http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.914230
ORIGINAL ARTICLE
Ameliorative effect of propolis against cyclophosphamide-induced toxicity in mice Sabry A. El-Naggar1,2, Abeer A. Alm-Eldeen2, Mousa O. Germoush1, Kamal F. El-Boray1,3, and Hassan A. Elgebaly1 Department of Biology, College of Science, Aljouf Univesity, Sakakah, KSA, 2Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt, and 3Department of Aquaculture, National Institute of Oceanography and Fisheries, Suez, Egypt
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Abstract
Keywords
Context: Cyclophosphamide (CTX) is a common anticancer agent used for the treatment of several malignancies. However, upon treatment, it induces severe toxicity due to its oxidative stress capability. Propolis, a natural product collected by honey bees, has shown several biological activities, such as free radical scavenging and antioxidant agent. Objective: This study elucidates the protective effects of propolis against CTX-induced changes in mice. Materials and methods: Forty-eight male Swiss albino mice were divided into four groups; group 1 was intraperitoneally (i.p.) injected with 200 mL of phosphate buffer saline (PBS), group 2 was injected with 100 mg/kg/d propolis, group 3 was injected with a single dose of CTX (200 mg/ kg), and group 4 was injected with a single dose of CTX (200 mg/kg) followed by propolis (100 mg/kg) for 7 consecutive days. After 12 d, mice were bled and then sacrificed to analyze the hematological, biochemical, and histological parameters. Results: The results indicated that CTX-injected mice showed an increase in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine and a decrease in the total number of white blood cells (WBCs) and platelets. Moreover, dramatically changes in the histological architectures of the liver and kidney were observed. The mice that were injected with CTX/propolis showed an improvement in the levels of ALT, AST, urea, creatinine, WBCs, and platelets. Moreover, the histological picture of the liver and kidney was significantly improved. Conclusions: In conclusion, propolis might be considered an effective agent in ameliorating the toxicity resulted from CTX treatment.
Cyclophosphamide, immunosuppression, kidney, liver, propolis, toxicity
Introduction Cyclophosphamide (CTX) is a common anticancer chemotherapeutic agent used alone or in a combination with other agents for the treatment of several human malignancies (Bass & Mastrangelo, 1998). CTX is used also in preconditioning the host for immunotherapy in several pre-clinical trials (Salem et al., 2010). In this regard, it has been reported that CTX preconditioning increases the numbers of the dendritic cells which directed the immune response (Salem et al., 2009, 2010). Other studies showed that CTX activates the immuno-response via inducing the mobilization of the hematopoietic stem cells and dendritic cells from bone marrow (Montgomery & Cottler-Fox, 2007; Salem et al., 2010). Along with these beneficial effects of CTX, several adverse side effects have been reported such as pneumonitis and pulmonary fibrosis (Ochoa et al., 2012) and induction of the genotoxicity (Paul et al., 2011; Rehman et al., 2012).
Correspondence: Sabry A. El-Naggar, Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt. Tel: +96 6563977876. E-mail: [email protected], [email protected]
History Received 31 December 2013 Revised 28 March 2014 Accepted 8 April 2014 Published online 7 October 2014
Furthermore, it has been reported that CTX increases the proportion of myeloid derived suppressor cells in blood and lymphoid organs and this suppresses the function of the immune system to fight against cancerous cells (DiazMontero et al., 2009). McDermott and Powell (1996) has also been reported that CTX treatment induces the oxidative stress by the generation of the free radicals and reactive oxygen species. Because of this excessive production of free radicals, it has been reported that CTX treatment reduces the levels of glutathione and glutathione peroxidase, catalase, and superoxide dismutase activities in testis (Etteldorf et al., 1976; Fukutani et al., 1981). Earlier studies have shown that the therapeutic dose of CTX could cause liver toxicity (Snover et al., 1989). CTX undergoes a metabolic activation by the cytochrome P450 mixed functional oxidase system to produce two metabolites, phosphoramide mustard and acrolein, which are responsible for the induction of oxidative stress (Ludeman, 1999). Oxidative stress is responsible for CTX hepatotoxicity (Selvakumar et al., 2005). Propolis, a natural product, is commonly used in folk medicines. It is a resinous substance collected by honey bee workers from the plant sources. It contains more than 150 polyphenolic compounds like flavonoids and phenolic acid
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and their esters (Greenaway et al., 1991). Several in vivo and in vitro studies showed that propolis has several biological activities such as free radical scavenging (Pascual et al., 1994), antioxidant (Jaiswal et al., 1997), antitumor (Awale et al., 2008; Ishihara et al., 2009; Ohta et al., 2008; Scheller et al., 1989) and immunomodulatory effects (Ivanovska et al., 1995). Takino and Mochida (1982) reported that the biological activity of propolis is mainly due to the presence of the flavonoid content. Recently, Akyol et al. (2012) stated that caffeic acid phenethyl ester (CAPE), which is the major compound that is extracted from the propolis, has antiinflammatory, antiproliferative, antioxidant, cytostatic, antiviral, antibacterial, antifungal, and antineoplastic properties. It has been reported also that CAPE can regulate antioxidant enzymes, inhibit lipid peroxidation, and reduce hepatic damage (Ates et al., 2006; Bhadauria et al., 2007). Furthermore, CAPE is considered as a potent antiplatelet agent and can induce apoptosis of human pancreatic cancer cells involving caspase and mitochondrial dysfunction (Chen et al., 2007, 2008). Propolis has biological effects that act in synergy with chemotherapy drugs such as 5-fluorouracil (Suzuki et al., 2002). Importantly, Santos and Cruz (2001) showed the antioxidant properties of propolis could reduce the side effects caused by some anticancer drugs without any detriment to the therapeutic effects. Moreover, it has been shown that propolis is a promising adjuvant with chemotherapy (Padmavathi et al., 2006) and with immunization (Chu, 2006). To our knowledge, there are no reports available on the protective effect of propolis on CTX-induced alterations. Therefore, the present study aimed to address the amelioration effect and the protective role of propolis after CTX-treatment.
Materials and methods The experiment adhered to the guidelines of the ethical committee of Aljouf University, Sakakah, KSA.
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King Saud University, KSA. The mice were maintained under standard laboratory conditions (temperature 24 ± 2 C, natural light-dark cycle), and had free access to drinking water and commercial standard pellet diet. After acclimatization for one week to laboratory conditions, the animals were randomly divided into four groups, 12 mice each. Group 1 (G1), control group includes mice that were injected with PBS; group 2 (G2), positive control includes mice that injected with 100 mg/kg/d propolis, group 3 (G3) includes mice that were injected i.p. with a single dose of CTX (200 mg/kg) dissolved in PBS; and group 4 (G4) includes mice that were injected i.p. with a single dose of CTX (200 mg/kg) followed by propolis (100 mg/kg) after 24 h of CTX injection for 7 consecutive days (Boutabet et al., 2011; Salem et al., 2010). The animals were left for 4 d then, at day 12, the mice were sacrificed, weighed, and the liver, kidney, and spleen were removed and weighed. The serum obtained after centrifugation (1500 rpm for 10 min at 4 C) was used for various serum biochemical assays. Hematological and biochemical assays Hematological profile Red blood cells (RBCs), hemoglobin content (Hb g/dl), white blood cell (WBCs) counts, and the total platelets counts were measured from fresh blood samples obtained from the orbital plexus of the eyes of all groups under the study using the electronic blood counter. Differential WBCs were carried out from blood smears on days 0, 2, 4, 8, and 12. Biochemical assays Determination of serum alanine and aspartate aminotransferases Serum alanine and aspartate aminotransferases were assayed using commercial diagnostic kits (Diagnostic systems, Medford, NY) according to the method described by Reitman and Frankel (1957).
Chemicals CTX was purchased from (Sigma-Aldrich, St. Louis, MO). Propolis was purchased from the commercial market at Sakaka city, Aljouf, KSA. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine kits were purchased from Doa El-Shamal Company in Sakaka City, KSA.
Determination of serum urea and creatinine levels
Propolis extraction
Livers and kidneys tissues were immediately removed from all groups under experimentation, sliced, and fixed in bouin fixative for 24 h. The specimens were then dehydrated, cleared, and embedded in paraffin. Serial sections of 5 mm thick were cut by means of a rotary microtome (Cut 4662). Sections were then processed for hematoxylin and eosin staining (Bancroft & Cook, 1994).
Ethanolic propolis extract was prepared by the methods described by Khalil (2006) and Attalla and Ayman (2008). Briefly, propolis (10 g) was crushed into small pieces in a mortar and mixed vigorously with 34.85 ml 80% (V/V) ethanol during 48 h at 37 ± 1 C. After extraction, the ethanol extract of propolis was filtered through Whatman No. 4 paper. The solvent was air-dried and the extract was weighed and suspended in 0.9% sterile saline for further processing. Animals and treatment Adult male albino mice (8-weeks old, weighing approximately 23 g) were purchased from the Faculty of Science,
Serum urea and creatinine levels were determined using commercial diagnostic kits according to the method described by Trinder (1969). Histopathological investigation
Statistical analysis Data were expressed as mean ± SD of a number of experiments (n ¼ 12). The statistical significance was evaluated by Student’s t-test using SPSS 15.0 software package (SPSS, Chicago, IL). Values were considered statistically significant when p50.05.
Therapeutic efficacy of propolis against toxicity induced by CTX injection
DOI: 10.3109/13880209.2014.914230
Results
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Effect on body weight and the relative weights of the spleen and liver The data showed that there are non-significant changes (p 0.05) in the body weights between the mice of the normal control (G1) that injected with PBS and the mice of the positive control group (G2) that were injected with propolis at day 12 (the end of the experiment). The body weights of the mice that were injected with CTX (G3) or with CTX along with propolis (G4) showed a significant decrease (p 0.05) when compared with the control groups (G1 and G2) (Table 1). The data showed also that there is a nonobservable change in the relative weights of the liver in the normal, positive control, CTX, and CTX along with propolisinjected groups (G1, G2, G3, and G4), respectively. However, there is an observable increase in the relative weight of the spleen in the positive control, CTX, and CTX along with propolis-injected groups (G2, G3, and G4), respectively, compared with the normal control group (G1) (Table 1). Effect on serum biochemical parameters The present study showed that there is a non-significant change (p 0.05) in the levels of ALT, AST, urea, and creatinine between the mice of the normal and positive control groups (G1 and G2). These parameters showed a significant increase (p 0.05) in their levels in the CTXinjected mice (G3) compared with their levels in the serum of the control groups (G1 and G2). The mice that were injected with CTX along with propolis (G4) showed a significant decrease (p 0.05) in these values compared with the CTXinjected mice (G3). However, they are still significantly high (p 0.05) compared with the control groups (G1 and G2) (Table 2). Effect on hematological parameters Total number of WBCs The data indicated that the mice of the normal control group (G1) and the positive control group (G2) have a nonsignificant difference (p 0.05) in the total number of the WBCs in the examined days. A significant decrease (p 0.05) in the total number of WBCs in CTX-injected mice (G3) and both CTX- and propolis-injected mice (G4)
compared with the control groups (G1 and G2) until the 8th day. At the same time, there is a non-significant difference (p 0.05) in the total number of WBCs in CTX- and propolisinjected mice (G4) compared with the CTX-injected mice of (G3) (Table 3). At the 12th day, the mice that were injected with CTX (G3) showed that they still have a significant decrease (p 0.05) in the total number of WBCs compared with the control groups (G1 and G2). Interestingly, the mice that injected with CTX along with propolis (G4) showed a significant increase (p 0.05) in the total number of WBCs compared with CTX-injected mice (G3) and the normal control group (G1) while showing a non-significant increase (p 0.05) in the total number of WBCs compared with the positive control group (G2) (Table 3). Total number of red blood cells (RBCs) and hemoglobin level The data showed that there is a non-significant difference (p 0.05) in the total number of RBCs or in the hemoglobin level in the control and CTX-injected groups (G1, G2, and G3), respectively. However, the mice that were injected with CTX along with (G4) showed a significant decrease (p 0.05) in the total number of RBCs and the Hb level compared with the control and CTX-injected groups (G1, G2, and G3), respectively (Table 4). Table 2. Effect of propolis on CTX-induced changes in serum biochemical parameters of liver and kidney (Mean ± SD, n ¼ 12).
Parameters
Parameters
Control
CTX alone
CTX and propolis
Total body 23.7 ± 2.3 24 ± 4.4 21.4 ± 3.2a 21.3 ± 3.5b weight (g) The relative liver 7.2 ± 0.6 6.3 ± 0.9 6.1 ± 0.5 5.8 ± 0.2 weight (%) The relative spleen 0.7 ± 0.05 0.8 ± 0.2c 1.0 ± 0.05c 2.3 ± 0.4c weight (%) a
p50.05: CTX-injected group (G3) was compared with the control groups (G1 and G2). p50.05: CTX- and propolis-injected groups (G4) was compared with the control groups (G1 and G2). c p50.05: propolis, CTX, and CTX and propolis-injected groups (G2, G3, and G4) were compared with the normal control group (G1). b
Control
Propolis alone
CTX alone
CTX and propolis
ALT (IU/L) 20.6 ± 0.5 21 ± 1.4 63.4 ± 1.6a 32.4 ± 0.9b,c AST (IU/L) 113 ± 1.4 114.3 ± 3.7 191.6 ± 2.4a 132.4 ± 0.9b,c Urea (mmol/L) 13.6 ± 0.5 14.25 ± 1.5 45 ± 0.7a 27.4 ± 0.5b,c Creatinine (mmol/L) 1 ± 0.07 1.25 ± 0.20 2.76 ± 0.05a 1.58 ± 0.05b,c ALT, alanine aminotransferase; AST, aspartate aminotransferase. The values represent mean + SD, number of samples is 12 mice per group. a p50.05: CTX-injected group (G3) was compared with the control groups (G1 and G2). b p50.05: CTX- and propolis-injected group (G4) was compared with CTX-injected group (G3). c p50.05: CTX and propolis-injected group (G4) was compared with the control groups (G1 and G2). Table 3. Effect of propolis on CTX-induced changes in WBC number (mean ± SD, n ¼ 12).
Table 1. Effect of propolis on CTX-induced changes in the total body weight, the relative liver, and spleen weights (mean ± SD, n ¼ 12). Propolis alone
3
Day Day Day Day Day
0 2 4 8 12
Control
Propolis alone
CTX alone
CTX and propolis
7.1 ± 0.8 6.8 ± 0.5 7.2 ± 0.6 7.2 ± 0.6 6.6 ± 1.3
7.3 ± 0.4 6.9 ± 0.6 7.0 ± 0.4 6.6 ± 0.2 7.5 ± 1.4
7.4 ± 0.5 2.9 ± 0.3a 3 ± 0.9a 3 ± 0.9a 5.1 ± 0.2b,c
7.5 ± 0.2 3.0 ± 0.9a 3.2 ± 0.2a 6.6 ± 1.2a 8.5 ± 0.7b,d
The values represent mean + SD, number of samples is 12 mice per group. a p50.05: CTX and CTX with propolis-injected groups (G3 and G4) were compared with the control groups (G1 and G2) at days 2, 4, and 8. b p50.05: CTX and CTX with propolis-injected groups (G3 and G4) were compared with the normal control group (G1) at day 12. c p50.05: CTX-injected group (G3) was compared with the positive control group (G2) at day 12. d p50.05: CTX- and propolis-injected group (G4) was compared with CTX-injected group (G3) at day 12.
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Total number of blood platelets The data showed that mice of the positive control group which is injected with propolis only (G2) have a significant increase (p 0.05) in the total number of the platelets compared with the normal control group (G1). Moreover, mice that were injected with CTX (G3) showed a significant increase (p 0.05) in the total number of the platelets compared with the control groups (G1 and G2). Interestingly, mice Table 4. Effect of propolis on CTX-induced changes in total RBCs, Hb content, and differential leucocytes.
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Parameters RBCs (106/ml) HGB (g/dl) PLT (103/ml) % Lmph. % Mon. % Gr.
Control
Propolis alone
CTX alone
CTX and propolis
7 ± 0.3 13 ± 1.1 318 ± 2 64 2.8 33.5
7.1 ± 0.1 12.3 ± 1.0 667 ± 2.1 63 2.6 34.4
6.9 ± 0.1 12.2 ± 0.5 549 ± 7 62 2.8 35.5
5.9 ± 0.2a 10.3 ± 0.35b 1228 ± 9c 69 1.5 29.5
The values represent mean + SD, number of samples is 12 mice per group. a p50.05: CTX and propolis-injected group (G4) was compared with the control groups (G1 and G2) and CTX-injected group (G3). b p50.05: CTX- and propolis-injected group (G4) was compared with the control groups (G1 and G2) and CTX-injected group (G3). c p50.05: CTX- and propolis-injected group (G4) was compared with the control groups (G1 and G2) and CTX-injected group (G3).
injected with CTX along with propolis (G4) showed a significant increase (p 0.05) in the total number of the platelets comparing with the control and CTX-injected groups (G1, G2, and G3) (Table 4). The percentage of differential leucocytes The present study showed that there is a non-significant change (p 0.05) in differential leucocytes in the control groups and CTX-injected groups (G1, G2, and G3). Mice which were injected with propolis along with CTX (G4) showed a slightly increase in the percentage of the lymphocytes and a slightly decrease in the percentage of the monocytes and the granulocytes compared with mice in the control and CTX-injected groups (G1, G2, and G3) (Table 4). Effect on the histopathological picture of the liver and kidney Histological examination of the liver sections from the control groups (G1 and G2) revealed a similar architectural appearance. They have cords of hepatocytes arranged radially extended from the central vein to a periphery of the hepatic lobules (Figure 1a). Histopathological examination of the liver sections from mice that were treated with CTX (G3) showed hepatic degeneration and pale-stained areas. Necrosis of many hepatocytes with pyknosis, swollen of some hepatocytes with nuclear chromatolysis, and infiltration of
Figure 1. Hematoxylin and eosin-stained liver sections. (a) Control groups (G1 and G2) showing normal hepatic architecture with radial arrangement of hepatocytes; (b)–(d) CTX-treated group (G3) showing hepatic degeneration and pale stained areas (arrows) and coagulative necrosis of many hepatocytes with piknotic nuclei (arrowheads) (b), cellular degeneration of some hepatocytes (arrow) with nuclear chromatolysis (arrowhead) (c), cellular infiltration of eosinophils (arrows) (d), and (e) CTX- and propolis-treated group (G4) showing that hepatocytes regained normal architecture with normal chromatolized and central nuclei. Also, regeneration of hepatocytes can be noticed by the presence of binucleate cells (arrows).
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DOI: 10.3109/13880209.2014.914230
Therapeutic efficacy of propolis against toxicity induced by CTX injection
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lymphocytes were observed (Figure 1b–d). Microscopic examination of the liver sections from the mice that treated with both CTX and propolis showed a significant amelioration of distorted hepatic lobules. In addition, more or less normal hepatic structure and normal stain ability were observed. Hepatocytes regained normal architecture with normal chromatolized and central nuclei. Regeneration of hepatocytes can be observed by the presence of binucleate cells (Figure 1e). Histological examination of the kidney sections from the control groups (G1 and G2) revealed normal similar architecture showing the renal cortex with normal renal tubules and glomerulei (Figure 2a). Histopathological examination of the kidney sections from the mice that were treated with CTX (G3) showed shrinkage of the glomerulei with distinct vacuolated and degenerated epithelial lining cells of the renal tubules (Figure 2b). Kidney sections from mice that were treated with CTX and propolis disappearance of the glomerular shrinkage, improvement of the renal cortex tissue architecture, and mild degeneration of renal epithelial cells were observed (Figure 2c).
Discussion This study was conducted to evaluate the possible ameliorative role of the propolis after treatment with the chemotherapeutic agent CTX. The results showed that there are no significant changes in the body weights between mice of the normal control group injected with PBS and mice of the positive control group which was injected with propolis. The body weights of the mice that were injected with CTX or CTX along with propolis showed a significant decrease compared with the normal control group although there was a non-observable change in the relative weights of the liver in all groups. In contrast, there is an observable increase in the relative weights of kidney in the positive control, CTX, and CTX along with propolis-injected groups. The decrease in the body weight after CTX injection would be due to its side effects on the different body organs and systems (Johnstone et al., 2002). Propolis treatment after CTX injection could partially protect the body from losing weight. The nonobservable change in the relative weight of the liver in all groups could be due to treatment with a single dose of CTX, which partially decreased the relative liver weights after days 2 and 4 of CTX injection (data not shown), and gradually returned to their normal relative weights at day 12. Santos and Cruz (2001) showed that the antioxidant properties of propolis could reduce the side effects caused by chemotherapy drugs without any detriment to the therapeutic effects. Mokhtar and Afrah (2009) also reported the effect of propolis antagonized the harmful effects of aluminum chloride on testis. Generally, in order to overcome the toxic adverse side effects of anticancer drugs, some antioxidant agents are considered useful to modulate oxidative stress. Accordingly, a treatment regimen with potential antioxidant agents could be an approach to ameliorate chemotherapeutic toxicity (Jalali et al., 2012; Liu et al., 2013; Santos & Cruz, 2001). In the present study, a significant increase in the levels of ALT, AST, urea, and creatinine in the serum of CTX-injected
Figure 2. Hematoxylin and eosin-stained kidney sections. (a) Control groups (G1 and G2) showing normal renal tubules and glomerulei (arrow), (b) CTX-treated group (G3) showing shrinkage of the glomerulei (arrows) with distinct vacuolated and degenerated epithelial lining cells of the renal tubules (arrow heads), (c) CTX- and propolis-treated group (G4) showing the disappearance of glomerular shrinkage (arrow) and mild degeneration of the renal epithelial cells (head arrows).
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mice was observed compared with levels in the control groups. Mice that were injected with propolis along with CTX showed a significant decrease in these values compared with the CTX-injected mice, although they are still significantly high comparing with the control groups. Histological examination of the liver and kidney sections from mice that were treated with CTX showed hepatic degeneration and shrinkage of the glomerulei with distinct vacuolated and degenerated epithelial lining cells of the renal tubules. This histological picture was significantly ameliorated after propolis injection. These finding showed the severe toxic effect of the CTX on liver and kidney functions. Treatment with propolis could ameliorate the side effects caused by CTX injection, although it could not return their values to normal levels. These findings are consistent with the earlier studies which showed that therapeutic dose of CTX could cause the liver toxicity (Snover et al., 1989). Ludeman (1999) reported that CTX undergoes a metabolic activation by cytochrome P450 mixed functional oxidase system to produce the two metabolites, phosphoramide mustard and acrolein, which are responsible for the induction of oxidative stress. Oxidative stress is responsible for CTX hepatotoxicity (Selvakumar et al., 2005). Ates et al. (2006) and Bhadauria et al. (2007) stated that CAPE, which is the major compound that is extracted from the propolis, regulated antioxidant enzymes, inhibited lipid peroxidation, and reduced hepatic damage. The present finding also stated that propolis has no toxic effect at the dose (100 mg/kg). This is in agreement with a previous study showing that 1200 mg/kg of propolis had no toxic effect (Burdock, 1998; Sforcin, 2007). The present study revealed a significant decrease in the total number of WBCs in CTX-injected mice compared with the control group in all the examined days. In CTX- and propolis-injected mice, and until the 8th day, the mice kept a significant decrease in the total number of WBCs compared with the control groups. From the 8th day, they showed a significant improvement and, interestingly, at the 12th day, they showed a significant increase in the total number of WBCs compared with control mice. Furthermore, the mice that were injected with CTX along with propolis showed a slight increase in the percentage of lymphocytes and a slight decrease in the percentage of monocytes and granulocytes although these values showed a non-significant change in CTX-injected groups comparing with the control group. According to the results of our study, propolis may act as an immunostimulant. It shows the importance of propolis to enhance the immune system which is in agreement with a previous study reported by Sforcin (2007). Most chemotherapeutic drugs have immune suppressive side effects. Due to treatment with chemotherapy, the dividing hematopoietic cells are affected leading to neutropenia and lymphopenia which in turn decreases immunity (Salem et al., 2012). The data showed that there is a non-significant difference in the total number of RBCs or in the Hb level in CTXinjected mice; however, mice that were injected with CTX along with propolis showed a significant decrease in their values compared with the control groups. Furthermore, it showed that the mice which were injected with propolis only or with propolis along CTX showed a significant increase in the total number of the platelets compared with the
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normal control group. These results showed that propolis may exhibit potent antiplatelet activity. In fact, recently, propolis has been demonstrated to play an important role in preventing oxidative stress, apoptosis and necrosis induced by lead (El-Masry et al., 2011).
Conclusion Collectively, propolis may be considered as a potential natural product that can be used to protect and ameliorate the adverse side effects, such as toxicity and immunosuppression, after chemotherapeutic treatment.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Akyol S, Ginis Z, Armutcu F, et al. (2012). The potential usage of caffeic acid phenethyl ester (CAPE) against chemotherapy-induced and radiotherapy-induced toxicity. Cell Biochem Funct 30:438–43. Ates B, Dogru MI, Ul MG, et al. (2006). Protective role of caffeic acid phenethyl ester in the liver of rats exposed to cold stress. Fund Clin Pharm 20:283–9. Attalla F, Ayman A. (2008). Protective effects of propolis against the amitraz hepatotoxicity in mice. J Pharmacol Toxicol 3:402–8. Awale S, Li F, Onozuka H, et al. (2008). Constituents of Brazilian red propolis and their preferential cytotoxic activity against human pancreatic PANC-1 cancer cell line in nutrient-deprived condition. Bioorg Med Chem 16:181–9. Bancroft JD, Cook HC. (1994). Manual of Histological Techniques and their Diagnostic Application. Edinburgh, UK: Churchill Livingstone, 457 p. Bass KK, Mastrangelo MJ. (1998). Immunopotentiation with low-dose cyclophosphamide in the active specific immunotherapy of cancer. Cancer Immunol Immunother 47:1–12. Bhadauria M, Nirala SK, Shukla S. (2007). Propolis protects CYP2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride. Mol Cell Biochem 302:215–24. Boutabet K, Kebsa W, Alyane M, Lahouel M. (2011). Polyphenolic fraction of Algerian propolis protects rat kidney against acute oxidative stress induced by doxorubicin. Indian J Nephrol 21:101–6. Burdock GA. (1998). Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 36:347–63. Chen TG, Lee JJ, Lin KH, et al. (2007). Antiplatelet activity of caffeic acid phenethyl ester is mediated through a cyclic GMP-dependent pathway in human platelets. Chin J Physiol 50:121–6. Chen MJ, Chang WH, Lin CC, et al. (2008). Caffeic acid phenethyl ester induces apoptosis of human pancreatic cancer cells involving caspase and mitochondrial dysfunction. Pancreatology 8:566–76. Chu WH. (2006). Adjuvant effect of propolis on immunization by inactivated Aeromonas hydrophila in carp (Carassius auratus gibelio). Fish Shelfish Immunol 21:113–17. Diaz-Montero CM, Salem ML, Nishimura MI, et al. (2009). Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin-cyclophosphamide chemotherapy. Cancer Immunol Immunother 58:49–59. El-Masry TA, Emara AM, El-Shitany NA. (2011). Possible protective effect of propolis against lead induced neurotoxicity in animal model. J Evol Biol Res 3:4–11. Etteldorf JN, West CD, Pitcock JA, Williams DL. (1976). Gonadal function, testicular histology, and meisosis following cyclophosphamide therapy in patients with nephrotic syndrome. J Pediatr 88:206–9. Fukutani K, Ishida H, Shinohara M, et al. (1981). Suppression of spermatogenesis in patient with Beheet’s disease treated with cyclophosphamide and colchicine. Fertil Steril 36:76–80. Greenaway W, May J, Scaysbrook T, Whatley FS. (1991). Identification by gas chromatography mass spectrometry of 150 compounds in propolis. Z Naturforsch 46:111–21.
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DOI: 10.3109/13880209.2014.914230
Therapeutic efficacy of propolis against toxicity induced by CTX injection
Ishihara M, Naoi K, Hashita M, et al. (2009). Growth inhibitory activity of ethanol extracts of Chinese and Brazilian propolis in four human colon carcinoma cell lines. Oncol Rep 22:349–54. Ivanovska N, Nechev H, Stefanova Z, et al. (1995). Influence of cinammic acid on lymphatic proliferation, cytokine release and Klebsiella infection in mice. Apidologie 26:73–81. Jaiswal AK, Venugopal R, Mucha J, et al. (1997). Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H: Quinone oxidoreductase (NQO1) gene. Cancer Res 57:440–6. Jalali AS, Hasanzadeh S, Malekinejad H. (2012). Achillea millefolium inflorescence aqueous extract ameliorates cyclophosphamide-induced toxicity in rat testis: Stereological evidences. Chin J Nat Med 10: 247–54. Johnstone RW, Ruefli AA, Lowe SW. (2002). Apoptosis: A link between cancer genetics and chemotherapy. Cell 108:153–64. Khalil ML. (2006). Biological activity of bee propolis in health and disease. Asian Pac J Cancer Prev 7:22–31. Liu CC, Hsu JM, Kuo LK, Chuu CP. (2013). Caffeic acid phenethyl ester as an adjuvant therapy for advanced prostate cancer. Med Hypotheses 80:617–19. Ludeman SM. (1999). The chemistry of the metabolites of cyclophosphamide. Curr Pharm Des 5:627–43. McDermott EM, Powell RJ. (1996). Incidence of ovarian failure in systemic lupus erythematosus after treatment with pulse cyclophosphamide. Ann Rheum Dis 55:224–9. Mokhtar IY, Afrah FS. (2009). Propolis protection from reproductive toxicity caused by aluminium chloride in male rats. Food Chem Toxicol 47:1168–75. Montgomery M, Cottler-Fox M. (2007). Mobilization and collection of autologous hematopoietic progenitor/stem cells. Clin Adv Hematol Oncol 5:127–36. Ochoa R, Bejarano PA, Glu¨ck S, Montero AJ. (2012). Pneumonitis and pulmonary fibrosis in a patient receiving adjuvant docetaxel and cyclophosphamide for stage 3 breast cancer: A case report and literature review. J Med Case Rep 30:413–19. Ohta T, Kunimasa K, Kobayashi T, et al. (2008). Propolis suppresses tumor angiogenesis by inducing apoptosis in tube-forming endothelial cells. Biosci Biotech Biochem 72:2436–40. Padmavathi R, Senthilnathan P, Chodon D, Sakthisekaran D. (2006). Therapeutic effect of paclitaxel and propolis on lipid peroxidation and antioxidant system in 7,12 dimethylbenz(a)anthracene-induced breast cancer in female Sprague Dawley rats. Life Sci 24:2820–5. Pascual C, Gonzalez R, Torricella RG. (1994). Scavenging action of propolis extract against oxygen radicals. J Ethnopharmacol 41:9–13.
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Paul R, Kulkarni P, Ganesh N. (2011). Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide induced genotoxicity in human lymphocyte culture. J Exp Ther Oncol 9:221–30. Rehman MU, Tahir M, Ali F, et al. (2012). Cyclophosphamide-induced nephrotoxicity, genotoxicity, and damage in kidney genomic DNA of Swiss albino mice: The protective effect of ellagic acid. Mol Cell Biochem 365:119–27. Reitman S, Frankel S. (1957). A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 28:56–63. Salem ML, Diaz-Montero CM, Al-Khami AA, et al. (2009). Recovery from cyclophosphamide-induced lymphopenia results in expansion of immature dendritic cells which can mediate enhanced prime-boost vaccination antitumor responses in vivo when stimulated with the TLR3 agonist poly(I:C). J Immunol 182:2030–40. Salem ML, AL-Khami AA, El-Naggar SA, et al. (2010). Cyclophosphamide induces dynamic alterations in the host microenvironments resulting in a FLT3L-dependent expansion of dendritic cells. J Immunol 184:1737–47. Salem ML, Al-Khami AA, El-Nagaar SA, et al. (2012). Kinetics of rebounding of lymphoid and myeloid cells in mouse peripheral blood, spleen and bone marrow after treatment with cyclophosphamide. Cell Immunol 276:67–74. Santos HD, Cruz WS. (2001). A terapia nutritional com vitaminas, antioxidantes e o tratamento quimioterapico e oncolgico. Rev Bras Cancerologia 47:303–8. Scheller S, Krol W, Swiacik J, et al. (1989). Antitumoral property of ethanolic extract of propolis in mice-bearing Ehrlich carcinoma, as compared to bleomycin. Z Naturforsch 44:1063–5. Selvakumar E, Prahalathan C, Mythili Y, Varalakshmi P. (2005). Mitigation of oxidative stress in cyclophosphamide-challenged hepatic tissue by DL-alpha-lipoic acid. Mol Cell Biochem 272:179–85. Sforcin JM. (2007). Propolis and the immune system: A review. J Ethnopharmacol 113:1–14. Snover DC, Weisdorf S, Bloomer J, et al. (1989). Nodular regenerative hyperplasia of the liver following bone marrow transplantation. Hepatology 9:443–8. Suzuki I, Hayashi I, Takaki T, et al. (2002). Antitumor and anticytopenic effects of aqueous extracts of propolis in combination with chemotherapeutic agents. Cancer Biother Radiopharm 17:553–62. Takino Y, Mochida S. (1982). Propolis its chemical constituents and biological activities. Honeybee. Science 3:145–52. Trinder P. (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem 6: 24–7.
ORIGINAL ARTICLE
Ameliorative effect of propolis against cyclophosphamide-induced toxicity in mice Sabry A. El-Naggar1,2, Abeer A. Alm-Eldeen2, Mousa O. Germoush1, Kamal F. El-Boray1,3, and Hassan A. Elgebaly1 Department of Biology, College of Science, Aljouf Univesity, Sakakah, KSA, 2Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt, and 3Department of Aquaculture, National Institute of Oceanography and Fisheries, Suez, Egypt
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Abstract
Keywords
Context: Cyclophosphamide (CTX) is a common anticancer agent used for the treatment of several malignancies. However, upon treatment, it induces severe toxicity due to its oxidative stress capability. Propolis, a natural product collected by honey bees, has shown several biological activities, such as free radical scavenging and antioxidant agent. Objective: This study elucidates the protective effects of propolis against CTX-induced changes in mice. Materials and methods: Forty-eight male Swiss albino mice were divided into four groups; group 1 was intraperitoneally (i.p.) injected with 200 mL of phosphate buffer saline (PBS), group 2 was injected with 100 mg/kg/d propolis, group 3 was injected with a single dose of CTX (200 mg/ kg), and group 4 was injected with a single dose of CTX (200 mg/kg) followed by propolis (100 mg/kg) for 7 consecutive days. After 12 d, mice were bled and then sacrificed to analyze the hematological, biochemical, and histological parameters. Results: The results indicated that CTX-injected mice showed an increase in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine and a decrease in the total number of white blood cells (WBCs) and platelets. Moreover, dramatically changes in the histological architectures of the liver and kidney were observed. The mice that were injected with CTX/propolis showed an improvement in the levels of ALT, AST, urea, creatinine, WBCs, and platelets. Moreover, the histological picture of the liver and kidney was significantly improved. Conclusions: In conclusion, propolis might be considered an effective agent in ameliorating the toxicity resulted from CTX treatment.
Cyclophosphamide, immunosuppression, kidney, liver, propolis, toxicity
Introduction Cyclophosphamide (CTX) is a common anticancer chemotherapeutic agent used alone or in a combination with other agents for the treatment of several human malignancies (Bass & Mastrangelo, 1998). CTX is used also in preconditioning the host for immunotherapy in several pre-clinical trials (Salem et al., 2010). In this regard, it has been reported that CTX preconditioning increases the numbers of the dendritic cells which directed the immune response (Salem et al., 2009, 2010). Other studies showed that CTX activates the immuno-response via inducing the mobilization of the hematopoietic stem cells and dendritic cells from bone marrow (Montgomery & Cottler-Fox, 2007; Salem et al., 2010). Along with these beneficial effects of CTX, several adverse side effects have been reported such as pneumonitis and pulmonary fibrosis (Ochoa et al., 2012) and induction of the genotoxicity (Paul et al., 2011; Rehman et al., 2012).
Correspondence: Sabry A. El-Naggar, Department of Zoology, Faculty of Science, Tanta University, Tanta, Egypt. Tel: +96 6563977876. E-mail: [email protected], [email protected]
History Received 31 December 2013 Revised 28 March 2014 Accepted 8 April 2014 Published online 7 October 2014
Furthermore, it has been reported that CTX increases the proportion of myeloid derived suppressor cells in blood and lymphoid organs and this suppresses the function of the immune system to fight against cancerous cells (DiazMontero et al., 2009). McDermott and Powell (1996) has also been reported that CTX treatment induces the oxidative stress by the generation of the free radicals and reactive oxygen species. Because of this excessive production of free radicals, it has been reported that CTX treatment reduces the levels of glutathione and glutathione peroxidase, catalase, and superoxide dismutase activities in testis (Etteldorf et al., 1976; Fukutani et al., 1981). Earlier studies have shown that the therapeutic dose of CTX could cause liver toxicity (Snover et al., 1989). CTX undergoes a metabolic activation by the cytochrome P450 mixed functional oxidase system to produce two metabolites, phosphoramide mustard and acrolein, which are responsible for the induction of oxidative stress (Ludeman, 1999). Oxidative stress is responsible for CTX hepatotoxicity (Selvakumar et al., 2005). Propolis, a natural product, is commonly used in folk medicines. It is a resinous substance collected by honey bee workers from the plant sources. It contains more than 150 polyphenolic compounds like flavonoids and phenolic acid
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and their esters (Greenaway et al., 1991). Several in vivo and in vitro studies showed that propolis has several biological activities such as free radical scavenging (Pascual et al., 1994), antioxidant (Jaiswal et al., 1997), antitumor (Awale et al., 2008; Ishihara et al., 2009; Ohta et al., 2008; Scheller et al., 1989) and immunomodulatory effects (Ivanovska et al., 1995). Takino and Mochida (1982) reported that the biological activity of propolis is mainly due to the presence of the flavonoid content. Recently, Akyol et al. (2012) stated that caffeic acid phenethyl ester (CAPE), which is the major compound that is extracted from the propolis, has antiinflammatory, antiproliferative, antioxidant, cytostatic, antiviral, antibacterial, antifungal, and antineoplastic properties. It has been reported also that CAPE can regulate antioxidant enzymes, inhibit lipid peroxidation, and reduce hepatic damage (Ates et al., 2006; Bhadauria et al., 2007). Furthermore, CAPE is considered as a potent antiplatelet agent and can induce apoptosis of human pancreatic cancer cells involving caspase and mitochondrial dysfunction (Chen et al., 2007, 2008). Propolis has biological effects that act in synergy with chemotherapy drugs such as 5-fluorouracil (Suzuki et al., 2002). Importantly, Santos and Cruz (2001) showed the antioxidant properties of propolis could reduce the side effects caused by some anticancer drugs without any detriment to the therapeutic effects. Moreover, it has been shown that propolis is a promising adjuvant with chemotherapy (Padmavathi et al., 2006) and with immunization (Chu, 2006). To our knowledge, there are no reports available on the protective effect of propolis on CTX-induced alterations. Therefore, the present study aimed to address the amelioration effect and the protective role of propolis after CTX-treatment.
Materials and methods The experiment adhered to the guidelines of the ethical committee of Aljouf University, Sakakah, KSA.
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King Saud University, KSA. The mice were maintained under standard laboratory conditions (temperature 24 ± 2 C, natural light-dark cycle), and had free access to drinking water and commercial standard pellet diet. After acclimatization for one week to laboratory conditions, the animals were randomly divided into four groups, 12 mice each. Group 1 (G1), control group includes mice that were injected with PBS; group 2 (G2), positive control includes mice that injected with 100 mg/kg/d propolis, group 3 (G3) includes mice that were injected i.p. with a single dose of CTX (200 mg/kg) dissolved in PBS; and group 4 (G4) includes mice that were injected i.p. with a single dose of CTX (200 mg/kg) followed by propolis (100 mg/kg) after 24 h of CTX injection for 7 consecutive days (Boutabet et al., 2011; Salem et al., 2010). The animals were left for 4 d then, at day 12, the mice were sacrificed, weighed, and the liver, kidney, and spleen were removed and weighed. The serum obtained after centrifugation (1500 rpm for 10 min at 4 C) was used for various serum biochemical assays. Hematological and biochemical assays Hematological profile Red blood cells (RBCs), hemoglobin content (Hb g/dl), white blood cell (WBCs) counts, and the total platelets counts were measured from fresh blood samples obtained from the orbital plexus of the eyes of all groups under the study using the electronic blood counter. Differential WBCs were carried out from blood smears on days 0, 2, 4, 8, and 12. Biochemical assays Determination of serum alanine and aspartate aminotransferases Serum alanine and aspartate aminotransferases were assayed using commercial diagnostic kits (Diagnostic systems, Medford, NY) according to the method described by Reitman and Frankel (1957).
Chemicals CTX was purchased from (Sigma-Aldrich, St. Louis, MO). Propolis was purchased from the commercial market at Sakaka city, Aljouf, KSA. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, and creatinine kits were purchased from Doa El-Shamal Company in Sakaka City, KSA.
Determination of serum urea and creatinine levels
Propolis extraction
Livers and kidneys tissues were immediately removed from all groups under experimentation, sliced, and fixed in bouin fixative for 24 h. The specimens were then dehydrated, cleared, and embedded in paraffin. Serial sections of 5 mm thick were cut by means of a rotary microtome (Cut 4662). Sections were then processed for hematoxylin and eosin staining (Bancroft & Cook, 1994).
Ethanolic propolis extract was prepared by the methods described by Khalil (2006) and Attalla and Ayman (2008). Briefly, propolis (10 g) was crushed into small pieces in a mortar and mixed vigorously with 34.85 ml 80% (V/V) ethanol during 48 h at 37 ± 1 C. After extraction, the ethanol extract of propolis was filtered through Whatman No. 4 paper. The solvent was air-dried and the extract was weighed and suspended in 0.9% sterile saline for further processing. Animals and treatment Adult male albino mice (8-weeks old, weighing approximately 23 g) were purchased from the Faculty of Science,
Serum urea and creatinine levels were determined using commercial diagnostic kits according to the method described by Trinder (1969). Histopathological investigation
Statistical analysis Data were expressed as mean ± SD of a number of experiments (n ¼ 12). The statistical significance was evaluated by Student’s t-test using SPSS 15.0 software package (SPSS, Chicago, IL). Values were considered statistically significant when p50.05.
Therapeutic efficacy of propolis against toxicity induced by CTX injection
DOI: 10.3109/13880209.2014.914230
Results
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Effect on body weight and the relative weights of the spleen and liver The data showed that there are non-significant changes (p 0.05) in the body weights between the mice of the normal control (G1) that injected with PBS and the mice of the positive control group (G2) that were injected with propolis at day 12 (the end of the experiment). The body weights of the mice that were injected with CTX (G3) or with CTX along with propolis (G4) showed a significant decrease (p 0.05) when compared with the control groups (G1 and G2) (Table 1). The data showed also that there is a nonobservable change in the relative weights of the liver in the normal, positive control, CTX, and CTX along with propolisinjected groups (G1, G2, G3, and G4), respectively. However, there is an observable increase in the relative weight of the spleen in the positive control, CTX, and CTX along with propolis-injected groups (G2, G3, and G4), respectively, compared with the normal control group (G1) (Table 1). Effect on serum biochemical parameters The present study showed that there is a non-significant change (p 0.05) in the levels of ALT, AST, urea, and creatinine between the mice of the normal and positive control groups (G1 and G2). These parameters showed a significant increase (p 0.05) in their levels in the CTXinjected mice (G3) compared with their levels in the serum of the control groups (G1 and G2). The mice that were injected with CTX along with propolis (G4) showed a significant decrease (p 0.05) in these values compared with the CTXinjected mice (G3). However, they are still significantly high (p 0.05) compared with the control groups (G1 and G2) (Table 2). Effect on hematological parameters Total number of WBCs The data indicated that the mice of the normal control group (G1) and the positive control group (G2) have a nonsignificant difference (p 0.05) in the total number of the WBCs in the examined days. A significant decrease (p 0.05) in the total number of WBCs in CTX-injected mice (G3) and both CTX- and propolis-injected mice (G4)
compared with the control groups (G1 and G2) until the 8th day. At the same time, there is a non-significant difference (p 0.05) in the total number of WBCs in CTX- and propolisinjected mice (G4) compared with the CTX-injected mice of (G3) (Table 3). At the 12th day, the mice that were injected with CTX (G3) showed that they still have a significant decrease (p 0.05) in the total number of WBCs compared with the control groups (G1 and G2). Interestingly, the mice that injected with CTX along with propolis (G4) showed a significant increase (p 0.05) in the total number of WBCs compared with CTX-injected mice (G3) and the normal control group (G1) while showing a non-significant increase (p 0.05) in the total number of WBCs compared with the positive control group (G2) (Table 3). Total number of red blood cells (RBCs) and hemoglobin level The data showed that there is a non-significant difference (p 0.05) in the total number of RBCs or in the hemoglobin level in the control and CTX-injected groups (G1, G2, and G3), respectively. However, the mice that were injected with CTX along with (G4) showed a significant decrease (p 0.05) in the total number of RBCs and the Hb level compared with the control and CTX-injected groups (G1, G2, and G3), respectively (Table 4). Table 2. Effect of propolis on CTX-induced changes in serum biochemical parameters of liver and kidney (Mean ± SD, n ¼ 12).
Parameters
Parameters
Control
CTX alone
CTX and propolis
Total body 23.7 ± 2.3 24 ± 4.4 21.4 ± 3.2a 21.3 ± 3.5b weight (g) The relative liver 7.2 ± 0.6 6.3 ± 0.9 6.1 ± 0.5 5.8 ± 0.2 weight (%) The relative spleen 0.7 ± 0.05 0.8 ± 0.2c 1.0 ± 0.05c 2.3 ± 0.4c weight (%) a
p50.05: CTX-injected group (G3) was compared with the control groups (G1 and G2). p50.05: CTX- and propolis-injected groups (G4) was compared with the control groups (G1 and G2). c p50.05: propolis, CTX, and CTX and propolis-injected groups (G2, G3, and G4) were compared with the normal control group (G1). b
Control
Propolis alone
CTX alone
CTX and propolis
ALT (IU/L) 20.6 ± 0.5 21 ± 1.4 63.4 ± 1.6a 32.4 ± 0.9b,c AST (IU/L) 113 ± 1.4 114.3 ± 3.7 191.6 ± 2.4a 132.4 ± 0.9b,c Urea (mmol/L) 13.6 ± 0.5 14.25 ± 1.5 45 ± 0.7a 27.4 ± 0.5b,c Creatinine (mmol/L) 1 ± 0.07 1.25 ± 0.20 2.76 ± 0.05a 1.58 ± 0.05b,c ALT, alanine aminotransferase; AST, aspartate aminotransferase. The values represent mean + SD, number of samples is 12 mice per group. a p50.05: CTX-injected group (G3) was compared with the control groups (G1 and G2). b p50.05: CTX- and propolis-injected group (G4) was compared with CTX-injected group (G3). c p50.05: CTX and propolis-injected group (G4) was compared with the control groups (G1 and G2). Table 3. Effect of propolis on CTX-induced changes in WBC number (mean ± SD, n ¼ 12).
Table 1. Effect of propolis on CTX-induced changes in the total body weight, the relative liver, and spleen weights (mean ± SD, n ¼ 12). Propolis alone
3
Day Day Day Day Day
0 2 4 8 12
Control
Propolis alone
CTX alone
CTX and propolis
7.1 ± 0.8 6.8 ± 0.5 7.2 ± 0.6 7.2 ± 0.6 6.6 ± 1.3
7.3 ± 0.4 6.9 ± 0.6 7.0 ± 0.4 6.6 ± 0.2 7.5 ± 1.4
7.4 ± 0.5 2.9 ± 0.3a 3 ± 0.9a 3 ± 0.9a 5.1 ± 0.2b,c
7.5 ± 0.2 3.0 ± 0.9a 3.2 ± 0.2a 6.6 ± 1.2a 8.5 ± 0.7b,d
The values represent mean + SD, number of samples is 12 mice per group. a p50.05: CTX and CTX with propolis-injected groups (G3 and G4) were compared with the control groups (G1 and G2) at days 2, 4, and 8. b p50.05: CTX and CTX with propolis-injected groups (G3 and G4) were compared with the normal control group (G1) at day 12. c p50.05: CTX-injected group (G3) was compared with the positive control group (G2) at day 12. d p50.05: CTX- and propolis-injected group (G4) was compared with CTX-injected group (G3) at day 12.
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Total number of blood platelets The data showed that mice of the positive control group which is injected with propolis only (G2) have a significant increase (p 0.05) in the total number of the platelets compared with the normal control group (G1). Moreover, mice that were injected with CTX (G3) showed a significant increase (p 0.05) in the total number of the platelets compared with the control groups (G1 and G2). Interestingly, mice Table 4. Effect of propolis on CTX-induced changes in total RBCs, Hb content, and differential leucocytes.
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Parameters RBCs (106/ml) HGB (g/dl) PLT (103/ml) % Lmph. % Mon. % Gr.
Control
Propolis alone
CTX alone
CTX and propolis
7 ± 0.3 13 ± 1.1 318 ± 2 64 2.8 33.5
7.1 ± 0.1 12.3 ± 1.0 667 ± 2.1 63 2.6 34.4
6.9 ± 0.1 12.2 ± 0.5 549 ± 7 62 2.8 35.5
5.9 ± 0.2a 10.3 ± 0.35b 1228 ± 9c 69 1.5 29.5
The values represent mean + SD, number of samples is 12 mice per group. a p50.05: CTX and propolis-injected group (G4) was compared with the control groups (G1 and G2) and CTX-injected group (G3). b p50.05: CTX- and propolis-injected group (G4) was compared with the control groups (G1 and G2) and CTX-injected group (G3). c p50.05: CTX- and propolis-injected group (G4) was compared with the control groups (G1 and G2) and CTX-injected group (G3).
injected with CTX along with propolis (G4) showed a significant increase (p 0.05) in the total number of the platelets comparing with the control and CTX-injected groups (G1, G2, and G3) (Table 4). The percentage of differential leucocytes The present study showed that there is a non-significant change (p 0.05) in differential leucocytes in the control groups and CTX-injected groups (G1, G2, and G3). Mice which were injected with propolis along with CTX (G4) showed a slightly increase in the percentage of the lymphocytes and a slightly decrease in the percentage of the monocytes and the granulocytes compared with mice in the control and CTX-injected groups (G1, G2, and G3) (Table 4). Effect on the histopathological picture of the liver and kidney Histological examination of the liver sections from the control groups (G1 and G2) revealed a similar architectural appearance. They have cords of hepatocytes arranged radially extended from the central vein to a periphery of the hepatic lobules (Figure 1a). Histopathological examination of the liver sections from mice that were treated with CTX (G3) showed hepatic degeneration and pale-stained areas. Necrosis of many hepatocytes with pyknosis, swollen of some hepatocytes with nuclear chromatolysis, and infiltration of
Figure 1. Hematoxylin and eosin-stained liver sections. (a) Control groups (G1 and G2) showing normal hepatic architecture with radial arrangement of hepatocytes; (b)–(d) CTX-treated group (G3) showing hepatic degeneration and pale stained areas (arrows) and coagulative necrosis of many hepatocytes with piknotic nuclei (arrowheads) (b), cellular degeneration of some hepatocytes (arrow) with nuclear chromatolysis (arrowhead) (c), cellular infiltration of eosinophils (arrows) (d), and (e) CTX- and propolis-treated group (G4) showing that hepatocytes regained normal architecture with normal chromatolized and central nuclei. Also, regeneration of hepatocytes can be noticed by the presence of binucleate cells (arrows).
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DOI: 10.3109/13880209.2014.914230
Therapeutic efficacy of propolis against toxicity induced by CTX injection
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lymphocytes were observed (Figure 1b–d). Microscopic examination of the liver sections from the mice that treated with both CTX and propolis showed a significant amelioration of distorted hepatic lobules. In addition, more or less normal hepatic structure and normal stain ability were observed. Hepatocytes regained normal architecture with normal chromatolized and central nuclei. Regeneration of hepatocytes can be observed by the presence of binucleate cells (Figure 1e). Histological examination of the kidney sections from the control groups (G1 and G2) revealed normal similar architecture showing the renal cortex with normal renal tubules and glomerulei (Figure 2a). Histopathological examination of the kidney sections from the mice that were treated with CTX (G3) showed shrinkage of the glomerulei with distinct vacuolated and degenerated epithelial lining cells of the renal tubules (Figure 2b). Kidney sections from mice that were treated with CTX and propolis disappearance of the glomerular shrinkage, improvement of the renal cortex tissue architecture, and mild degeneration of renal epithelial cells were observed (Figure 2c).
Discussion This study was conducted to evaluate the possible ameliorative role of the propolis after treatment with the chemotherapeutic agent CTX. The results showed that there are no significant changes in the body weights between mice of the normal control group injected with PBS and mice of the positive control group which was injected with propolis. The body weights of the mice that were injected with CTX or CTX along with propolis showed a significant decrease compared with the normal control group although there was a non-observable change in the relative weights of the liver in all groups. In contrast, there is an observable increase in the relative weights of kidney in the positive control, CTX, and CTX along with propolis-injected groups. The decrease in the body weight after CTX injection would be due to its side effects on the different body organs and systems (Johnstone et al., 2002). Propolis treatment after CTX injection could partially protect the body from losing weight. The nonobservable change in the relative weight of the liver in all groups could be due to treatment with a single dose of CTX, which partially decreased the relative liver weights after days 2 and 4 of CTX injection (data not shown), and gradually returned to their normal relative weights at day 12. Santos and Cruz (2001) showed that the antioxidant properties of propolis could reduce the side effects caused by chemotherapy drugs without any detriment to the therapeutic effects. Mokhtar and Afrah (2009) also reported the effect of propolis antagonized the harmful effects of aluminum chloride on testis. Generally, in order to overcome the toxic adverse side effects of anticancer drugs, some antioxidant agents are considered useful to modulate oxidative stress. Accordingly, a treatment regimen with potential antioxidant agents could be an approach to ameliorate chemotherapeutic toxicity (Jalali et al., 2012; Liu et al., 2013; Santos & Cruz, 2001). In the present study, a significant increase in the levels of ALT, AST, urea, and creatinine in the serum of CTX-injected
Figure 2. Hematoxylin and eosin-stained kidney sections. (a) Control groups (G1 and G2) showing normal renal tubules and glomerulei (arrow), (b) CTX-treated group (G3) showing shrinkage of the glomerulei (arrows) with distinct vacuolated and degenerated epithelial lining cells of the renal tubules (arrow heads), (c) CTX- and propolis-treated group (G4) showing the disappearance of glomerular shrinkage (arrow) and mild degeneration of the renal epithelial cells (head arrows).
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mice was observed compared with levels in the control groups. Mice that were injected with propolis along with CTX showed a significant decrease in these values compared with the CTX-injected mice, although they are still significantly high comparing with the control groups. Histological examination of the liver and kidney sections from mice that were treated with CTX showed hepatic degeneration and shrinkage of the glomerulei with distinct vacuolated and degenerated epithelial lining cells of the renal tubules. This histological picture was significantly ameliorated after propolis injection. These finding showed the severe toxic effect of the CTX on liver and kidney functions. Treatment with propolis could ameliorate the side effects caused by CTX injection, although it could not return their values to normal levels. These findings are consistent with the earlier studies which showed that therapeutic dose of CTX could cause the liver toxicity (Snover et al., 1989). Ludeman (1999) reported that CTX undergoes a metabolic activation by cytochrome P450 mixed functional oxidase system to produce the two metabolites, phosphoramide mustard and acrolein, which are responsible for the induction of oxidative stress. Oxidative stress is responsible for CTX hepatotoxicity (Selvakumar et al., 2005). Ates et al. (2006) and Bhadauria et al. (2007) stated that CAPE, which is the major compound that is extracted from the propolis, regulated antioxidant enzymes, inhibited lipid peroxidation, and reduced hepatic damage. The present finding also stated that propolis has no toxic effect at the dose (100 mg/kg). This is in agreement with a previous study showing that 1200 mg/kg of propolis had no toxic effect (Burdock, 1998; Sforcin, 2007). The present study revealed a significant decrease in the total number of WBCs in CTX-injected mice compared with the control group in all the examined days. In CTX- and propolis-injected mice, and until the 8th day, the mice kept a significant decrease in the total number of WBCs compared with the control groups. From the 8th day, they showed a significant improvement and, interestingly, at the 12th day, they showed a significant increase in the total number of WBCs compared with control mice. Furthermore, the mice that were injected with CTX along with propolis showed a slight increase in the percentage of lymphocytes and a slight decrease in the percentage of monocytes and granulocytes although these values showed a non-significant change in CTX-injected groups comparing with the control group. According to the results of our study, propolis may act as an immunostimulant. It shows the importance of propolis to enhance the immune system which is in agreement with a previous study reported by Sforcin (2007). Most chemotherapeutic drugs have immune suppressive side effects. Due to treatment with chemotherapy, the dividing hematopoietic cells are affected leading to neutropenia and lymphopenia which in turn decreases immunity (Salem et al., 2012). The data showed that there is a non-significant difference in the total number of RBCs or in the Hb level in CTXinjected mice; however, mice that were injected with CTX along with propolis showed a significant decrease in their values compared with the control groups. Furthermore, it showed that the mice which were injected with propolis only or with propolis along CTX showed a significant increase in the total number of the platelets compared with the
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normal control group. These results showed that propolis may exhibit potent antiplatelet activity. In fact, recently, propolis has been demonstrated to play an important role in preventing oxidative stress, apoptosis and necrosis induced by lead (El-Masry et al., 2011).
Conclusion Collectively, propolis may be considered as a potential natural product that can be used to protect and ameliorate the adverse side effects, such as toxicity and immunosuppression, after chemotherapeutic treatment.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Akyol S, Ginis Z, Armutcu F, et al. (2012). The potential usage of caffeic acid phenethyl ester (CAPE) against chemotherapy-induced and radiotherapy-induced toxicity. Cell Biochem Funct 30:438–43. Ates B, Dogru MI, Ul MG, et al. (2006). Protective role of caffeic acid phenethyl ester in the liver of rats exposed to cold stress. Fund Clin Pharm 20:283–9. Attalla F, Ayman A. (2008). Protective effects of propolis against the amitraz hepatotoxicity in mice. J Pharmacol Toxicol 3:402–8. Awale S, Li F, Onozuka H, et al. (2008). Constituents of Brazilian red propolis and their preferential cytotoxic activity against human pancreatic PANC-1 cancer cell line in nutrient-deprived condition. Bioorg Med Chem 16:181–9. Bancroft JD, Cook HC. (1994). Manual of Histological Techniques and their Diagnostic Application. Edinburgh, UK: Churchill Livingstone, 457 p. Bass KK, Mastrangelo MJ. (1998). Immunopotentiation with low-dose cyclophosphamide in the active specific immunotherapy of cancer. Cancer Immunol Immunother 47:1–12. Bhadauria M, Nirala SK, Shukla S. (2007). Propolis protects CYP2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride. Mol Cell Biochem 302:215–24. Boutabet K, Kebsa W, Alyane M, Lahouel M. (2011). Polyphenolic fraction of Algerian propolis protects rat kidney against acute oxidative stress induced by doxorubicin. Indian J Nephrol 21:101–6. Burdock GA. (1998). Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol 36:347–63. Chen TG, Lee JJ, Lin KH, et al. (2007). Antiplatelet activity of caffeic acid phenethyl ester is mediated through a cyclic GMP-dependent pathway in human platelets. Chin J Physiol 50:121–6. Chen MJ, Chang WH, Lin CC, et al. (2008). Caffeic acid phenethyl ester induces apoptosis of human pancreatic cancer cells involving caspase and mitochondrial dysfunction. Pancreatology 8:566–76. Chu WH. (2006). Adjuvant effect of propolis on immunization by inactivated Aeromonas hydrophila in carp (Carassius auratus gibelio). Fish Shelfish Immunol 21:113–17. Diaz-Montero CM, Salem ML, Nishimura MI, et al. (2009). Increased circulating myeloid-derived suppressor cells correlate with clinical cancer stage, metastatic tumor burden, and doxorubicin-cyclophosphamide chemotherapy. Cancer Immunol Immunother 58:49–59. El-Masry TA, Emara AM, El-Shitany NA. (2011). Possible protective effect of propolis against lead induced neurotoxicity in animal model. J Evol Biol Res 3:4–11. Etteldorf JN, West CD, Pitcock JA, Williams DL. (1976). Gonadal function, testicular histology, and meisosis following cyclophosphamide therapy in patients with nephrotic syndrome. J Pediatr 88:206–9. Fukutani K, Ishida H, Shinohara M, et al. (1981). Suppression of spermatogenesis in patient with Beheet’s disease treated with cyclophosphamide and colchicine. Fertil Steril 36:76–80. Greenaway W, May J, Scaysbrook T, Whatley FS. (1991). Identification by gas chromatography mass spectrometry of 150 compounds in propolis. Z Naturforsch 46:111–21.
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DOI: 10.3109/13880209.2014.914230
Therapeutic efficacy of propolis against toxicity induced by CTX injection
Ishihara M, Naoi K, Hashita M, et al. (2009). Growth inhibitory activity of ethanol extracts of Chinese and Brazilian propolis in four human colon carcinoma cell lines. Oncol Rep 22:349–54. Ivanovska N, Nechev H, Stefanova Z, et al. (1995). Influence of cinammic acid on lymphatic proliferation, cytokine release and Klebsiella infection in mice. Apidologie 26:73–81. Jaiswal AK, Venugopal R, Mucha J, et al. (1997). Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H: Quinone oxidoreductase (NQO1) gene. Cancer Res 57:440–6. Jalali AS, Hasanzadeh S, Malekinejad H. (2012). Achillea millefolium inflorescence aqueous extract ameliorates cyclophosphamide-induced toxicity in rat testis: Stereological evidences. Chin J Nat Med 10: 247–54. Johnstone RW, Ruefli AA, Lowe SW. (2002). Apoptosis: A link between cancer genetics and chemotherapy. Cell 108:153–64. Khalil ML. (2006). Biological activity of bee propolis in health and disease. Asian Pac J Cancer Prev 7:22–31. Liu CC, Hsu JM, Kuo LK, Chuu CP. (2013). Caffeic acid phenethyl ester as an adjuvant therapy for advanced prostate cancer. Med Hypotheses 80:617–19. Ludeman SM. (1999). The chemistry of the metabolites of cyclophosphamide. Curr Pharm Des 5:627–43. McDermott EM, Powell RJ. (1996). Incidence of ovarian failure in systemic lupus erythematosus after treatment with pulse cyclophosphamide. Ann Rheum Dis 55:224–9. Mokhtar IY, Afrah FS. (2009). Propolis protection from reproductive toxicity caused by aluminium chloride in male rats. Food Chem Toxicol 47:1168–75. Montgomery M, Cottler-Fox M. (2007). Mobilization and collection of autologous hematopoietic progenitor/stem cells. Clin Adv Hematol Oncol 5:127–36. Ochoa R, Bejarano PA, Glu¨ck S, Montero AJ. (2012). Pneumonitis and pulmonary fibrosis in a patient receiving adjuvant docetaxel and cyclophosphamide for stage 3 breast cancer: A case report and literature review. J Med Case Rep 30:413–19. Ohta T, Kunimasa K, Kobayashi T, et al. (2008). Propolis suppresses tumor angiogenesis by inducing apoptosis in tube-forming endothelial cells. Biosci Biotech Biochem 72:2436–40. Padmavathi R, Senthilnathan P, Chodon D, Sakthisekaran D. (2006). Therapeutic effect of paclitaxel and propolis on lipid peroxidation and antioxidant system in 7,12 dimethylbenz(a)anthracene-induced breast cancer in female Sprague Dawley rats. Life Sci 24:2820–5. Pascual C, Gonzalez R, Torricella RG. (1994). Scavenging action of propolis extract against oxygen radicals. J Ethnopharmacol 41:9–13.
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Paul R, Kulkarni P, Ganesh N. (2011). Avocado fruit (Persea americana Mill) exhibits chemo-protective potentiality against cyclophosphamide induced genotoxicity in human lymphocyte culture. J Exp Ther Oncol 9:221–30. Rehman MU, Tahir M, Ali F, et al. (2012). Cyclophosphamide-induced nephrotoxicity, genotoxicity, and damage in kidney genomic DNA of Swiss albino mice: The protective effect of ellagic acid. Mol Cell Biochem 365:119–27. Reitman S, Frankel S. (1957). A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 28:56–63. Salem ML, Diaz-Montero CM, Al-Khami AA, et al. (2009). Recovery from cyclophosphamide-induced lymphopenia results in expansion of immature dendritic cells which can mediate enhanced prime-boost vaccination antitumor responses in vivo when stimulated with the TLR3 agonist poly(I:C). J Immunol 182:2030–40. Salem ML, AL-Khami AA, El-Naggar SA, et al. (2010). Cyclophosphamide induces dynamic alterations in the host microenvironments resulting in a FLT3L-dependent expansion of dendritic cells. J Immunol 184:1737–47. Salem ML, Al-Khami AA, El-Nagaar SA, et al. (2012). Kinetics of rebounding of lymphoid and myeloid cells in mouse peripheral blood, spleen and bone marrow after treatment with cyclophosphamide. Cell Immunol 276:67–74. Santos HD, Cruz WS. (2001). A terapia nutritional com vitaminas, antioxidantes e o tratamento quimioterapico e oncolgico. Rev Bras Cancerologia 47:303–8. Scheller S, Krol W, Swiacik J, et al. (1989). Antitumoral property of ethanolic extract of propolis in mice-bearing Ehrlich carcinoma, as compared to bleomycin. Z Naturforsch 44:1063–5. Selvakumar E, Prahalathan C, Mythili Y, Varalakshmi P. (2005). Mitigation of oxidative stress in cyclophosphamide-challenged hepatic tissue by DL-alpha-lipoic acid. Mol Cell Biochem 272:179–85. Sforcin JM. (2007). Propolis and the immune system: A review. J Ethnopharmacol 113:1–14. Snover DC, Weisdorf S, Bloomer J, et al. (1989). Nodular regenerative hyperplasia of the liver following bone marrow transplantation. Hepatology 9:443–8. Suzuki I, Hayashi I, Takaki T, et al. (2002). Antitumor and anticytopenic effects of aqueous extracts of propolis in combination with chemotherapeutic agents. Cancer Biother Radiopharm 17:553–62. Takino Y, Mochida S. (1982). Propolis its chemical constituents and biological activities. Honeybee. Science 3:145–52. Trinder P. (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann Clin Biochem 6: 24–7.
