Mutagenesis vol. 18 no. 4 pp. 337-343,
July 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Naringin, a citrus flavonone, protects against radiation-induced chromosome damage in mouse bone marrow
Department of Radiobiology, Kasturba Medical College, Manipal 576 119, India
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Abstract |
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Free radicals are responsible for the induction of damage to the cellular DNA that leads to the formation of chromosome aberrations. Antioxidants are known to scavenge free radicals, thereby decreasing the degree of such effects. Radiation is a well-known inducer of free radicals and compounds that can scavenge free radicals may reduce radiation-induced DNA damage. Naringin, a bioflavonoid predominant in grapefruit and other citrus fruits, has been found to scavenge free radicals, therefore it may also reduce radiation-induced damage. The aim of the present study was to evaluate the radioprotective action of 2 mg/kg naringin in the bone marrow of mice exposed to different doses of 60Co
-radiation by scoring the frequency of asymmetrical chromosomal aberrations. The irradiation of mice resulted in a dose-dependent elevation in the frequency of aberrant cells, acentric fragments, chromatid and chromosome breaks, dicentrics and exchanges. All these aberrations were elevated with scoring time up to 24 h post-irradiation and declined thereafter, except chromatid breaks, which were maximum at 12 h post-irradiation. Treatment of mice with 2 mg/kg body wt naringin before exposure to various doses of -radiation resulted in a significant reduction in the frequencies of aberrant cells and chromosomal aberrations like acentric fragments, chromatid and chromosome breaks, centric rings, dicentrics and exchanges. The evaluation of free radical scavenging activity of naringin revealed a dose-dependent scavenging of hydroxyl, superoxide and 2,2
-diphenyl-1-picryl hydrazyl radical. Naringin at 5 µM scavenged the 2,2-azino-bis-3-ethyl benzothiazoline-6-sulphonic acid cation radical very efficiently, where a 90% scavenging was observed. Our study demonstrates that naringin can protect mouse bone marrow cells against radiation-induced chromosomal damage. |
Introduction |
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Chromosomal aberrations can be detected during post-irradiation mitosis and are best scored during metaphase. Chromosomal aberrations can be numerical (change in number) as well as structural (Savage, 1975
). Exposure to ionizing radiation or environmental mutagens and carcinogens may lead to genomic instability. The use of certain chemical agents may either arrest or reduce the mutagenic and/or carcinogenic potential of certain environmental toxicants, including radiation.
Since the discovery by Patt et al. (1949) that cysteine protected rats and mice against radiation-induced sickness and mortality, several attempts have been made to reduce radiation-induced damage (Yuhas and Storer, 1969; Modig et al., 1977). However, the practical applicability of the majority of these compounds to humans remains limited, owing to their high toxicity at their optimum protective doses (Sweeny, 1979).
Little attention has been paid to ordinary metabolites, which form part of the daily human diet (Hertog et al., 1993a,b). It is likely that an effect of such agents may be beneficial for patients undergoing radiotherapy, simply because their concentration may be more easily and safely manipulated than those of other chemicals. The patients may also tolerate them better than other more promising exotic drugs since they form part of the daily human diet. The flavonoids are polyphenolic compounds with antioxidant properties and are widely distributed in foods of plant origin such as vegetables, fruits, tea and wine (Hertog et al., 1993b). Their antioxidant effects are greater than vitamins C and E, selenium and zinc and they modify the body’s reaction to compounds such as allergens, viruses and carcinogens.
Naringin (glycoside) is a ‘bioflavonoid’ derivative of grapefruit peel and related citrus species. It has been reported to protect against oxygen free radical-stimulated K+ permeability (Maridonneau-Parini, 1986) and is predominantly found in Citrus paradisi, Citrus sinensis, Citrus unshiu, Citrus nobilis, Citrus tachibana, Citrus junos, Artemisia selengensis and Artemisia stolonifera (Swiader and Zarawska, 1996), roots of Cudrania cochinchinensis var. geronatogea (Lin et al., 1996), aerial parts of Thymus herba barona (Corticchiato et al., 1995), fruits of Pon cirus spp. (Kim< et al., 1994), Swertia polyphylla (Dubeois and Seneden, 1995) and related citrus species. Like most flavanoids, naringin has metal chelating, antioxidant and free radical scavenging properties (Jung et al., 1983; Kroyer, 1986; Chen et al., 1990) and has been reported to offer protection against mutagenesis (Francis et al., 1989). The antioxidant and antimutagenic activity of naringin stimulated us to investigate its radioprotective effect against the induction of chromosome aberrations in the bone marrow of mice exposed to different doses of whole body 60Co -radiation.
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Materials and methods |
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The animal care and handling were done according to the guidelines set by the World Health Organisation (Geneva, Switzerland) and INSA (Indian National Science Academy, New Delhi, India). Eight- to ten-week-old male Swiss albino mice weighing 30–36 g were selected from an inbred colony maintained under controlled conditions of temperature (23 ± 2°C), humidity (50 ± 5%) and light (10 light/14 h dark). The animals were provided with sterile food and water ad libitum. Four animals were housed in a polypropylene cage containing sterile paddy husk (procured locally) as bedding throughout the experiment. The study was cleared by the institutional animal ethical committee.
All chemicals were procured from Sigma Chemical Co. (St Louis, MO) unless otherwise stated.
Preparation of the drug
Naringin, 4H-1-benzopryran-4-one,7-[ [2-O-(deoxy-
-L-mannopyranosyl) -ß-D-glucopyranosyl]osyl]-2,3-dihydro-5-hydroxy-2-(4-hydroxyphenyl(S)), was procured from Acros Organics Ltd (Belgium) and dissolved in sterile double-distilled water (DDW) freshly each time immediately before use.
Mode of administration
The animals were administered naringin or DDW i.p. 45 min before irradiation.
Experimental protocol
The animals were divided into the following groups. DDW + irradiation group: the animals in this group received 0.01 ml/g body wt sterile DDW before exposure to various doses of -radiation. NIN + irradiation group: the animals in this group were injected with 2 mg/kg body wt naringin before exposure to various doses of -radiation.
Irradiation
Forty-five minutes after DDW or drug administration, the prostrate and immobilized animals (achieved by inserting cotton plugs in the restrainer) were whole body exposed to 0, 0.5, 1, 2, 3 or 4 Gy of 60Co -radiation from a teletherapy source (Theratron; Atomic Energy Agency, Canada) in a specially designed well-ventilated acrylic box. A batch of eight animals (four each from the DDW and drug-treated groups) was irradiated each time at a dose rate of 1.66 Gy/min at a source to animal distance (mid point) of 87 cm.
Preparation of metaphase plates
The animals from both groups were injected with 2.5 mg/kg body wt colchicine (C-9754, lot 82H0005) 2 h before killing to arrest the cells in metaphase. The metaphase plates were prepared as described by Jagetia (1993). Briefly, the animals were killed at 12, 24 and 48 h post-irradiation by cervical dislocation, the femora of each animal were removed, cleaned and the marrow was flushed out into normal saline. The cells were centrifuged and subjected to a mild hypotonic treatment (0.56% ammonium oxalate). After incubation, the cells were centrifuged and the resultant pellet was fixed in Carnoy's fixative (3:1 methanol:acetic acid). After proper fixation the suspension was dropped onto pre-cleaned coded chilled slides to avoid observer bias. The slides were air dried and the cells were stained with aqueous Giemsa (catalogue no. 0546750; BDH, UK). One-hundred well-spread metaphase plates were scored from each animal for the presence of asymmetrical chromosome aberrations. Four animals were used for each radiation dose for each group.
The data regarding the aberrant cells, chromatid and chromosome breaks, dicentrics, centric rings, exchanges, acentric fragments (apart from fragments, double minutes and acentric rings were also included in this class), total aberrations, polyploidy and severely damaged cells (SDC, cells bearing 10 or more aberrations) were collected according to the criteria of Savage (1975). The statistical analysis was carried out using the Mann–Whitney U-test and the data were fitted to a linear or linear quadratic model. Solo 4 (BMDP Inc.) statistical software was used for data analysis.
Estimation of free radical scavenging in vitro
Hydroxyl radical scavenging activity
The scavenging of •OH free radicals was measured by the method described by Halliwell et al. (1987). Briefly, the reaction mixture contained 2.8 mM deoxyribose, 0.05 M KH2PO4–NaOH buffer, pH 7.4, 0.1 mM FeCl3, 0.1 mM EDTA, 1 mM H2O2, 0.1 mM ascorbate and 50–1000 nM naringin in a final volume of 2 ml. The reaction mixture was incubated for 30 min at ambient temperature, followed by the addtion of 2 ml of trichloroacetic acid (2.8% w/v) and thiobarbituric acid. The reaction mixture was kept in a boiling water bbath for 30 min, cooled and the absorbance was read at 532 nm in a UV-visible double beam spectrophotometer (UV-260; Shimadzu Corp., Japan).
Superoxide anion scavenging activity
The scavenging of superoxide anion was measured as described by Hyland et al. (1983). Briefly, the reaction mixture contained various concentrations of naringin (50–1000 nM), nitroblue tetrazolium and alkaline dimethyl sulfoxide (DMSO). The blank consisted of pure DMSO instead of alkaline DMSO. The absorbance was read at 560 nm using a UV-visible double beam spectrophotometer (UV-260; Shimadzu Corp., Japan).
DPPH scavenging activity
The principle for reduction of the 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical is that the antioxidant reacts with the stable free radical and converts it to 2,2-diphenyl-1-picryl hydrazine (Scheme
). To an ethanolic solution of DPPH (0.05 mM), an equal volume of naringin (5–400 µM) dissolved in water was added to a final volume of 1.0 ml. An equal amount of methanol was added to the control. After 20 min, absorbance was recorded at 517 nm in a UV-visible double beam spectrophotometer (UV-260; Shimadzu Corp., Japan).
Total antioxidant activity assay
Total antioxidant potential was determined by 2,2-azino-bis(3-ethyl benzothiazoline-6-sulphonic acid) diammonium salt (ABTS) assay as described by Miller et al. (1996). This technique measures the relative ability of antioxidant substances to scavenge the ABTS•+ cation radical generated in the aqueous phase. The reaction mixture contained 0.17 mM ABTS, 5–40 µM naringin and buffer in a total volume of 3.5 ml. The absorbance was measured at 734 nm in a UV-visible double beam spectrophotometer (UV-260; Shimadzu Corp., Japan).
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Results |
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The results are expressed as per cent aberrant cells, chromatid and chromosome breaks, dicentrics, centric rings, exchanges, acentric fragments, total aberrations, polyploidy and SDC in Tables I– III. The free radical scavenging activity has been expressed as per cent inhibition in Figure 1.
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Chromosome aberrations
Aberrant cells Exposure of mice to 0–4 Gy resulted in a dose-dependant elevation in the frequency of aberrant cells at all post-irradiation scoring times. This elevation has been found to be linear, with the maximum aberrations in the animals exposed to 4 Gy. The greatest number of aberrant cells was observed at 24 h post-irradiation (Tables I– III). The pattern of induction of aberrant cells in the NIN + irradiation group was similar to that in the DDW + irradiation group, except that the frequency of aberrant cells was significantly lower at all post-irradiation scoring times in the former group when compared with the latter group (Tables I– III).
Chromatid and chromosome breaks
The irradiation of animals resulted in the induction of chromatid and chromosome breaks. The incidence of chromatid breaks was highest at 12 h, while that of chromosome breaks was highest at 24 h post-irradiation. The lowest frequencies of these aberrations were observed at 48 h post-exposure. The dose–effect relationship for both chromatid and chromosome breaks was linear (Tables I– III). Treatment of mice with naringin before exposure to different doses of -radiation resulted in a significant reduction in the frequency of both chromatid and chromosome breaks at all the post-exposure times when compared with the concurrent DDW + irradiation group (Tables I– III). The chromatid breaks were approximately 2-fold lower in the NIN + irradiation group when compared with the DDW + irradiation group (Tables I and II).
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Dicentrics, centric rings and exchanges Irradiation of animals with 0–4 Gy caused a dose-dependant elevation in the frequency of dicentrics, centric rings and exchanges in the DDW + irradiation group at all the post-irradiation times studied. The elevation in the dicentrics, centric rings and chromatid exchanges was higher at 24 h when compared with 12 h in the DDW + irradiation group (Tables I and II) and showed a decline thereafter (Table III). A similar pattern was observed for chromatid exchanges. The trend for the induction of centric rings, dicentrics and exchanges was similar in the NIN + irradiation group. However, naringin pretreatment significantly reduced the frequency of dicentrics, centric rings and exchanges and this reduction was by at least 2-fold when compared with the concurrent DDW + irradiation group (Tables I and II). The dose–response relationship was linear quadratic for both groups at all post-irradiation times.
Acentric fragments and total aberrations The pattern of yield of acentric fragments (including double minutes and acentric rings) and total aberrations was similar to that of chromosome breaks. The acentric fragments and total aberrations showed an increase with increasing irradiation dose in both the DDW + irradiation and NIN + irradiation groups at all post-irradiation times. However, the administration of naringin before exposure to 0–4 Gy significantly reduced radiation-induced acentric fragments and total aberrations in comparison with the DDW + irradiation group (Tables I– III). The dose–response relationship for all these aberrations was linear at all post-irradiation times for both groups studied. The acentric fragments and total aberrations showed a peak at 24 h and declined thereafter (Tables I– III). The reduction in acentric fragments and total aberrations in the NIN + irradiation group was approximately 2-fold when compared with the DDW + irradiation group at all times studied.
SDC Exposure of mice to 0–2 Gy did not induce SDC, however, once the exposure dose was increased to 3 Gy, a dose-dependent elevation in the frequency of SDC was recorded in the DDW + irradiation group at 12 and 24 h post-:irradiation, but not at 48 h, where they could only be scored after 4 Gy irradiation. The naringin treatment reduced the induction of SDC when compared with the concurrent DDW + irradiation group. However, a significant difference was only observed after 4 Gy exposure at 24 h post-irradiation.
Polyploidy Exposure of mice to 0–1 Gy radiation did not induce polyploidy. However, a further increase in the irradiation dose resulted in a dose-dependent increase in the frequency of polyploid cells in the DDW + irradiation group at all post-irradiation scoring times (Tables I– III). Naringin treatment before irradiation significantly reduced the occurrence of polyploid cells, especially at 4 Gy, and the dose–response relationship was linear quadratic. Significant protection against the induction of polyploid cells by naringin was observed only for 24 h post-irradiation at 2–4 Gy irradiation (Table II).
Aberrant cell proportion The proportion of cells bearing chromatid or chromosome type aberrations was also scored, which increased in a dose-dependent manner in both the DDW + irradiation and NIN + irradiation groups. The cells bearing chromatid breaks were highest at 12 h, while the cells bearing chromosome type aberrations were highest at 24 h post-exposure. The trend for cells bearing chromatid or chromosome type aberrations in the naringin-pretreated group was similar to that of the DDW + irradiation group. However, their frequencies were lower than that of the DDW + irradiation group. The cells bearing both chromatid and chromosome type aberrations showed the greatest frequency at 24 h post-exposure, and naringin pretreatment reduced the proportion of these cells. However, the frequencies of cells bearing both types of aberrations simultaneously were considerably less.
Free radical scavenging
The data are shown as per cent inhibition of free radical generation in Figure 1. Naringin inhibited the generation of •OH and O2•– radicals in a dose-dependent manner and a maximum inhibition was observed at 800 nM; thereafter a steady-state was attained (Figure 1a and b). The inhibition of DPPH by naringin was also dose-dependent up to 200 µM and a steady-state was obtained thereafter (Figure 1c). The total antioxidant activity was measured using the ABTS assay and the lowest concentration of 5 µM was found to inhibit 90% generation of the ABTS•+ radical cation. However, a further increase in the drug concentration caused a dose-dependent depletion in the inhibition of ABTS•+ radicals (Figure 1d).
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Discussion |
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Radiation is known to damage DNA and other molecules, causing gene mutation. Gene mutation in germ cells is expressed in descendants, whereas it is expressed in the individual if it occurs in somatic cells. Radiation has also been reported to produce immediate and delayed effects (Hallenbeck, 1994
). Scoring of chromosome aberrations gives direct assessment of genotoxicity of various physical and chemical agents. The use of certain chemicals may help to reduce/inhibit the genotoxicity, which in turn may inhibit mutagenesis and carcinogenesis.
Chromosome aberrations are highly quantifiable manifestations of radiation-induced damage to DNA that may be observed in the first post-irradiation mitosis, and studies conducted in plants employed scoring of chromosome aberrations as a method to quantify levels of radioprotection by various SH compounds (Mikaelsen, 1952; Wolff, 1954). The administration of 2 mg/kg naringin prior to irradiation resulted in a significant decline in the frequency of aberrant cells. This dose of naringin was selected on the basis of survival studies in which the highest number of animals survived after irradiation to 10 Gy when compared with the other doses of naringin (data not shown). Similarly, a micronucleus study has also shown that 2 mg/kg naringin was the best dose for protection against radiation-induced micronucleus formation (Jagetia and Reddy, 2002). However, other SH compounds, like MPG and WR-2721, have been reported to reduce the frequency of radiation-induced aberrant cells (Gupta and Uma Devi, 1985, 1986; Thomas and Uma Devi, 1987). Similarly, another non-SH compound DMAP [(E)4- (4-N,N-dimethylaminophenyl)but-3-en-2-one] has been reported to reduce the frequency of aberrant cells in mouse bone marrow (Jacob, 1993).
Naringin was equally effective in protecting against radiation-induced chromosome damage in mouse bone marrow as evidenced by a reduction in the frequency of asymmetrical aberrations like chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges, acentric fragments and total aberrations. Other plant flavonoids, like vicenin and orientin, have also been reported to protect mouse bone marrow cells against radiation-induced chromosome aberrations (Vrinda and Uma Devi, 2001). Thiol compounds like MPG and WR-2721 have also been reported to protect mouse chromosomes against radiation-induced damage (Thomas and Uma Devi, 1987). Liv.52, an indigenous herbal preparation, has also been found to protect against radiation-induced asymmetrical aberrations in mouse bone marrow (Jagetia and Ganapathi, 1991).
Most of the chromosome aberrations increased with scoring time up to 24 h in both the DDW + irradiation and NIN + irradiation groups and declined thereafter, except for the chromatid breaks, which were highest at 12 h post-irradiation. The bone marrow consists of asynchronous cell populations, and cells that are exposed in the G2 and M phases of the cell cycle will manifest chromatid aberrations at 12 h after irradiation, while those which are in G1 and early S phase of the cell cycle will contain chromosome breaks at 24 h, by which time the exposed cell would have divided. This is the reason for the higher number of chromosome breaks at 24 h post-irradiation. The decline in chromosome aberrations at 48 h post-irradiation is expected, as the irradiated cell would already have divided at least once after the induction of aberrations, resulting in a dilution of the observed chromosome aberrations. A similar effect has been observed earlier with Liv.52 in mouse bone marrow (Jagetia and Ganapathi, 1991). Chromosome aberrations have been reported to be reduced by half with successive cell divisions (Carrano and Heddle, 1973).
Chromosome aberrations have been used as sensitive monitors of cellular damage in studies of several radioprotectors (Sasaki and Matsubara, 1977; Midander, 1982; Virsik and Harder, 1982; Gupta and Uma Devi, 1985; Littlefield et al., 1988). DMSO has been reported to confer protection against radiation-induced chromosome aberrations in Vicia faba seeds (Kaul, 1969). Several other studies have shown DMSO to efficiently protect against various manifestations of radiation-induced damage to mammalian DNA in both cellular and chemical systems (Chapman et al., 1973, 1975a,b, 1979; Okada et al., 1983; Ward et al., 1985; Corn et al., 1987). Antipain, an inhibitor of serine and thiol proteases, such as trypsin, papain and cathepsin, has also been shown to protect against X-ray-induced chromosomal aberrations in human lymphocytes (Afzal et al., 1989). Tempol, a non-thiol stable nitroxide free radical, has also been reported to significantly protect against radiation-induced chromosome aberrations like dicentrics, rings and tri-radials in human peripheral blood lymphocytes (Johnstone et al., 1995).
The exact mechanism of action of chromosome protection by naringin is not known. However, scavenging of radiation-induced free radicals may be one of the important mechanisms of radiation protection by naringin, which is evident as a dose-dependent scavenging of •OH, O2•– and DPPH free radicals in vitro in the present study. Naringin also inhibited the induction of ABTS•+ radicals in vitro efficiently and a maximum inhibition of ABTS•+ radicals (90%) was observed at the lowest concentration of 5 µM. These observations confirm earlier studies in which naringin was reported to scavenge hydroxyl and superoxide free radicals and lipid peroxides (Kroyer, 1986; Chen et al., 1990; Kim et al., 1994). Recently, naringin has been reported to elevate catalase, superoxide dismutase and glutathione peroxidase mRNA synthesis (Jeon et al., 2001, 2002). The elevation of these enzymes by naringin may also be responsible for the observed protection against radiation-induced chromosome damage. Naringin has also been reported to protect against DNA damage (Russo et al., 2000). Therefore, the reduction in chromosomal damage in the present study may be due to a free radical scavenging activity of naringin as well as protection against radiation-induced DNA damage. DNA double-strand breaks are reported to be responsible for the induction of various types of chromosome aberrations (Natarajan et al., 1980; Bryant, 1988). Our study demonstrates that naringin treatment is able to reduce chromosome damage significantly, as evidenced by the low frequency of all types of aberrations in the naringin-pretreated group. The protection offered by naringin may principally be due to scavenging of radiation-induced free radicals.
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Acknowledgements |
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We wish to thank Prof. M.S.Vidyasagar and Dr J.G.R.Solomon (Department of Radiotherapy and Oncology, Kasturba Medical College, Manipal, India) for providing the necessary irradiation facilities and help in radiation dosimetry, respectively. The financial assistance from Arya Vysya Sangha, a Charitable Trust (Chitradurga, Karnataka, India), and RVRR Biotech Ltd., Hyderabend, A.P., India is thankfully acknowledged.
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Notes |
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1To whom correspondence should be addressed. Tel: +91 8252 571201; Fax: +91 8252 570062; Email: gc.jagetia{at}kmc.manipal.edu
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Received on May 20, 2002; accepted on February 20, 2003.
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