Capsaicin Abate Doxorubicin Induced Hepatotoxicity in Wistar Rats

INTRODUCTION

Doxorubicin (DOX) is a quinone-containing anthracycline, also known as Adriamycin, has been used in oncologic practice since the late 1960s. DOX is a wide spectrum anticancer drug used for the treatment of solid tumors which develop in the breast, bile ducts, endometrial tissue, esophagus and liver, osteosarcomas, softtissue sarcomas and non-Hodgkin’s lymphoma. The clinical application of DOX is limited because its life threatening adverse effects on various organs systems such as cardiotoxicity, neurotoxicity, hepatotoxicity, nephrotoxicity and testicular toxicity. Treatment with DOX leads to an imbalance between free oxygen radi-cals/ reactive oxygen species (ROS) and antioxidants. Modulation in oxidant-antioxidant sys-tems results in tissue injuries which reveal the lipid peroxidation and protein oxidation in tis-sue. Endogenous antioxidant enzymes like superoxide dismutase (SOD) and catalase can augment the effects of ROS but quickly become overwhelmed by large quantities of ROS. Several studies have illustrated that in combination with inflammatory events, ROS, oxidative stress injury and lipid peroxidation are frequently associated with liver damage induced by toxic drugs such as DOX. Regulation of these mediators is utmost therapeutic necessity to inhibit DOX induced deleterious effects in various organs.^1-3

Many studies were demonstrated for antioxidants from the natural medicine with target to suppress oxidative stress injury developed by DOX. Many antioxidants of natural sources were screened to prevent the DOX-induced cell damage without altering its anti-tumor efficacy in the animal studies.³ Capsaicin (trans-8- methyl-N-vanillyl-6-nonenamide) is the active pungent phytoconstituent present in the fruits of plants fromthe genus Capsicum, belongs to family, Solanaceae.⁴ Recent literature datum of capsaicin demonstrated the potent pharmacological activities such as analgesic, antiobesity, antipruritic, anti-inflammatory, antiapoptotic, antineoplastic, free radical scavenging and neuroprotective effects. Also, recent studies explored its clinical efficacy in treatment of vascular-related disorders, metabolic syndrome, and gastro protective effects.⁵ The dearth of key medicines for prevention of anticancer drug induced manifestations influences the urge for further research on different pharmacological activities of capsaicin. The present study was undertaken to evaluate the effect of capsaicin in alleviating DOX induced hepatotoxicity in experimental rats.

MATERIALS AND METHODS

Animals: For the present study, wistar rats of either sex (250-300g) were procured from animal house of Shree Devi College of Pharmacy, Mangalore. They were acclimatized to con-trolled laboratory conditions of temperature (25±20 C), 30-70% humidity and 12h light-dark cycles. The rats were randomized into experimental and control groups and housed four each in sanitized polypropylene cages containing sterile paddy husk as bedding. They had free ac-cessed to standard pellets as basal diet and water ad libitum. All the studies conducted were approved by Institutional Animal Ethical Committee, Shree Devi College of pharmacy (SDCP/IAEC/03/2018), Mangalore, Karnataka, according to prescribed guidelines of com-mittee for the purpose of control and supervision of experiments in animals (CPCSEA), Gov-ernment of India.

Chemicals: Capsaicin was procured from Yucca Enterprises, Mumbai. DOX was procured from Cipla Ltd, Verna, Goa and other chemicals used were of analytical grade and purchased from standard companies. Biochemical kits for the estimation of biomarkers/enzymes were procured from Robonik India, Pvt Ltd, Mumbai.

Experimental Design: ^6-8 The present study was designed for 22 days. After 7-8 days of ac-climatization, the experimental rats were randomly divided into four equal groups in separate polypropylene cages, six animals in each. The healthy adult wistar rats of either sex were di-vided in to four groups of six animals each as following.

Group-I: (Normal) - Animals treated with 1% Dimethyl Sulfoxide (DMSO) for 21 days.

Group- II: (DOX Alone) – DOX total cumulative dose of 15 mg/kg i.p for 2 weeks in six divided dosage.

Group- III: (CAP Alone) - Animals treated with CAP at a dosage of 10 mg/kg p.o for 21 days.

Group- IV: (CAP+DOX) – CAP 10 mg/kg p.o for 21 days + DOX total cumulative dose of 15 mg/kg i.p for 2 weeks in six divided dosages.

Samples collection:^6,7

After 24 hour after the last administration the rat was anesthetized with ketamine (70 mg/kg i.p.) and xylazine (10 mg/kg i.p.). The blood was collected by retro-orbital puncture and the serum was separated by centrifugation (3000 rpm for 15 min). The serum (supernatant) was collected and used for the estimation of biomarkers. Then rats were decapitated, the liver tis-sues were isolated, separated from their surrounding fat and connective tissue and washed with ice-cold saline quickly. Each liver was divided into two parts. Half portion was used for antioxidant parameters study and other half portion was processed for histopathological in-vestigation.

Estimation of serum biomarkers:

The levels of hepatic biomarkers alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) from serum (supernatant) were analyzed by using commercial kits (Robonik) by using semi autoanalyser (Robonik).

Endogenous antioxidants assessments:

Half portion of liver of each rat was subjected to homogenization in ice-cold 10 % Trichloro acetic acid phosphate buffer and saline (0.05 M, pH7.4). The liver homogenates were centri-fuged for 15000 rpm for 15 mins. The obtained supernatants were used for estimations of GSH, SOD and Catalase by colorimetric method. The GSH level of hepatic tissue was analyzed by using Ellmanns method.⁹ Catalase enzyme activity was measured by Aebi method.10 SOD enzyme activity of hepatic tissue was analyzed by the method described by Kakkar et al.¹¹

Histopathological Study:

Specimens of liver tissues of each group were fixed in 10% buffered formalin and processed with paraffin wax. For histopathological feature examination, 5μm sections were stained with hematoxylin and eosin for the examination using light microscope.¹²

Statistical analysis:

The data obtained by the various parameters was statistically evaluated by one way Analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparison tests. The mean val-ues ±SEM were calculated for each parameter.

RESULTS

Effect of CAP on serumbiomarkers in DOX induced hepatotoxicity: DOX group explored significant (P<0.001) rise in AST, ALT and ALP level compared to normal control. Animals exposed with CAP+DOX group showed significant (P<0.001) de-cline in AST, ALT and ALP level compared to DOX group. Animals treated with CAP (alone) exhibit almost near values of serum markers as that of normal group (Table1 and Fig-ure1).

Effect of CAP on endogenous antioxidants in DOX induced hepatotoxicity:

A Significant (P<0.001) decrease in the level of SOD, GSH and catalase in DOX group com-pared to normal group were observed. CAP (Alone) group and CAP+DOX group showed moderately significant (P<0.01) increase in the content of SOD, GSH and catalase compared DOX group (Table 2 and Figure2).

Histopathological study:

A normal histological architecture of hepatic tissue was observed with hepatocytes, central vein, liver sinusoids, and parenchymal cells in normal group animals. Animals exposed with DOX group showed distended hepatocytes, fatty degeneration and severe tissue degranulation. Animals treated with CAP+DOX demonstrated protective efficiency of CAP by exhibiting decreased inflammatory infiltration, and hepatic sinusoids appear closely normal. Animals administered with CAP (alone) demonstrate nearly same architecture of hepatic tissue as that of normal group. (Figure3).

DISCUSSION

The current study was designed to evaluate the protective effect of CAP on DOX induced hepatotoxicity in wistar rats. In recent days, antineoplastic agents contribute significantly to the global burden of life threatening adverse effects.¹³

Most of the studies evidenced that hepatic damage results due to elevation in serum biomark-ers enzymes and oxidative stress and generation of ROS. In present study, hepatotoxicity is caused due to generation of oxygen derived free radicals as they cause direct injury to cell membranes, which kills hepatic membranes.^14-15

Serum biomarkers such as ALT, AST, and ALP are regarded as sensitive parameters in he-patic injury as these are situated in cytoplasm region and entered into circulation after cellular damage and they play a vital role in evaluating function and integrity of hepatic cells. In the current work, animals administered with single dose of DOX were showed a significant elevation in the levels AST, ALT, and ALP. These findings illustrate hepatic injury and cellular disruption followed by necrosis in hepatocytes due to serum enzymes. CAP exhibits a signif-icant decline in AST, ALT, and AST in DOX treated rats. The decline in levels of serum biomarker findings in CAP administered animals might be due to its free radical scavenging action that reduces leakage of enzymes into bloodstream and stabilizes membrane permeability.^14,16

Elevation of ROS destroys the lipid components on cytoplasmic membrane, denature of proteins and nucleic acids. DOX is highly reactive with SH groups and reduces GSH content. These mechanisms lead to depletion in GSH, SOD, and catalase values in the pathogenesis of hepatic tissue injury. Several antioxidant compounds were developed and analyzed in various experimental and clinical studies to ameliorate DOX-induced toxicities.^14,17 CAP administration implicates significant attenuation effects by an elevation in GSH, catalase, and SOD in DOX-treated rats. Thus, CAP significantly attenuated ROS and oxidative stress inju-ries in liver caused by DOX by its antioxidant action.

In the present study, histopathological findings of hepatic tissues reported that DOX administration induces distended hepatocytes, fatty degeneration, inflammatory and severe tissue degranulation. It was already demonstrated that histopathological modulations occur in DOX induced liver toxicities. These observations might be correlated with an increase in serum biomarkers and oxidative stress. CAP pretreated experimental rats were showed high potency on histopathological changes in DOX induced hepatic damage. The beneficial alterations brought in histopathological degenerations of liver tissue by CAP might be due to its ability to diminish the free radicals in oxidative stress injury and to scavenge the peroxyl and hydroxyl radicals in hepatic tissues.^14,16

CONCLUSION

The present study implicates that DOX administration leads to hepatic injury through genera-tion of ROS and oxidative stress evidenced by rise in liver biomarkers, depletion in endogen-ous antioxidant values and severe histopathological manifestations. The oral administration with CAP demonstrates protective role by its antioxidant and free radical scavenging actions against DOX induced hepatotoxicity in rats.

CONFLICT OF INTEREST

The authors declare no conflicts of interests.