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Mangiferin protects human peripheral blood lymphocytes against γ-radiation–induced DNA strand breaks: a fluorescence analysis of DNA unwinding assay
Nutrition Research 26 (2006) 303 – 311 www.elsevier.com/locate/nutres
Mangiferin protects human peripheral blood lymphocytes against c-radiation–induced DNA strand breaks: a fluorescence analysis of DNA unwinding assay Ganesh Chandra Jagetia4, Venkatasubbaiah A. Venkatesha Department of Radiobiology, Kasturba Medical College, Manipal-576 104, India Received 9 July 2005; revised 2 June 2006; accepted 13 June 2006
Abstract Ionizing radiations induce free radicals that lead to a cascade of events causing damage to cellular DNA. It is generally accepted that cell death induced by ionizing irradiation is due to DNA doublestrand breaks. Therefore, agents that can neutralize free radicals may be able to reduce DNA damage effectively and protect cell death. Effect of 0, 5, 10, 20, 50, or 100 lg/mL mangiferin, a glucosylxanthone present in mango (Mangifera indica), has been studied in human peripheral blood lymphocytes (HPBLs) exposed to 3 Gy c-radiation by fluorescence analysis of DNA unwinding assay. This assay detects DNA single- and double-strand breaks and alkali-labile sites by their effect on the rate of DNA denaturation in alkali, which was monitored by the fluorescence intensity of an intercalating dye, Hoechst 33258 (bisbenzimide). Estimation of DNA damage by fluorescence analysis of DNA unwinding assay showed that mangiferin as such did not have adverse effect on DNA damage, and it reduced the radiation-induced DNA damage in a concentration-dependent manner. In addition, a maximum undamaged double-stranded DNA was observed for 50 lg/mL of mangiferin. Therefore, further experiments were carried out using this concentration, wherein lymphocytes were exposed to 0, 1, 2, 3, or 4 Gy c-radiation 30 minutes after mangiferin treatment. Irradiation of HPBLs caused a radiation dose-dependent increase in the DNA strand breaks and a reduction in the undamaged double-stranded DNA, whereas treatment of lymphocytes with 50 lg/ mL mangiferin before irradiation significantly reduced DNA strand breaks and subsequently enhanced the undamaged double-stranded DNA at 4 hours posttreatment, indicating repair of radiation-induced DNA strand breaks. Mangiferin treatment restored the undamaged double-stranded DNA to almost normal level after 1 Gy irradiation, whereas it was 50% for 4 Gy at 4 hours postirradiation. Our observations suggest that mangiferin reduces initial DNA damage and enhances DNA repair in the HPBLs exposed to 1 to 4 Gy c-radiation and could serve as a protector against the radiation-induced DNA damage during planned and unplanned radiation exposures. D 2006 Elsevier Inc. All rights reserved. Keywords:
Human peripheral blood lymphocytes; Mangiferin; Irradiation; DNA strand breaks
1. Introduction
4 Corresponding author. Tel.: +91 820 2922122 (work); fax: +91 820 2571919. E-mail addresses: [email protected], [email protected] (G.C. Jagetia). 0271-5317/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2006.06.011
Interactions of ionizing radiation in mammalian cells induce a wide range of molecular damage. These can lead to diverse cellular responses, such as cell inactivation, chromosomal rearrangement, and mutations, which may contribute to the development of cancer and hereditary diseases.
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The random energy deposition by ionizing radiations induces a wide array of DNA lesions. Ionizing radiations induce damage to DNA by direct ionization and through generation of hydroxyl radicals that attack DNA [1] resulting in single-strand breaks, double-strand breaks, and oxidative damage to sugar and base residues that can be converted into strand breaks subsequently [2]. Because of induction of DNA double-strand breaks, the ionizing radiation is extremely effective in producing chromosomal aberrations leading to genomic instability [3]. Since the discovery by Patt et al [4] that cysteine protected rats and mice against radiation-induced sickness and mortality, several attempts have been made to reduce the radiation-induced damage [5,6]. However, the practical applicability of most of these compounds in humans remained limited because of their high toxicity at their optimum protective doses [7]. Therefore, a need for continued search for nontoxic radioprotector is felt. Use of various agents has been reported to reduce radiation-induced DNA damage in various systems. Zinc aspartate inhibited the radiation-induced spermatogonia killing in mice [8]. A grapefruit flavonone, naringin, has been found to protect radiation-induced chromosomal aberrations and micronuclei formation in bone marrow cells of mice [9,10]. Dietary agents that are already consumed by humans have not received the attention they deserve for their potential radioprotective effects [11]. It is likely that, if such agents are radioprotective, they may be more easily and safely used in patients undergoing radiotherapy than other radioprotective chemicals. Patients may also better tolerate them than other more promising exotic drugs because they form part of the daily human diet since time immemorial. Mango, Mangifera indica, is widely used in India and in Southeast Asia as a food supplement in the form of a pickle, and the fruit is eaten as such or is drunk in the form of juice. Mango has been widely used as an antiinflammatory, antipyretic, diuretic, antidiarrheal, antiviral, antibacterial, and hypoglycemic in the traditional system of medicine in India [12-18]. It has also been found to inhibit a-glucosidase and a-amylase activities in vitro [19,20]. The flavonoids from M indica have been found to be antihypolipidemic in rats [21]. Mangiferin (MGN), a C-glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-b-d-glucoside) purified from M indica and Anemarrhena asphodeloides has been found to inhibit proliferation of neoplastic cells in vitro and in vivo and antagonize the cytopathic effect of HIV [22]. It has also been reported to reduce intestinal neoplasms in rats [23]. Mangiferin is active against the herpes simplex virus 1, and it increases the antioxidant status in rats [18,24]. Humans are exposed to ionizing radiations for diagnostic or therapeutic purposes and in occupational settings. Apart from these man-made exposures, humans are also exposed to cosmic radiations that arise from solar flares and other cosmic interactions during air and space travel. Ionizing radiations are known to damage important biomolecules like
DNA, RNA, proteins, and lipids. DNA damage disturbs the fidelity of DNA replication causing genomic instability that may result in mutagenesis and carcinogenesis [25]. It is necessary to protect humans against the deleterious effects of ionizing radiations. Use of nutritional factors may have an advantage over other chemicals as radioprotectors because of their daily consumption and wide acceptability in humans. Pretreatment with MGN has been found to reduce the radiation-induced micronuclei formation in human peripheral blood lymphocytes (HPBLs) and increase the survival of animals exposed to different doses of c-radiation [26-28]. However, the effect of MGN on the radiationinduced molecular DNA damage has not been investigated, and its effective dose is far lower than the toxic dose [28]. Therefore, the present study was undertaken to investigate the effect of MGN on the radiation-induced DNA strand breaks in the cultured HPBLs exposed to various doses of c-radiation. 2. Methods and materials Mangiferin (catalogue no. M 3547), RPMI-1640 medium, Histopaque 1119, cyclohexane diamine tetraacetate, b-mercaptoethanol, Hoechst 33258 (bisbenzamide), and myo-inositol were obtained from Sigma Chemical Company, St. Louis, USA. The other chemicals were procured from Ranbaxy Fine Chemicals, India. 2.1. Preparation of drug Mangiferin (Fig. 1) was dissolved at a concentration of 10 mg/mL in dimethyl sulfoxide (DMSO) immediately before use. This stock was further diluted with RPMI medium as per the requirement before incubation of cells. Dimethyl sulfoxide was used as vehicle control. 2.2. Blood collection The peripheral blood was collected with informed consent from a healthy, nonsmoking, and nonalcoholic donor through venipuncture. The Kasturba Medical College ethical committee has approved this study. The blood was diluted 1:1 with phytohemagglutinin-free RPMI-1640 medium to understand the DNA strand break repair kinetics.
Fig. 1. Chemical structure of MGN.
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2.3. Determination of optimum dose (experiment 1) The blood was diluted and distributed into several culture flasks. The flasks were divided into the following groups: 1.
2.
Mangiferin + Sham-irradiation: The diluted blood of this group was treated with 0, 5, 10, 20, 50, or 100 lg/mL MGN and served as control group. Mangiferin + Irradiation: The diluted blood of this group was treated with 0, 5, 10, 20, 50, or 100 lg/mL MGN 30 minutes before exposure to 3 Gy c-radiation [26,27].
2.3.1. Irradiation Thirty minutes after DMSO or MGN treatment, various flasks containing HPBLs were kept on ice. The flasks were exposed to 0 or 3 Gy 60Co c-radiation delivered as a single fraction from a Tele Cobalt therapy source (Theratron, Atomic Energy Agency, Ontario, Canada) at a dose rate of 1 Gy/min. 2.4. Determination of radioprotective effect (experiment 2) Because 50 lg/mL MGN provided the greatest protection against the radiation-induced DNA strand breaks in the previous experiment, a separate experiment was carried out to study its radioprotective effects against different doses of c-radiation, where HPBLs were divided into the following groups: 1.
2.
DMSO + Irradiation: The diluted blood of this group was treated with DMSO 30 minutes before exposure to various doses of c-radiation and served as control group. MGN + Irradiation: The diluted blood of this group was treated with 50 lg/mL MGN 30 minutes before exposure to different doses of c-radiation.
2.4.1. Irradiation The irradiation procedure was exactly similar to that described above, except that the diluted blood from both groups was exposed to 0, 1, 2, 3, or 4 Gy of c-radiation. The DNA strand break repair kinetics in both groups was studied by fluorescence analysis of DNA unwinding (FADU) at 0, 1, 2, 4, 6, 12, or 24 hours for experiment 1, whereas at 0 or 4 hours postirradiation for experiment 2. Triplicate cultures were used for each dose of MGN or radiation for both the groups in each experiment, and results were reconfirmed by repetition of the experiments. Application of the test of homogeneity between the original and repeat experiments did not reveal significant differences; therefore, results of both experiments were combined.
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denaturation in alkali, monitored by the fluorescence intensity of an intercalating dye. 2.5.1. Lymphocyte isolation The lymphocytes from the above-mentioned groups were isolated at different postirradiation times by layering the irradiated diluted blood on Histopaque1119 and by centrifugation at 800 rpm for 15 minutes at room temperature. The buffy coat rich in lymphocytes was aspirated carefully and transferred into a fresh tube containing phosphatebuffered saline (PBS). The cells were washed twice with PBS, and the lymphocyte count was determined by trypan blue dye exclusion method to check the cell viability. Because no dead cells were reported, 3 106 isolated cells were distributed into 1.5 mL PBS and were divided equally (0.5 mL) in 3 tubes designated as P, B, or T and treated as described below before starting the FADU assay. P: lymphocytes, reagent mixture but without dye. B: lymphocytes, PBS and dye but without reagent mixture. T: lymphocytes, PBS, reagent mixture and dye. 2.5.2. Assay procedure Briefly, the cells were incubated with 1 mL of reagent mixture (containing 0.3 mL of 6 mmol myo-inositol, 0.1 mL of 2% urea, 0.1 mL of 5% SDS (sodium dodecyl sulphate), 0.4 mL of 5 M NaOH, and 0.1 mL of 2 mmol cyclohexane diamine tetraacetate in deionized double-distilled water) for 1 hour at 158C. The samples were flash-frozen for 1 minute, and 0.4 mL neutralizing mixture was added at room temperature (1 M glucose, 14 mmol b-mercaptoethanol). The samples were immediately sonicated for 5 seconds (Sonics Vibra-cell, Newtown, Conn, USA) to inhibit reassociation of complementary strands and to reduce the molecular weight of DNA. Each cell sample was transferred to a quartz cuvette and mixed gently with 1 mL Hoechst 33258 (5 lg/mL) by single inversion, and the fluorescence was read at an excitation wavelength of 350 nm and an emission wavelength of 450 nm using a spectrofluorimeter (SFM 25, Kontron Instruments, Neufahrn, Germany) at room temperature. The percent-undamaged DNA (ds-DNA) and DNA strand breaks (DNA SBs) in the treatment groups were calculated as follows: Percent undamaged ds-DNA = (PB/TB) 100 Percent DNA SBs = 100 percent undamaged ds-DNA Strand scission factor (SSF) = 1(log F t/F 0) where F t and F 0 are the percent ds-DNA of treated and untreated samples, respectively.
2.5. Fluorescence analysis of DNA unwinding assay
2.6. Statistical analysis
The FADU assay was performed in HPBLs as described by Birnboim and Jevcak [29] with minor modifications. This method detects single- and double-strand breaks and alkali labile sites by their effect on the rate of DNA
The statistical significance between the treatments was determined using one-way analysis of variance (ANOVA) and Fisher exact test. Differences indicated by P b .05 were considered statistically significant. Solo 4 statistical package
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Table 1 Effect of various concentrations of MGN on the yield of DNA strand breaks in HPBLs exposed to 3 Gy c-irradiation MGN (lg/mL)
Undamaged ds-DNA (%) F SEM Postirradiation repair time (h) 0
0a 5 10 20 50 100
SIR 92.49 F 0.56 92.64 F 0.77 92.46 F 0.35 92.66 F 0.28 93.11 F 0.83 93.20 F 0.72
MGN + IR 27.62 F 0.50 28.73 F 0.49 32.48 F 0.394 34.74 F 0.684 37.63 F 0.6744 38.03 F 0.4144
1 SIR 92.50 92.66 92.58 92.72 93.30 93.35
F F F F F F
0.60 0.57 0.33 0.58 1.08 0.27
2
MGN + IR 36.51 F 0.38 39.26 F 0.584 43.56 F 0.64444 46.57 F 0.7044 59.06 F 0.67444 59.40 F 0.68444
SIR 92.51 92.71 92.73 92.80 93.63 93.64
F F F F F F
0.16 0.16 1.23 1.19 0.47 1.20
MGN + IR 46.71 F 0.77 50.91 F 0.304 53.05 F 0.4544 57.09 F 0.4844 66.22 F 0.51444 66.60 F 1.25444
4 SIR 92.56 92.86 92.71 92.86 93.87 93.75
F F F F F F
0.77 1.02 0.62 0.28 0.11 0.65
MGN + IR 50.78 F 0.37 53.27 F 0.73 56.47 F 0.524 61.24 F 0.6344 78.12 F 0.73444 78.50 F 0.71444
The healthy donor blood was cultured in phytohemagglutinin-free RPMI-1640 medium (1:1 vol/vol) with or without MGN for different times. SIR indicates sham-irradiation; IR, irradiation. Values with no symbols are not significant when compared with the non–MGN-treated irradiated group. One-way ANOVA with Bonferroni post hoc test was used to calculate statistical significance. Triplicate cultures were used for each concentration of MGN which was given 30 min before irradiation. a DMSO (dimethyl sulfoxide) alone. 4 P b .05 when compared with non–MGN-treated irradiated group. 44 P b .01 when compared with non–MGN-treated irradiated group. 444 P b .001 when compared with non–MGN-treated irradiated group.
(BMDP Statistical software Inc, Los Angeles, Calif, USA.) was used for statistical analysis. 3. Results The results are expressed as undamaged ds-DNA (%) F SEM in Tables 1 and 2 and Figs. 2 and 3 and as SSF in Table 3 and Fig. 4. 3.1. Determination of optimum dose (experiment 1) The spontaneous frequency of undamaged ds-DNA remained unaltered when HPBLs were treated with the various concentrations of MGN (92.49% F 0.56%) at all postirradiation periods (Table 1). Immediate assessment of DNA damage (0 hour) after exposure of HPBLs to 3 Gy c-radiation caused a drastic reduction in the undamaged dsDNA (27.62% F 0.5%). The repair of DNA damage progressed with time, and a maximum repair (50.78% F 0.37%) was observed at 4 hours postirradiation. Allowing additional time did not enhance DNA repair, which remained almost static even up to 24 hours postirradiation (Table 1). Treatment of HPBLs with different concentrations of MGN 30 minutes before irradiation resulted in a progressive repair of DNA strand breaks up to 4 hours postirradiation ( P b .05 for 5 lg/mL MGN at 1, 2, and 4 hours; P b .001, 0.01, and 0.05 for 10 lg/mL MGN at 1, 2, and 4 hours; P b .01 for 20 lg/mL MGN at 1, 2, and 4 hours; P b .001 for 50 and 100 lg/mL MGN at 1, 2, and 4 hours postirradiation) when compared with irradiated (0 lg MGN) control group. This repair of DNA strand breaks was significantly greater in the MGN + irradiation group when compared with 3 Gy irradiation (0 lg MGN) group (Table 1; Fig. 2). Incubation of cells up to 24 hours did not significantly elevate the amount of undamaged ds-DNA in both MGN + sham-irradiation or MGN + 3 Gy irradiation groups. The highest amount of undamaged ds-DNA or
lowest DNA strand breaks were observed in HPBLs treated with 50 lg/mL MGN at 4 hours postirradiation, increase in MGN concentration did not further augment the repair of DNA strand breaks (Table 1). 3.2. Determination of radioprotective effect (experiment 2) Evaluation of DNA damage in the previous experiment indicated that a maximum repair was found at 4 hours postirradiation at an optimum dose of 50 lg/mL MGN. Therefore, DNA strand breaks were measured at 4 hours postirradiation in the lymphocytes treated with 50 lg/mL MGN before exposure to 0, 1, 2, 3, or 4 Gy of c-radiation (Table 2; Fig. 3). A maximum amount of undamaged DNA (91.29% F 1.11%) was observed for the DMSO + shamirradiation (0 Gy) group. Mangiferin pretreatment resulted in a nonsignificant elevation in the undamaged ds-DNA (93.02% F 0.82%) at 0 hour. A similar effect was also discernible for 4 hours postirradiation. Irradiation of HPBLs to various doses of c-radiation resulted in a dose-dependent elevation in DNA strand breaks as evidenced by a progressive reduction in undamaged ds-DNA in the DMSO + irradiation group ( P b .001 at all irradiation doses in comparison with the DMSO + sham irradiation group) (Table 2; Fig. 3). The highest DNA strand breaks (80%) were observed for 4 Gy at 0 hour postirradiation in the DMSO + irradiation group. The assessment of DNA damage at 4 hours postirradiation showed repair of DNA strand breaks that declined in an irradiation dose-dependent manner, and the lowest repair of DNA strand breaks was observed for 4 Gy in the DMSO + irradiation group ( P b .01, 0.001, 0.001, and 0.001 at 1, 2, 3, or 4 Gy, respectively, in comparison with the DMSO + sham irradiation group at 4 hours) (Table 2; Fig. 3). Treatment of HPBLs with 50 lg/mL MGN for 30 minutes before exposure to different doses of c-radiation significantly inhibited the DNA strand breaks induction at 0 hour postirradiation ( P b .01, 0.05, 0.05, and 0.05 at 1, 2, 3, or 4 Gy c-radiation in
G.C. Jagetia, V.A. Venkatesha / Nutrition Research 26 (2006) 303 – 311
6 SIR 92.58 92.93 92.94 93.10 94.06 93.80
F F F F F F
0.94 0.10 0.55 0.65 0.84 0.81
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12 MGN + IR
SIR
50.23 54.34 57.00 61.79 78.17 78.45
92.60 93.01 93.10 93.18 94.08 93.85
F F F F F F
0.62 0.734 1.114 0.9644 0.57444 1.5244
F F F F F F
0.44 0.14 0.72 0.32 0.62 0.11
comparison with concurrent DMSO + irradiation group). Mangiferin pretreatment caused a significant enhancement in the repair of DNA strand breaks at 4 hours, which was approximately 30% higher than the concurrent DMSO + irradiation group (Table 2). The repair of DNA strand breaks reached an almost normal level (91.07%) at 4 hours after 1 Gy irradiation in the MGN + irradiation group when compared with the DMSO + irradiation group (72% at 4 hours). This inhibition in the generation of radiation-induced DNA strand breaks was approximately 1.3-fold for 1, 2, 3, or 4 Gy irradiation, respectively, at 4 hours in MGN + irradiation group (Table 2). 3.2.1. Strand scission factor Irradiation of HPBLs caused a dose-dependent increase in the SSF (Fig. 4). Incubation of HPBLs up to 4 hours postirradiation resulted in the repair of DNA strand breaks, which was evidenced by a significant reduction in the SSF
24 MGN + IR
SIR
50.58 54.50 57.41 61.79 78.32 78.55
92.66 93.02 93.22 93.28 94.16 93.9
F F F F F F
1.22 1.0144 0.854 0.934 0.7144 1.3544
MGN + IR F F F F F F
0.77 0.11 0.50 0.32 0.85 0.17
50.75 54.86 57.60 61.90 78.54 78.85
F F F F F F
1.10 0.7744 0.564 0.3844 0.78444 0.9444
in both DMSO + irradiation and MGN + irradiation groups when compared with the corresponding 0-hour groups (Fig. 4). This reduction in the SSF was significant in the MGN + irradiation group in comparison with the corresponding DMSO + irradiation group at 4 hours postirradiation, indicating an enhancement of DNA strand break repair by MGN (Table 3; Fig. 4). Treatment of HPBLs with a nutritional factor, MGN, reduced the radiation-induced DNA strand breaks that are responsible for genomic instability and cancer. Therefore, MGN may help to reduce the deleterious effects of ionizing radiations during planned and unplanned exposures. 4. Discussion Diagnostic and treatment procedures, space and frequent air travel, industrial procedures, and cosmic radiations have resulted in an increased human exposure to ionizing
Table 2 Effect of 50 lg/mL MGN on the DNA strand break repair in HPBLs exposed to different doses of c-irradiation at 4 h postirradiation repair time Exposure dose (Gy)
Undamaged DNA (%) F SEM DMSO + IR
MGN + IR Postirradiation repair time (h)
0 91.29 50.29 42.36 27.42 20.34 0.98
4 92.12 72.46 63.00 50.44 35.99 0.95
0 F 1.11 F 0.80 1 F 1.11 F 1.364 2 F 1.18 F 1.284 3 F 0.88 F 1.004 4 F 0.74 F 0.744 r 2 Value Values with no symbols are not significant when compared with the corresponding 0-h group. One-way ANOVA with Bonferroni post hoc test was used to calculate statistical significance. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 min before irradiation. 4 P b .01 when compared with corresponding 0-h group. 44 P b .001 when compared with corresponding 0-h group. y P b .01 when compared with corresponding DMSO + IR group at 4 h postirradiation repair time. z P b .05 when compared with corresponding DMSO + IR group at 4 h postirradiation repair time.
0 93.02 54.74 46.60 37.90 26.21 0.99
F 0.82 F 1.08 F 1.36 F 0.85 F 1.06
4 94.07 91.07 82.23 77.23 50.25 0.87
F F F F F
0.76 0.7644y 1.454,z 1.484,y 1.764,z
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Fig. 2. Effect of various concentrations of MGN on the yield of DNA strand breaks in HPBLs exposed to 3 Gy c-irradiation. Closed squares, percent DNA strand breaks (SBs) at 0 hour; open squares, percent DNA SBs at 1 hour; closed triangles, percent DNA SBs at 2 hours; open triangles, percent DNA SBs at 4 hours post–3 Gy c-radiation. The blood for all groups was suspended 1:1 vol/vol in phytohemagglutinin-free RPMI-1640 medium and incubated up to 4 hours. The cells were harvested at various repair times, and the percent DNA SBs were determined by FADU assay. Triplicate cultures were used for each concentration of MGN, which was given 30 minutes before irradiation.
radiation. Ionizing radiations are known to inflict insult to important biomolecules like DNA, RNA, proteins, and lipids and cause DNA strand breaks by direct or indirect effect [30-33]. Out of these, induction of DNA strand breaks by ionizing radiation has a serious consequence as these are converted into chromosome aberrations leading to the increased instability of genome, mutagenesis, and carcinogenesis [34]. Most of the mammalian cells possess nonhomologous end-joining (NHEJ) pathway to restore the integrity of the genome by the repair of DNA strand breaks [35,36]. However, this may not be enough when the cells suffer insult from ionizing radiation. The exogenous supply of natural antioxidant may be helpful to minimize radiation effects. The usage of common nutrient factors for radioprotection may be of immense help as they are consumed daily, have wide acceptability, have better tolerance, do not have side effects, and can be safely manipulated for human use. Therefore, an attempt has been made to investigate the radioprotective effect of MGN in the cultured HPBLs exposed to different doses of c-radiation by assessing the radiation-induced DNA strand breaks using the FADU assay. Most naturally occurring DNA strand breaks, particularly the medically relevant are produced by ionizing radiations and some chemotherapeutic agents, result from oxidative processes. The predominant repair pathway for repairing DNA strand breaks induced by ionizing radiation is NHEJ
[37,38]. The optimal repair conditions vary depending upon the chemical structure of the strand break end being rejoined [35]. In addition to strand breaks induced by c-radiation, the nucleotides proximal to the strand break site may have also become damaged, and the extent of such damage limits the repair by NHEJ pathway [39]. Rapid rejoining of strand breaks during in vitro incubation of peripheral lymphocytes at 378C was observed following a dose of 1 Gy 60Co c-radiation [29]. Various authors have used DNA strand breaks repair studies to see the inhibition of DNA repair by arsenate and antimony compounds in Chinese hamster ovary cells [40,41]. The same principle has been used in our study to demonstrate the effect of MGN in reducing the DNA damage and enhancing the repair of radiation-induced DNA strand breaks in human lymphocytes. The FADU assay has been used to detect strand breakage in HPBLs induced by some metal ions [42]. As far as authors are aware, other investigators have not used the application of FADU assay in the evaluation of radiation protection by MGN. The sensitivity of the FADU method is comparable to the alkaline elution method of measurement of DNA strand breaks [43]. The principle and application of the assay has been described by Birnboim and Jevcak [29]. The fluorescent dye selectively binds to the double-stranded DNA in the presence of single-stranded DNA, whose short duplex regions are destabilized by alkali. DNA lesions other than in
Fig. 3. Effect of 50 lg/mL MGN on the yield of DNA SBs in HPBLs exposed to different doses of c-irradiation at 4 hours postirradiation repair time. Closed squares, DMSO + irradiation at 0 hour; open squares, DMSO + irradiation at 4 hours; closed triangles, MGN + irradiation at 0 hour; open triangles, MGN + irradiation at 4 hours postirradiation. The blood for all groups was suspended 1:1 vol/vol in phytohemagglutinin-free RPMI-1640 medium and incubated to 4 hours. The cells were harvested, and the percent DNA SBs were determined by FADU assay. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 minutes before irradiation.
G.C. Jagetia, V.A. Venkatesha / Nutrition Research 26 (2006) 303 – 311 Table 3 Effect of 50 lg/mL of MGN on the SSF of HPBLs before exposure to various doses of c-radiation at 4 h postirradiation repair incubation time Exposure dose (Gy)
SSF F SEM DMSO + IR
MGN + IR
Postirradiation repair time (h) 1 2 3 4
0 0.26 0.33 0.52 0.65
F F F F
0.005 0.004 0.006 0.006
4 0.10 0.16 0.26 0.41
F F F F
0.002 0.003 0.003 0.005
0 0.23 0.30 0.39 0.55
F F F F
0.003 0.004 0.006 0.005
4 0.01 0.06 0.08 0.27
F F F F
0.0003 0.0003 0.0006 0.002
SSF = 1(log F t/F 0), where F t and F 0 are the percent undamaged DNA (ds-DNA) of treated and untreated samples, respectively. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 min before irradiation.
situ strand breaks will not affect the rate of strand separation or be labile in alkali [44]. Our experimental data indicate that the FADU method is highly sensitive to analyze the DNA strand breaks induced by c-radiation, and other authors reported that they could detect the damage produced by 1-10 cGy to 3 Gy of ionizing radiation [29,45]. Exposure of lymphocytes to c-radiation increased the DNA strand breaks. Ionizing radiations have been reported to increase the DNA strand breaks in various cells earlier [46-48]. Pretreatment of lymphocytes with MGN significantly reduced the radiation-induced DNA strand breaks in a drug dose-dependent manner up to 50 lg/mL. A time-dependent decline in DNA strand breaks was observed up to 4 hours when the cells were allowed to repair the damage in both DMSO + irradiation and MGN + irradiation groups. However, a significantly higher DNA repair was observed for MGN + irradiation group. A time-dependent elevation in DNA repair has been reported earlier up to 4 hours in various mammalian cells [49,50]. Irradiation of HPBLs to various doses of c-radiation resulted in a dose-dependent increase in DNA strand breaks. An identical effect of c-radiation in various cells has been reported earlier [46-48]. Pattern of DNA strand break induction in the MGN + irradiation group is similar to the DMSO + irradiation group, except that they have been significantly lower in this group. This observation on MGN has not been reported earlier. Further MGN treatment also restored the undamaged DNA to almost spontaneous level in lymphocytes exposed to 1 Gy at 4 hours postirradiation. The bark extract of M indica has been reported to reduce bleomycin-induced DNA damage, which mimics the damage induced by c-radiation [51]. These observations are in agreement with our earlier studies on micronuclei, where MGN has been reported to reduce the radiation-induced micronuclei in human lymphocytes [27]. In contrast, metals like nickel, arsenate, and antimony compounds have been reported to inhibit DNA repair in Chinese hamster ovary cells [40,41]. Mangiferin even reduced the normal wear and tear of DNA as evidenced by a nonsignificant decline in spontaneous DNA strand breaks. Treatment of lymphocytes
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with various concentrations of MGN did not alter the spontaneous frequency of micronucleated binucleate cells as it was within reference range [27]. Some natural products, namely curcumin, resveratrol, indole-3-carbinol, and ellagic acid, have been reported to reduce the MNNG (N-methyl-NVnitro-N-nitrosoguanidine)-induced DNA strand breaks in Chinese hamster lung fibroblast cells [52]. Similarly, vitamins C and E by virtue of antioxidant properties have been found to reduce benzo-a-pyrene–induced DNA strand breaks in HPBLs [53]. The mechanism of radioprotection of MGN is not very clear and cannot be attributed to a single mechanism. The protection afforded by MGN to radiation-induced DNA damage could be attributed to several mechanisms. Scavenging of free radicals by MGN may have played an important and significant role in protecting the DNA from the radiationinduced damage. Mangiferin has been reported to scavenge hydroxyl, DPPH (2,2-diphenyl-1-picryl hydrazine), and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt) free radicals in a concentrationdependent manner [27]. Another important mechanism of MGN may be the inhibition of radiation-induced depletion in nonprotein sulfhydryl (NPSH) groups because it has been reported to prevent NPSH loss [54]. Lipid peroxidation and the intermediary peroxyl radicals induce DNA base damage and mutation in human fibroblast cells [55]. The reduction in lipid peroxidation by MGN may also have reduced the DNA
Fig. 4. Effect of 50 lg/mL of MGN on the SSF of HPBLs before exposure to various doses of c-radiation at 4 hours postirradiation repair incubation time. Closed and open squares, DMSO + irradiation at 0 and 4 hours postirradiation time; closed and open triangles, MGN + irradiation at 0 and 4 hours postirradiation time. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 minutes before irradiation. Strand scission factor was calculated using the formula SSF = 1(log F t/F 0), where F t and F 0 are the percent ds-DNA of treated and untreated samples, respectively.
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damage in our study. Mangiferin has also been found to scavenge lipid peroxidation in a concentration-dependent manner in human lymphocytes [27]. Mangiferin inhibited phospholipid peroxidation and protein glycation and scavenged superoxide and peroxynitrite radicals [56]. Mangiferin, with established antioxidant and free radical scavenging activities, may have multidimensional mode of cytoprotection in HPBLs against radiation-induced DNA damage. The scavenging of free radicals by MGN might have inhibited the generation of complex strand breaks or multiple/clustered damaged sites, which may have favored the NHEJ pathway to repair the strand breaks, thereby protecting the HPBLs against radiation damage. Our study demonstrates that MGN, a dietary nutrient, protects HPBLs against the radiation-induced DNA damage as is evidenced by the reduction in DNA strand breaks in the MGN-treated irradiated HPBLs. Mangiferin may have protected against the radiation-induced DNA strand breaks by scavenging of free radicals, increasing NPSH levels and antioxidant status. Use of MGN may reduce the radiationinduced toxic effects in patients undergoing radiotherapy as well as during occupational exposures, space and air flights, accidental exposures, and nuclear terror attacks. Acknowledgment We thank Shoba U. Kamath (Department of Biochemistry, Kasturba Medical College, Manipal, India) for the help in using SFM 25. We also thank M.S. Vidyasagar (professor and head) and 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 Arya Vysya Sangha, a community-based charitable trust, Chitradurga, India, is gratefully acknowledged. References [1] O’Neill P, Fielden EM. Primary radical process in DNA. Adv Radiat Biol 1993;17:53 - 120. [2] Wallace SS. Detection and repair of DNA base damages produced by ionizing radiation. Environ Mol Mutagen 1988;12:431 - 77. [3] Natarajan AT, Darroudi F, Mullenders LH, Meijers F. The nature and repair of DNA lesions that lead to chromosomal aberrations induced by ionizing radiations. Mutat Res 1986;160:231 - 6. [4] Patt EB, Tyree RL, Straube DE, Smith DE. Cysteine protects against X-irradiation. Science 1949;110:213 - 4. [5] Modig HG, Margereta E, Revesz L. Effect of radioprotective aminothiols on the induction and repair of single strand breaks in the DNA of irradiated mammalian cells. Acta Radiol 1977;16:245 - 56. [6] Yuhas JM, Storer JB. Chemoprotection against three modes of radiation death in mouse. Int J Radiat Biol 1969;15:233 - 7. [7] Sweeney TR. A survey of compounds from the Antiirradiation Drug Development Program of the U.S. Army Medical Research and Development Command. Washington (DC)7 Government Printing Office; 1979. p. 308 - 18. [8] Krishnamurthy H, Jagetia GC, Jyothi P. Radioprotective effect of zinc aspartate on mouse spermatogenesis: a flow cytometric evaluation. Mutat Res 1998;401:111 - 220.
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G.C. Jagetia, V.A. Venkatesha / Nutrition Research 26 (2006) 303 – 311 [31] Dahm-Daphi J, Sass C, Alberti W. Comparison of biological effects of DNA damage induced by ionizing radiation and hydrogen peroxide in CHO cells. Int J Radiat Biol 2000;76:67 - 75. [32] Chaudhry MA, Weinfeld M. Reactivity of human apurinic/apyrimidinic endonuclease and Escherichia coli exonuclease III with bistranded abasic sites in DNA. J Biol Chem 1997;272:15650 - 5. [33] Ames BN. Cancer prevention: novel nutrient and pharmaceutical developments. Ann N Y Acad Sci 1999;889:87 - 106. [34] Peiffer P. The mutagenic potential of DNA double-strand break repair. Toxicol Lett 1998;96/97:119 - 29. [35] Pastwa E, Neumann RD, Winters TA. In vitro repair of complex unligatable oxidatively induced DNA double-strand breaks by human cell extracts. Nucleic Acids Res 2001;29:2 - 10. [36] Weiss JF, Landauer MR. Radioprotection by antioxidants. Ann N Y Acad Sci 2000;899:44 - 60. [37] Utsumi H. DNA double-strand break repair and radiation effects. Tanpakushitsu Kakusan Koso 2001;46:1089 - 96 [in Japanese]. [38] Utsumi H, Elkind MM. Requirement for repair of DNA double-strand breaks by homologous recombination in split-dose recovery. Radiat Res 2001;155:680 - 6. [39] Pastwa E, Neumann RD, Mezhevaya K, Winters TA. Repair of radiation-induced DNA double strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks. Radiat Res 2003;159:251 - 61. [40] Takahashi S, Takeda E, Kubota Y, Okayasu R. Inhibition of repair of radiation induced DNA-double strand break repair by nickel and arsenate. Radiat Res 2000;154:686 - 91. [41] Takahashi S, Sato H, Kubota Y, Utsumi H, Bedford JS, Okayasu R. Inhibition of DNA-double strand break repair by antimony compounds. Toxicol 2002;180:249 - 56. [42] McLean JR, McWilliams RS, Kaplan GJ, Birnboim HC. Rapid detection of DNA strand breaks in human peripheral blood cells and animal organs following treatment with physical and chemical agents. Proceedings of the International Symposium on Chemical Mutagenesis, Human Population Monitoring and Genetic Risk Assessment. 1980, Progress in Mutation Research, vol. 1. Amsterdam7 Elsevier/ North-Holland Biomedical Press; 1981. [43] Baumstark-Khan C, Grienbach U, Rink H. Comparison of DNA strand break induction in CHO cells measured by alkaline elution and by fluorometric analysis of DNA unwinding (FADU). Free Radic Res Commun 1992;16:381 - 9.
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Mangiferin protects human peripheral blood lymphocytes against c-radiation–induced DNA strand breaks: a fluorescence analysis of DNA unwinding assay Ganesh Chandra Jagetia4, Venkatasubbaiah A. Venkatesha Department of Radiobiology, Kasturba Medical College, Manipal-576 104, India Received 9 July 2005; revised 2 June 2006; accepted 13 June 2006
Abstract Ionizing radiations induce free radicals that lead to a cascade of events causing damage to cellular DNA. It is generally accepted that cell death induced by ionizing irradiation is due to DNA doublestrand breaks. Therefore, agents that can neutralize free radicals may be able to reduce DNA damage effectively and protect cell death. Effect of 0, 5, 10, 20, 50, or 100 lg/mL mangiferin, a glucosylxanthone present in mango (Mangifera indica), has been studied in human peripheral blood lymphocytes (HPBLs) exposed to 3 Gy c-radiation by fluorescence analysis of DNA unwinding assay. This assay detects DNA single- and double-strand breaks and alkali-labile sites by their effect on the rate of DNA denaturation in alkali, which was monitored by the fluorescence intensity of an intercalating dye, Hoechst 33258 (bisbenzimide). Estimation of DNA damage by fluorescence analysis of DNA unwinding assay showed that mangiferin as such did not have adverse effect on DNA damage, and it reduced the radiation-induced DNA damage in a concentration-dependent manner. In addition, a maximum undamaged double-stranded DNA was observed for 50 lg/mL of mangiferin. Therefore, further experiments were carried out using this concentration, wherein lymphocytes were exposed to 0, 1, 2, 3, or 4 Gy c-radiation 30 minutes after mangiferin treatment. Irradiation of HPBLs caused a radiation dose-dependent increase in the DNA strand breaks and a reduction in the undamaged double-stranded DNA, whereas treatment of lymphocytes with 50 lg/ mL mangiferin before irradiation significantly reduced DNA strand breaks and subsequently enhanced the undamaged double-stranded DNA at 4 hours posttreatment, indicating repair of radiation-induced DNA strand breaks. Mangiferin treatment restored the undamaged double-stranded DNA to almost normal level after 1 Gy irradiation, whereas it was 50% for 4 Gy at 4 hours postirradiation. Our observations suggest that mangiferin reduces initial DNA damage and enhances DNA repair in the HPBLs exposed to 1 to 4 Gy c-radiation and could serve as a protector against the radiation-induced DNA damage during planned and unplanned radiation exposures. D 2006 Elsevier Inc. All rights reserved. Keywords:
Human peripheral blood lymphocytes; Mangiferin; Irradiation; DNA strand breaks
1. Introduction
4 Corresponding author. Tel.: +91 820 2922122 (work); fax: +91 820 2571919. E-mail addresses: [email protected], [email protected] (G.C. Jagetia). 0271-5317/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2006.06.011
Interactions of ionizing radiation in mammalian cells induce a wide range of molecular damage. These can lead to diverse cellular responses, such as cell inactivation, chromosomal rearrangement, and mutations, which may contribute to the development of cancer and hereditary diseases.
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The random energy deposition by ionizing radiations induces a wide array of DNA lesions. Ionizing radiations induce damage to DNA by direct ionization and through generation of hydroxyl radicals that attack DNA [1] resulting in single-strand breaks, double-strand breaks, and oxidative damage to sugar and base residues that can be converted into strand breaks subsequently [2]. Because of induction of DNA double-strand breaks, the ionizing radiation is extremely effective in producing chromosomal aberrations leading to genomic instability [3]. Since the discovery by Patt et al [4] that cysteine protected rats and mice against radiation-induced sickness and mortality, several attempts have been made to reduce the radiation-induced damage [5,6]. However, the practical applicability of most of these compounds in humans remained limited because of their high toxicity at their optimum protective doses [7]. Therefore, a need for continued search for nontoxic radioprotector is felt. Use of various agents has been reported to reduce radiation-induced DNA damage in various systems. Zinc aspartate inhibited the radiation-induced spermatogonia killing in mice [8]. A grapefruit flavonone, naringin, has been found to protect radiation-induced chromosomal aberrations and micronuclei formation in bone marrow cells of mice [9,10]. Dietary agents that are already consumed by humans have not received the attention they deserve for their potential radioprotective effects [11]. It is likely that, if such agents are radioprotective, they may be more easily and safely used in patients undergoing radiotherapy than other radioprotective chemicals. Patients may also better tolerate them than other more promising exotic drugs because they form part of the daily human diet since time immemorial. Mango, Mangifera indica, is widely used in India and in Southeast Asia as a food supplement in the form of a pickle, and the fruit is eaten as such or is drunk in the form of juice. Mango has been widely used as an antiinflammatory, antipyretic, diuretic, antidiarrheal, antiviral, antibacterial, and hypoglycemic in the traditional system of medicine in India [12-18]. It has also been found to inhibit a-glucosidase and a-amylase activities in vitro [19,20]. The flavonoids from M indica have been found to be antihypolipidemic in rats [21]. Mangiferin (MGN), a C-glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-b-d-glucoside) purified from M indica and Anemarrhena asphodeloides has been found to inhibit proliferation of neoplastic cells in vitro and in vivo and antagonize the cytopathic effect of HIV [22]. It has also been reported to reduce intestinal neoplasms in rats [23]. Mangiferin is active against the herpes simplex virus 1, and it increases the antioxidant status in rats [18,24]. Humans are exposed to ionizing radiations for diagnostic or therapeutic purposes and in occupational settings. Apart from these man-made exposures, humans are also exposed to cosmic radiations that arise from solar flares and other cosmic interactions during air and space travel. Ionizing radiations are known to damage important biomolecules like
DNA, RNA, proteins, and lipids. DNA damage disturbs the fidelity of DNA replication causing genomic instability that may result in mutagenesis and carcinogenesis [25]. It is necessary to protect humans against the deleterious effects of ionizing radiations. Use of nutritional factors may have an advantage over other chemicals as radioprotectors because of their daily consumption and wide acceptability in humans. Pretreatment with MGN has been found to reduce the radiation-induced micronuclei formation in human peripheral blood lymphocytes (HPBLs) and increase the survival of animals exposed to different doses of c-radiation [26-28]. However, the effect of MGN on the radiationinduced molecular DNA damage has not been investigated, and its effective dose is far lower than the toxic dose [28]. Therefore, the present study was undertaken to investigate the effect of MGN on the radiation-induced DNA strand breaks in the cultured HPBLs exposed to various doses of c-radiation. 2. Methods and materials Mangiferin (catalogue no. M 3547), RPMI-1640 medium, Histopaque 1119, cyclohexane diamine tetraacetate, b-mercaptoethanol, Hoechst 33258 (bisbenzamide), and myo-inositol were obtained from Sigma Chemical Company, St. Louis, USA. The other chemicals were procured from Ranbaxy Fine Chemicals, India. 2.1. Preparation of drug Mangiferin (Fig. 1) was dissolved at a concentration of 10 mg/mL in dimethyl sulfoxide (DMSO) immediately before use. This stock was further diluted with RPMI medium as per the requirement before incubation of cells. Dimethyl sulfoxide was used as vehicle control. 2.2. Blood collection The peripheral blood was collected with informed consent from a healthy, nonsmoking, and nonalcoholic donor through venipuncture. The Kasturba Medical College ethical committee has approved this study. The blood was diluted 1:1 with phytohemagglutinin-free RPMI-1640 medium to understand the DNA strand break repair kinetics.
Fig. 1. Chemical structure of MGN.
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2.3. Determination of optimum dose (experiment 1) The blood was diluted and distributed into several culture flasks. The flasks were divided into the following groups: 1.
2.
Mangiferin + Sham-irradiation: The diluted blood of this group was treated with 0, 5, 10, 20, 50, or 100 lg/mL MGN and served as control group. Mangiferin + Irradiation: The diluted blood of this group was treated with 0, 5, 10, 20, 50, or 100 lg/mL MGN 30 minutes before exposure to 3 Gy c-radiation [26,27].
2.3.1. Irradiation Thirty minutes after DMSO or MGN treatment, various flasks containing HPBLs were kept on ice. The flasks were exposed to 0 or 3 Gy 60Co c-radiation delivered as a single fraction from a Tele Cobalt therapy source (Theratron, Atomic Energy Agency, Ontario, Canada) at a dose rate of 1 Gy/min. 2.4. Determination of radioprotective effect (experiment 2) Because 50 lg/mL MGN provided the greatest protection against the radiation-induced DNA strand breaks in the previous experiment, a separate experiment was carried out to study its radioprotective effects against different doses of c-radiation, where HPBLs were divided into the following groups: 1.
2.
DMSO + Irradiation: The diluted blood of this group was treated with DMSO 30 minutes before exposure to various doses of c-radiation and served as control group. MGN + Irradiation: The diluted blood of this group was treated with 50 lg/mL MGN 30 minutes before exposure to different doses of c-radiation.
2.4.1. Irradiation The irradiation procedure was exactly similar to that described above, except that the diluted blood from both groups was exposed to 0, 1, 2, 3, or 4 Gy of c-radiation. The DNA strand break repair kinetics in both groups was studied by fluorescence analysis of DNA unwinding (FADU) at 0, 1, 2, 4, 6, 12, or 24 hours for experiment 1, whereas at 0 or 4 hours postirradiation for experiment 2. Triplicate cultures were used for each dose of MGN or radiation for both the groups in each experiment, and results were reconfirmed by repetition of the experiments. Application of the test of homogeneity between the original and repeat experiments did not reveal significant differences; therefore, results of both experiments were combined.
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denaturation in alkali, monitored by the fluorescence intensity of an intercalating dye. 2.5.1. Lymphocyte isolation The lymphocytes from the above-mentioned groups were isolated at different postirradiation times by layering the irradiated diluted blood on Histopaque1119 and by centrifugation at 800 rpm for 15 minutes at room temperature. The buffy coat rich in lymphocytes was aspirated carefully and transferred into a fresh tube containing phosphatebuffered saline (PBS). The cells were washed twice with PBS, and the lymphocyte count was determined by trypan blue dye exclusion method to check the cell viability. Because no dead cells were reported, 3 106 isolated cells were distributed into 1.5 mL PBS and were divided equally (0.5 mL) in 3 tubes designated as P, B, or T and treated as described below before starting the FADU assay. P: lymphocytes, reagent mixture but without dye. B: lymphocytes, PBS and dye but without reagent mixture. T: lymphocytes, PBS, reagent mixture and dye. 2.5.2. Assay procedure Briefly, the cells were incubated with 1 mL of reagent mixture (containing 0.3 mL of 6 mmol myo-inositol, 0.1 mL of 2% urea, 0.1 mL of 5% SDS (sodium dodecyl sulphate), 0.4 mL of 5 M NaOH, and 0.1 mL of 2 mmol cyclohexane diamine tetraacetate in deionized double-distilled water) for 1 hour at 158C. The samples were flash-frozen for 1 minute, and 0.4 mL neutralizing mixture was added at room temperature (1 M glucose, 14 mmol b-mercaptoethanol). The samples were immediately sonicated for 5 seconds (Sonics Vibra-cell, Newtown, Conn, USA) to inhibit reassociation of complementary strands and to reduce the molecular weight of DNA. Each cell sample was transferred to a quartz cuvette and mixed gently with 1 mL Hoechst 33258 (5 lg/mL) by single inversion, and the fluorescence was read at an excitation wavelength of 350 nm and an emission wavelength of 450 nm using a spectrofluorimeter (SFM 25, Kontron Instruments, Neufahrn, Germany) at room temperature. The percent-undamaged DNA (ds-DNA) and DNA strand breaks (DNA SBs) in the treatment groups were calculated as follows: Percent undamaged ds-DNA = (PB/TB) 100 Percent DNA SBs = 100 percent undamaged ds-DNA Strand scission factor (SSF) = 1(log F t/F 0) where F t and F 0 are the percent ds-DNA of treated and untreated samples, respectively.
2.5. Fluorescence analysis of DNA unwinding assay
2.6. Statistical analysis
The FADU assay was performed in HPBLs as described by Birnboim and Jevcak [29] with minor modifications. This method detects single- and double-strand breaks and alkali labile sites by their effect on the rate of DNA
The statistical significance between the treatments was determined using one-way analysis of variance (ANOVA) and Fisher exact test. Differences indicated by P b .05 were considered statistically significant. Solo 4 statistical package
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Table 1 Effect of various concentrations of MGN on the yield of DNA strand breaks in HPBLs exposed to 3 Gy c-irradiation MGN (lg/mL)
Undamaged ds-DNA (%) F SEM Postirradiation repair time (h) 0
0a 5 10 20 50 100
SIR 92.49 F 0.56 92.64 F 0.77 92.46 F 0.35 92.66 F 0.28 93.11 F 0.83 93.20 F 0.72
MGN + IR 27.62 F 0.50 28.73 F 0.49 32.48 F 0.394 34.74 F 0.684 37.63 F 0.6744 38.03 F 0.4144
1 SIR 92.50 92.66 92.58 92.72 93.30 93.35
F F F F F F
0.60 0.57 0.33 0.58 1.08 0.27
2
MGN + IR 36.51 F 0.38 39.26 F 0.584 43.56 F 0.64444 46.57 F 0.7044 59.06 F 0.67444 59.40 F 0.68444
SIR 92.51 92.71 92.73 92.80 93.63 93.64
F F F F F F
0.16 0.16 1.23 1.19 0.47 1.20
MGN + IR 46.71 F 0.77 50.91 F 0.304 53.05 F 0.4544 57.09 F 0.4844 66.22 F 0.51444 66.60 F 1.25444
4 SIR 92.56 92.86 92.71 92.86 93.87 93.75
F F F F F F
0.77 1.02 0.62 0.28 0.11 0.65
MGN + IR 50.78 F 0.37 53.27 F 0.73 56.47 F 0.524 61.24 F 0.6344 78.12 F 0.73444 78.50 F 0.71444
The healthy donor blood was cultured in phytohemagglutinin-free RPMI-1640 medium (1:1 vol/vol) with or without MGN for different times. SIR indicates sham-irradiation; IR, irradiation. Values with no symbols are not significant when compared with the non–MGN-treated irradiated group. One-way ANOVA with Bonferroni post hoc test was used to calculate statistical significance. Triplicate cultures were used for each concentration of MGN which was given 30 min before irradiation. a DMSO (dimethyl sulfoxide) alone. 4 P b .05 when compared with non–MGN-treated irradiated group. 44 P b .01 when compared with non–MGN-treated irradiated group. 444 P b .001 when compared with non–MGN-treated irradiated group.
(BMDP Statistical software Inc, Los Angeles, Calif, USA.) was used for statistical analysis. 3. Results The results are expressed as undamaged ds-DNA (%) F SEM in Tables 1 and 2 and Figs. 2 and 3 and as SSF in Table 3 and Fig. 4. 3.1. Determination of optimum dose (experiment 1) The spontaneous frequency of undamaged ds-DNA remained unaltered when HPBLs were treated with the various concentrations of MGN (92.49% F 0.56%) at all postirradiation periods (Table 1). Immediate assessment of DNA damage (0 hour) after exposure of HPBLs to 3 Gy c-radiation caused a drastic reduction in the undamaged dsDNA (27.62% F 0.5%). The repair of DNA damage progressed with time, and a maximum repair (50.78% F 0.37%) was observed at 4 hours postirradiation. Allowing additional time did not enhance DNA repair, which remained almost static even up to 24 hours postirradiation (Table 1). Treatment of HPBLs with different concentrations of MGN 30 minutes before irradiation resulted in a progressive repair of DNA strand breaks up to 4 hours postirradiation ( P b .05 for 5 lg/mL MGN at 1, 2, and 4 hours; P b .001, 0.01, and 0.05 for 10 lg/mL MGN at 1, 2, and 4 hours; P b .01 for 20 lg/mL MGN at 1, 2, and 4 hours; P b .001 for 50 and 100 lg/mL MGN at 1, 2, and 4 hours postirradiation) when compared with irradiated (0 lg MGN) control group. This repair of DNA strand breaks was significantly greater in the MGN + irradiation group when compared with 3 Gy irradiation (0 lg MGN) group (Table 1; Fig. 2). Incubation of cells up to 24 hours did not significantly elevate the amount of undamaged ds-DNA in both MGN + sham-irradiation or MGN + 3 Gy irradiation groups. The highest amount of undamaged ds-DNA or
lowest DNA strand breaks were observed in HPBLs treated with 50 lg/mL MGN at 4 hours postirradiation, increase in MGN concentration did not further augment the repair of DNA strand breaks (Table 1). 3.2. Determination of radioprotective effect (experiment 2) Evaluation of DNA damage in the previous experiment indicated that a maximum repair was found at 4 hours postirradiation at an optimum dose of 50 lg/mL MGN. Therefore, DNA strand breaks were measured at 4 hours postirradiation in the lymphocytes treated with 50 lg/mL MGN before exposure to 0, 1, 2, 3, or 4 Gy of c-radiation (Table 2; Fig. 3). A maximum amount of undamaged DNA (91.29% F 1.11%) was observed for the DMSO + shamirradiation (0 Gy) group. Mangiferin pretreatment resulted in a nonsignificant elevation in the undamaged ds-DNA (93.02% F 0.82%) at 0 hour. A similar effect was also discernible for 4 hours postirradiation. Irradiation of HPBLs to various doses of c-radiation resulted in a dose-dependent elevation in DNA strand breaks as evidenced by a progressive reduction in undamaged ds-DNA in the DMSO + irradiation group ( P b .001 at all irradiation doses in comparison with the DMSO + sham irradiation group) (Table 2; Fig. 3). The highest DNA strand breaks (80%) were observed for 4 Gy at 0 hour postirradiation in the DMSO + irradiation group. The assessment of DNA damage at 4 hours postirradiation showed repair of DNA strand breaks that declined in an irradiation dose-dependent manner, and the lowest repair of DNA strand breaks was observed for 4 Gy in the DMSO + irradiation group ( P b .01, 0.001, 0.001, and 0.001 at 1, 2, 3, or 4 Gy, respectively, in comparison with the DMSO + sham irradiation group at 4 hours) (Table 2; Fig. 3). Treatment of HPBLs with 50 lg/mL MGN for 30 minutes before exposure to different doses of c-radiation significantly inhibited the DNA strand breaks induction at 0 hour postirradiation ( P b .01, 0.05, 0.05, and 0.05 at 1, 2, 3, or 4 Gy c-radiation in
G.C. Jagetia, V.A. Venkatesha / Nutrition Research 26 (2006) 303 – 311
6 SIR 92.58 92.93 92.94 93.10 94.06 93.80
F F F F F F
0.94 0.10 0.55 0.65 0.84 0.81
307
12 MGN + IR
SIR
50.23 54.34 57.00 61.79 78.17 78.45
92.60 93.01 93.10 93.18 94.08 93.85
F F F F F F
0.62 0.734 1.114 0.9644 0.57444 1.5244
F F F F F F
0.44 0.14 0.72 0.32 0.62 0.11
comparison with concurrent DMSO + irradiation group). Mangiferin pretreatment caused a significant enhancement in the repair of DNA strand breaks at 4 hours, which was approximately 30% higher than the concurrent DMSO + irradiation group (Table 2). The repair of DNA strand breaks reached an almost normal level (91.07%) at 4 hours after 1 Gy irradiation in the MGN + irradiation group when compared with the DMSO + irradiation group (72% at 4 hours). This inhibition in the generation of radiation-induced DNA strand breaks was approximately 1.3-fold for 1, 2, 3, or 4 Gy irradiation, respectively, at 4 hours in MGN + irradiation group (Table 2). 3.2.1. Strand scission factor Irradiation of HPBLs caused a dose-dependent increase in the SSF (Fig. 4). Incubation of HPBLs up to 4 hours postirradiation resulted in the repair of DNA strand breaks, which was evidenced by a significant reduction in the SSF
24 MGN + IR
SIR
50.58 54.50 57.41 61.79 78.32 78.55
92.66 93.02 93.22 93.28 94.16 93.9
F F F F F F
1.22 1.0144 0.854 0.934 0.7144 1.3544
MGN + IR F F F F F F
0.77 0.11 0.50 0.32 0.85 0.17
50.75 54.86 57.60 61.90 78.54 78.85
F F F F F F
1.10 0.7744 0.564 0.3844 0.78444 0.9444
in both DMSO + irradiation and MGN + irradiation groups when compared with the corresponding 0-hour groups (Fig. 4). This reduction in the SSF was significant in the MGN + irradiation group in comparison with the corresponding DMSO + irradiation group at 4 hours postirradiation, indicating an enhancement of DNA strand break repair by MGN (Table 3; Fig. 4). Treatment of HPBLs with a nutritional factor, MGN, reduced the radiation-induced DNA strand breaks that are responsible for genomic instability and cancer. Therefore, MGN may help to reduce the deleterious effects of ionizing radiations during planned and unplanned exposures. 4. Discussion Diagnostic and treatment procedures, space and frequent air travel, industrial procedures, and cosmic radiations have resulted in an increased human exposure to ionizing
Table 2 Effect of 50 lg/mL MGN on the DNA strand break repair in HPBLs exposed to different doses of c-irradiation at 4 h postirradiation repair time Exposure dose (Gy)
Undamaged DNA (%) F SEM DMSO + IR
MGN + IR Postirradiation repair time (h)
0 91.29 50.29 42.36 27.42 20.34 0.98
4 92.12 72.46 63.00 50.44 35.99 0.95
0 F 1.11 F 0.80 1 F 1.11 F 1.364 2 F 1.18 F 1.284 3 F 0.88 F 1.004 4 F 0.74 F 0.744 r 2 Value Values with no symbols are not significant when compared with the corresponding 0-h group. One-way ANOVA with Bonferroni post hoc test was used to calculate statistical significance. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 min before irradiation. 4 P b .01 when compared with corresponding 0-h group. 44 P b .001 when compared with corresponding 0-h group. y P b .01 when compared with corresponding DMSO + IR group at 4 h postirradiation repair time. z P b .05 when compared with corresponding DMSO + IR group at 4 h postirradiation repair time.
0 93.02 54.74 46.60 37.90 26.21 0.99
F 0.82 F 1.08 F 1.36 F 0.85 F 1.06
4 94.07 91.07 82.23 77.23 50.25 0.87
F F F F F
0.76 0.7644y 1.454,z 1.484,y 1.764,z
308
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Fig. 2. Effect of various concentrations of MGN on the yield of DNA strand breaks in HPBLs exposed to 3 Gy c-irradiation. Closed squares, percent DNA strand breaks (SBs) at 0 hour; open squares, percent DNA SBs at 1 hour; closed triangles, percent DNA SBs at 2 hours; open triangles, percent DNA SBs at 4 hours post–3 Gy c-radiation. The blood for all groups was suspended 1:1 vol/vol in phytohemagglutinin-free RPMI-1640 medium and incubated up to 4 hours. The cells were harvested at various repair times, and the percent DNA SBs were determined by FADU assay. Triplicate cultures were used for each concentration of MGN, which was given 30 minutes before irradiation.
radiation. Ionizing radiations are known to inflict insult to important biomolecules like DNA, RNA, proteins, and lipids and cause DNA strand breaks by direct or indirect effect [30-33]. Out of these, induction of DNA strand breaks by ionizing radiation has a serious consequence as these are converted into chromosome aberrations leading to the increased instability of genome, mutagenesis, and carcinogenesis [34]. Most of the mammalian cells possess nonhomologous end-joining (NHEJ) pathway to restore the integrity of the genome by the repair of DNA strand breaks [35,36]. However, this may not be enough when the cells suffer insult from ionizing radiation. The exogenous supply of natural antioxidant may be helpful to minimize radiation effects. The usage of common nutrient factors for radioprotection may be of immense help as they are consumed daily, have wide acceptability, have better tolerance, do not have side effects, and can be safely manipulated for human use. Therefore, an attempt has been made to investigate the radioprotective effect of MGN in the cultured HPBLs exposed to different doses of c-radiation by assessing the radiation-induced DNA strand breaks using the FADU assay. Most naturally occurring DNA strand breaks, particularly the medically relevant are produced by ionizing radiations and some chemotherapeutic agents, result from oxidative processes. The predominant repair pathway for repairing DNA strand breaks induced by ionizing radiation is NHEJ
[37,38]. The optimal repair conditions vary depending upon the chemical structure of the strand break end being rejoined [35]. In addition to strand breaks induced by c-radiation, the nucleotides proximal to the strand break site may have also become damaged, and the extent of such damage limits the repair by NHEJ pathway [39]. Rapid rejoining of strand breaks during in vitro incubation of peripheral lymphocytes at 378C was observed following a dose of 1 Gy 60Co c-radiation [29]. Various authors have used DNA strand breaks repair studies to see the inhibition of DNA repair by arsenate and antimony compounds in Chinese hamster ovary cells [40,41]. The same principle has been used in our study to demonstrate the effect of MGN in reducing the DNA damage and enhancing the repair of radiation-induced DNA strand breaks in human lymphocytes. The FADU assay has been used to detect strand breakage in HPBLs induced by some metal ions [42]. As far as authors are aware, other investigators have not used the application of FADU assay in the evaluation of radiation protection by MGN. The sensitivity of the FADU method is comparable to the alkaline elution method of measurement of DNA strand breaks [43]. The principle and application of the assay has been described by Birnboim and Jevcak [29]. The fluorescent dye selectively binds to the double-stranded DNA in the presence of single-stranded DNA, whose short duplex regions are destabilized by alkali. DNA lesions other than in
Fig. 3. Effect of 50 lg/mL MGN on the yield of DNA SBs in HPBLs exposed to different doses of c-irradiation at 4 hours postirradiation repair time. Closed squares, DMSO + irradiation at 0 hour; open squares, DMSO + irradiation at 4 hours; closed triangles, MGN + irradiation at 0 hour; open triangles, MGN + irradiation at 4 hours postirradiation. The blood for all groups was suspended 1:1 vol/vol in phytohemagglutinin-free RPMI-1640 medium and incubated to 4 hours. The cells were harvested, and the percent DNA SBs were determined by FADU assay. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 minutes before irradiation.
G.C. Jagetia, V.A. Venkatesha / Nutrition Research 26 (2006) 303 – 311 Table 3 Effect of 50 lg/mL of MGN on the SSF of HPBLs before exposure to various doses of c-radiation at 4 h postirradiation repair incubation time Exposure dose (Gy)
SSF F SEM DMSO + IR
MGN + IR
Postirradiation repair time (h) 1 2 3 4
0 0.26 0.33 0.52 0.65
F F F F
0.005 0.004 0.006 0.006
4 0.10 0.16 0.26 0.41
F F F F
0.002 0.003 0.003 0.005
0 0.23 0.30 0.39 0.55
F F F F
0.003 0.004 0.006 0.005
4 0.01 0.06 0.08 0.27
F F F F
0.0003 0.0003 0.0006 0.002
SSF = 1(log F t/F 0), where F t and F 0 are the percent undamaged DNA (ds-DNA) of treated and untreated samples, respectively. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 min before irradiation.
situ strand breaks will not affect the rate of strand separation or be labile in alkali [44]. Our experimental data indicate that the FADU method is highly sensitive to analyze the DNA strand breaks induced by c-radiation, and other authors reported that they could detect the damage produced by 1-10 cGy to 3 Gy of ionizing radiation [29,45]. Exposure of lymphocytes to c-radiation increased the DNA strand breaks. Ionizing radiations have been reported to increase the DNA strand breaks in various cells earlier [46-48]. Pretreatment of lymphocytes with MGN significantly reduced the radiation-induced DNA strand breaks in a drug dose-dependent manner up to 50 lg/mL. A time-dependent decline in DNA strand breaks was observed up to 4 hours when the cells were allowed to repair the damage in both DMSO + irradiation and MGN + irradiation groups. However, a significantly higher DNA repair was observed for MGN + irradiation group. A time-dependent elevation in DNA repair has been reported earlier up to 4 hours in various mammalian cells [49,50]. Irradiation of HPBLs to various doses of c-radiation resulted in a dose-dependent increase in DNA strand breaks. An identical effect of c-radiation in various cells has been reported earlier [46-48]. Pattern of DNA strand break induction in the MGN + irradiation group is similar to the DMSO + irradiation group, except that they have been significantly lower in this group. This observation on MGN has not been reported earlier. Further MGN treatment also restored the undamaged DNA to almost spontaneous level in lymphocytes exposed to 1 Gy at 4 hours postirradiation. The bark extract of M indica has been reported to reduce bleomycin-induced DNA damage, which mimics the damage induced by c-radiation [51]. These observations are in agreement with our earlier studies on micronuclei, where MGN has been reported to reduce the radiation-induced micronuclei in human lymphocytes [27]. In contrast, metals like nickel, arsenate, and antimony compounds have been reported to inhibit DNA repair in Chinese hamster ovary cells [40,41]. Mangiferin even reduced the normal wear and tear of DNA as evidenced by a nonsignificant decline in spontaneous DNA strand breaks. Treatment of lymphocytes
309
with various concentrations of MGN did not alter the spontaneous frequency of micronucleated binucleate cells as it was within reference range [27]. Some natural products, namely curcumin, resveratrol, indole-3-carbinol, and ellagic acid, have been reported to reduce the MNNG (N-methyl-NVnitro-N-nitrosoguanidine)-induced DNA strand breaks in Chinese hamster lung fibroblast cells [52]. Similarly, vitamins C and E by virtue of antioxidant properties have been found to reduce benzo-a-pyrene–induced DNA strand breaks in HPBLs [53]. The mechanism of radioprotection of MGN is not very clear and cannot be attributed to a single mechanism. The protection afforded by MGN to radiation-induced DNA damage could be attributed to several mechanisms. Scavenging of free radicals by MGN may have played an important and significant role in protecting the DNA from the radiationinduced damage. Mangiferin has been reported to scavenge hydroxyl, DPPH (2,2-diphenyl-1-picryl hydrazine), and ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt) free radicals in a concentrationdependent manner [27]. Another important mechanism of MGN may be the inhibition of radiation-induced depletion in nonprotein sulfhydryl (NPSH) groups because it has been reported to prevent NPSH loss [54]. Lipid peroxidation and the intermediary peroxyl radicals induce DNA base damage and mutation in human fibroblast cells [55]. The reduction in lipid peroxidation by MGN may also have reduced the DNA
Fig. 4. Effect of 50 lg/mL of MGN on the SSF of HPBLs before exposure to various doses of c-radiation at 4 hours postirradiation repair incubation time. Closed and open squares, DMSO + irradiation at 0 and 4 hours postirradiation time; closed and open triangles, MGN + irradiation at 0 and 4 hours postirradiation time. Triplicate cultures were used for each dose of radiation. Mangiferin was given 30 minutes before irradiation. Strand scission factor was calculated using the formula SSF = 1(log F t/F 0), where F t and F 0 are the percent ds-DNA of treated and untreated samples, respectively.
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damage in our study. Mangiferin has also been found to scavenge lipid peroxidation in a concentration-dependent manner in human lymphocytes [27]. Mangiferin inhibited phospholipid peroxidation and protein glycation and scavenged superoxide and peroxynitrite radicals [56]. Mangiferin, with established antioxidant and free radical scavenging activities, may have multidimensional mode of cytoprotection in HPBLs against radiation-induced DNA damage. The scavenging of free radicals by MGN might have inhibited the generation of complex strand breaks or multiple/clustered damaged sites, which may have favored the NHEJ pathway to repair the strand breaks, thereby protecting the HPBLs against radiation damage. Our study demonstrates that MGN, a dietary nutrient, protects HPBLs against the radiation-induced DNA damage as is evidenced by the reduction in DNA strand breaks in the MGN-treated irradiated HPBLs. Mangiferin may have protected against the radiation-induced DNA strand breaks by scavenging of free radicals, increasing NPSH levels and antioxidant status. Use of MGN may reduce the radiationinduced toxic effects in patients undergoing radiotherapy as well as during occupational exposures, space and air flights, accidental exposures, and nuclear terror attacks. Acknowledgment We thank Shoba U. Kamath (Department of Biochemistry, Kasturba Medical College, Manipal, India) for the help in using SFM 25. We also thank M.S. Vidyasagar (professor and head) and 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 Arya Vysya Sangha, a community-based charitable trust, Chitradurga, India, is gratefully acknowledged. References [1] O’Neill P, Fielden EM. Primary radical process in DNA. Adv Radiat Biol 1993;17:53 - 120. [2] Wallace SS. Detection and repair of DNA base damages produced by ionizing radiation. Environ Mol Mutagen 1988;12:431 - 77. [3] Natarajan AT, Darroudi F, Mullenders LH, Meijers F. The nature and repair of DNA lesions that lead to chromosomal aberrations induced by ionizing radiations. Mutat Res 1986;160:231 - 6. [4] Patt EB, Tyree RL, Straube DE, Smith DE. Cysteine protects against X-irradiation. Science 1949;110:213 - 4. [5] Modig HG, Margereta E, Revesz L. Effect of radioprotective aminothiols on the induction and repair of single strand breaks in the DNA of irradiated mammalian cells. Acta Radiol 1977;16:245 - 56. [6] Yuhas JM, Storer JB. Chemoprotection against three modes of radiation death in mouse. Int J Radiat Biol 1969;15:233 - 7. [7] Sweeney TR. A survey of compounds from the Antiirradiation Drug Development Program of the U.S. Army Medical Research and Development Command. Washington (DC)7 Government Printing Office; 1979. p. 308 - 18. [8] Krishnamurthy H, Jagetia GC, Jyothi P. Radioprotective effect of zinc aspartate on mouse spermatogenesis: a flow cytometric evaluation. Mutat Res 1998;401:111 - 220.
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