|Year : 2006 | Volume
| Issue : 1 | Page : 12-17
Prevention of Radiation-Induced Clastogenic Effect by Diltiazem in Mouse Bone Marrow
Archana Jindal, V Nunia, PK Goyal
Radiation and Cancer Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur, India
P K Goyal
Radiation Biology Laboratory, Department of Zoology, University of Rajasthan, Jaipur 302 004
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Intraperitoneal administration of diltiazem (DTZ), half an hour prior to whole-body gamma irradiation (5.0 Gy), showed the protection of animals from radiation-induced micronuclei in bone marrow. The frequency of micronuclei elevated from 6 to 24 hrs. post-irradiation in both irradiated groups but declined thereafter. The counts of micronuclei were found to be significantly lower in DTZ treated irradiated group and it restored to normal on 7th day, while the normal number of micronuclei observed in control group only after 28 days post-irradiation. These data demonstrate that DTZ protects mice bone marrow against radiationinduced micronucleus formation.
|How to cite this article:|
Jindal A, Nunia V, Goyal P K. Prevention of Radiation-Induced Clastogenic Effect by Diltiazem in Mouse Bone Marrow. Indian J Nucl Med 2006;21:12-7
|How to cite this URL:|
Jindal A, Nunia V, Goyal P K. Prevention of Radiation-Induced Clastogenic Effect by Diltiazem in Mouse Bone Marrow. Indian J Nucl Med [serial online] 2006 [cited 2020 Feb 29];21:12-7. Available from: http://www.ijnm.in/text.asp?2006/21/1/12/43434
| Introduction|| |
The application of ionizing radiation to many fields of human activities has promoted rapid progress in the practice as well as in the underlying philosophy of radiation protection. Along with the refinement of physical devices for radiation dosimetry of occupational exposures, biological methods for dose assessment have been developed. Of the biological methods adopted for dosimetry purposes, cytogenetic analysis has been the most popular one. The occurrence of chromosome aberrations has been used as the most sensitive biological indicator of irradiation ,,, . These days attention has been turned to the micronucleus assay because of the close relationship between the micronucleus formation and the presence of chromosomal aberrations ,, . Micronucleus formation in mammalian bone marrow can be studied with a high resolution power ,, as well as at very low doses of radiations ,, .
Since the pioneering work of Patt et al  demonstrating that cysteine protected mice and rats against radiation-induced sickness and mortality, several sulphydryl compounds have been thoroughly investigated  . Non-sulphydryl compounds have also been used for radiation protection. However, their success has always been limited owing to the lower degree of protection.
Diltiazem is a calcium channel blocker used in cardio-vascular therapy, and it acts by inhibiting the influx of Ca 2+ through specialized channels into cells and thus influence numerous functions  . There are only few reports on the protective action of calcium channel blockers ,,, . But prevention of radiation induced micronucleus formation by calcium channel blockers has not been studied extensively. Under what conditions diltiazem can attain this efficacy needs evaluation.
Therefore, the present study has been undertaken to assess the radioprotective effect of diltiazem on the bone marrow of mice exposed to a sublethal dose of a-radiation by studying the frequency of micronuclei.
| Materials and Methods|| |
Animal care and handling
The animal care and handling were done according to the guidelines set by the World Health Organization, Geneva, Switzerland and the INSA (Indian National Science Academy, New Delhi, India).
Swiss albino mice, 6-8 weeks old weighing 22±2 g., from an inbred colony were used for the present study. These animals were maintained under controlled conditions of temperature and light (Light : dark, 10 hrs : 14 hrs.). They were provided standard mice feed (procured from Hindustan Levers Ltd., India) and water ad libitum. Tetracycline water once a fortnight was given as preventive measures against infections. Four animals were housed in a polypropylene cage containing sterile paddy husk (procured locally) as bedding throughout the experiment. The study was approved by the Animal Ethical Committee of our Department.
Cobalt teletherapy unit (ATC-C9) of the Radiotherapy Department, Cancer Treatment Centre, SMS Medical College & Hospital, Jaipur was used for irradiation. Unanaesthetised animals were restrained in well-ventilated perspex boxes and exposed to 5.0 Gy gamma radiation at source to surface distances (SSD) of 77.5 cm to deliver the dose - rate of 1.24 Gy/ min. Secondary Standard Dosimeter has been used for this Purpose  .
Diltiazem (procured from Dr. Reddy's Laboratory, Hyderabad, India) was dissolved in double distilled water (DDW). According to the body weight of the animals it was injected in mice intra-peritonially (i.p.) as 100 mg/kg b.wt.[Figure 1]
Chemical toxicity of diltiazem
The chemical toxicity of diltiazem was determined by division of animals into various groups of 10 each. Each group of animals was administered with various doses viz. 25, 50, 100, 200, 400, 800, 1200 or 1600 mg/kg b.wt. of the diltiazem intraperitoneally. Mortality of the animals was observed upto 30 days post drug treatment and acute LD 50/30 of the diltiazem was calculated. No mortality was seen till 200 mg/kg b.wt. diltiazem was given. 20%, 40%, 50% and 100% mortality was observed when the administered dose was given 400, 800, 1200 and 1600 mg/kg b.wt. respectively.
Determination of optimum dose of DTZ against radiation
Dose selection of diltiazem was done on the basis of our previously conducted drug tolerance and animal survival study  . For this purpose, various doses of Diltiazem (25, 50, 100, 200 mg/kg b.wt.) were tested for their effect on the tolerance to 8.0 Gy gamma irradiation in 8 mice, and percentage survival (12.5, 37.5, 87.5 and 12.5 % respectively) of the animals were observed. Thus, the optimum and tolerable dose of diltiazem (100 mg/kg b.wt.) was selected, and the same was used in this experiment for further study.
Modification of radiation response
The mice were selected for this study divided into four groups. Animals in Group-I were injected intraperitoneally (i.p.) with DDW (volume equal to DTZ solution) to serve as normal, while animals in Group-II were given diltiazem (i.p.) in a dose of 100 mg/kg b.wt. Animals of Group-III received an equal volume of DDW (as in Group-I) and were exposed to 5.0 Gy gamma rays. Animals in Group- IV (experimental) were given diltiazem (as in Group-II) 30 minutes before exposure to 5.0 Gy gamma irradiation.
All animals were observed daily for 30 days for any sign of sickness, morbidity, behavioral toxicity and mortality. The percentage of surviving animals on each day was used for analysis of survival.
Five animals from each group were killed by cervical dislocation at 6, 12, 24 hrs and 2, 3, 7, 14 and 28 days postirradiation. The micronuclei were prepared according to the method of Schmid  with certain modifications described by Jagetia and Jacob  . Briefly, the femora of animals were dissected out and the bone marrow was flushed out into Dulbecco's Modified Eagle's Medium (DMEM) separately. The suspension was centrifuged. A few drops of fetal calf serum (FCS) was added and the pellet was mixed thoroughly. Smears were drawn onto precleaned coded slides using a drop of resultant suspension in FCS. The slides were air dried and fixed in absolute methanol. The results were confirmed by repetition of the experiment.
The slides were stained with May-Grunwald (Sigma Chemical CO. USA) and Giemsa (BDH, England).The slides were mounted in DPX mountant and observed under a fluoroscent microscope. 2000 nucleated cells were scored for each animal and a total of 10000 cells were scored for each autopsy interval. The results are expressed as the percentage of cells with micronuclei.
Glutathione (GSH) assay : The hepatic level of glutathione (GSH) was determined in 6 animals by the method of Moron et al  . The GSH content in blood was measured spectrophotometrically using Ellman's reagent with 5-5', dithiobis-2-nitrobenzoic acid [DTNB] as a colouring reagent, according to the method Beutler et al  . The absorbance was read at 412 nm using a UV-VIS Systronics Spectropotometer 108.
Lipid peroxidation assay : The lipid peroxidation (LPx) level in liver and serum of 6 animals was measured by the assay of thiobarbituric acid reactive substances (TBARS) using the method of Ohkhawa et al  in which the absorbance was read at 532 nm using a UV-VIS systronic spectrophotometer 108.
| Statistical Analysis|| |
The statistical significance of the differences observed between normal and DDW + irradiated control as well as between control and DTZ + irradiated experimental set ups were evaluated by using the Student's 't' test.
| Results|| |
The diltiazem alone treatment (Group-II) did not show any acute toxic symptoms or mortality at the dose of 100 mg/kg b.wt. In addition, the treated animals did not show any significant change in biochemical determinants, behaviour, urination or defecation patterns. Similar results were noted in the animals receiving DDW alone (Group-I).
No significant difference in the frequency of micronuclei in the bone marrow of mice was observed between the normal and DTZ (drug alone) treated animals. The number of micronuclei increased from 6 hr. (1.25 ± 0.036) with the highest frequency (5.05 ± 0.270) at 24 hrs. post-irradiation in the irradiated control group. The counts of micronuclei later declined abruptly at day 2 and continued to decrease throughout the remaining post-irradiation period upto 28 days.
The frequency finally reached to normal level by the end of experiment [Table 1].
In DTZ pretreated irradiated animals (Group-IV), pattern of micronuclei formation remained essentially similar to that of untreated irradiated animals, but the frequency of micronuclei was significantly reduced at all the post-irradiation time intervals studied. At 24 hrs. and day 2 post-irradiation, the difference between control and experimental groups was found as highly significant, with the almost 3 times lower magnitude of micronuclei in DTZ pretreated irradiated group than in the untreated irradiated group. In the drug treated mice, the counts of micronuclei decreased to nearly normal levels from day 3 post-irradiation.
No significant alterations in the hepatic and blood GSH contents were observed between normal and DTZ treated animals. However, a statistically significant ( p <0.001) decrease in GSH was evident in control animals. Experimental animals showed a significant increase in GSH content (blood and liver) with respect to control, but the values remained below normal [Figure 2]. The LPx level showed a significant elevation in control animals while a significant decrease was noted in the DTZ pretreated irradiated animal [Figure 3].
| Discussion|| |
Micronucleus assay could be used to assess the chromosome changes in the cell as micronuclei are formed from an acentric fragment or a whole chromosome that lag behind during the cell division. The advantage of micronuclei assay lies in its simplicity, as the scoring of micronuclei is rapid and does not require much expertise, unlike chromosome aberration scoring, which is laborious, time consuming and needs considerable expertise for scoring.
There are chromosomal changes that might be involved in causation of tumors. These changes seem to be primarily rearrangements  . At least some of these structural changes require breaks. Thus, if people who are to acquire a tumor in later life are particularly prone to chromosome breakage. This characteristic feature might be reflected by higher scores of micronuclei. Evidence that micronuclei may serve as predictors of carcinogenic risks was presented by many workers ,,.
The results from the present study demonstrate the radioprotective effect of diltiazem on radiation-induced micronuclei formation while diltiazem itself does not have any marked effect on the bone marrow cells as far as induction of micronuclei is concerned.
The increase in the frequency of micronuclei from 6 hrs. to 24 hrs. in mice bone marrow after exposure to 5 Gy is in accordance with the findings ,,,, of others who have reported an increase in the frequency of micronuclei in mouse bone marrow from 1/4 to day 1 after exposure to 0.5 to 3.0 Gy of X or gamma rays.
Micronuclei begins appearing at the end of first mitotic division after irradiaton. Although additional micronuclei may appear in the next few divisions and accumulate for a certain time  . The decline in the counts of micronuclei after day 2 may be due to the delaying effect of radiation on the progression of the cell cycle as well as dilution of the micronucleated cells with normal cells and the loss of aberrant cells. The persistence of micronuclei till day 14 post-irradiation may be due to the latent damage suffered by the stem cell compartment which may not be enough to impair the reproductive capacity of the cells and hence passed to the next generation.
The administration of DTZ prior to irradiation resulted in a significant decline in the counts of micronuclei from 6 hrs. and the frequency of micronuclei was nearly 3 times lower at day 1 and 2 post-irradiation and the difference between DTZ treated and untreated was found to be significant. This clearly points out that the presence of DTZ before irradiation, has reduced radiation-induced damage at chromosomal level. It exhibits that DTZ provided enough protection to the chromosomal DNA against the induction of strand breaks by radiation which are supposed to be responsible for micronuclei formation.
There appears to be a close correlation between the increase in lipid peroxidation and the depletion of GSH and antioxidant enzymes. Under normal conditions, the antioxidant defense system of the body protects against free radicals and oxidative stress  . The oxidative stress caused by the radiation-induced free radicals can cause a drastic fall in GSH , , overwhelm the cellular defense, and lead to membrane lipid peroxidation and loss of protective thiols  .Such an effect is demonstrated in the present study by a significant reduction in the liver and blood GSH following radiation exposure. This could be due to the enhanced utilization of the antioxidant system in an attempt to detoxify the free radicals generated by radiation. The calcium channel blocker, diltiazem used in the present study, might prevent cellular injury due to membrane impairment caused by inhibition of perfusion injury  , and by direct inactivation of free radicals  . The increased GSH levels by DTZ pretreatment may facilitate the reduction of oxidative free radicals by H+ donation. This allows the restoration of GSH by GSH reductase activity. The lower depletion of liver and blood GSH in the DTZ-pretreated and irradiated animals could be due to the higher availability of GSH, which increases the ability to cope with the free radicals generated by radiation.
The effect of diltiazem is comparable to that of established protectors. This could help in the damage repair, thus further reducing the radiation-induced clastogenic changes. The protection of chromosomes by DTZ may explain the higher stem cell survival. The exact mechanism of action of diltiazem is not known, however, it may scavenge free radicals produced by radiation and thus inhibit radiation-induced damage to the cellular DNA. Diltiazem upon irradiation undergoes a tautomeric conversion, from keto to -enol group and a potential -SH group is formed. Such -SH group may combine with -SH group of proteins by forming a disulphide bond which acts as a shield to protect DNA against radiation-induced damage. Irradiation inhibits cell proliferation and pretreatment of mouse with DTZ protects against radiation-induced inhibition of cell proliferation as well as micronucleus induction.
| Conclusion|| |
It can be concluded that DTZ protects mouse bone marrow cells and significantly reduces the radiation-induced micronuclei formation. It inhibits GSH depletion and LPx elevation caused due to irradiation. Since significant protection is obtained at low non-toxic dose, which has an advantage over the other available radioprotectors. However, further study is necessary to study the exact mechanism of its action.
| Acknowledgements|| |
The authors are grateful to Prof. D. P. Agarwal and Dr. A. Chaugle of the Department of Radiothepary, SMS Medical College & Hospital, Jaipur for providing the necessary irradiation facilities and help in dosimetry.
| References|| |
|1.||Lloyd DC, Purrott RJ. Chromosome aberration analysis in radiological protection dosimetry. Radiat Prot Dosim; 1: 19-28,1981. |
|2.||Lloyd DC. Biological dosimetry in radiological protection recent developments. J Soc Radiat Prot; 4: 5-11, 1984. |
|3.||Bauchinger M. Cytogenetic effects in human lymphocytes as a dosimetry system. In : Biological Dosimetry (Eds. Easert W. G. and Mendelsohn, M. L.) Springer Verlag, Berlin, 1984, p 15. |
|4.||Ganasondari A, Uma Devi P & Rao MNA. Protection against radiation induced chromosome damage in mouse bone marrow by Ocimum sanctum. Mutation Res; 373: 271-276, 1997. |
|5.||Heddle JA & Carrano AV. The DNA content of micronuclei induced in mouse bone marrow by gamma irradiation, Evidence that micronucleus arise from acentric chromosomal fragments. Mutation Res; 44, 63-69, 1977. |
|6.||Yamamoto KI & Kikuchi Y. A comparison of diameters of micronuclei induced by clastogens and spindle poisons. Mutation Res, 71, 127-131, 1980. |
|7.||Bisht KS & Uma Devi P. Modification of radiation induced chromosomal damage and micronucleus induction in mouse bone marrrow by misonidazole and hyperthermia. Acta Oncol, 34, 913-918, 1995. |
|8.||Boller K & Schmid W. Chemische mutagennese beim sauger. Das knochenmark des chinesischem Hamster als in vitro - Test system Hamatologische Befunde nach Behandlung mit. Trenimoss, Humangenetic. 11, 34-54,1970. |
|9.||LedeburVon M & Schmid W. The micronucleus test: methodological aspects. Mutation Res, 19, 109-117, 1973. |
|10.||Matter BE & Grauwiler J. Micronuclei in mouse bone marrow cells : a simple in vivo model for the evaluation of drug-induced chromosomal aberrations. Mutation Res, 23, 239-249, 1974. |
|11.||Mitchell JC & Norman A. The induction of micronuclei in human lymphocytes by low doses of radiation. Int J Radiat Bio,.52, 527-535, 1987. |
|12.||Uma Devi P & Sharma AS. Mouse bone marrow response to low doses of whole-body gamma irradiation : induction of micronuclei. Int J Radiat Biol, 57, 97-101, 1990. |
|13.||Jagetia GC & Ganapathi NG. Radiation induced micronucleus formation in mouse bone marrow after low dose exposures. Mutation Res, 304, 237-242, 1994. |
|14.||Patt HM, Tyree EB, Straube RL, et al. Cysteine protection against X-irradiation. Science, 110, 213-214, 1949. |
|15.||Sweeney TR. A survey of compounds from the antiradiation drug development program of the U. S. Army Medical Research and Development Command. Government Printing Office, Washington, DC, Publication. 1979 : 0-308-318. |
|16.||Nayler WB. Calcium antagonists. London:Academic Press. 1988. |
|17.||Miller DD, Waters DD, Dangoisse V, et al. Syptometic coronary artery spasm following radiotherapy for Hodgkin's disease. Chest, 83, 284-85, 1983. |
|18.||Floersheim GL. Radioprotective effects of calcium antagonists used alone and other types of radioprotectors. Radiat Res, 133, 80-87, 1993. |
|19.||Galler J, Garcia de la Rubia P, Gonzalez GG, et al. Irradiation of the anterior segment of the eye by ultra violet radiation.: Influence of nerve blockage and calcium antagonists. Curr Eye Res, 14, 827-35, 1995. |
|20.||Goel HC, Ganguly SK, Parsad J, et al. Radioprotective effects of diltiazem on cytogenetic damage and survival in gamma ray exposed mice. Indian J Exp Biol, 34, 1194-1200, 1996. |
|21.||Dardenne JC, Wambersie A. Extension of the range of measurement of a farmer secondary standard dosimeter. J Belge Radiol, 55(3): 361-364, 1972. |
|22.||Goyal PK, Yadav R, Nunia V, et al. Radioprotective effect of calcium blocker, Diltiazem, on survival in gamma rays exposed mice. Radiat Prot Env, 24, 7-9, 2001. |
|23.||Schmid W, The micronucleus test. Mutation Res.; 31: 9-12, 1975. |
|24.||Jagetia GC & Jacob PS. Vinblastine treatment induces dose dependent increase in the frequency of micronuclei in mice bone marrow. Mutation Res, 280, 87-92, 1992. |
|25.||Moron MS, Depierre JW, Mannervik B. Levels of GSH, GR and GST activities in rat lung and liver. Biochem Biophys, 582, 6778, 1979. |
|26.||Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med, 61, 882 888, 1963. |
|27.||Ohkhawa H, Ohishi N, Yogi K. Assay for lipid peroxidation in animals tissue by thiobarbituric acid reaction. Anal Biochem, 95, 351-358, 1979. |
|28.||Sandberg AA. A chromosomal hypothesis of oncogenesis. Cancer Genet Cytogenet, 8, 277-285, 1983. |
|29.||Stich HF, Acton AB, Palacic B. Towards an automated micronucleus assay as an internal dosimeter for carcinogen exposed human population groups. Recent Results Cancer Res, 120, 94105, 1990. |
|30.||Stich HF, Rosin MP, Brunnemann KD. Oral lesions, genotoxicity and nitrosamines in betel quid chewers with no obvious increase in oral cancer risk. Cancer Lett, 31, 15-25, 1986. |
|31.||Ronen A & Heddle JA, Site specific induction of nuclear anomalies (apoptotic bodies and micronuclei) by carcinogens in mice. Cancer Res, 44, 1536-1540, 1984. |
|32.||Heddle JA. A rapid in vivo test for chromosomal damage. Mutation Res, 18, 187-190, 1973. |
|33.||Jenson D & Ramel C. Factors affecting the induction of micronuclei at low doses of X-rays, MMS and dimethylnitrosamine in mouse erythroblasts. Mutation Res, 58, 51-65, 1978. |
|34.||Cole RJ, Taylor N, Cole J, et al. Short-term tests for transplacentally active carcinogens. I. Micronucleus formation in fetal and maternal mouse erythroblasts. Mutation Res, 80, 141-157, 1981. |
|35.||Uma Devi P, Bisht KS. Misonidazole does not sensitize normal bone marrow cells to radiation. Radobiol Radiother, 28, 473475, 1987. |
|36.||Jagetia GC & Aruna R. The herbal preparation abana protects against radiation-induced micronuclei in mouse bone marrow. Mutation Res, 393, 157-163, 1997. |
|37.||Kappus H, In: Sies H, editor. Oxidative Stress. New York: Academic Press. 1985. |
|38.||Navarroj J, Obrador E, Pellicer JA, et al. Blood glutathione phosphate solution of radiation-induced stress in mice and humans. Free Rad Biol Med, 22, 1203-1209, 1997. |
|39.||Pellmar TC, Rney D, Lepinski DL. Role of glutathione in repair of free radical damage in hippocampus in vitro. Brain Res, 583, 194-200, 1992. |
|40.||Konnings AWT, In: Walden Jr TL, Hughes HN, editors. Prostaglandins and Lipid Metabolism in Radiation Injury. New York: Plennum Press. 1987. |
|41.||Opie LH. Perfusion injury and its pharmacology modification. Circul, 80, 1049-1062, 1989. |
|42.||Koller PT, Bergmann R. Reduction of lipid peroxidation in reperfused isolated rabbit hearts by diltiazem. Circul Res, 65, 838-846, 1989. |
[Figure 1], [Figure 2], [Figure 3]