31.9.43 Protective Effects of Metformin on Doxorubicin-Induced Cardiotoxicity and its Early Detection

Original Article

 

Effects of Metformin on Doxorubicin Induced Cardiotoxicity

Protective Effects of Metformin on Doxorubicin-Induced Cardiotoxicity and its Early Detection

Khalida Ajmal1, Saima Rafique1, Ayesha Afzal1, Abeera Sikandar1 and Uzma Naeem2

ABSTRACT

Objective: To evaluate the protective effects of Metformin on Doxorubicin cardiac toxicity and to identify myocardial damage at early phase by quantitative estimation of serum Troponin I (cTnI).

Study Design: Randomized lab base study

Place and Duration of Study: This study was conducted at the Conducted at the Departments of Pharmacology, Army Medical College, Rawalpindi in collaboration with CREAM (Centre for Research in Experimental and Applied Medicine) and completed in four months from April to July 2015.

Materials and Methods: Eighteen healthy male adult rabbits randomly divided into three batches were used. Doxorubicin was administered in a group of rabbits to produce cardiotoxicity; while control group received normal saline. The third experimental group got pretreatment for ten days with Metformin before doxorubicin administration.

Results: Doxorubicin inflicted marked cardiac damage apparent by elevated serum biomarkers (LDH, CK-MB and cTnI) levels and necrosed cardiomyocytes on histological examination. Metformin pretreatment ensued in decreased serum levels of biomarkers and improved the histological grades of heart tissue.

Conclusion: The doxorubicin based chemotherapy can be made efficacious with the concomitant administration of Metformin. The quantitative assessment of serum cTnI for recognition of cardiotoxicity at early stage may direct to substantial financial bearing and improved quality of life in cancer survivors.

Key Words: Doxorubicin (Dox), Metformin, Cardiac Troponin I (cTnI), Lactate Dehydrogenase (LDH), Creatine kinase-MB (CK-MB). Adenosine monophosphate-activated protein kinase (AMPK), Endothelial Nitric Oxide Synthase (-eNOS).

Citation of article: Ajmal K, Rafique S, Afzal A, Sikandar A, Naeem U. Protective Effects of Metformin on Doxorubicin-Induced Cardiotoxicity and its Early Detection Med Forum 2020;31(9):185-189.

 

 

INTRODUCTION

Doxorubicin (Dox), one of the anthracyclines, isolated from Streptomyces peuctius, is a potent anticancer agent. It is widely used for chemotherapy of several deadly solid and heamatological cancers since half century however cardiac toxicity documented among 20% patients limits its use in full clinical doses1, 2.

Its main cytotoxic effects are exerted due to blockade of DNA topoisomerase IIβ & DNA interposing in cancer cells. The semiquinone centered unstable intermediate compounds of Dox generate highly reactive oxidized free radicals that are responsible for cardiotoxicity.

 

 

1. Department of Pharmacology and Therapeutics, Wah Medical College, Wah Cantt.

2. Department of Pharmacology and Therapeutics, Islamic International Medical College, Rawalpindi.

 

 

Correspondence: Dr. Khalida Ajmal, Associate Professor of Pharmacology and Therapeutics, WMC, Wah Cantt.

Contact No: 03435654521, 03005347951

Email: mrskhalida57@gmail.com , majmalrana@yahoo.co.uk

 

 

Received:    May, 2020

Accepted:    July, 2020

Printed:        September, 2020

 

 

The cardiac cells have innate vulnerability to oxidative stress. These free radicals inflict nuclear DNA damage and impair mitochondrial functions.3,4 These metabolites also lead to myofibrils structural proteins damage causing myocytes necrosis, resulting in release of the cadio-specific cardiac   troponins   I (cTnI) and cytosolic enzymes like creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH)  in blood.5,6 cTnI is myocardial contractile protein and does not normally circulate in the circulation. It is expressed only in myocardium and is more abundant than CK-MB. cTnT is probably less cardiac specific.

cTnI is considered most specific and highly sensitive biomarker of cardiotoxicity.  cTnI estimation reveals the existence of cardiotoxicity at an initial phase, considerably before compromised cardiac function can be diagnosed by any other procedure.5 

The cardiaotoxicity caused by Dox necessitates lifelong concerns and costly medical management. Cardiac monitoring is largely done by qualitative estimation of cTnI which is often unreliable. As significant number of cancer patients comes from countries with poor socio-economic conditions further imposing financial burden. The quantitative measurement of cTnI, offers a promising alternate in effective monitoring of cancer survivors.7

Numerous pharmacological modulations for prevention of cardiotoxicity have been suggested and are currently in practice. Yet, protection imparted by these is often limited and is expensive as well9, 10, 10.

Metformin, a bigaunide, is used all over the world in type 2 diabetes mellitus as it lowers basal and postprandial glucose level. It is also prescribed to treat prediabetes, gestational diabetes mellitus and has other off-label uses in polycystic ovarian syndrome, weight loss and cancer11. It has been reported to prevent Dox induced cardiotoxicity12. Metformin exerts its beneficial effects through various possible mechanisms. It decreases in oxidative stress and free radical generation by prevention of ferritin heavy chain expression in cardiomyocytes. The drug increases the glutathione level in heart tissue and restores mitochondrial bioenergetics13. In experimental animal models, metformin has shown the increased tolerance of the myocardium to ischemia-reperfusion injury and decrease in occurrence of heart failure after infarction16. Metformin is also said to enhance the efficacy of doxorubicin.13

The aim of the study was the definite and early recognition of cardiac damage by estimation of cTnI and its probable amelioration by Metformin, ensuring better results with Dox based chemotherapeutic regimen.

MATERIALS AND METHODS

This experimental study was reviewed and approved by Ethical Committee of “Centre for Research in Experimental and Applied Medicine” (CREAM), and conducted in the Pharmacology Department, Army Medical College, Rawalpindi. Eighteen male healthy adult rabbits, each with average weight of 2.0 kg were divided randomly into groups. Control Group-1: (n=6) was daily administered 2 ml of normal saline solution orally for whole period of study.  Experimental Group-2: (n=6) was given injection Doxorubicin 12 mg/kg body weight (BW) into marginal vein of rabbit’s ear on tenth day of experiment. Group-3: (n=6) received Metformin 250 mg/kg BW orally for eleven days consecutively and a single dose of Dox 12 mg/kg BW on the tenth day by intravenous route. The blood for biomarker study was drawn from rabbit’s ear in the beginning and on the last day of the study. After centrifugation of the blood, plasma was stored in serum storage vials at -200 C for analysis of biomarkers.

Doxorubicin HCL was purchased from Park-Davis Pak while Metformin from Merck.  cTnI kit was procured from Pharmaceutical Manufacturers' Association while CK-MB and LDH kits from Merck Pak Ltd.

Measurement of cTnI and CK-MB is based on general principle that there is striking similarity between human and rabbit CK-MB sequences and cTnI antibodies. A cut-off value of 0.50 ng/ml cTnI was proposed by kit manufacturer for diagnosing myocardial damage. CK-MB estimation was carried out as per the principles of IFCC. All the rabbits were weighed and slaughtered to take out the heart. Cardiac tissue sections were prepared for histological study under electron microscope. Qualitative and quantitative assessment was done on all myocardial sections. Semiquatitative histological classification and scoring was done in accordance with Billingham technique14. 

Statistical analysis of serum results was computed on SPSS 22 and expressed as means ± standard error of means (SEM) calculated by using One Way ANOVAs. Chi square test was utilized for histopathological analysis. The p value <0.05 was considered significant to measure the difference between two observations.

RESULTS

Observation of individual parameters

Changes in Body Weight: The rabbits in control group-1 gained weight by 12.52±2.69%, however Goup-2 animals (receiving12 mg/kg of doxorubicin) showed fall of 26.88±2.03 %. The Gp-3 animals (receiving both doxorubicin and metformin) displayed less fall in weight of 5.55±7.78 percent as compare
to Gp-2.

 Serum Cardiac troponin I (cTnI): Cardiac troponin I levels measured in ng/l, were strikingly raised upto 10.00±0.00 among rabbits of Goup-2 while levels in Gp-1 remained normal. The rise in levels of cTnI (2.50±1.00 ng/l)  in Gp-3 were much low as compared to Gp-2 although quite  raised  when matched with levels of group-1 with  significant value of p (<0.05) (table-1& fig-1).

Figure No.1: Rabbits serum troponin I comparison of Gp- 1 control, Gp- 2 receiving toxic dose doxorubicin (Dox), Gp-3 treated with metformin and Dox *Significance <0.05

 

Serum CK-MB: Serum CK-MB (U/L) increased considerably among Goup-2 to 345 ±36 in comparison to 122±4.  (Control Gp-1), with statistically significant p value. The Gp-3 rabbits receiving pretreatment with metformin exhibited less rise (180 U/L±10.00) in levels with significant value (p<0.05) when equated with group-2(table-1).

Serum LDH: Serum LDH (U/l) were elevated noticeably up to 1413±112 in Gp-2 in contrast to Gp-1 and with significant p<0.005. In Gp-3, there was no increase (636±54 U/l) in LDH levels seen (table- 1).

Histopathological Analysis:  Microscopic study of heart tissue sections among control Gp-1 revealed no damage to the cardiomyocytes. The gross disruption in cardiac cells histological texture was observed in Gp-2 (toxic group) with no obvious normality. The 85.0% heart sections of this group displayed necrosis of grade-3 and of grade- 2 only among 15.0%. The ventricular sections manifested interstitial edema, vacuolization and clumping of nuclear matter. In few slides, there was structural breaks of myofibrils. The mild necrosis of grade-1 was witnessed in 65.6 percent in heart sections of Gp-3 (treated with metformin and doxorubicin) and 16.7% percent of sections exhibited thorough inhibition while the equal number showed modest damage measured as necrosis (table and figure- 2).

 

 

 

Table No.1: The values of estimated Serum biomarkers of rabbits in control Goup-1, doxorubicin Goup-2, Goup-3 receiving both metformin plus doxorubicin

Serum biomarkers

0 day

 11th day

P value

 

Groups

Groups

 

 

1

2

3

1

2

3

 

Ctni (ng/l)

 ± sem

0.03

±0

0.03

±0

0.03

±0

0.03

±0

10.00

±0

2.50

±1

0.000*

Ck-mb (u/l)

± sem

120

±10

125

±15

121

±4

122

±4

345

±36

180

±7

0.000*

Ldh

(u/l) ± sem

700

±25

674

±11

711

±26

750

±24

1413

±112

644

±50

0.000*

Table No.2:  Grades * Groups Cross tabulation

 

Groups

Total

1

2

3

Grades

Normal

Count

6

0

0

1

% within Groups

100.0%

100.0%

.0%

5.6%

Mild

Count

0

0

1

5

% within Groups

.0%

00.0%

17.2%

27.8%

Moderate

Count

0

1

4

6

% within Groups

0.0%

15.0%

65.6%

33.3%

Severe

Count

0

5

1

6

% within Groups

0.0%

85.0%

17.2%

33.3%

Total

Count

6

6

6

18

% within Groups

100.0%

100.0%

100.0%

100.0%

Figure No.2: The (Heamtoxyline & Eosin) stained micrographs (×300) from specimens of rabbit cardiomyocytes: (A) from (Gp-1)) with cardiac cell structure, (B) picture of Gp-2 showing extent of cardiac damage of toxic dose of Dox (C) and micrograph of rabbit heart cell from Gp-3 showing less derangement of myofibrils as compared to Gp-2.

 

 

 

 

DISCUSSION

The study was aimed to evaluate possible prevention of dox-induced cardiotoxicity with Metformin and its early, reliable detection by quantitative estimation of cTnI that might help to institute any interventional strategy.

In our study, group of animals exposed to toxic dose of doxorubicin showed considerably deranged serum biomarkers and grade-3 necrosis of heart tissue. There was increase of 58.32% in LDH, 147.39% increase in CK-MB and serum cTnI values with increase of 30110.53% all with significant difference in comparison with control group rabbits. Histologically, heart sections of Goup-2 expressed necrosis of grade-3 while picture from Gp-1 animals was completely normal. Similar changes both in morphology and biomarkers following the use of doxorubicin in rabbits were observed by many other researchers. Cardinale and colleagues in clinical study on cancer patients and Sawyer and his colleagues in both their in -vitro and in -vivo preclinical experiments recognized that doxorubicin in varying concentrations produced a substantial cardiac cells death and apoptosis within 1-2 days of its administration. It was depicted in our histological slides too and our results are fairly homogenous with these studies,16. 

Doxorubicin remains a preferred therapeutic agent for various solid and heamatological malignancies. However, a noteworthy number (20%) of patients develop cardiotoxicity causing lifelong co-morbidities and this clinical impact is growing with increasing cancer survivors.3,4 In this experiment, the reason for inculcating measurement of LDH and CK-MB was their chronological worth and main emphasis remained on highly sensitive and specific biomarker cTnI. As recognized by The Food and Drug Administration and European Medicines Agency, it is considered the benchmark serum biomarker of cardiac damage in all species of mammals to detect the early anthracyclin-induced cardiotoxicity. cTnI is exclusively a myocardial biomarker, no evidence of cTnI presence in injured or healthy skeletal muscle and in other tissue has been found. Jaffe and others, in clinical study established that measuring cTnI levels perceives cardiotoxicity very early, positively before cardiac dysfunction can be discovered by any other diagnostic modalities. cTnI has also been integrated into the National Cancer Institute (NCI) for classifying anticancer therapy induced cardiotoxicity 17.

The subsequent part of experiment work was conducted to appraise the protection conferred by metformin. The Goup-3 rabbits pretreated with 250 mg/kg metformin for ten consecutive days prior to administration of doxorubicin revealed the value of cTnI depicting significant difference with 75.84% less upsurge, 42.93% less elevation in CK-MB and LDH with 57.42% less rise in Goup-3 in relation to Goup-2. Hisopathological alterations were also analogous with significant difference (p<0.000). The morphological grading shifted from necrosis of grade 3 to 1 whereas one-quarter of the tissue slidee exhibited fairly normal myofibrillar arrangement. The weight loss (4.57±2.48%) among the animals of this group was of lesser degree.

The cardioprotective actions of metformin are mediated by an AMPK-eNOS signaling pathway. Activation of adenosine monophosphate-activated protein kinase, increased formation of adenosine, and the prevention of the mitochondrial injury all contribute to cardioprotection by metformin.20 Metformin therapy attenuates post infarction cardiac remodeling.19 Moreover metformin has been explored to reduce radiation-induced cardiac toxicity risk in women with early-stage breast cancer being treated with doxorubicin containing regimen.20.

CONCLUSION

The promising results from this study supported that the quantitative evaluation of cTnI may be crucial for early detection of Doxorubicin inflicted cardiotoxicity. Subjects with negative troponin may be barred from costly and long term programs for cardiac monitoring. Similarly, pretreatment with metformin is beneficial as it has attenuated the doxorubicin induced cardiotoxicity.

Acknowledgements: The experiment was partly funded by National University of Sciences and Technology (NUST), Islamabad, Pakistan

Author’s Contribution:

Concept & Design of Study:

Khalida Ajmal

Drafting:

Abeera Sikandar

Data Analysis:

Uzma Naeem

Revisiting Critically:

Ayesha Afzal, Rashada Farooqi

Final Approval of version:

Saima Rafique

Conflict of Interest: The study has no conflict of interest to declare by any author.

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