Genotoxic And Cardio-Protective Effects Of Kalanchoe Integra In Doxorubicin-Induced Cardiotoxicity In A Rat Model

Abstract

Background: Advancement in cancer therapy has improved survival among patients. However, the use of anticancer drugs like the anthracyclines (e.g., doxorubicin) are not without untoward or side effects. Notable among these is its cardiotoxic effect which ranges from mild transient blood pressure changes to potentially serious heart failure. Kalanchoe integra (KI) is used in folklore medicine in the management of several clinical conditions. The components of KI predict its cardioprotective potential which could be beneficial among its numerous uses. The aim of the study was to determine the cardio-protective effect of KI against doxorubicin-induced cardiotoxicity using a rat model. Methods: The leaves of Kalanchoe integra (KI) were collected, air-dried, pulverized and extracted using 70% ethanol. HPLC fingerprinting analyses of KI was carried out using an Agilent 1100 system (Santa Clara, CA, USA), composed of quaternary pump, autosampler, diode array detector (DAD), and HP ChemStation Software. Chromatographic separation was carried out on a Tskgel ODS C18 (250 x 4.6 mm i.d., 5 μm particle size) analytical column maintained at 40 oC. The single cell gel electrophoresis assay (SCGE, also known as comet assay) was also employed to ascertain the genotoxic effects of the KI extract. The comet assay was used to study the potential genotoxic effect of KI on the liver, kidney, epithelium of the rectum and bone marrow. A total number of 42 Sprague-Dawley rats (150-200g) were put into 7 groups (n=6). Group I: Vehicle control, received normal saline (1 ml/kg p.o) for 30 days. Group II: Toxic control, received doxorubicin (DOX) (20 mg/kg i.p.) once on the 29th day. Group III: KI control, received KI (300 mg/kg p.o) for 30 days. Group IV: Vitamin E control, received Vitamin E (100 mg/kg p.o) for 30 days. Group V: KI treated-1, received KI (300 mg/kg p.o) for 30 days and DOX (20 mg/kg i.p) on the 29th day. Group VI: KI treated-2, received KI (600 mg/kg p.o) for 30 days and DOX (20mg/kg i.p) on the 29th day. Group VII: Vitamin E treated, received Vitamin E (100 mg/kg p.o) for 30 days and DOX (20mg/kg i.p) on the 29th day. Aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), lactate dehydrogenase (LDH), enzymatic antioxidants such as glutathione peroxidase (GPX), superoxide dismutase (SOD), and catalase (CAT) were assayed from blood taken via cardiac puncture 36 hours after last treatment administration. Excised hearts from rats from each group were taken through histopathological examination. Results: In the HPLC fingerprint analysis, 13 peaks were identified. The first peak with retention time of 3.4min had a peak area of 1.1754 x10 4 mAU and overall percentage peak area of 19.2%. Also peak with retention time of 24.0 min had the highest peak area of 3.223 x104 mAU and overall percentage peak area of 52.63% which is directly proportional to high concentration of that compound in the plant extract. The criterion for toxicity was a statistically significant increase in the number of deoxyribonucleic acid (DNA) comets in the organs and tissues studied compared with controls. Results showed that the KI extract was non-genotoxic. Pre-treatment with KI protected rats against doxorubicin- induced cardiotoxicity as evidenced by the low levels of AST, ALT, ALP, CK and LDH compared with the controls (p < 0.05). SOD, CAT and GPX levels were also high for KI extracts; further showing that KI protected rats against doxorubicin induced cardiotoxicity. Histological examinations revealed that in the KI pretreated groups there were no signs of abnormal myocardial fibers. The myocardia were of normal shape, size and configuration. It was also observed that there was no evidence of vacuolation, neither was there any sign of necrosis or inflammation. The observations contrasted with what was observed in the doxorubicin only group (without treatment). Rats in the doxorubicin only group showed signs of abnormal hypertrophic myocardial fibers, and the myocardia also had small and large vacuoles. Conclusion: The current study showed that the ethanolic (70%) leaf extract of KI offered cardio protection in rats administered with doxorubicin. This is evident in AST, ALT, ALP, CK, LDH, GPX, SOD and CAT levels of assayed blood samples. KI also protected the heart of the rats against histopathological changes such as necrosis, abnormal myocardial fibers, and edema. The study also showed that KI is non-genotoxic.