent study based on endomyocardial biopsies, it has been found that cardiac muscle damage begins before clinical signs become evident . The heart is more susceptible to DOX-induced lipid peroxidation and toxicity because of its high energy requirement and high mitochondrial density. Further, the heart also lacks the antioxidant enzymes needed to detoxify superoxide anions and hydrogen peroxide; thus, the generated free radicals accumulate and cause severe lipid peroxidation, leading to extensive destruction of the cardiac cellular mitochondrial membranes, endoplasmic reticulum, and nucleic acid [1,5]. The pathophysiological background of DOX cardiotoxic effect is multifactorial and not completely elucidated [5,6]. This damage produced by DOX is dose-related and may lead to cardiomyopathy [7,8]. N-acetyl cysteine (NAC) is an antioxidant substance that plays an important role in the protection of cell constituents from oxidative stress . The standard treatment for DOX induced systolic heart failure is with angiotensin-converting enzyme inhibitors. Few trials have suggested that N-acetyl cysteine (NAC) may reduce the incidence of left ventricular dysfunction in high risk patients after DOX chemotherapy .
Aim of the Work
This study was aimed to assess the effects of DOX on male rat cardiac muscle and the possible role of NAC.
Materials and Methods
Forty Wister adult male albino rats with average weight 100-150 g were used in this study. The animals were kept in the scientific research center, Faculty of Medicine, Ain Shams University for one week for acclimatization. The animals were randomly divided into three groups. Each group was kept in galvanized wired cage through the duration of experiment. All the experimental procedures were carried out according to the recommendation and the guidelines of the institutional animal ethics committee at Faculty of Medicine, Ain Shams University. Group I: it was formed of 20 animals served as control group. Further it was divided into two subgroups; subgroup Ia and Subgroup Ib. Subgroup Ia: it was formed of 10 animals received intraperitoneal injection of 0.5 ml of 0.9% physiologic saline three times a week for 2 weeks. Subgroup Ib: it was formed of 10 animals received NAC by nasogastric at a dose of 250 mg/Kg/ day for 2 weeks . Group II: it was formed of 10 animals that received DOX dissolved in 0.5 ml 0.9% physiologic saline intraperitoneally at a dose of 2.5 mg/Kg/ three times a week (Sunday, Tuesday and Thursday) for 2 weeks (cumulative dose: 15 mg/Kg) . Group III: it was formed of 10 animals that received DOX similar to the group II and NAC by nasogastric at a dose of 250 mg/Kg/ day for 2 weeks . Doxorubicin was purchased in the form of vials 50 mg/25 ml. It is manufactured by EBEWE Pharma Ges.m.b.H. Nfg.KG, Austria. N-acetyl cysteine was purchased in the form of sachets: 600 mg/ sachet. It is manufactured by Sedico Egypt. All animals were kept in standard conditions, well aerated room and exposed to 12 hour dark/light cycle. They were freely allowed to water and food ad libitum. At the end of the second week, all animals were sacrificed and the heart specimens were dissected out and divided into two ways of processing.
For light microscopic study
The specimens were prepared as usual and paraffin sections at 7-9 μm were cut . The following histological stains were done:
• H&E stain,
• Masson's trichrome. All sections were stained and prepared for examinations.
For electron microscopic study
Specimens were taken from the wall of left ventricle at a size of 1 mm3. They were fixed in glutraldehyde, then in osmic acid, dehydrated in ascending grades of alcohol, cleared in propylene and embedded in resin. Semithin sections were cut for selection of the proper field for ultrathin sections. Ultrathin sections were stained by urenyl citrate and prepared for electron microscopic examinations. Specimens were examined and photographed using a JEM 1200 EXII transmission electron microscope, JEOL, Tokyo, Japan (TEM) at the Faculty of Science, Ain Shams University . Morphometric and statistical study • The mean thickness of cardiac muscle fibers was measured in 5 non-overlapping fields (magnification X400) in 5 different sections in H&E stained sections. • The mean area percentage of collagen among cardiac muscle fibers was measured in 5 non-overlapping fields (magnification X400) in 5 different sections in Masson's trichrome stained sections. These data were calculated by image analysis program (Leica, QwinQgo)(Microsystems Imaging Solutions Ltd, Cambridge, UK) in Histology Department, Faculty Of Medicine Ain Shams University. The data were expressed as mean and standard deviation. Student's "t" test was used to compare the morphometric data and the P value was calculated using SPSS program (version 17; SPSS Inc., Chicago, Illinois, USA). Statistical significance was determined at a level of P<0.05. So, P>0.05 was considered non-significant and P value <0.05 was considered significant (n=6) .
Light microscopic results
Both subgroups Ia and Ib showed similar results by light and electron microscope. These were also confirmed statistically.
Light microscopic results: by examination of H&E stained sections the cardiac muscle fibers were arranged in branching and anastomosing manner. The fibers were separated by connective tissue that contains the coronary blood vessels (Figure 1). Each cardiac muscle fibers showed acidophilic sarcoplasm and transverse striations. It also contained centrally vesicular oval nucleus. The connective tissue between the fibers had many fibroblasts with elongated nucleus (Figure 2).
In group II examination of H&E stained sections; the cardiac muscle fibers showed deeply stained sarcoplasm and congested coronary vessels in between the muscle fibers (Figure 3). Moreover, disrupted and destructed cardiac muscle fibers were seen and deeply stained nuclei were observed in other fibers together with extravasated blood (Figure 4). Cardiac muscle fibers showed alternating deeply stained and pale stained sarcoplasm, loss of myofibers and deeply stained pyknotic nuclei were also observed (Figures 5 and 6). Moreover, cellular infiltration in between the cardiomyocytes and around a blood vessel was seen (Figure 7). In H&E stained sections of group III, the cardiac muscle fibers appeared in branching and anastomosing arrangement separated by connective tissue containing blood vessels (Figure 8). The cardiac myocytes showed acidophilic striations and oval vesicular centrally located nuclei (Figure 9).
By Masson's trichrome stained sections the greenish collagen fibers were apparently few and minimum among the cardiac muscle fibers in group I (Figure 10) while in group II an apparent increase in collagen was seen between cardiac muscle fibers (Figure 11). Moreover, an apparent decrease in greenish collagen fibers appeared between the cardiac myocytes in group III (Figure 12).
Electron microscopic results
The cardiac myocytes of control group I showed myofibrils, numerous mitochondria and central euchromatic nuclei. The myofibrils consisted of actin and myosin that showed alternating dark and light bands. The myofibrils were formed of repeated successive sarcomeres each one formed of the distance between two adjacent Z lines. H zone and M line bisected it were found at the middle. Numerous mitochondria were distributed between the myofibrils. Diad systems of cardiac muscle fibers were noticed at the Z lines of sarcomeres (Figure 13). Intercalated disc was located between adjacent cardiac muscle fibers. It showed step like structure with many desmosomes and fascia adherents. Less electron dense regions of the disc had abundant gap junctions (Figure 14).
Electron microscopic examination of group II showed the cardiac muscle fibers with thinning out of some myofibrils and vacuolations of the sarcoplasm. The vacuolations were distributed among the myofibrils and more around the centrally located nuclei. Moreover, the cardiomyocytes showed irregular wavy nuclear envelop around the nucleus (Figure 15). Telocytes appeared in the interstitium in close relation to the vacuolated cardiac muscle fibers and disrupted intercalated disc was also observed (Figure 16). Telocytes sent their processes (telopodes) in close relation to both the sarcolemma of cardiac muscle fibers and blood vessels (Figure 17). Many cells with euchromatic nucleus and others with myofibrils were seen in between the cardiac myocytes (Figure 18).
Group III by electron microscopic showed the cardiac muscle with numerous mitochondria and myofibrils (Figure 19). The intercalated disc was nearly similar to that of the control (Figure 20).
Morphometric and statistical results
The mean thickness of cardiac muscle fibers of subgroup Ia was 12.8 ± 0.86 μm. Subgroup Ib expressed no statistically significant difference compared with subgroup Ia. There was significant increase in group II in comparison to subgroup Ia, Ib and group III. There was no significant change in the mean thickness of cardiac muscle fibers group III compared with subgroup Ia and Ib (Table 1 and Figure 21).
The mean area percentage of collagen by Masson's trichrome stain of subgroup Ia was 4.7 ± 0.57. Subgroup Ib expressed no statistically significant difference compared with subgroup Ia. There was significant increase in group II in comparison to subgroup Ia, Ib and group III. There was no significant change in group III compared with subgroup Ia and Ib (Table 1 and Figure 22).
In spite of being an effective anti-cancer agent, DOX usage at maximal therapeutic dose is life threatening due to its accumulation in the circulation which may induce irreversible cardiomyopathy and heart failure . Following DOX treatments, aberrations to myocardial architecture and function include cardiac cell hypertrophy and death, heightened susceptibility to myocardial infarction (MI), cardiomyopathy, and left ventricular dysfunction [17-19].
There are few reported studies about N-acetyl cysteine NAC on non-ischemic cardiomyopathy, such as DOX-induced cardiomyopathy and heart failure. Our study provides the important evidence of the beneficial effects of NAC on cardiac dysfunction resulting from DOXinduced heart injury in terms of myocardial fibrosis.
In group II the animals received DOX, the cardiac muscle fibers showed deeply stained sarcoplasm and vacuolated in other fibers areas. This could be explained by some authors who reported that free radicals created by DOX with the mitochondrial and endoplasmic reticulum membranes . Moreover, cellular infiltration was observed around the blood vessels of connective tissue and between cardiac muscle fibers. Cardiac muscle swelling also was confirmed by morphometric and statistical analysis. These could be explained by the oxidative stress produced by DOX that leaded to release of cytokines attract inflammatory cells and vasodilate the vessels. These were in accordance to some researchers who explained that one of the features of DOX is the induction of apoptosis of both terminally differentiated cardiomyocytes and cardiac progenitor cells, resulting in the loss of myocardial tissue and intrinsic regenerative capacity, respectively [21,22]. Other authors , said that doxorubicin impairs the insulinlike growthfactor-1 system and causes insulin-like growth factor-1 resistance in cardiomyocytes.
In our study fibrosis was observed in DOX induced cadiomyopathy. The fibrosis was confirmed by morphometric and statistical analysis. These findings were explained by many authors . They recorded that TGF-β1 is a protein secreted by cardiac myofibroblast and fibroblast that controls proliferation and is responsible for cardiac muscle hypertrophy, and interstitial fibrosis. Previous reports showed that TGF-β1 gene expression is increased in the left ventricular myocardium of patients with idiopathic hypertrophic cardiomyopathy or dilated cardiomyopathy and in animals after myocardial infarction . Fibrosis plays a major role in adverse cardiac remodeling in DOX induced cardiomyopathy and post myocardial infarction . These findings suggest the possible involvement of TGF-β1 gene and protein in the regulation of DOX cardiac fibrosis. The inflammatory response caused by injury initiated the processes of tissue repair which could cause tissue fibrosis.
Mitochondrial damage and thinning out of myofibrils in the same group (DOX treated group). These went side by side with many authors[27,28] who stated that oxidative stress of membranous organelles especially mitochondria lead to ATP depletion and more consumption of glycogen to maintain the homeostasis of cardiac muscle fibers.
In the present study telocytes were demonstrated in DOX treated group. This was in accordance with many authors. They reported the role of telocytes in cardiac tissue regeneration and repair [29,30]. They added that stem cell-based tissue regeneration therapy is thought to be a potential and promising method to treat some diseases in different tissues and organs, such as the heart. Recently, other authors  confirmed the growing evidences that there is a close contact between telocytes, stem cells and progenitor cells in stem cell niches in the heart. Others  explained that the 3D interstitial network constructed by telocytes not only provided a mechanical support for stem cells and progenitor cells but also promoted proliferation, differentiation, maturation, and migration of stem cells. They explained that the main mechanisms of interaction between these cells are atypical junctions and paracrine effects.
In group III the animals received both DOX and NAC for 2 weeks, the cardiac muscle showed improvement. There were improvement in vacuolations and fibrosis. Moreover, the glycogen content appeared similar to that of the control. The cardiac muscle showed picture similar to that of the control. Thus, the use of substances with antioxidant properties has been proven effective at protecting the cardiac muscle against damage caused by reactive oxygen species (ROS) generation. In this sense, several studies have shown that NAC can attenuate the effects caused by DOX . They reported that NAC was able to restore the anti- and prooxidant balance of the cardiac muscle and prevent the harmful effects of DOX treatment. Morphometric and statistical analysis confirmed the histological picture of NAC against DOX as regard the fibrosis and swelling. These were in accordance to many authors [34,35]. They reported that NAC is a precursor of glutathione (GSH), an important enzyme of the cellular antioxidant system that is able to stimulate and sustain its intracellular levels, which detoxify ROS.
Doxorubicin induced cardiomyopathy, once developed, carries a poor prognosis and is frequently fatal. The present study showed that N-acetyl cysteine had beneficial effects that could ameliorate the severity of cardiac injury induced by Doxorubicin.
Extensive investigations are recommended to confirm and fully clarify the mechanism of N-acetyl cysteine effects on preventing Doxorubicin induced cardiomyopathy
- Hajra S, Basu A, Singha Roy S, Patra AR, Bhattacharya S (2017) Attenuation of doxorubicin-induced cardiotoxicity and genotoxicity by an indole-based natural compound 3,3’-diindolylmethane (DIM) through activation of Nrf2/ARE signaling pathways and inhibiting apoptosis. Free Radic Res 51: 812-827.
- Elsherbiny NM, Salama MF, Said E, El-Sherbiny M, Al-Gayyar MM (2016) Crocin protects against doxorubicin-induced myocardial toxicity in rats through down-regulation of inflammatory and apoptic pathways. Chem Biol Interact 247: 39-48.
- Polegato BF, Minicucci MF, Azevedo PS, Carvalho RF, Chiuso-Minicucci F, et al. (2015) Acute doxorubicin-induced cardiotoxicity is associated with matrix metalloproteinase-2 alterations in rats. Cell PhysiolBiochem 35: 1924-1933.
- Ezzat SM, El Gaafary M, El Sayed AM, Sabry OM1, Ali ZY, et al. (2016) The cardenolide glycoside Acovenoside A affords protective activity in doxorubicin-induced cardiotoxicity in mice. J Pharmacol Exp Ther 358: 262-270.
- Singh P, Sharma R, McElhanon K, Allen CD, Megyesi JK, et al. Sulforaphane protects the heart from doxorubicin-induced toxicity. Free Radic Biol Med 86: 90-101.
- Pei XM, Yung BY, Yip SP, Ying M, Benzie IF, et al. (2014) Desacyl ghrelin prevents doxorubicin-induced myocardial fibrosis and apoptosis via the GHSR-independent pathway. Am J Physiol Endocrinol Metab 306: E311-E323.
- Martinel Lamas DJ, Nicoud MB, Sterle HA, Carabajal E, Tesan F, et al. (2015) Selective cytoprotective effect of histamine on doxorubicin-induced hepatic and cardiac toxicity in animal models. Cell Death Discov 1: 15059.
- Imbaby S, Ewais M, Essawy S, Farag N (2014) Cardioprotective effects of curcumin and nebivolol against doxorubicin-induced cardiac toxicity in rats. Hum Exp Toxicol 33: 800-813.
- Padilha Mendonça MC, Ferreira LB, Rizoli C, Batista ÂG, Marostica MR, et al. (2018) N-Acetylcysteine reverses silver nanoparticle intoxication in rats. Nanotoxicology 26: 1-13.
- Mansour HH, Kiki SM, Hasan HF (2015) Protective effect of N-acetylcysteine on cyclophosphamide-induced cardiotoxicity in rat. Environ Toxicol Pharmacol 40: 417-422.
- Yu W-C, Tian L-Y, Cheng W (2015) Efficacy study of edaravone and acetylcysteine towards bleomycin-induced rat pulmonary fibrosis. Int J Clin Exp Med 8: 8730-8739.
- Arafa MH, Mohammad NS, Atteia HH, Abd-Elaziz HR (2014) Protective effect of resveratrol against doxorubicin-induced cardiac toxicity and fibrosis in male experimental rats. J PhysiolBiochem 70: 701-711.
- Kim S, Christopher L, Bancroft J (2018) Theory andpractice of histological techniques (8thedn), Churchill Livingstone, London.
- Hayat MA (1989) Principle and techniques of electron microscopy biological applications (3rdedn), Macmillan Press, Scientific Medical LTD, London.
- Goldstone LA (1983) Understanding medical statistics (1stedn), London William Mairmann. Medical Books limited.
- Migrino RQ, Aggarwal D, Konorev E, Brahmbhatt T, Bright M, et al. (2008) Early detection of doxorubicin cardiomyopathy using two-dimensional strain echocardiography. Ultrasound Med Biol 34: 208-214.
- Geisberg CA, Sawyer DB (2010) Mechanisms of anthracycline cardiotoxicity andstrategies to decrease cardiac damage. Curr Hypertens Rep 12: 404-410.
- Raj S, Franco VI, Lipshultz SE (2014) Anthracycline-induced cardiotoxicity: a review of pathophysiology, diagnosis, and treatment. Curr Treat Options Cardiovasc Med 16: 10-14.
- Psaltis PJ, Carbone A, Leong DP, Lau DH, Nelson AJ, et al. (2011) Assessmentof myocardial fibrosis by endoventricularelectromechanical mapping inexperimental nonischemic cardiomyopathy. Int J Cardiovasc Imaging 27: 25-37.
- Zhan S, Fan X, Zhang F, Wang Y, Kang L, et al. (2015) A proteomic study of Shengmaiinjection's mechanism on preventing cardiac ischemia-reperfusion injury viaenergy metabolism modulation. Mol BioSyst 11: 540-548.
- De Angelis A, Piegari E, Cappetta D, Marino L, Filippelli A, et al. (2010) Anthracycline cardiomyopathyis mediated by depletion of the cardiac stem cell pool and is rescued by restoration of progenitor cell function. Circulation 121: 276-292.
- Piegari E, De Angelis A, Cappetta D, Russo R, Esposito G, et al. (2013) Doxorubicin inducessenescence and impairs function of human cardiac progenitor cells. Basic Res Cardiol 108: 334.
- Vejpongsa P, Yeh ET (2014) Prevention of Anthracycline-Induced Cardiotoxicity: Challenges and Opportunities. J Am Coll Cardiol 64: 938-945.
- Yarmohmmadi F, Rahimi N, Faghir-Ghanesefat H (2017) Protective effects of agmatine on doxorubicin-induced chronic cardiotoxicity in rat. Eur J Pharmacol 796: 39-44.
- Singh P, Sharma R, McElhanon K (2015) Sulforaphane protects the heart from doxorubicin-induced toxicity. Free Radic Biol Med 86: 90-101.
- Merino H, Singla DK (2014) Notch-1 mediated cardiac protection following embryonic and induced pluripotent stem cell transplantation in doxorubicin-induced heart failure. PLoS One 9: e101024.
- Lv X, Yu X, Wang Y, Wang F, Li H, et al. (2012) Berberine inhibitsdoxorubicin-triggered cardiomyocyte apoptosis via attenuating mitochondrialdysfunction and increasing Bcl-2 expression. PLoS One 7: e47351.
- Chen YL, Loh SH, Chen JJ, Tsai CS (2012) Urotensin II prevents cardiomyocyteapoptosis induced by doxorubicin via Akt and ERK. Eur J Pharmacol 680: 88-94.
- Gherghiceanu M, Popescu LM (2012) Cardiac telocytes-their junctions and functional implications. Cell Tissue Res 348: 265-279.
- Llucià-Valldeperas A, Bragós R, Bayés-Genís A (2019) Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential. J Vis Exp 18: 143.
- Xiang Q, Yang B, Li L, Qiu B, Qiu C, et al. (2019) Critical role of Lin28-TNFR2 signalling in cardiac stem cell activation and differentiation. J Cell Mol Med.
- Quaife-Ryan GA, Sim CB, Porrello ER, Hudson JE (2016) Resetting the epigenome for heart regeneration. Semin Cell Dev Biol 58: 2-13.
- Reddy PS, Rani GP, Sainath SB, Meena R, Supriya C (2011) Protective effects of N-acetylcysteine against arsenic-induced oxidative stress and reprotoxicityinmale mice. Journal of Trace Elements in Medicine and Biology 25: 247-253.
- Wang FG, Liu SR, Shen YQ, Zhuang RX, Xi JJ, et al. (2014) Protective effects of N-acetylcysteine on cisplatin- induced oxidative stress and DNA damage in HepG2 cells. Exp Ther Med 8: 1939-1945.
- Priya S, Vijayalakshmi P, Vivekanandan P, Karthikeyan S (2011) N-acetylcysteine attenuates dimethylnitrosamine induced oxidative stress in rats. Eur J Pharmacol 654: 181-186.
Citation: Mostafa HKK, Raafat MH (2019) Effect of N-Acetyl Cysteine on Doxorubicin Induced Cardiotoxicity in Adult Male Albino Rat: Histological Study. J Cytol Histol 10: 536. DOI: 10.4172/2157-7099.1000536
Copyright: © 2019 Mostafa HKK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Select your language of interest to view the total content in your interested language
Share This Article
- Total views: 486
- [From(publication date): 0-0 - Dec 15, 2019]
- Breakdown by view type
- HTML page views: 441
- PDF downloads: 45