Mutagenesis vol. 18 no. 4 pp. 387-393,
July 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Evaluation of the radioprotective effect of Aegle marmelos (L.) Correa in cultured human peripheral blood lymphocytes exposed to different doses of 
-radiation: a micronucleus study
Department of Radiobiology, Kasturba Medical College, Manipal 576 119, India
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Abstract |
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The radioprotective effect of a hydroalcoholic extract of Aegle marmelos (AME) was evaluated in cultured human peripheral blood lymphocytes (HPBLs) by the micronucleus assay. The optimum protective dose of the extract was selected by treating HPBLs with 1.25, 2.5, 5, 6.25, 10, 20, 40, 60, 80 and 100 µg/ml AME before exposure to 3 Gy
-radiation and then evaluating the micronucleus frequency in cytokinesis blocked HPBLs. Treatment of HPBLs with different doses of AME reduced the frequency of radiation-induced micronuclei significantly, with the greatest reduction in micronucleus induction being observed for 5 µg/ml AME. Therefore, this dose of AME was considered as the optimum dose for radioprotection and further studies were carried out treating the HPBLs with 5 µg/ml AME before exposure to different doses (0, 0.5, 1, 2, 3 and 4 Gy) of -radiation. The irradiation of HPBLs with different doses of -radiation caused a dose-dependent increase in the frequency of lymphocytes bearing one, two and multiple micronuclei, while treatment of HPBLs with 5 µg/ml AME significantly reduced the frequency of lymphocytes bearing one, two and multiple micronuclei when compared with the irradiated control. The dose–response relationship for both groups was linear. To understand the mechanism of action of AME separate experiments were conducted to evaluate the free radical scavenging of OH, O2–, DPPH, ABTS+ and NO in vitro. AME was found to inhibit free radicals in a dose-dependent manner up to a dose of 200 µg/ml for the majority of radicals and plateaued thereafter. Our study demonstrates that AME at 5 µg/ml protected HPBLs against radiation-induced DNA damage and genomic instability and its radioprotective activity may be by scavenging of radiation-induced free radicals and increased oxidant status. |
Introduction |
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Ionizing radiations interact with mammalian cells by inducing a wide range of detrimental effects, with the most important damage occurring to the cellular DNA. It is a classical mutagen and the DNA of the exposed cells undergoes single- and double-strand breaks and damage to the bases and sugars, ultimately leading to chromosomal aberrations (Wallace, 1988
). Depending on the severity of the damage, the cell may be killed and eliminated or may incorporate the damage (mutagenesis) and proliferate, leading to carcinogenesis. Therefore, prevention of cytogenetic damage in the exposed cell may lead to a reduction in chromosomal instability, mutagenesis and carcinogenesis.
The use of chemical agents to provide protection against radiation injury has been a major field of study and, historically, the discovery of the radioprotective effects of cysteine in rats and mice by Patt et al. (1949) paved the way for research on radiation protection in humans. Since then, there has been an explosion in studies on radioprotection, and compounds with varied structures and physiological functions have been tested for their radioprotective abilities over the past 50 years. However, the practical applicability of the majority of these synthetic compounds remained limited, owing to their high toxicity at their optimum protective doses (Sweeny, 1979). Therefore, a need has been felt to find non-toxic and effective alternatives to the synthetic compounds.
Aegle marmelos, commonly known as bael, is a spiny tree belonging to the family Rutaceae. The leaves, roots, bark, seeds and fruits of A.marmelos are edible. The medicinal properties of this plant have been described in the Ayurveda. In fact, as per Charaka (1500 B.C.), no drug has been longer or better known or appreciated by the inhabitants of India than the bael (CHEMEXCIL, 1992). The leaves of bael are astringent, a laxative, a febrifuge and an expectorant and are useful in ophthalmia, deafness, inflammations, catarrh, diabetes and asthmatic complaints. The unripe fruits are bitter, acrid, sour, astringent, a digestive and stomachic and are useful in diarrhea, dysentery and stomachalgia. The roots of A.marmelos are one of the ingredients of dashamula (10 roots), a medicine commonly used by Ayurvedic practitioners. The leaves are bitter and are used as a remedy for ophthalmia, ulcers, dropsy, cholera and beri beri associated with a weakness of the heart. Fresh aqueous and alcoholic leaf extracts of A.marmelos are reported to have a cardiotonic effect, like digitalis, and to decrease the requirement for circulatory stimulants (Nadkarni, 1976). An aqueous decoction of the leaves has been shown to possess a significant hypoglycemic effect (Karunanayake et al., 1984). Aegle leaf extract has been reported to regenerate damaged pancreatic ß-cells in diabetic rats (Das et al., 1996). It is found to be as effective as insulin in the restoration of blood glucose and body weight to normal levels (Seema et al., 1996).
The diverse medicinal properties attributed to A.marmelos stimulated us to investigate its radioprotective activity. Thus, the aim of the present study was to evaluate the radioprotective activity of various concentrations of a leaf extract of A.marmelos (AME) in cultured human peripheral blood lymphocytes exposed to different doses of -radiation.
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Materials and methods |
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The identification of A.marmelos (L.) Correa, family Rutaceae, was carried out by Dr Gopal Krishna Bhat (a well-known taxonomist of this area) (Department of Botany, Poorna Prajna College, Udupi, India). The leaves of the tree A.marmelos were collected locally during the months April–May, cleaned, dried in the shade and powdered. A sample of 100 g of leaf powder was extracted with 50% ethanol in a Soxhlet apparatus. The extract was freeze dried and stored at –80°C until further use. An approximate yield of 26% was obtained.
Chemicals
Phytohemagglutinin (PHA) was procured from Difco Laboratories (Detroit, MI), while cytochalasin-B, RPMI, fetal calf serum (FCS), L-glutamine, gentamycin sulfate, dimethyl sulfoxide (DMSO), deoxyribose, EDTA, ascorbic acid, nitroblue tetrazolium, 2,2-diphenyl-1-picryl hydrazyl free radical (DPPH), sodium nitroprusside, Greiss reagent and 2,2-azinobis(3-ethyl benzothiazoline-6-sulphonic acid) diammonium salt (ABTS) were procured from Sigma Chemical Co. (St Louis, MO). Ferric chloride, sodium bicarbonate, sodium carbonate, sodium chloride, potassium chloride, potassium hydrogen phosphate, disodium hydrogen phosphate and hydrogen peroxide were procured from Ranbaxy Fine Chemicals (New Delhi, India).
Preparation of drug and other solutions
The AME was dissolved in phosphate-buffered saline (PBS) at a concentration of 10 mg/ml immediately before use, filter sterilized and diluted to the required concentrations. Cytochalasin B (Sigma catalog no. C-6762) was dissolved in DMSO at a concentration of 10 mg/ml, stored at –80°C and diluted with PBS immediately before use. One vial of PHA was reconstituted in 5 ml sterile double-distilled water as directed by the manufacturer and 500 µl of reconstituted PHA was added to 1 l of RPMI medium before filter sterilization.
Lymphocyte culture
The whole blood was collected from a healthy non-smoking donor in heparinized vacutainers (Becton Dickinson, Bedford, MA). The details of human peripheral blood lymphocyte (HPBL) culture are given elsewhere (Jagetia et al., 2001). Briefly, erythrocytes were allowed to sediment and the buffy coat-containing nucleated cells were used for lymphocyte culture. Usually, 106 nucleated cells were inoculated into each culture tube containing RPMI 1640 medium supplemented with 10% FCS, L-glutamine and PHA as the mitogen and the following experiments were conducted.
Experiment 1: Selection of optimum dose
Several HPBL cultures were set up and the optimum radioprotective dose of AME was selected by dividing the cultures into two groups as follows.
AME + sham irradiation The cultures of this group were treated with 0.0, 2.5, 5, 6.25, 10, 20, 40, 60, 80 and 100 µg/ml AME before sham irradiation (0 Gy).
AME + irradiation
The cultures of this group were treated with 0.0, 2.5, 5, 6.25, 10, 20, 40, 60, 80 and 100 µg/ml AME before exposure to 3 Gy -radiation.
Experiment 2: Radioprotective effect
Since 5 µg/ml AME provided the greatest effect, a separate experiment was carried out to study the radioprotective effect of AME in HPBLs, in which several HPBL cultures were set up and divided into the following groups.
PBS + irradiation
The cultures of this group were treated with 10 µl/ml sterile PBS before exposure to different doses of -radiation.
AME + irradiation
The cultures of this group were treated with 5 µg/ml AME before exposure to different doses of -radiation.
Irradiation
Thirty min after AME treatment, the HPBL cultures of experiment 1 were exposed to 3 Gy, while those of experiment 2 were exposed to 0, 0.5, 1, 2, 3 and 4 Gy 60Co -radiation from a Tele Cobalt therapy source (Theratron; Atomic Energy Agency, Ontario, Canada) at a dose rate of 1 Gy/min. Immediately after exposure (within 5 min), the cell cultures were transferred to a CO2 incubator (Nuaire, Plymouth, MN) and allowed to grow up to 72 h at 37°C.
Micronucleus assay
The micronucleus (MN) assay was carried out according to the method of Fenech and Morley (1985) and the protocol described by Kirsch-Volders et al. (2000) for the in vitro MN assay was followed. The details of the procedure are described elsewhere (Jagetia et al., 2001). Briefly, 5 µg/ml cytochalasin B was added to each culture 44 h after irradiation to inhibit cytokinesis. The cultures were harvested 72 h after irradiation by centrifugation. The lymphocytes were subjected to a mild hypotonic (0.56% ammonium oxalate) treatment for 4 min, centrifuged and fixed in Carnoy’s fixative (3:1 methanol:acetic acid). The cells were centrifuged again, resuspended in a small volume of fixative and spread on precleaned coded slides to avoid observer bias. Quadruplicate cultures were used for each drug concentration or irradiation dose for each group. The slides containing cells were stained with 0.125% acridine orange (Gurr catalog no. 34001 9704640E; BDH, Poole, UK) in Sorensen’s buffer (pH 6.8) and washed twice in buffer. The buffer mounted slides were observed under a fluorescence microscope equipped with the 450–490 nm BP filter set with excitation at 453 nm (Photomicroscope III; Carl Zeiss, Germany) using a 40x neofluar objective for the presence of MN in binucleate lymphocytes (BNC). A minimum of 1000 BNC with well-preserved cytoplasm were scored from each culture and the frequency of micronucleated binucleate cells (MNBNC) was determined. MN identification was done according to the criteria of Countryman and Heddle (1976) and Kirsch-Volders et al. (2000).
The statistical significance among various treatments between the two groups was determined using one-way ANOVA and Fisher’s exact test. Bonferroni’s post hoc test was applied after one-way ANOVA to determine the statistical significance between various doses of AME. The Solo 4 statistical package (BMDP Statistical Software Inc., Los Angeles, CA) was used for statistical analysis.
Experiment 3: Estimation of free radical scavenging in vitro
Hydroxyl radical scavenging activity
Scavenging of the hydroxyl (—OH) free radical was measured by the method of Halliwell et al. (1987). Briefly, the reaction mixture contained deoxyribose (2.8 mM), KH2PO4–NaOH buffer, pH 7.4 (0.05 M), FeCl3 (0.1 mM), EDTA (0.1 mM), H2O2 (1 mM), ascorbate (0.1 mM) and AME (10–500 µg/ml) in a final volume of 2 ml. The reaction mixture was incubated for 30 min at ambient temperature followed by addition of 2 ml of trichloroacetic acid (2.8% w/v) and thiobarbituric acid. The reaction mixture was kept in a boiling water bath for 30 min, cooled and the absorbance was read at 532 nm in a UV-Vis double beam spectrophotometer (UV-260; Shimadzu Corp, Tokyo, Japan).
Superoxide anion scavenging activity
Scavenging of the superoxide (O2–) anion radical was measured as described by Hyland et al. (1983). Briefly, the reaction mixture contained various concentrations of AME (10–500 µg/ml), nitroblue tetrazolium and alkaline DMSO. The blank consisted of pure DMSO instead of alkaline DMSO. The absorbance was read at 560 nm using a UV-Vis double beam spectrophotometer (UV-260).
DPPH scavenging activity
The principle for reduction of the DPPH free radical is that the antioxidant reacts with the stable free radical DPPH and converts it to 2,2-diphenyl-1-picryl hydrazine. The ability to scavenge the stable free radical DPPH is measured as a decrease in absorbance at 517 nm (Mensor et al., 2001). To an ethanolic solution of DPPH (0.05 mM) was added an equal volume of AME (10–500 µg/ml) dissolved in water, 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-Vis double beam spectrophotometer (UV-260).
Total antioxidant activity assay
Total antioxidant potential was determined by the 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 ABTS (0.00017 M), AME (10–500 µg/ml) and buffer in a total volume of 3.5 ml. The absorbance was measured at 734 nm in a UV-Vis double beam spectrophotometer (UV-260).
Nitric oxide scavenging activity
Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reaction as described previously. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide (Marcocci et al., 1994; Sreejayan and Rao, 1997), which interacts with oxygen to produce nitrite ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with oxygen leading to reduced production of nitric oxide (Marcocci et al., 1994). Sodium nitroprusside (5 mM) in PBS was mixed with different concentrations of AME (20–400 µg/ml) and incubated at 25°C for 150 min. The samples from the above were reacted with Greiss reagent (1% sulfanilamide, 2% H3PO4 and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during diazotization of nitrite with sulfanilamide and subsequent coupling with napthylethylenediamme was read at 546 nm and referred to the absorbance of standard solutions of potassium nitrite treated in the same way with Griess reagent.
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Results |
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Experiment 1: Selection of optimum dose
Treatment of HPBLs up to a dose of 80 µg/ml did not significantly alter the spontaneous frequency of MNBNC in the AME + sham irradiation group. However, a further increase in the AME dose up to 100 µg/ml resulted in a significant elevation in the frequency of MNBNC, with a peak level of MNBNC being observed in the AME + sham irradiation group (Table I). Treatment of HPBLs with different doses of AME before exposure to 3 Gy
-radiation caused a significant decline in radiation-induced MN formation up to 40 µg/ml when compared with the non-drug-treated 3 Gy exposed cultures. A further increase in the drug dose up to 80 µg/ml AME did not alter the frequency of radiation-induced MN significantly and a still further increase up to 100 µg/ml only marginally elevated the frequency of MNBNC, however, the difference was non-significant when compared with the non-drug-treated 3 Gy irradiated cultures (Table I). Among all the doses of AME screened the lowest frequency of MN was observed for 5 µg/ml AME (Figure 1), therefore this dose was considered to be the optimum protective dose and further experiments were carried out using this dose.
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Experiment 2: Radioprotective effect
The frequency of MNBNC increased in a dose-dependent manner in the PBS + irradiation group and the dose–response relationship was linear (Figure 2). Pretreatment of HPBLs with AME before exposure to different doses of
-radiation resulted in a significant decline in the frequency of MNBNC when compared with the PBS + irradiation group. Treatment of HPBLs with AME before exposure to different doses of -radiation caused a decline in the frequency of MNBNC by 1.5-fold at all the exposure doses when compared with the concurrent PBS + irradiation group (Table II).
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, PBS + irradiation; •, AME + irradiation. (a) Total MNBNC; (b) MNBNC with one MN; (c) MNBNC with two MN; (d) MNBNC with multiple MN.
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The frequency of MNBNC with one, two and multiple MN was scored separately. The frequency of MNBNC with one MN was elevated in a dose-dependent manner in the PBS + irradiation group and the dose–response relationship was linear (Figure 2). Pretreatment of HPBLs with 5 µg/ml AME resulted in a significant inhibition of MNBNC bearing one MN when compared with the PBS + irradiation group. The frequency of MNBNC with one MN was 1.4-fold lower in the HPBLs exposed to 0.5–2 Gy, while it was 1.5-fold lower than in the concurrent PBS + irradiation group after exposure to 3 and 4 Gy, respectively (Table II). The dose–response relationship was linear (Figure 2).
MNBNC with two MN was elevated in a dose-dependent manner in the PBS + irradiation group (Figure 2). AME pretreatment resulted in a significant decline in the frequency of MNBNC with two MN when compared with the PBS + irradiation group (Table II). The highest frequency of MNBNC with two MN was observed at 4 Gy irradiation. The frequency of MNBNC with two MN was lower by 1.7-, 1.6-, 1.7-, 1.6-, 1.5- and 1.3-fold in the HPBLs treated with 5 µg/ml AME before exposure to 0, 0.5, 1, 2, 3 and 4 Gy, respectively, when compared with the concurrent PBS + irradiation group. The dose–response relationship was linear in both the PBS + irradiation and AME + irradiation groups (Figure 2).
The MNBNCs with multiple (≥3) MN were conspicuous by their absence for PBS + sham irradiation (Table II). However, a further increase in the irradiation dose resulted in a dose-dependent elevation in the frequency of MNBNC with multiple MN. Pretreatment of HPBLs with AME resulted in a significant decline in the frequency of MNBNC with multiple MN at all the exposure doses when compared with the concurrent PBS + irradiation group (Figure 2). There was a 2-fold decline in MNBNC with multiple MN at 0.5 and 1 Gy, while it was 1.6-fold for 2 and 3 Gy and 1.5-fold for 4 Gy, when compared with the concomitant PBS + irradiation group. The dose–response relationship was linear for both the PBS + irradiation and AME + irradiation groups.
Experiment 3: Free radical scavenging
The data are shown as percent scavenging of free radical generation in Figure 3. AME inhibited the generation of OH and O2– radicals in a dose-dependent manner and maximum scavenging was observed at 500 µg/ml (Figure 3a and b). AME was ineffective in scavenging the DPPH radical up to a concentration of 100 µg/ml, however, a further increase in AME concentration resulted in a dose-dependent elevation of DPPH scavenging up to 500 µg/ml (Figure 3c). The total antioxidant activity was measured using the ABTS assay. Inhibition of the ABTS+ radical showed dose-dependent scavenging up to 200 µg/ml and plateaued thereafter (Figure 3d). Similarly, AME also showed dose-dependent scavenging of nitric oxide up to 60 µg/ml, after which the elevation in nitric oxide scavenging reached an almost steady-state. However, the highest scavenging was observed at 400 µg/ml (Figure 3e).
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Discussion |
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The interaction of ionizing radiation with cells causes genomic instability leading to mutagenesis and carcinogenesis, therefore, there is a need to protect the genome against such effects of ionizing radiation. Plants have been companions of mankind since time immemorial and the antiquity of the use of plants for human health care by man is unknown. Plant/natural products may be useful in providing protection against the harmful effects of ionizing radiation. Therefore, an attempt has been made to screen the radioprotective activity of AME in cultured HPBLs. The MN assay is a simple cytogenetic tool to assess the genomic damage in a dividing cell population. MN are formed from an acentric fragment or a whole chromosome that fails to be incorporated into the daughter nuclei following mitosis due to a defective kinetochore (Countryman and Heddle, 1976
). Therefore, scoring of MN can help to evaluate the radioprotective action of various agents (Midander and Révész, 1980; Jagetia and Ganapathi, 1989; Jagetia et al., 1994; Umegaki et al., 1994; Gasiev et al., 1996; Jagetia and Aruna, 1997; Vrinda and Uma Devi, 2001; Jagetia and Baliga, 2002b).
Treatment of HPBLs with 1.25–6.25 µg/ml AME did not alter the spontaneous frequency of MNBNC, however, a further increase in the AME concentration up to 100 µg/ml resulted in an elevation in MNBNC frequency. A similar effect has been observed earlier for Syzygium cumini extract in HPBLs (Jagetia and Baliga, 2002b) and naringin, a naturally occurring grapefruit flavanone, in mice (Jagetia and Reddy, 2002). Treatment of HPBLs with various concentrations of AME before exposure to 3 Gy irradiation resulted in a significant decline in radiation-induced MN up to a dose of 10 µg/ml; thereafter the decline was non-significant and the highest dose of 100 µg/ml resulted in a marginal but non-significant elevation in MNBNC frequency. The highest reduction in the frequency of MNBNC was found at a concentration of 5 µg/ml, which was 1.5-fold lower than the non-drug-treated 3 Gy irradiated cultures. Therefore, this dose of AME was considered as the optimum protective dose. A similar effect was earlier observed for S.cumini extract and naringin (Jagetia and Baliga, 2002b; Jagetia and Reddy, 2002). Radioprotective agents have been reported to give protection up to a particular dose, but may even be toxic thereafter (Thomson, 1962; Nagata et al., 1972; Jagetia and Baliga, 2002a,b; Jagetia et al., 2002; Jagetia and Reddy, 2002). Our findings support the hypothesis that radioprotectors have an optimum dose for their action.
Exposure of HPBLs to different doses of -radiation resulted in a dose-dependent elevation in MNBNCs and the dose–response relationship was linear. Treatment of HPBLs with 5 µg/ml AME significantly reduced the frequency of MNBNCs when compared with the PBS + irradiation group. This reduction in MNBNC was 1.5-fold at all exposure doses when compared with the concurrent PBS + irradiation group. As far as the authors are aware, there have been no reports regarding the use of AME as a radioprotective agent in vitro and this is the first report of the screening of AME for radioprotective activity. However, an extract of S.cumini has been reported to protect HPBLs against radiation-induced MN formation (Jagetia and Baliga, 2002b). Other natural products, like vitamins A, C and E, which are present in several vegetables have been reported to reduce radiation-induced MN formation in cultured HPBLs exposed to -rays (Gasiev et al., 1996). Similarly, ß-carotene has also been reported to prevent X-ray-induced MN induction in HPBLs (Umegaki et al., 1994). The flavonoids of Ocimum sanctum, orientin and vicenin, have also been reported to protect against radiation-induced MN formation in human lymphocytes (Vrinda and Uma Devi, 2001).
The irradiation of lymphocytes with different doses of -radiation not only increased MNBNC with one MN but also MNBNC bearing two and multiple MN. Irradiation has been reported to increase the frequency of cells bearing one, two or multiple MN in lymphocytes and various cultured cell lines (Catena et al., 1997; Jagetia and Adiga, 1997, 2000; Adiga and Jagetia, 1999; Belyakov et al., 1999; Jagetia and Aruna, 1999, 2000; Jagetia and Nayak, 2000; Vrinda and Uma Devi, 2001; Jagetia and Baliga, 2002c). Treatment of HPBLs with 5 µg/ml AME before exposure to different doses of -radiation not only reduced the frequency of MNBNC bearing one MN significantly but also those with two and multiple MN, indicating that AME treatment has been able to inhibit the multiple sites of damage to DNA and complex chromosome aberrations in the HPBLs. Similarly, treatment of HPBLs with S.cumini extract has been reported to reduce the frequency of MNBNC bearing two or more MN (Jagetia and Baliga, 2002b). Other drugs, like (E)4-[4-N,N-dimethylaminophenyl]but-3-en-2-one, a synthetic compound (Jagetia et al., 1994), an extract of abana and naringin (Jagetia and Aruna, 1997; Jagetia and Reddy, 2002), have been reported to reduce the frequency of polychromatic erythrocytes bearing two or more MN in mouse bone marrow exposed to different doses of -radiation.
The exact mechanism of action of AME is not known. However, scavenging of free radicals by AME may be one of the important mechanisms of protection against radiation-induced damage to the cellular DNA. This contention is supported by the experiments on free radical scavenging, where AME has been found to scavenge OH, O2–, DPPH, ABTS+and NO radicals in a dose-dependent manner up to a particular dose and plateaued at higher concentrations. The flavonoids present in AME (Rastogi and Mehrotra, 1990) have been reported to scavenge free radicals like hydroxyl and superoxide and also inhibit lipid peroxidation (Maridonneau-Pairini et al., 1986; Uddin and Ahmad, 1995; Korina and Afansév, 1997; Abalea et al., 1999). Bael has been reported to contain aegeline, aegelenine, marmelosine, marmelin, o-methyl hayordinol, alloimperratorin methyl ester, o-isopentanyl hayordinol, linoleic acid, cineole, p-cymene, citronella, citral, cuminaldehyde, D-limonene, eugenol, tannins, phlobatannins, flavon-3-ols, leucoanthocyanins, anthocyanins and flavonoid glycoside. Most of these compounds have been reported to possess antioxidative and free radical scavenging activities (Korina and Afanasév, 1997). Further, eugenol, which is present in AME, has also been reported to be a good antioxidant and to inhibit lipid peroxidation (Vidhya and Devraj, 1999; Ogata et al., 2000). Alternatively, AME could have increased non-protein sulfhydryl (NPSH) levels, imparting protection against radiation-induced damage to the genome. Cineol, an important constituent of AME, has been reported to restore NPSH levels to normal (Santos and Rao, 2001). The depletion of intracellular glutathione (GSH) has been implicated as one of the causes of radiation-induced damage, while increased levels of intracellular GSH are responsible for a radioprotective action (Révész, 1985). Eugenol and the terpenes and flavonoids that are present in AME are good antioxidants and modulators of xenobiotic metabolizing enzymes, especially phase II enzymes like glutathione S-transferase, and GSH (Kong Ah-Ng et al., 2000) and the radioprotection observed in the present study may be attributed to the up-regulation of GST and GSH. AME has been reported to increase levels of GSH, glutathione reductase, glutathione peroxidase, superoxide dismutase and catalase in mice in a dose-dependent manner (Singh et al., 2000). AME has been found to inhibit radiation-induced lipid peroxidation and the decline in GSH levels in irradiated mice (data not shown). Free radical scavenging and an increase in GSH and antioxidant enzymes by AME seems to be an important mechanism in protecting HPBLs against radiation-induced MN formation.
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Acknowledgements |
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We thank Dr M.S.Vidyasagar (Professor and Head) 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. Financial assistance by the Christian Concern Mission, Hyderabad and Atomic Energy Regulation Board (AERB), Department of Atomic Energy, Government of India, New Delhi, 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 February 10, 2003; accepted on April 17, 2003.
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