Protective effect of carvedilol alone and coadministered with diltiazem and prednisolone on doxorubicin and 5‐fluorouracil‐induced hepatotoxicity and nephrotoxicity in rats

Abstract This study investigated the protective effects of carvedilol alone and coadministered with prednisolone and diltiazem on doxorubicin (DOX) and 5‐fluorouracil (5‐FU)‐induced toxicity. Each of 2 pools of 70 female rats were randomly allotted into 10 groups of 7 animals each and treated as follows: Group 1: normal saline (10 mL/kg); Group 2: normal saline and DOX (40 mg/kg)/5‐FU (20 mg/kg) alone; Group 3: gallic acid (200 mg/kg) and DOX/5‐FU; Group 4: carvedilol (0.075 mg/kg) and DOX/5‐FU; Group 5: carvedilol (0.15 mg/kg) and DOX/5‐FU; Group 6: carvedilol (0.30 mg/kg) and DOX/5‐FU; Group 7: diltiazem (3.43 mg/kg) and DOX/5‐FU; Group 8: diltiazem (3.43 mg/kg), carvedilol (0.15 mg/kg), and DOX/5‐FU; Group 9: prednisolone (0.57 mg/kg) and DOX/5‐FU; and Group 10: prednisolone (0.57 mg/kg), carvedilol (0.15 mg/kg), and DOX/5‐FU. Treatments were done p.o. for 16/14 days for the DOX/5‐FU models. DOX/5‐FU was administered i.p. to the rats in Groups 2‐10 on day 14/10‐14. On day 17/15 (DOX/5‐FU), blood samples were collected, and liver and kidneys of rats were harvested for antioxidant and histopathological assessments. Carvedilol alone and coadministered with prednisolone significantly (P < .05) decreased alanine aminotransferase level compared with administration of DOX alone. Carvedilol alone and coadministered with diltiazem significantly (P < .05) decreased creatinine level compared with administration of DOX/5‐FU alone. Carvedilol alone and coadministered with diltiazem and prednisolone significantly (P < .05) increased the level of hepatic superoxide dismutase and catalase, and decreased malondialdehyde compared with DOX administration alone. Histopathological observations correlated with results of biochemical and antioxidant analyses. Carvedilol administered alone and coadministered with diltiazem and prednisolone reduced the effect of DOX/5‐FU‐induced hepatic and renal toxicities due to enhanced in vivo antioxidant activity. The protective effect was more prominent in the doxorubicin model compared with the 5‐fluorouracil test. Coadministration of carvedilol with either diltiazem or prednisolone did not show better protection relative to carvedilol alone.

problems associated with antineoplastic agents include resistance, secondary malignancy, cost, and lack of selectiveness resulting in negative impact on normal cells of the body. Doxorubicin is used to treat different forms of cancer, including ovarian, breast, lung, uterine and cervical cancers, Hodgkin's disease, and soft tissue and primary bone sarcomas. 1 The use of this drug is limited by toxic effects on body organs, causing cardiac, pulmonary, hepatic, renal, hematological, and testicular toxicities. 2  , an antimetabolite, has played an important role in the management of colon and breast cancers, and cancers involving the head and neck. 3 Despite the many advantages, its clinical application has been greatly limited due to drug resistance and organ toxicity.
The incidence of drug-induced hepatotoxicity and nephrotoxicity has been increasing with the ever-increasing number of drugs and with easy availability of over the counter medications, with hepatotoxicity being important cause of morbidity and mortality and the most common reason for new drugs withdrawal. 4 Most drug-induced nephrotoxicities exert toxic effects by one or more common pathological mechanisms, including altered intraglomerular hemodynamics, tubular cell toxicity, inflammation, crystal nephropathy, rhabdomyolysis, and thrombotic microangiopathy. 5 Redressive measures/interventions are needed to reduce vital organ toxicities posed by doxorubicin, 5-FU, and other clinically useful chemotherapeutic agents that are normally used at relatively high doses and for an appreciable period of time. Developing new drugs or discovering the other clinical potentials of existing drugs will help in this regard.
Prednisolone (phospholipase A 2 inhibitor) has been demonstrated to have anti-inflammatory property, and ameliorate symptoms and improve biochemical and histologic abnormalities in many types of liver diseases, including autoimmune hepatitis, cirrhosis patients with septic shock, and liver transplantation. [6][7][8][9] Calcium ions (Ca 2+ ) are major regulators of vital cellular functions and interference with Ca 2+ homeostasis contributes to cell injury and death in a number of pathological conditions. 10,11 Diltiazem is a calcium channel blocker and calcium antagonism/blockade has been exploited in the management of cell injury. 12 Carvedilol is a third-generation nonselective beta-blocker with vasodilatory property due to alpha 1 blockade. 13 It has also been reported to possess antioxidant property in terms of free radical scavenging and inhibition of lipid peroxidation. 14,15 This study aimed to determine the protective effect of carvedilol alone and coadministered with diltiazem and prednisolone against doxorubicin and 5-FU-induced liver and kidney toxicities.

| Drugs and chemicals
The following drugs and chemicals were used in this study: Doxorubicin (Get Well Pharmaceuticals, Gurgaon, India), 5-Fluorouracil

| Animals
Seventy female Wistar rats weighing 150-200 g were obtained from the Laboratory Animal Centre of the College of Medicine, University of Lagos, Lagos, Nigeria. The animals were housed at 25°C with 12 hours light/dark cycle, allowed to acclimatize for 14 days before commencement of the experiment, and had free access to standard feed (Livestock Feeds Plc., Lagos, Nigeria) and water. The experimental protocol was in conformity with the guidelines of the United States National Academy of Sciences Guide for the Care and Use of Laboratory Animals. 16

| Treatment
Seventy female rats were randomly allotted into 10 groups of 7 animals each and treated as follows: Group 1 (Control): Normal saline (10 mL/kg). Group 2: Normal saline (10 mL/kg) and doxorubicin alone (40 mg/kg).       Animals in Group 1 were administered normal saline only for 16 days, while others were separately administered normal saline (Group 2); gallic acid (Group 3); carvedilol (Groups 4-6); diltiazem (Group 7); diltiazem and carvedilol (Group 8); prednisolone (Group 9); and prednisolone and carvedilol (Group 10) at doses stated above for 16 days. On day 14, doxorubicin was administered to the rats in Groups 2-10, 2 hours after treatment with the other drugs. 17 The clinical dose of carvedilol was calculated as the average of doses used for indications of carvedilol, the subclinical dose was half of the clinical dose and the supraclinical dose was twice the clinical dose. A day after the end of administration (day 17), blood samples were collected into plain sample bottles for analysis. Rats were sacrificed by cervical dislocation, laparatomized, and the liver and kidneys were harvested for antioxidant indices and histopathological assessments.
In respect of the 5-FU model, animals in Group 1 were administered normal saline (10 mL/kg) only for 14 days, while others were separately administered normal saline (Group 2); gallic acid (Group 3); carvedilol (Groups 4-6); diltiazem (Group 7); diltiazem and carvedilol (Group 8); prednisolone (Group 9); and prednisolone and carvedilol (Group 10) at the same doses used for the doxorubicin model for 14 days. 5-fluorouracil (20 mg/kg, i.p.) was administered to the rats in Groups 2-10 from days 10 to 14, 2 hours after treatment with the other drugs. 18 A day after administration stopped (day 15), blood samples were collected into plain sample bottles for analysis.
Rats were sacrificed humanely under inhaled diethyl ether anesthesia, laparatomized and the liver and kidneys were harvested for antioxidant indices and histopathological assessments.

| Biochemical analysis
Blood sample was collected from each rat at the end of the drug administration period, via retro-orbital artery bleeding under anesthesia, into plain sample bottles for biochemical analysis. Blood collected into plain bottles was allowed to clot at room temperature and centrifuged to obtain the serum. The sera were analyzed using Randox diagnostic kits (Randox Laboratories Ltd., London, England) to assess aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities, and the determination of concentrations of serum total protein, albumin, urea, creatinine, high-density lipoprotein (HDL), low-density lipoprotein (LDL), cholesterol, and triglycerides (TG), according to established protocols. 19

| Statistical analysis
Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test using GraphPad Prism 5 (GraphPad Software Inc., CA). Results were expressed as mean AE SEM and values were considered significant at P < .05.  (Table 1).
A significant (P < .05) increase in creatinine level in animals administered doxorubicin relative to those that received normal saline was observed ( Table 2). Administration of carvedilol (all doses), in the presence of doxorubicin, significantly (P < .05) decreased creatinine level compared with animals administered doxorubicin alone.
In the presence of doxorubicin, coadministration of diltiazem and carvedilol, as well as prednisolone and carvedilol also significantly (P < .05) decreased creatinine level relative to rats administered doxorubicin alone ( Table 2).

| Antioxidant indices (doxorubicin model)
In respect of the liver, Table 3  In respect of the kidneys, results in Table 4
Administration of carvedilol alone and coadministered with diltiazem or prednisolone significantly (P < .05) decreased AST level relative to rats that received 5-FU alone.
As shown in Table 6, a significant (P < .05) increase in creatinine level was observed in rats administered 5-FU compared with the control group. In the presence of 5-FU, administration of carvedilol alone (all doses) and coadministered with diltiazem significantly (P < .05) reduced the creatinine level compared with the rats that received 5-FU only.

| Antioxidant indices (5-FU model)
In respect of the liver, significant (P < .05) decreases in CAT, SOD, GSH, and GPx levels and a significant (P < .05) increase in MDA T A B L E 3 Effect of carvedilol alone and coadministered with prednisolone and diltiazem (in the presence of doxorubicin) on antioxidant indices in the liver level were observed in rats administered 5-FU relative to the normal saline (control) group (Table 7). Carvedilol (0.075 mg/kg) coadminis- In respect of the kidney, 5-FU administration significantly (P < .05) decreased GSH and increased MDA levels compared with rats administered normal saline (control) ( Table 8). Carvedilol     Although the presence of diltiazem did not add any advantage as against using carvedilol alone in this study, the ability of diltiazem to protect or reduce injury caused by toxicants had earlier been reported by Bojani c et al. 28 This study revealed that doxorubicin and 5-FU, known nephrotoxicants, 17