British Journal of Radiology (2006) 79, 415-418
© 2006 British Institute of Radiology
doi: 10.1259/bjr/40692384
Radioprotective effects of hesperidin against gamma irradiation in mouse bone marrow cells
S J Hosseinimehr, PhD1 and
A Nemati, MSc2
1 Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandarn University of Medical Sciences, Sari, 2 Novin Medical Radiation Institute, Tehran, Iran
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Abstract
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The radioprotective effects of hesperidin (HES), a flavonone glucoside, were investigated by using the micronucleus test for anticlastogenic and cell proliferation activity. A single intraperitoneal (ip) administration of hesperidin at doses of 10 mg kg–1, 20 mg kg–1, 40 mg kg–1, 80 mg kg–1 and 160 mg kg–1 45 min prior to gamma irradiation (2 Gy) reduced the frequencies of micronuleated polychromatic erythrocytes (MnPCEs). All five doses of HES significantly reduced the frequencies of MnPCEs and increased PCE/PCE+NCE ratio in mice bone marrow compared with non-drug-treated irradiated control (p<0.0001). There was a drug dose–response effect of HES in reducing MnPCE and increasing the PCE/PCE+NCE ratio in bone marrow cells. The maximum reduction in MnPCE was observed in mice treated with HES at a dose of 80 mg kg–1. The total MnPCE values were 2.85 fold less in the 80 mg kg–1 HES group after being exposed to 2 Gy of 
-rays than those in the respective irradiated control. Our study demonstrates that hesperidin has powerful protective effects on the radiation-induced DNA damage and on the decline in cell proliferation in mouse bone marrow.
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Introduction
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Ionizing radiation generates reactive oxygen species in the cells. These free radicals can induce damage to critical macromolecules such as DNA. The cellular DNA damage leads to mutation and cancer [1]. High levels of gamma irradiation can induce mortality in mammals. With respect to radiation damage to humans, it is important to protect biological systems from radiation-induced genotoxicity or lethality. The main radioprotective class is thiol synthetic compounds such as amifostine. Amifostine is a powerful radioprotective agent compared with other agents, but this drug is limited in the use in clinical practice due to side effects and toxicity [2–4]. The search for less-toxic radiation protectors has spurred interest in the development of natural products. Recently, we reported that citrus extract protects mouse bone marrow cells against gamma irradiation. The citrus extracts contained high amounts of flavonoids [5]. Flavonoids are a family of polyphenolic compounds found in fruits and vegetables. Flavonoids have wide biological properties including antibacterial, antiviral, anticancer, immunostimulant and antioxidant effects [6]. Hesperidin (HES) is a flavonone glycoside, belonging to the flavonoid family. This natural product is found in citrus species. HES was reported to have many biological effects including anti-inflammatory, antimicrobial, anticarcinogenic and antioxidant effects, and decreasing capillary fragility [7]. HES, in combination with a flavon called diosmin is used as Daflon® (Servier, France) to treat chronic venous insufficiency in Europe [8]. Other biological effects of HES are unknown. In continuation of this line of investigation, the in vivo radioprotective activity of HES was investigated by using gamma rays as an oxidative DNA damaging agent, and evaluating any reduction in the frequency of micronuclated polychromatic erythrocytes (MnPCEs) in mouse bone marrow exposed to gamma rays.
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Materials and methods
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Animals
Male NMRI mice weighing 25±3 g were purchased from the Razi Institute (Tehran, Iran). Mice were housed in accordance to the principles outlined in "The Guide for The Care and Use of Laboratory Animals" prepared by Tehran University of Medical Sciences in the university animal house, and given standard mouse pellets and water ad libitum. All animals were kept under controlled lighting conditions (light:dark, 12:12 h) and temperature (22±1°C).
Chemicals and treatment
HES was from Aldrich (USA). HES was dissolved in phosphate buffered saline (pH 7.6). Mice were injected intraperitoneally for all experiments. For selection of the optimum dose of HES for radioprotection, five doses (10 mg kg–1, 20 mg kg–1, 40 mg kg–1, 80 mg kg–1, 160 mg kg–1) of HES were administrated to the experimental animals 45 min before gamma irradiation. The control animals received the same volume of normal saline or buffer phosphate. Five mice were used for each treatment group.
Irradiation
Whole-body irradiation was performed with a cobalt-60 -radiation source (Teratron 780, Canada). Mice were placed in ventilated Plexiglas® cages and irradiated in groups of five mice, simultaneously. The source-to-skin distance was 80 cm with a dose rate of 1.03 Gy min–1 at room temperature (23±2°C). The mice were irradiated with a total dose of 2 Gy -rays.
Micronucleus assay
The mouse bone marrow micronucleus test was carried out according to the method described by Schmid for evaluation of the chromosomal damage in experimental animals [5, 9]. The animals were euthanized by cervical dislocation 24 h after irradiation. The bone marrow from both femurs was flushed in the form of a fine suspension into a centrifuge tube containing fetal calf serum (FCS). The cells were dispersed by gentle pipetting and collected by centrifuge at 2000 rpm for 5 min at 4°C. The cell pellet was resuspended in a drop of FCS and bone marrow smears were prepared. The slides were coded to avoid observer bias. After 24 h air-drying, the smears were stained with May-Grunwald/Giemsa, as described by Schmid. With this method, polychromatic erythrocytes (PCEs) stain reddish-blue and normochromatic erythrocytes (NCEs) stain orange, while nuclear material is dark purple. For each experimental point, five mice were used and a total of 5000 PCEs were scored for each experimental point to determine the percentage of micronucleated polychromatic erythrocytes (MnPCEs), micronucleated normochromatic erythrocytes (MnNCEs) and ratio of PCE to (PCE + NCE). The ratio of PCE to (PCE + NCE) was determined for each experimental group to assess radiation effects with or without HES on bone marrow proliferation [10].
Statistical analysis
The data are presented as mean±standard deviation (SD). One-way analysis of variance (ANOVA) analysis and Tukey's HSD test were used for multiple comparisons of data.
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Results
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The effect of gamma irradiation with or without HES on the induction of MnPCEs and the PCE/PCE+NCE ratio in bone marrow cells, 24 h after -irradiation, is shown in Table 1
. The frequency of micronuclei was increased in all groups of mice irradiated with 2 Gy -irradiation compared with the control treated with normal saline or phosphate buffer (p<0.0001). The frequencies of MnPCE found in the HES treated groups were significantly lower than that of the group treated with radiation alone. The total MnPCE values were 1.15, 1.36, 1.66, 2.85 and 1.8 fold less in the 10 mg kg–1, 20 mg kg–1, 40 mg kg–1, 80 mg kg–1 and 160 mg kg–1 HES group after being exposed to 2 Gy of -rays, respectively, than those in the respective irradiated control (Figure 1). All five doses were effective in significantly reducing (p<0.0001) the frequency of MnPCE induced by 2 Gy irradiation and there was a significant difference between the effects of various doses of HES. There was a drug dose–response effect of HES in the reduction of MnPCE in bone marrow cells. The maximum reduced MnPCE was observed in mice treated with HES at a dose of 80 mg kg–1 (Figure 1). The frequency of MnPCE in the latter group was 5.01±0.32%, much lower than in the group receiving radiation alone (14.29±0.5). With a further increase in the HES dose to 160 mg kg–1, there was a reduced effect of HES on the frequency of MnPCE induced by -irradiation. The ratio of PCE/PCE+NCE reduced significantly after exposure to 2 Gy of -irradiation (p<0.0001). Determination of ratio of PCE/PCE+NCE in the gamma irradiated mice showed a pronounced cytotoxic effect of radiation on bone marrow proliferation. Treatment of mice with HES arrested the radiation-induced decline in the PCE/PCE+NCE ratio (Table 1), and this increase in the PCE/PCE+NCE ratio in the HES+irradiated group (at doses 20 mg kg–1, 40 mg kg–1, 80 mg kg–1, 160 mg kg–1) was higher than that of the irradiated-alone group (p<0.001). The highest PCE/PCE+NCE ratio was observed in HES treated mice with 80 mg kg–1 before -irradiation (Figure 2). There was no significant difference between phosphate buffer-control and HES treated mice at this dose before -rays. In this study, HES did not indicate any genotoxic and toxic effects at 40 mg kg–1 and 80 mg kg–1, but genotoxicity was observed at 160 mg kg–1 compared with phosphate buffer (p<0.001).
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Table 1. Effects of hesperidin(HES) on the formation of radiation-induced micronulei polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) and the ratio of PCE/PCE+NCE in mice bone marrow exposed to 2 Gy -irradiation
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Figure 1. Effect of various doses of hesperidin(HES) on the frequency of micronucleated polychromatic erythrocytes (MnPCEs) in the bone marrow of mice exposed to -radiation (R) at a dose of 2 Gy.
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Figure 2. Effects of various doses of hesperidin(HES) on the radiation induced polychromatic erythrocyte (PCEs)/PCE+ normochromatic erythrocytes (NCEs) ratio in the bone marrow of mice exposed to -radiation (R) at dose 2 Gy.
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Discussion
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We previously reported that citrus extracts could protect mice bone marrow cells against gamma irradiation and cyclophosphamide, when injected prior to exposure and cyclophosphamide treatment. These citrus extracts contain high levels of flavonoids [5, 11]. The results of this study demonstrated the protective effects of HES, a flavonone, against genotoxicity and toxicity induced by -irradiation in mouse bone marrow cells. However, synthetic compounds, mainly thiol compounds, have good radioprotective effects, but they are limited in their use by side effects. Natural compounds, including flavonoids, may play a role in scavenging free radicals, such as hydroxyl radicals generated by -rays in cells. Ionizing radiation generates free radical damage in DNA and induces genotoxic effects and death in the cells [1, 12]. There is a possibility that pre-treatment with flavonoids could induce protection against oxidative stress. Orientin and Vicenin, two flavonoids, protect mice against chromosomal aberration induced by -irradiation when administrated before 2 Gy -rays [13]. HES is the predominant flavonoid in lemons and oranges. The peel and membranous parts of these fruits have the highest HES concentrations. Sweet or navel oranges (Citrus sinensis) and lemons (Citrus limon) are the richest dietary sources of HES [14]. In this study, HES had a dose-dependent protective effect on reducing MnPCEs induced by gamma irradiation. The greatest protective effect was observed at a dose of 80 mg kg–1. HES did not show any genotoxic or toxic effects at doses up to 80 mg kg–1 in mice bone marrow cells. Treating mice with HES at dose 80 mg kg–1 before exposure to 2 Gy radiation reduced the frequency of MnPCEs almost 2.85 fold. The percentage PCE/PCE+NCE ratio declined in irradiated mice, since this ratio gives a direct index of cell division. HES protected mice against radiation-induced decline in cell proliferation, as evidenced by the increased PCE/PCE+NCE ratio. The molecular mechanism of the radioprotective effects of HES is not clear. It has been reported that flavonoids have antioxidant and chelating properties. These polyphenols are excellent scavengers of free radicals due to the high reactivity of their hydroxyl substituents [12]. Free-radical scavenging is apparently responsible for the inhibitory effect of flavonoids such as rutin, morin, quercetin and genestin on the clastogenic activity induced by -irradiation in mice [15]. Among naturally occurring flavonoids, HES has been pharmacologically evaluated as a potential anticarcinogenic agent because of its antioxidant activity [8, 16]. Other biological effects include immune-modulation, treatment of venous insufficiency and scavenging of peroxynitrite as a reactive oxidant [7, 17]. HES has also protected against photo-induced breakage of DNA [18]. It is possible that HES protects bone marrow cells with its antioxidant activity. However, the antioxidant capacity of HES is not as high as that of other flavonoids such as quercetin and myricetin [19, 20]. Other mechanisms probably contribute to its radioprotective effects. Thus, further experiments are needed to explain the molecular mechanism of HESs protective effects.
In conclusion, our results demonstrate that HES gives significant protection to mice bone marrow against the clastogenic effects of gamma irradiation.
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Acknowledgments
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We would like to thank Mr Mahmoudzadeh, Dr Zahmatkesh, Dr Akhlaghpour and Dr Abassi from the Novin Medical Radiation Institute for their assistance.
Received for publication April 22, 2005.
Revision received September 20, 2005.
Accepted for publication October 12, 2005.
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Abstract
Introduction
