Abstract: The present study investigated the protective role of royal jelly against mutagenic effect of adriamycin and/or cobalt gamma radiation in rats. The pretreatment of royal jelly for ten days before adriamycin treatment or/and gamma exposure showed a decrease in their mutagenic effect. Royal jelly administration decreased the different types of chromosomal aberrations induced by adriamycin without significant levels in 2nd, 4th, 7th and 14th days after adriamycin treatment, except the total structural aberrations with gaps in 7th day decreased significantly at p<0.05. Also the royal jelly pretreatment induced decreases of DNA fragmentation induced by adriamycin with a highly significant level (p<0.001) in 4th day after adriamycin treatment and this significant decrease continued for two weeks after adriamycin treatment. The result showed that there was no significant decrease in the number of aberrations in 2nd and 4th days after irradiation, while the total number of structural aberrations decreased significantly at p<0.01 in 7th day and at p<0.05 in 14th days after gamma exposure. Also, the royal jelly pretreatment induced a significant decrease of the DNA fragmentation in 2nd day after gamma exposure at a level p<0.01 and this significant increase reached to p<0.001 level in 4th, 7th and 14th after exposure. The statistical analysis showed that the royal jelly pretreatment decreased the numbers of all aberrations induced in combined treatment (adriamycin plus gamma radiation) group without any significant level, except the total number of structural aberrations in 2nd day where it decreased at p<0.05. The royal jelly pretreatment induced a decrease of DNA fragmentation without significant level in 2nd and 4th days after combined treatment and this decrease reached to the significant level at p<0.001 in 7th day and at p<0.05 in 14th day.
INTRODUCTION
The cancer is the most serious disease that causes death all over the world; it is also known as malignant tumors and neoplasms. It is defined as an abnormal growth of cells which tend to proliferate in an uncontrolled way (Grandis and Sok, 2004).
Adriamycin is widely used as an antineoplastic drug in the treatment of various tumors. It is used for the treatment of solid tumors such as those arising in the breast, bile ducts, liver, esoohagus, soft tissue sarcomas and non-Hodgkin’s lumphoma (Prahalathan et al., 2006). Also, cobalt gamma radiation is a type of ionizing radiation which has been used to treat human malignancies. Gamma radiation therapy was used either alone or in combination with surgery and/or chemotherapeutic modalities (Eriksson, 2006).
The intracellular effect of adriamycin includes free radical formation (Wagner et al., 2005), inhibition of DNA topoisomerase II (Gruber et al., 2005) and nucleotide intercalation (Jain et al., 2005). The DNA-damaging action of adriamycin correlated well with its antiproliferative effect on the cells. This action is appearing as DNA fragmentation, chromosomal aberrations and sister chromatid exchanges.
The radiotherapy treatment of human cancer is often limited by its several side effects; one of its most serious effects is the formation of the secondary tumors (Orta et al., 2006). Gamma radiation induced the increasing of intracellular reactive oxygen species in un-irradiated cells as well as the irradiated cells. The formation of reactive oxygen species may lead to the induction of genomic instability and neoplastic transformation (Goldberg and Lehnert, 2002; Prevost et al., 2004).
The DNA-damaging action of adriamycin or/and gamma is appearing as numerical aberrations (Islam et al., 1993; Touil et al., 2000) and structural aberrations, especially chromatid gaps and breaks (Klimova et al., 1990a, b; Ponnaiya et al., 1997; Hone et al., 2006), sister chromatid exchange (Ratain and Rowley, 1992; Osmak and Horvat, 1992), micronuclei formation (Antoshchina et al., 2005) and formation of small DNA fragments (Fram et al., 1983; Campa et al., 2004, 2005).
Many of the natural and artificial substances are used to reduce the mutagenic effect of chemotherapeutic drugs like adriamycin and radiotherapy like cobalt gamma radiation. These substances have antitumor effect beside their protective effect, so they increase the efficiency of the therapy without any side effects on the normal cells (Swellam et al., 2003). Royal jelly, one of honey products, stimulated cell survival, cell growth and cell differentiation and it also had a cytotoxic effect on the carcinoma cells (Salazar-Olivo and Paz-Gonzalez, 2005). Royal jelly has an anti-tumor effect (Bincoletto et al., 2005) and antimetastatic effect (Kimura et al., 2003).
The aim of this study is to investigate the protective role of royal jelly against chromosomal aberrations and DNA fragmentation induced in rat after adriamycin treatment or/and cobalt gamma exposure.
MATERIALS AND METHODS
Experimental Animals
This study was carried out between January 2005 and January 2006; 150 adult
male Wistar rats weighting 150-200 g were used in this study. The animals were
provided by the animal house of National Research Centre (NRC), Dokki, Cairo,
Egypt. Food and water were given ad libitum. The animals were classified
into seven groups (Table 1).
Anticancer Drug
Adriamycin (doxorubicin hydrochloride, Pharmacia Co.) is an anthracycline
antibiotic. Adriamycin 50 mg vial was dissolved in 25 mL double distilled water
to get a concentration of 2 mg mL-1.
Irradiation
A cobalt-60 source of gamma radiation was used. The animals were irradiated
in Eastern Regional Radioisotope Center for The Arab Countries, Dokki, Cairo,
Egypt. The animals were irradiated once with a dose of 200 rad (2 Gy).
Royal Jelly
Pure Royal jelly capsules were used in this study (Pharco Pharmaceuticals
Co., Egypt). Each capsule (1000 mg) was dissolved in 10 mL double distilled
water to get a concentration of 100 mg mL-1.
| Table 1: | The classification of animal groups under investigation |
| (1): Royal jelly was given by gavage; (2): Adriamycin was given by intraperitoneal injection; (3): Whole body radiation | |
Chromosome Aberration Study Chromosome Preparation
The chromosomes were prepared for microscopic examination according to Yosida
et al. (1971). The animals were injected intraperitoneally with 0.5%
colchicine (15 mg kg-1). After 2 h, the animals were sacrificed by
cervical dislocation. The femurs were removed and cleaned from muscles. Bone-marrow
cells were aspirated from the femur using saline solution (0.9% NaCl). The bone-marrow
cells were centrifuged at 1200 rpm for 10 min. The cell pellet was resuspended
in 5 mL of warmed hypotonic solution (0.075 M KCl) and incubated for 30 min
at 37°C. The cells were centrifuged at 1200 rpm for 10 min. The pellet was
resuspended in 5 mL of the fixative (3:1 methanol/glacial acetic acid) and centrifuged
at 1200 rpm for 10 min. The cells were fixed at least three times before dropping
onto slides, dipped in 70% ethyl alcohol and dried over a hot plate.
Slides Staining
The slides were stained with 10% Giemsa solution (Giemsa stain/Sorenson's
buffer) for 30 min and washed in Sorenson's buffer for 1 min. Then, the slides
were immersed in distilled water and air dried.
Scoring
For chromosomal aberration, 50 well spread metaphases were examined for
each animal. Both structural aberrations (deletions, breaks, gaps and fragments)
and numerical aberrations (polyploidy and aneuploidy) were recorded.
DNA Fragmentation
DNA fragmentation was quantified by diphenylamine (DPA) method according
to Gibb et al. (1997) with little modifications. About 0.2 g of rat liver
was homogenized in 5 mL of saline solution (0.9% NaCl). Two milliliter of the
liver homogenization were centrifuged at 1500 rpm for 10 min at 4°C. The
cell pellet was resuspended in 1600 μL of 0.01 M PBS and then 1400 μL
of ice cold lysis buffer (5 mM Tris + 20 mM EDTA + 0.5% Triton X-100, pH 8.0)
were added to the suspension and incubated on ice for 15 min to isolate DNA.
The suspension was centrifuged at 13000 rpm for 20 min at 4°C.
After centrifugation, the supernatant which contains the fragmented DNA was transferred to a new tube. The pellet which contains the intact DNA was resuspended in 3 mL of TE buffer (10 mM Tris + 1 mM EDTA). Three milliliter of 10% Trichloroacetic Acid (TCA) were added to both fragmented and intact DNA and incubated at room temperature for 10 min.
The TCA precipitations were centrifuged at 2000 rpm for 15 min at 4°C. After discarding the supernatants, DNA hydrolyte by resuspended the pellets in 1400 μL of 5% TCA and then boiled at 100°C for 15 min and allowed to cool at room temperature. The suspensions were centrifuged at 1500 rpm for 15 min at 4°C and 1 mL of supernatants was transferred to new glass tubes. Two milliliter of diphenylamine (DPA) were added to the supernatants and then incubated overnight at 30°C. Blank was prepared by adding 1 mL of 5% TCA to 2 mL of DPA.
The amounts of both fragmented and intact DNA were determined by spectrophotometer at 600 nm. The fragmentation of DNA was calculated according to the equation:
Statistical Analysis
Chi-square test was used for statistical analysis of the results for the
chromosomal aberrations, while the t-test of the difference between means was
used for statistical analysis of results for DNA fragmentation (Othman, 2000).
RESULTS
Chromosomal Aberrations
The structural chromosomal aberrations investigated in this study included
gaps (Fig. 2), deletions (Fig. 3), breaks
(Fig. 4, 5) and fragments (Fig.
6), while the numerical abnormalities included the polyploid (Fig.
7) and aneuploid cells (Fig. 8, 9).
Figure 1 shows a metaphase spread of rat bone marrow cell
showing normal chromosomes.
From Table 2, it was observed that the total numbers of structural and numerical chromosomal aberrations induced in rat bone-marrow cells after treatment with adriamycin increased with significantly levels in all subgroups. The statistical comparison between the subgroups treated with adriamycin alone and those treated with adriamycin after royal jelly pretreatment showed that the different types of aberrations decreased without significant level, except the total structural aberrations with gap in 7th day subgroup that decreased significantly at p<0.05.
Table 3 showed that the total numbers of different structural and numerical chromosomal aberrations induced in rat bone-marrow cells after exposure to gamma radiation increased significantly at p<0.001 in all subgroups. Regarding to the effect of royal jelly pretreatment, it was shown that there was no significant decrease in the number of aberrations in 2nd and 4th days after irradiation, while the number of the breaks in 7th day and the number of the deletions in 14th day decreased significantly at p<0.05. The total number of structural aberrations decreased significantly at p<0.01 in 7th day and at p<0.05 in 14th day after gamma irradiation.
| Fig. 1: | Metaphase spread of rat bone marrow cell showing normal chromosomes. (Giemsa stain, X 1000) |
| Fig. 2: | Metaphase spread of rat bone marrow cell showing a chromatid gap (G). (Giemsa stain, X 1000) |
| Fig. 3: | Metaphase spread of rat bone marrow cell showing deletion (D). (Giemsa stain, X 1000) |
| Fig. 4: | Metaphase spread of rat bone marrow cell showing a chromosome break (cB). (Giemsa stain, X 1000) |
| Fig. 5: | Metaphase spread of rat bone marrow cell showing a chromatid break (B). (Giemsa stain, X 1000) |
| Fig. 6: | Metaphase spread of rat bone marrow cell showing a fragment (F). (Giemsa stain, X 1000) |
| Fig. 7: | Metaphase spread of rat bone marrow cell showing a polyploid cell (Giemsa stain, X 1000) |
| Fig. 8: | Metaphase spread of rat bone marrow cell showing an aneuploid cell (2n-1) (Giemsa stain, X 1000) |
| Fig. 9: | Metaphase spread of rat bone marrow cell showing an aneuploid cell (2n+1) (Giemsa stain, X 1000) |
| Table 2: | The effect of royal jelly on chromosomal aberrations induced by adriamycin in rat bone-marrow cells |
| ADR: Adriamycin; R.j: Royal jelly; Del: Deletion; Br: Break; Frag: Fragment; Poly: Polyploidy; Aneu: Aneuploidy. a: p<0.05; b: p<0.01; c: p<0.001, a, b and c: The significant levels of the comparison between the control and treated groups. d: p<0.05, d: The significant level of the comparison between adriamycin and adriamycin plus royal jelly groups | |
| Table 3: | The effect of royal jelly on chromosomal aberrations induced by cobalt gamma radiation in rat bone-marrow cells |
| RAD: Radiation; R.j: Royal jelly; Del: Deletion; Br: Break; Frag: Fragment; Poly: Polyploidy; Aneu: Aneuploidy. a: p<0.05; b: p<0.01; c: p<0.001, a, b and c: The significant levels of the comparison between the control and treated groups, d: p< 0.05; e: p<0.01, d and e: The significant levels of the comparison between 60Co gamma radiation and radiation plus royal jelly groups | |
| Table 4: | The effect of royal jelly on chromosomal aberrations induced by combined treatment in rat bone-marrow cells |
| Com:combined treatment; R.j: Royal jelly; Del: deletion; Br: Break; Frag: Fragment; Poly: Polyploidy; Aneu:Aneuploidy, a: p<0.05; b: p<0.01; c: p<0.001, a, b and c: The significant levels of the comparison between the control and treated groups. d: p<0.05 d: The significant level of the comparison between combined treatment and combined treatment plus royal jelly groups | |
The total number of structural and numerical chromosomal aberrations increased after combined treatment with a highly significant level at p<0.001 in all subgroups (Table 4), except the total number of structural aberrations without gap in 14th day increased significantly at p<0.01. The statistical comparison between the subgroups treated with combined treatment and the subgroups treated with combined treatment plus royal jelly showed that the numbers of all aberrations decreased without any significant level, except the total number of structural aberrations in 2nd day, it decreased with a low significant level (p<0.05).
DNA Fragmentation
Table 5 shows the effect of adriamycin with or without
royal jelly on DNA fragmentation. The statistical comparison between the negative
control group and adiamycin-treated subgroups showed the significant increase
of the means of DNA fragmentation in all days at p<0.001. The statistical
comparison between the subgroups treated with adriamycin alone and those treated
with adriamycin after royal jelly pretreatment showed a high significant decrease
at p<0.001 in the means of DNA fragmentation in 4th, 7th and 14th days after
adriamycin treatment but the significant level was absent in 2nd day.
The statistical comparison between the negative control and the subgroups exposed to gamma radiation showed the high significant increase of the means of DNA fragmentation in all days at p<0.001. The statistical comparison between the subgroups exposed to gamma radiation alone and those exposed to gamma radiation after royal jelly pretreatment showed the significant reduction in the means of DNA fragmentation in all days. In 2nd day after irradiation, the significant level of the reduction was at p<0.01, while in 4th, 7th and 14th days, the significant level was at p<0.001 (Table 6).
The high significant increase of the means of DNA fragmentation in all days was noted in the statistical comparison between the negative control and the subgroups after combined treatment (p<0.001).
| Table 5: | The effect of royal jelly on the DNA fragmentation induced by adriamycin in rat liver cells |
| ADR: Adriamycin; R.j: Royal jelly; SD: Standard Deviation, c: p<0.001,c: The significant level of the comparison between the control and treated groups. f:. p<0.001, f: The significant level of the comparison between adriamycin and adriamycin plus royal jelly groups | |
| Table 6: | The effect of royal jelly on DNA fragmentation induced by cobalt gamma radiation in rat liver cells |
| RAD: radiation; R.j: Royal jelly; SD: Standard Deviation, c: p<0.001,c: The significant level of the comparison between the control and treated groups. e:. p<0.01; f: p<0.001, e and f: The significant level of the comparison between 60Co gamma radiation and 60Co gamma plus royal jelly groups | |
| Table 7: | The effect of royal jelly on DNA fragmentation induced by combined treatment in rat liver cells |
| Comb.: combined treatment; R.j: Royal jelly; SD: standard deviation,c: p<0.001, c: The significant level of the comparison between control groups and treated groups, d:. p<0.05; e: p<0.01, d and e: The significant level of the comparison between combined treatment and combined treatment plus royal jelly groups | |
While the comparison between the subgroups after combined treatment and after combined treatment plus royal jelly showed a decrease without any significant level in 2nd and 4th days after treatment. The significant decrease at p<0.01 appeared in 7th day, while it was at p<0.05 in 14th day after treatment (Table 7).
DISCUSSION
Chemotherapy and radiotherapy are widely used for cancer treatment. The basic action mode of these both therapies is making lesions in the malignant cells led to the apoptosis. But they are also possibly related to the induction of carcinogenesis and mutagenesis in the normal cells. Some natural and artificial substances are used to reduce the toxic effect of chemotherapy and radiotherapy; these substances are known as antimutagenic agents or protective agents.
The present study was designed to investigate the protective role of royal jelly against mutagenicity induced in rats after treatment with adriamycin or/and exposure to cobalt gamma radiation. The chromosomal aberrations; numerical and structural aberrations and DNA fragmentation are the parameters used in this study.
Adriamycin and gamma radiation induced DNA double strand breaks, this action is correlated with their antiproliferative effect on the cells (Nishio et al., 1982). DNA double strand breaks can induce cell cycle arrest, induction of apoptosis or mitotic cell death caused by loss of genomic material (Olive, 1998). These different modes determine the therapeutic success of cancer treatment but, adversely, also side effects are possibly caused in normal tissue (Rothkamm and Löbrich, 2002). The non- or misrepair of DNA double-strand breaks lead to chromosomal aberrations especially the chromatid aberrations (Klimova et al., 1990a). DNA double strand breaks induced the cell cycle arrest by activation of p53 (Jackson and Pereira-Smith, 2006). The adriamycin-induced p53 activation arrested the cell cycle at G2-phase or S-phase (Minemoto et al., 2001) whereas exposure to gamma radiation induced the cell cycle arrest at G2/M checkpoint (Yuan et al., 2003). The activation of p53 is followed by the formation of DNA fragments (Wang et al., 2004). DNA fragmentation is used to detect the apoptosis (Zhan et al., 2001).
The present results showed that the separated and combined treatment with adriamycin and/or cobalt gamma radiation induced the significant increase in the frequencies of chromosomal aberrations, structural and numerical, in all treated groups when compared with those in negative controls.
The cytogenetic measurement of adriamycin mutagenic effect showed induction of the structural chromosomal aberrations in the previous studies, especially the chromatid aberrations (Klimova et al., 1990a, b; Khlusova et al., 1992). Regarding the numerical chromosomal aberrations, our findings agreed with result of Aly et al. (1999). They recorded a significant increase of trisomy chromosomes 7 and 17 in blood cultures after exposure to adriamycin.
Chromosomal aberrations are also considered to be highly sensitive biological indicators for the effects of ionizing radiation (Varanda and Takahashi, 1993). Ponnaiya et al. (1997) reported an increase in the chromatid-type gaps and breaks in human epithelial cells after exposure to gamma radiation. The numerical chromosomal aberrations were also induced by gamma ionized radiation (Devi and Satyamitra, 2005), especially an increase in the number of aneuploid cells. Newcomb et al. (1989) recorded tumor production in mice treated with ionized gamma radiation characterized by trisomy of chromosome 15, while, Kang et al. (2006) recorded the increase in the copy number of chromosomes 4 and 5 and loss in the copy number of chromosomes 11, 16 and 19.
The activation of p53 is followed by the formation of DNA fragments (Wang et al., 2004). DNA fragmentation is used to detect the apoptosis (Zhan et al., 2001). Adriamycin-induced DNA fragmentation was reported in rat cells by Jang et al. (2004) and Kim et al. (2005) and in mouse cells by Dominguez-Rodriguez et al. (2001). Also in this study, DNA fragmentation was increased in all days of experiment with a highly significant level. The effect of 60Co gamma radiation on the formation of DNA fragments was similar to the adriamycin effect. This result agreed with the results of Liu and Zhang (2003), who measured the DNA fragmentation in mouse bone-marrow cells after irradiated with 60Co gamma radiation and Campa et al. (2004), who reported the formation of DNA fragments in Chinese hamster V79.
The combined therapy has a genotoxic effect more than either treatment given alone (Jagetia and Nayak, 2000; Dutta et al., 2005; Bergs et al., 2006). The strong mutagenic effect of combined treatment showed in this study agreed with the results of Lichter and Lawrence (1995), Bilban-Jakopin and Bilban (2001) and Lègal et al. (2002).
Royal jelly, a substance produced by bee worker, used as a treatment for many diseases (Leigh, 1999) like reduction of total lipids and cholesterol level in the serum and liver, improvement of the highly coagulant status of blood (Shen et al., 1995; Vittek, 1995) and suppression of the development of atopic dermatitis-like skin lesions (Taniguchi et al., 2003) and it has anti-allergic effect (Oka et al., 2001; Okamoto et al., 2003), anti-fatigue effect (Kamakura et al., 2001) and immunomodulatory effect (Sver et al., 1996). Royal jelly also has anti-tumor effect (Tamura et al., 1987; Bincoletto et al., 2005; Kimura et al., 2003; Salazar-Olivo and Paz-Gonzalez, 2005).
In the present study, the pretreatment of royal jelly for ten days before adriamycin treatment decreased the percentages of total structural and numerical chromosomal aberrations induced by adriamycin in all days of experiment. Also the protective effect of royal jelly against genotoxicity of adriamycin appeared in the study of DNA fragmentation. When the royal jelly was administrated for ten days before adriamycin treatment, DNA fragmentation decreased with a highly significant level (p<0.001) which appeared after four days from the last treatment and continued for two weeks.
The protective effect of royal jelly appeared clear when it combined with cobalt gamma radiation (60Co). The total number of structural chromosomal aberrations decreased significantly after royal jelly pretreatment when it was compared with those in groups exposed to cobalt gamma radiation (60Co) alone. The numerical chromosomal aberrations also decreased in the royal jelly pretreatment group but did not reach any significant level. On the other hands, the royal jelly pretreatment induced a significant decrease in DNA fragmentation The royal jelly had a slightly protective effect against the genotoxicity of adriamycin/60Co gamma radiation combined treatment and its effect was less than that in separated treatment groups. There was no significant difference between the total numbers of chromosomal aberrations induced by combined treatment with and without royal jelly pre-treatment. On the other hands, the significant levels appeared between DNA fragmentation with and without royal jelly pretreatment was p<0.01 after one week and p<0.05 after two weeks from the last treatment. The slightly protective effect of royal jelly with the combined treatment may be due to the increase of the cell sensitivity to ionizing radiation by the chemotherapeutic drug, adriamycin, beside its direct effect on dividing cells (Lichter and Lawrence, 1995).
CONCLUSION
The protective effect of royal jelly may be due to its component vitamins, antioxidant vitamins A, E and C, vitamin D and vitamin B complex (Leigh, 1999). These vitamins themselves had anticancer effect (Pour and Lawson, 1984; Liu et al., 2000; Klaassen and Braakhuis, 2002; Giovannucci et al., 2006; Zou et al., 2006) and protective effects against the genotoxicity of chemotherapy and radiotherapy (Parchure et al., 1984; Chen and Pan, 1988; Sarma and Kesavan, 1993; Antunes and Takahashi, 1998, 1999; Tavares et al., 1998; Konopacka et al., 2002; Gülkaç et al., 2004; Kocak et al., 2004). Royal jelly also contained protein fractions (Salazar-Olivo and Paz-Gonzalez, 2005) that they had also anticancer effects.