|Year : 2017 | Volume
| Issue : 3 | Page : 141-146
Modification of radiation-induced murine thymic lymphoma incidence by curcumin
PS Dange, HD Yadav, Vimalesh Kumar, HN Bhilwade, BN Pandey, HD Sarma
Division of Radiation Biology and Health Sciences, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India
|Date of Web Publication||17-Oct-2017|
H D Sarma
Division of Radiation Biology and Health Sciences, Bhabha Atomic Research Centre, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: Curcumin is a known antioxidant, preventing radiation damage including carcinogenesis. However, concentration and feeding schedule of curcumin in modification of radiation induced thymic lymphoma incidence in vivo model has not been studied. Materials and Methods: We report here modification of incidence of γ-radiation-induced thymic lymphoma in mice fed with different doses of curcumin (0.05 to 1*) in diet. Results: Female Swiss mice (6-8 weeks) fed with normal diet and exposed to 3 Gy whole body60Co γ-irradiation (WBI) showed 85 * incidence of thymic lymphoma (TL) at 120 days post-irradiation. A concentration of 1 * curcumin was found the most effective in TL incidence prevention than other fed concentrations. The TL incidence was remarkably reduced when curcumin was fed to the mice before than after the radiation exposure. The incidence of TL was reduced to 63* in mice fed with 1* curcumin in diet for 3 weeks after exposure to WBI. On the other hand, when animals were fed with same concentration of curcumin for 2 weeks and 3 weeks before WBI, the TL incidence was reduced to 55* and 35*, respectively. Curcumin feeding resulted in significant prevention in micronucleus formation in the bone marrow, which was corroborated with inhibition in DNA damage quantified by comet assay. Moreover, significant prevention in DNA damage was also observed in the peripheral blood cells in curcumin fed and irradiated mice, which however, was not prominent in thymus. Curcumin was able to prevent apoptotic death in thymus and bone marrow 4 h after irradiation, which however, got attenuated at longer post-irradiation period (24 h). Conclusion: These results suggest modification of TL incidence by curcumin in irradiated mice involving DNA damage and apoptotic death mechanisms.
Keywords: Curcumin, radiation, radiation induced, thymic lymphoma
|How to cite this article:|
Dange P S, Yadav H D, Kumar V, Bhilwade H N, Pandey B N, Sarma H D. Modification of radiation-induced murine thymic lymphoma incidence by curcumin. J Radiat Cancer Res 2017;8:141-6
|How to cite this URL:|
Dange P S, Yadav H D, Kumar V, Bhilwade H N, Pandey B N, Sarma H D. Modification of radiation-induced murine thymic lymphoma incidence by curcumin. J Radiat Cancer Res [serial online] 2017 [cited 2017 Dec 11];8:141-6. Available from: http://www.journalrcr.org/text.asp?2017/8/3/141/216872
| Introduction|| |
Risk of cancer incidence is of paramount importance in the area of radiation protection., Various experimental models including mice have been studied to investigate the radiation-induced lymphomagenesis. Development of thymic lymphoma (TL) after fractionated dose of whole body radiation was first reported by Kaplan et al. which was subsequently studied in many laboratories,, including ours. Our previous studies showed that whole body radiation exposure of Swiss mice resulted in TL in ~80% animals, which was found to be dependent on the age and gender of mice. Moreover, we also observed inhibition of TL when animals were fed with antioxidants (curcumin, eugenol, and ascorbic acid) before irradiation. However, modification of TL by curcumin fed at different concentrations and schedule has not been reported. Hence, the present study is aimed to study the effect of curcumin feeding in different concentrations and before/after irradiation on incidence of TL. Moreover, magnitude of DNA damage and apoptosis in target organs such as bone marrow and thymus was also studied in curcumin-mediated modification of TL in the irradiated mice.
| Materials and Methods|| |
All animal experiments were conducted adhering the guidelines from the Institutional Animal Ethics Committee. Four-to-eight-week-old female Swiss mice were used in these experiments. These animals were housed in husk-bedded polypropylene cages and were maintained on a standard laboratory diet and water ad libitum under controlled temperature conditions and cycles of 12 h light/12 dark.
Animals were irradiated to 3 Gy (dose rate: 1 Gy/min, 15 cm × 15 cm) to whole body γ-radiation from a60 Co γ-rays (Bhabhatron II, Panacea Medical Technologies, Bengaluru, India) in specially designed well-ventilated Perspex Box. Dosimetry of radiation exposure was performed using 0.6 cc Farmer's Chamber.
Curcumin feeding and determination of thymic lymphoma incidence
Curcumin powder was procured from Sammy Laboratory, Bengaluru, India. Curcumin powder at concentrations of 0.05%, 0.1%, 0.5%, and 1.0% was thoroughly mixed with the pulverized animal diet and fed to the different experimental groups of mice in specially designed clay feed cups. Control animals were fed with pulverized normal diet. Feeding of curcumin mixed powdered diet was done to the experimental animals for the duration of either 3 weeks before or 2–3 weeks after exposure of the animals to 3 Gy whole body60 Co γ-irradiation (WBI). While feeding curcumin, each animal was housed in a single cage and was observed daily for the consumption of curcumin mixed diet. Average food intake of a mouse was observed to be approximately 5 g/day. Curcumin feeding was started to the experimental mice aging 4–8 weeks. The actual age of a mouse for the administration of curcumin mixed diet was determined in such a way that irrespective of the duration of curcumin feeding and the age of mice in all experimental and the control groups were same at the time of irradiation. Animals were sacrificed after 120 days of irradiation to check the TL development. The aggressiveness of TL incidence was categorized based on tumor weight as small (>0.04–0.1 g), moderate (>0.1–0.2 g), and large (>0.2 g). The percentage incidence of TL was rounded to nearest whole number.
In another set of experiment, to study chromosomal aberrations, DNA damage, and apoptosis, animals were fed with 1% curcumin for 3 weeks followed by radiation (3 Gy). For MN assay and DNA damage studies, animals were sacrificed 4 h after radiation. However, for apoptosis measurements animals were sacrificed after 4 h (TUNEL and caspase 3 assays) and 24 h (caspase 3 assay).
Estimation of DNA damage by alkaline comet assay
DNA damage at different time points was estimated by alkaline comet assay, which measures total strand breaks including single and double strand breaks. For the comet assay, ~50 μl of peripheral blood was collected from the tail vein of individual mice in heparinized vials, which was used to prepare two slides. For measurement of DNA damage in thymocytes, thymus was collected after sacrifice of the animal followed by preparation thymocytes single cell suspension as mentioned earlier. These samples were processed for comet assay., The images of comets were acquired using a fluorescence microscope (Axioplan, Carl Zeiss, Germany). Randomly selected fifty images per slide were analyzed for determination of % DNA in tail and tail moment using CASP software (www.casplab.com). Tail moment is a product of fraction of the DNA in the tail of the comet and the tail length.
Bone marrow micronuclei assay
At 24 h of irradiation, femur bones were dissected out from the control and irradiated animals after sacrifice. From these bones, bone marrow cells were aspirated and flushed out into fetal bovine serum. These obtained cells were pelleted down (centrifugation 1000 × g; 5 min) and loosened cell pellet was smeared on to the glass slides and air dried. The slides were stained using May-Grunwald-Giemsa and mounted using DPX mounting medium (Sigma, St Louis, MO, USA). Two slides were prepared from each animal. Slides were scored for the micronucleated polychromatic erythrocytes at ×1000 and MN frequency was calculated as mentioned earlier.
Determination of magnitude of apoptosis
For determination of magnitude of apoptosis, TUNEL and caspase 3 assays were performed. For TUNEL assay, 5 μ thick section was obtained from freshly collected thymus tissues from the control/treated animals using Cryotome (Microm International, Sweden). These slides were processed using TUNEL kit (Roche) as instructions provided in the kit. The slides were observed using confocal microscopy (LSM 510 Meta, Carl-Zeiss, Germany). For caspase 3 assay, lysates of thymus tissue were prepared, and activity of caspase-3 in control and irradiated thymocytes was measured using FluorAce Apopain Assay kit (Bio-Rad, USA). The fluorescence intensity of reaction mixture was estimated fluorimetrically (LS50B, Perkin Elmer, USA) and enzymatic activity was calculated following the protocol provided by the manufacturer of kit.
Where ever required, statistical analysis was performed using t-test. Values considered significantly different when P < 0.05.
| Results|| |
Thymic lymphoma incidence in irradiated female mice fed with curcumin
Effect of curcumin on TL incidence was monitored in whole body irradiated mice and fed with curcumin either before or after irradiation. In first set of experiment, in animals were fed with varying doses (0.05%–1%), curcumin for 3 weeks followed by irradiation [Table 1]. The TL incidence was 80% in animals irradiated with 3 Gy and fed with normal diet. In this group of animals, most of the TL was small (55%) in size. Compared to irradiated animals fed on normal diet, animals fed with 0.05% and 0.1% curcumin did not show any change in total percentage of TL. However, percentage small size of TL was lower in these curcumin fed animals. A marginal decrease (8%) in TL incidence was found when irradiated animals were prior fed with 0.5% curcumin. However, at the highest concentration of curcumin, i.e. 1%, 45% decrease TL incidence was observed, where most of the TL (30%) was large in size. Furthermore, effect of feeding of curcumin either before or after radiation on TL incidence was determined [Table 2]. Our studies showed that compared to 3 weeks feeding of curcumin before irradiation, 2-week feeding resulted in 20% higher TL incidence. It was interesting to observe that while prefed (3 weeks) curcumin resulted in significant prevention in small size tumors, postfeeding prevented mainly large size tumors.
|Table 1: Incidence of thymic lymphoma (*) in 6-8 week-old female Swiss mice fed with control (normal diet) or 0.05*-1* curcumin in diet for 3 weeks before 3 Gy of whole body γ irradiation and observed at 120 days postirradiation|
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|Table 2: Incidence of thymic lymphoma (*) in 6-8 weeks old female Swiss mice fed with control (normal diet) or 1* curcumin in diet for 3 weeks before or after 3 Gy of whole body γ irradiation and observed at 120 days postirradiation|
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Magnitude of DNA damage and micronuclei formation in irradiated animals fed with curcumin
To study the magnitude of DNA damage, MN formation was also estimated in bone marrow cells obtained from (i) control, (ii) 1% curcumin fed, (iii) 3 Gy, and (iv) curcumin + 3 Gy groups of animals [Figure 1]a. Moreover, using comet assay, DNA damage was also determined in peripheral blood cells and thymocytes of these groups of animals [Figure 1]b and [Figure 1]c. Compared to control animals, MN frequency was significantly increased after irradiation, which was attenuated in curcumin fed animals before irradiation. Our results showed that in case of whole body irradiation (3 Gy) resulted in increase in DNA damage in peripheral blood cells, which was significantly decreased when animals were fed with curcumin before irradiation. On the contrary, radiation-induced increased DNA damage in thymocytes was not prevented significantly when animals were fed to curcumin.
|Figure 1: (a) Magnitude of MN formation in bone marrow cells in control and treated animals; (b) DNA damage in peripheral blood cells and (c) in thymocytes by comet assay in different treatment groups. Data are presented as the mean ± standard error of the mean. *Significantly different (at P < 0.05) compared to radiation (3 Gy) group|
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Effect of curcumin feeding on magnitude of apoptosis in irradiated animals
Magnitude of apoptosis was determined in thymus tissue using TUNEL assay. Compared to control (sham-irradiated control and curcumin fed) animals, the thymus tissue obtained from irradiated animals showed higher number of TUNEL positive cells. However, decreased level of TUNEL positive cells were observed in irradiated animals fed with curcumin (1%) [Figure 2]a,[Figure 2]b,[Figure 2]c,[Figure 2]d. Magnitude of apoptosis was also measured in bone marrow cells and thymocytes by caspase 3 assay. Our results showed a significant increase in activity of caspase 3 in thymocytes and bone marrow cells obtained 4 h after whole body irradiation (3 Gy). However, prior feeding of animals with 1% curcumin showed substantial decrease in caspase-3 activity in both these cells. The magnitude of radiation-induced caspase 3 activity was drastically decreased in these cells at longer time point (i.e. 24 h). At longer time period (24 h), curcumin feeding did not affect the magnitude of caspase 3 activity in thymocytes, but in case of bone marrow cells, radiation induced caspase 3 activity was increased after curcumin feeding [Figure 3].
|Figure 2: TUNEL assay of thymus tissue obtained after 4 h radiation from different treatment groups of animals. Scale: 50 μm. (a) Control, (b) Curcumin (CCM), (c) 3 Gy, (d) 3Gy+Curcumim (CCM)|
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|Figure 3: Caspase 3 assay in (a) thymocytes and (b) bone marrow obtained from control and treated animals after 4 and 24 h after irradiation|
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| Discussion|| |
In the present study, we have examined the modification of radiation-induced murine TL incidence by curcumin. Curcumin has shown antioxidant effect and modification of radiation-induced mammary carcinogenesis. However, ability of curcumin to modify the radiation-induced TL and its underlying mechanism has not been studied except our previous study, in which the protective ability of curcumin against TL incidence was compared with some other antioxidants such as eugenol and ascorbic acid. Furthermore, the concentration and feeding schedule of curcumin for the prevention of radiation-induced TL were also optimized in this study. Our results showed decrease in TL incidence, which was dependent on the concentration of curcumin. Lower concentrations of curcumin (0.05% and 0.1%) were unable to prevent the TL incidence in irradiated mice. Even at 0.5% curcumin, the inhibition in TL incidence in irradiated animals was marginal (8%). Curcumin was able to prevent the radiation-induced TL incidence only at higher concentration (i.e. 1%), which may be associated with the requirement of certain level of serum curcumin for the prevention of radiation-induced TL incidence. These results are in agreement with observed prevention of radiation-induced mammary carcinoma at 1% of curcumin. Application of higher dose of radiation, i.e. 3 Gy may be one of the reasons for requirement of high concentration of curcumin for prevention of TL incidence. The risk of cancer is more relevant at relatively lower radiation doses, where other adverse effects of radiation are relatively downplay. In practically possible occupational, diagnostic, and accidental exposure conditions (at lower radiation doses and local irradiation), curcumin is expected to substantially prevent the cancer incidence even much lower concentrations. Such studies are worthy to be investigated in the future. The ability of curcumin to prevent radiation-induced TL both before and after radiation exposure suggests its higher potential to be applied in planned (occupational and therapeutic) as well as unplanned (accidental) exposure conditions. However, the postfeeding of curcumin showed lower potential for prevention of radiation-induced TL. In case of prior feeding, sufficient level of curcumin will be available in the serum to prevent damage while radiation exposure, thus lowering initial and subsequent damages. Our results also showed that curcumin was able to inhibit DNA damage and apoptotic death in target organs such as thymus and bone marrow. Whole body radiation exposure would result in extensive DNA damage and apoptotic death to the sensitive organs such as bone marrow and thymus. The bone marrow will attempt to replenish the thymocytes which have undergone apoptotic death. A successful replacement of damaged thymocytes will prevent TL incidence. Hence, prevention in damage of bone marrow will be more effective than the prevention of thymus itself. This line of argument gets supported by previous studies, which showed physical protection of bone marrow against radiation damage prevented TL incidence after irradiation. Our results broaden the knowledge about radiomodifying ability of curcumin against radiation damage and associated incidence of TL.
| Conclusion|| |
These results suggest modification of TL incidence by curcumin in irradiated mice involving DNA damage and apoptotic death mechanisms.
Author would like to thank the technical support of Mr. Manjoor Ali and Mrs. R. Vasumathy while confocal microscopy.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]