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ORIGINAL ARTICLE
Year : 2009  |  Volume : 5  |  Issue : 9  |  Page : 53-56

Modulatory action of 2-deoxy-D-glucose on mitomycin C-and 4-nitroquinoline-1-oxide-induced genotoxicity in Swiss albino mice In vivo


1 Department of Genetics, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai, India
2 Science Center, Manipal Academy of Higher Education, Manipal, Mangalore, India

Date of Web Publication21-Aug-2009

Correspondence Address:
Sathiyavedu Thyagarajan Santhiya
Department of Genetics, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.55144

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 > Abstract 

Background: 2-Deoxy-D-glucose (2-DG), a structural analog of glucose is an effective inhibitor of glucose metabolism and ATP production. It selectively accumulates in cancer cells and interferes with glycolysis leading to cell death. 2-DG is shown to differentially enhance the radiation-induced damage in cancer cells both under euoxic and hypoxic conditions. A combination of 2-DG and ionizing radiation selectively destroys tumors while protecting the normal tissue. 2-DG is being advocated as an adjuvant in the radiotherapy and chemotherapy of cancer.
Objective: The present investigation focuses on the modulatory effect of 2-DG on mitomycin C- (MMC) and 4-nitroquinoline-1-oxide (4-NQO)-induced cytogenetic damage in bone marrow cells of Swiss albino mice in vivo.
Materials and Methods: Experimental animals were pretreated with 2-DG (500 mg/kg, i.p.) for five consecutive days followed by MMC (2 mg/kg, i.p) or 4-NQO (15 mg/kg, i.p.), 24h prior to sacrifice. Control animals were given either the mixture of olive oil and acetone (3:1) or distilled water. Bone marrow cells were processed for the micronucleus assay and metaphase analysis for estimating cytogenetic damage.
Results: 2-DG significantly (P < 0.001) reduced the frequency of aberrant cells induced by MMC (~90%) and 4-NQO (~74%). Incidence of micronucleated polychromatic erythrocytes (MnPCEs) induced by the mutagens were reduced up to 68%.
Conclusion: 2-DG effectively reduces the MMC-and 4-NQO-induced genotoxicity.

Keywords: Genotoxicity, mitomycin C, 2-deoxy-D-glucose, 4-nitroquinoline-1-oxide


How to cite this article:
Mohapatra R, Ramesh A, Jayaraman G, Santhiya ST, Gopinath PM. Modulatory action of 2-deoxy-D-glucose on mitomycin C-and 4-nitroquinoline-1-oxide-induced genotoxicity in Swiss albino mice In vivo . J Can Res Ther 2009;5, Suppl S1:53-6

How to cite this URL:
Mohapatra R, Ramesh A, Jayaraman G, Santhiya ST, Gopinath PM. Modulatory action of 2-deoxy-D-glucose on mitomycin C-and 4-nitroquinoline-1-oxide-induced genotoxicity in Swiss albino mice In vivo . J Can Res Ther [serial online] 2009 [cited 2019 Aug 24];5:53-6. Available from: http://www.cancerjournal.net/text.asp?2009/5/9/53/55144


 > Introduction Top


Tumor cells, because of a higher rate of glucose requirement, depend on aerobic and anaerobic glycolysis, which sensitizes these cells to radiation and chemotherapeutic agents. Furthermore, cellular responses to therapeutic agents are influenced by several factors like effect on DNA, type of lesions induced, and capability to repair the damaged DNA and cell cycle kinetics.

2-Deoxy-D-glucose (2-DG), a synthetic glucose analog that exploits the metabolic state of malignant cells, inhibits the repair of radiation-induced damage in tumor cells while enhancing the repair capability in normal cells. [1],[2],[3] Damage of the adjoining normal tissue and non-target organs limits the therapeutic dose. Therefore, investigations on the effect of 2-DG alone or in combination with radiation and anticancer drugs on normal cells/tissues are of interest. Human peripheral blood leucocytes when exposed to 2-DG for few hours post-irradiation revealed a significant reduction in the cytogenetic damage. [3] A reduction in radiation-induced cytogenetic damage was also reported in cultured Chinese hamster cells exposed to X-rays. [4] Methyl methane sulfonate and gamma rays-induced chromosomal aberrations were reduced by 2-DG in cultured lymphocytes of Down's syndrome, Fanconi's anemia, retinoblastoma, and psoriasis patients. [5] In vitro protective effects of 2-DG on radiation, [6],[7] bleomycin-, and mitomycin C -induced cytogenetic damages were reported.[8] 2-DG also reduces radiation-induced in vivo cytogenetic damage in mice. [9],[10] A modifying effect of chemical-induced cytogenetic damage has also been reported in plants in vivo and in vitro . [11],[12],[13] While multiple mechanisms could be responsible for the protection of normal cells by 2-DG, a reduction in proliferation appears to be an important factor for the protection of normal cells by 2-DG.

MMC is a natural antitumor, antibiotic, and cytotoxic agent against solid tumors, gastric, pancreatic, and oesophageal carcinomas, and bladder cancer. The cytotoxicity of MMC is primarily through the formation of DNA adducts and interstrand cross-links. [14],[15] Although MMC is an effective anticancer drug, its clinical use is restricted owing to its toxicity. MMC induces chromosomal damage during the S phase. [16] 4-nitroquinoline-1-oxide, a synthetic mutagen and carcinogen, was discovered by Nakahara and Fukuoka (1959). It induces DNA single-strand breaks in mammalian cells. The mutagenic activity of 4-NQO provides a useful model for cancer chemoprevention studies. [17] 2-DG is known to be a modulator of radiation-induced damage; however, it was rarely studied with chemotherapeutic drugs. There is a need to study the protective action of 2-DG over normal cells in combination with therapeutic drugs. The present study reports the modulatory effect of 2-DG on MMC-and 4-NQO-induced chromosomal damage and micronuclei in vivo in Swiss albino mice.


 > Materials and Methods Top


Animals

Experiments were carried out with 10-to 12-week-old Swiss albino mice of either sex weighing 25-30 g procured from King Institute, Chennai, India, and were acclimatized for 1 week prior to the experimentation under standard environmental conditions of temperature 25 2C, and humidity 60 5% with a 12-h light/dark period; they were fed with standard pelleted diet (M/s. Hindustan Lever Ltd., Bengaluru, India) and water ad libitum . Animals used in the present study were maintained in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India, and approved by the Institute's ethical committee.

Chemicals

2-DG, mitomycin C, and 4-nitroquinoline-1-oxide were purchased from Sigma-Aldrich, Inc. (USA). Fetal bovine serum, colchicine, and May-Grunwald and Giemsa stains were obtained from Hi-Media laboratories, India. 2-DG and MMC were dissolved in sterile, distilled water while, 4-NQO was dissolved in a mixture of olive oil and acetone (3:1). A working solution of both the mutagens was freshly prepared by reconstituting the known mutagen in a required volume of the solvent (10ml/kg). The test compounds as well as the mutagens were administered to the animals intraperitoneally (i.p.).

Experimental design

Animals were randomized into experimental and control groups (one to seven) of six animals each. Animals in Group1 were control (0.5ml of double distilled water) and Group2 was solvent control (olive oil:acetone, 3:1) for 4-NQO; Group3 was treated with 2-DG (500 mg/kg) for 5 days. Group 4 animals were treated with MMC (2 mg/kg) for 24 h and animals of Group 5 were treated with 4-NQO (15 mg/kg) for 24h. Group6 animals were treated with 2-DG (500 mg/kg) for 5 days followed by MMC (2 mg/kg) for 24h and Group 7 animals were treated with 2-DG (500 mg/kg) for 5 days followed by 4-NQO for 24 h. After sacrifice, bone marrow preparations were processed for metaphase analysis [18] and micronucleus assay. [19] The computation of chromosome damage was based on analysis of 100 metaphases/animal for each experimental point. Two thousand five hundred polychromatic erythrocytes (PCEs) and a corresponding number of normochromatic erythrocytes (NCEs) were analyzed for the frequencies of MnPCEs and MnNCEs. The ratio of PCE to NCE (P/N) was recorded for inference on any cytotoxic effect on cell proliferation. [20]


 > Results Top


[Table 1] shows the data on the modulatory effect of 2-DG pretreatment on the mitotic index and chromosomal aberrations both in control and experimental groups of animals, and it is observed that 2-DG-treated mice had few dividing cells, which could be, classified as single aberrant cells, while, treatment with either MMC or 4-NQO recorded both single and multiple aberrant cells; some of the multiple aberrant cells showed pulverization. No significant difference could be observed in the frequency of aberrant cells in animals administered with 2-DG as against controls. However, an eightfold increase ( P < 0.001) in the number of aberrant cells over the solvent control in MMC (2 mg/kg) and fivefold ( P < 0.001) increase in 4-NQO (15 mg/kg)-treated animals was observed. Upon pretreatment with 2-DG, it was significantly brought down by about 10 folds ( P < 0.001) in MMC and about 4 folds ( P < 0.001) in 4-NQO-treated animals. [Table 2] shows the data on the frequency of MnPCEs. Both MMC (2 mg/kg) and 4-NQO (15 mg/kg) enhanced the MnPCE frequency by 10 folds and pretreatment with 2-DG significantly ( P < 0.001) reduced the MnPCE frequency by 3 folds in animals exposed to either MMC or 4-NQO. Hence, it can be inferred that 2-DG by itself has no effect on the micronuclei frequency.


 > Discussion Top


The protection of the adjoining normal tissue as well as non-target organs is one of the major concerns in cancer therapy. This led to the development of adjuvant therapy, which is aimed to enhance disease-free survival. The cellular repair of potentially lethal damage, restitution of chromosomal break, and rejoining of DNA breaks are shown to be ATP-requiring processes. The glucose transporter protein Glut-1 enables the diffusion of 2-DG into the cells. On entry in the cell, 2-DG competes with glucose in the glycolytic metabolism. Phosphorylation to 2-DG-6-PO 4 stops further metabolism and, 2-DG-6-PO 4 accumulates in the cell until dephosphorylation. This might inhibit the rate of transport and phosphorylation of glucose by negative feedback mechanism, thus leading to reduction and a complete blockade of ATP production. [21]

MMC is a potent anticancer agent and currently used as an adjunct in cancer chemotherapy. The bioreductive mechanism has suggested its efficacy in targeting cells of the hypoxic region of solid tumors. [22] Two distinct processes are responsible for the diverse biological effects exhibited by mitomycin C. DNA alkylation and the generation of reactive free radicals such as superoxide and hydroxyl radicals induce strand scission. This process is promoted by metal ions such as Cu(II) and can be inhibited by catalase, superoxoide dismutase, radical scavengers, and sequestering agents. The alkylation of DNA by MMC has been well documented and is known to involve both monoalkylation and dialkylation, coupled with cross-linking.

4-NQO is a strong mutagen in standard short-term assays and induces DNA single-strand breaks in mammalian cells. It is carcinogenic in multiple organs of rat [23] and its potentiality is initiated by the enzymatic reduction of its nitro group to 4HAQO. 4-NQO is a base substitution mutagen, which principally acts at guanine residues. [24],[25] Free radical-mediated genotoxicity may also contribute to the action of 4-NQO on cells. 4-NQO not only forms adduct with purine bases but also causes oxidative DNA damage due to its metabolite, namely, 4-hydroxylamino-quinoline-1-oxide. Catalase and specific chelating agents of copper ions were found to inhibit oxidative DNA damage. [26]

Both 4-NQO and MMC inhibit the mitotic index (MI) due to cytotoxicity of the mutagens. In this study, 2-DG does not inhibit the mitotic index. 2-DG pretreatment significantly ( P < 0.001) reduces chromosomal aberrations (CAs) [Table 1]. The reduction of CAs and micronuclei induced by gamma rays in the root meristematic cells of Allium cepa on post-treatment with 2-DG was reported. [27] Pretreatment with 2-DG was also found to be effective against radiation-induced damage in mice [9] and in PHA-stimulated peripheral human leukocytes. [3] A significant modulatory action was also reported against several chemical mutagens, namely, endoxan, [28] isoniazid, [29] 2,4-dichloro-phenoxy acetic acid, and methyl methane sulfonate. [12]

The protective effects of 2-DG can be related to its free radical scavenging activity. The NADPH generated by pentose phosphate pathway is a reducing equivalent for reduced glutathione (GSH), which has been shown to participate in the metabolic breakdown of H 2 O 2 and organic hydro peroxides.[30] The blockade of glycolysis by 2-DG enhances the level of glucose-6-phosphate which in turn increases NADPH ultimately utilized by glutathione reductase (GR), thus restoring the GSH level. GSH serves as the first line of defense against the pro-oxidant stress. Probably, an elevation in the GSH level in 2-DG-pretreated animals might give protection against free radical-mediated genetic damage.


 > Acknowledgements Top


The authors gratefully acknowledge the financial support received from Defense Research and Development Organization (DRDO) toward the project. The authors wish to acknowledge Dr. G N Hariharan, Principal Scientist, MSSRF, for suggestions and critical reading of the manuscript. The paper was presented at the Symposium on "Applications of 2-deoxy-D-glucose in the management of cancer," Institute of Nuclear Medicine and Allied Sciences, New Delhi, India, November 8-10, 2006.

 
 > References Top

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27.Damodaran TV, Gopinath PM, Jain VK. Modification of radiation -induced mitotic disturbances in the root of Allium cepa. VII Annl Conf Envtl. Mutagen Soc India; p. 41-2.  Back to cited text no. 27
    
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29.Girijamani K. Cytogenetic effects of isoniazid and 2-deoxy -D-glucose on leucocytes exhibiting chromosomal instability. M.Phil. Dissertation, University of Madras. 1984.  Back to cited text no. 29
    
30.Varshney R, Adhikari JS, Dwarakanath BS. Contribution of oxidative Stress to radio sensitization by 2-DG and 6-AN in human cancer cell line. Ind J of Exp Biol 2003;41:1384-91.  Back to cited text no. 30
    



 
 
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