<Text>Evaluation of protective effect of Amifostine on Dacarbazine induced genotoxicity </Text>

<Text>Abstract </Text>

<Text>Anticancer therapy with alkylating agents has been used for many years. Dacarbazine (DTIC) as an alkylating agent is used alone or in combination with other chemotherapy drugs. Using preventional strategies in order to inhibit formation of secondary cancers as a result of chemotherapy by DTIC is necessary. This study was undertaken to evaluate the genoprotective effects of amifostine on genotoxic effects of DTIC in cell culture condition. The comet assay method and HepG2 cells were used in this study. To determine the sufficient genotoxic concentration of dacarbazine, HepG2 cells were incubated with concentrations of 5, 10 and 20 µg/ml for 2 hours and effects were evaluated by the comet assay. The result of our investigation showed that in a two-hour period incubation of HepG2 cells with different concentration of DTIC, the concentration of 5 µg/ml could act as genotoxic factor which could be determined by comet assay method. In order to determine protective effects of amifostine on genotoxicity induced by DTIC, HepG2 cells were incubated with different concentrations of amifostine (2, 3 and 5 mg/ml) for 1 hour followed by 2-hour incubation period with DTIC (5µg/ml). One hour incubation of cells with different concentrations of amifostine before DTIC incubation indicated that at least 5 mg/ml concentration of amifostine can prevent genotoxic effects of DTIC on HepG2 cells in described condition. In conclusion amifostine could prevent DNA damage effect of DTIC on HepG2 cells. </Text>

<Text>Keywords: DNA damage, Comet assay, DTIC, Amifostine </Text>

<Text> </Text>

<Text>1. Introduction </Text>

<Text>Dacarbazine (DTIC) as an anticancer drug is used in combination with other chemotherapy drugs in treatment of several cancer types such as Hodgkin's disease, malignant melanoma, soft tissue sarcoma, neuroblastoma and fibrosarcomas (1-7). Although the clear mechanism of action of this drug is not known, it seems to acts as an alkylating agent (8-11). Liver has the key role in transformation of this prodrug to its reactive compound, methyl triazeno imidazole carboxamide (MTIC) which is able to attach an alkyl group to DNA. The repairing mechanisms of DNA are capable to repair these kinds of defects by a repairing enzyme called O-6-methylguanine methyltransferase (MGMT). In the absence of active enzyme in repairing process, mutation which could lead cells to death may occur (9). Several studies showed that DTIC could act as a purine analog in order to interact with sulfhydryl groups in inhibition of DNA, RNA and protein synthesis (9, 11). By transportation of this drug to different parts of body, it could affect most of normal healthy cells and numerous side effects such as nausea, vomiting, neutropenia, myelosuppression and alopecia will appear. Chemoprotective agents and symptomatic treatments are suggested to reduce these side effects. Development of secondary neoplasia as a result of chemotherapy especially with alkylating agents is common (12-15). Collins et al (2000) reported an acute myeloid leukemia as a secondary cancer of treatment process by DTIC (16). An organic thiophosphate called amifostine could protect normal cells against toxic effects of anticancer drugs and radiotherapy, while it's not effective on neoplastic cells. Amifostine as a prodrug is activated by membrane-bound alkaline phosphatase to its active metabolite WR-1065 (17-19). It acts as a scavenger of oxygen free radicals and is able to bind to platinum and alkylating agents (20). Higher concentrations of alkaline phosphatase in normal cells and higher pH of normal tissues in comparison with cancerous cells leads to selective uptake of WR-1065 by normal cells (19, 21, 22). </Text>

<Text>Several methods have been applied to evaluate the DNA damages (23, 24). Comet assay, known as Single Cell Gel method (SCG), and introduced as a micro electrophoresis method for direct observation of DNA damage. The mechanism by which comet assay detects DNA damage has been explained previously (25). The cells trapped in the agarose gel and lysed under the alkaline pH to release DNA from the cell. Under the effect of the electrical flow in electrophoresis, the DNA molecules move toward anode, to form the comets. The comet formation pattern is determined by the size of the DNA fragments and the number of broken ends (26). As the percent of damage increases, the free DNA fragments make longer tails. To perform this test, a suspension of the separated cells should be prepared. DNA damage should be assessed in the cells without giving them the opportunity of being exposed to any other genotoxic agents (27). Microscopic observation of DNA migration is possible using ethidium bromide staining and a flourescent microscope (28). </Text>

<Text>According to the wide application of DTIC in cancer treatment protocols, and its serious side effects especially secondary cancers, finding new strategies to prevent these side effects is necessary. With regard to the preventative effects of amifostine on normal cells, this study was performed to evaluate the genoprotective and dose dependent effects of this drug on genotoxicity of DTIC on the metabolically competent human hepatoma cell line (HepG2 cells). </Text>

<Text>2. Materials and methods </Text>

<Text>2.1. Materials </Text>

<Text>Dacarbazine and Amifostine were respectively purchased from Medac Co. (Germany) and Medlmmune Pharma BV. (Poland). Tris, Triton X-100, H2O2, NaCl, EDTA, NaOH and NaH2PO4 were purchased from Merck Co. (Germany), low melting point agarose (LMA), Na2HPO4, KCl and ethidium bromide were purchased from Sigma Co. (USA), normal melting point agarose (NMA) was purchased from Cinnagen Co. (Iran), RPMI-1640, FBS and antibiotic were purchased from PAA Co. (Australia). HepG2 cells were purchased from Pasture Institute (Iran). </Text>

<Text>2.2. Cell culture </Text>

<Text>2.3. Alkaline comet assay </Text>

<Text>2.3. Statistical analysis </Text>

<Text>Tail moment (percentage of DNA in tail × Tail length), Tail length (the length of the comet tail) and percent of DNA in tail (percentage of colored spots in tail) are the most frequently used factors in evaluation in the comet assay method. We used these factors for statistical analysis in this investigation (33, 34). </Text>

<Text>One-way analysis of variance (ANOVA) was used to compare the results of comet assay, followed by Tukey's multiple comparison post hoc tests. The P-values of 0.05 and less were considered as statistically significant. </Text>

<Text>3. Results </Text>

<Text>3.1. The comet assay results for different concentrations of DTIC </Text>

<Text>According to previous researches, the genotoxic effect of dacarbazine was tested (6, 35, 36). To determine the appropriate genotoxic concentration of dacarbazine, HepG2 cells were incubated with concentrations of 5, 10 and 20 µg/ml of the medicine for 2 hours before the comet assay stages (Fig. 1). The one-way analysis (ANOVA) for the results of tail length showed it has been increased significantly (P < 0.0001). According to the results of Tukey's multiple comparison post hoc test, all concentrations of dacarbazine have increased the tail length significantly (P < 0.001) comparing with the negative control group (Fig. 1A). The one-way analysis (ANOVA) for the results of percentage of DNA in Tail indicates it has been significantly increased (P < 0.0001). According to the results of the Tukey's multiple comparison post hoc test, in all concentrations of Dacarbazine the percentage of DNA in Tail have been increased significantly (P < 0.001) compared with the control group (Fig. 1B). One-way analysis result of the Tail moment for all groups showed this factor has increased significantly (P < 0.0001), more over the results of Tukey's multiple comparison post hoc test for all groups showed significant increase (P < 0.001) in this parameter compared with the control group (Fig. 1C). </Text>

<Text> With regard to the results of this stage the lowest concentration of DTIC (5 µg/ml) has significantly increased all studied factors in compare with the negative control group, so this concentration was selected for next stage of this study. </Text>

<Text>3.2. The comet assay results for different concentrations of amifostine combined with DTIC (5µg/ml) </Text>

<Text>In order to determine protective effects of amifostine on genotoxicity induced by DTIC, HepG2 cells were incubated with different concentrations of amifostine (2, 3 and 5 mg/ml) for 1 hour followed by 2-hour incubation period for 5µg/ml of DTIC (Fig.2). The result of the one-way analysis (ANOVA) of the tail length was increased significantly (P < 0.0001). According to the results of Tukey's multiple comparison post hoc test, all three concentrations of amifostine in combination with DTIC (5µg/ml) were able to inhibit the genotoxic effects of DTIC and decreased the tail length significantly (P<0.001 ) compared with the DTIC group (Fig. 2A). </Text>

<Text>The One-way analysis of the percentage of DNA in tail and the tail moment showed significant results (P < 0.0001). The results of Tukey's multiple comparison post hoc test showed at the concentrations of 3 and 5 mg/ml, these parameters have significantly decreased (P < 0.001), while at the concentration of 2 mg/ml of amifostine the percentage of DNA has decreased less than other concentrations (P < 0.05) compared with the DTIC group. (Fig. 2B and 2C) </Text>

<Text>3.3. The comet assay results for different concentrations of amifostine </Text>

<Text> Different concentrations of amifostine (2, 3 and 5 mg/ml) were tested by comet assay method after a one-hour period incubation. None of concentrations had genotoxic effect on HepG2 cells. </Text>

<Text>4. Discussion </Text>

<Text>The results of this study indicate that concentrations of 5, 10 and 20 µg/ml of DTIC are genotoxic for HepG2 cells during a two-hour period of incubation. The concentration of 5 µg/ml of DTIC was selected as the minimum genotoxic concentration for HepG2 cells at described condition. At the second part of this study, cells were incubated by three concentrations of amifostine (2, 3 and 5 mg/ml) for one hour followed by incubation of DTIC (5 µg/ml) for two hours. All these three concentration of amifostine were able to protect the genotoxic effects of DTIC on HepG2 cells as mentioned above. According to previous researches, alkylating agents such as DTIC are used in anticancer therapy (37-39). The most important side effect of these drugs is apparition of secondary neoplasia or cancers in additional sites (12-15). Alkylating agents can substitute alkyl groups on DNA leads to damage DNA and cause to break labile bonds of DNA, micronucleus formation and finally leading to chromosomal breaks and genome instability. These damages cause effects such as inhibition of the biosynthesis pathways, cell cycle arrest, teratogenicity and apoptosis (35, 40). Dacarbazine may inhibit DNA and RNA synthesis by acting as a purine analogue. This drug is bio-activated in liver by demethylation to MTIC and then to diazomethane, which attacks to nucleophilic groups on DNA (8, 35, 40, 41). Amifostine is FDA approved to reduce side effects of cisplatin in patients with advanced ovarian cancer (42). Nowadays, amifostine known as a selective cytoprotective agent of normal tissues against the toxicity of chemotherapy and radiotherapy (17). WR-2721 is a prodrug dephosphorylated by alkaline phosphatase (AP) in tissues in order to activate free thiol metabolites. The selective protection of non-malignant tissues is believed to be due to higher AP activity in normal tissues (40, 41, 43, 44). Previous researches evaluated the genotoxic effects of doxorubicin after 3, 6 and 9 hours exposure of 10 µg/ml doxorubicin. The results of this study showed time dependent genotoxicity of doxorubicin (45). Buschini et al. determined cytoprotective effect of amifostine (0, 50 and 100 µg/ml) on bleomycin genotoxicity by comet assay. In this study, amifostine could reduce bleomycin genotoxic effects (46). Blasiak and his co-workers evaluated cytoprotective effects of vitamin C and E and amifostine on idarubicin genotoxicity on lymphocyte cells. Vitamin C and amifostine (14 mM) reduced DNA damage induced by idarubicin while vitamin E increased DNA damage of idarubicin (47). The result of our investigation showed that in a two-hour period incubation of HepG2 cells with different concentration of DTIC, the concentration of 5 µg/ml could act as genotoxic factor which could be determined by comet assay method. One-hour incubation of cells with different concentrations of amifostine before DTIC (5 µg/ml) incubation indicated that studied concentration of amifostine are able to prevent genotoxic effects of DTIC on HepG2 cells in described condition. </Text>

<Text>5. Conclusion </Text>

<Text>It can be concluded from the discussion that amifostine could prevent genotoxic effect of DTIC on HepG2 cells so it could be suggested to insert in chemotherapy protocols containing DTIC in order to prevent formation of secondary cancers. </Text>

<Text>6. Acknowledgements </Text>

<Text>This paper was derived from a pharmacy doctorate thesis (No. 391441) in Isfahan University of Medical Sciences, Isfahan, Iran. We would like to acknowledge the research department of Isfahan University of Medical Sciences, Isfahan, I.R.Iran, for their co-operation and financial supports. </Text>

<Text>7. References </Text>

<Text>1. Yi JH, Yi SY, Lee HR, Lee SI, Kim JH, Park KW, et al. Dacarbazine-based chemotherapy as first-line treatment in noncutaneous metastatic melanoma: multicenter, retrospective analysis in Asia. Melanoma Re. 2011;21(3):223-7. </Text>

<Text>2. Batty N, Hagemeister FB, Feng L, Romaguera JE, Rodriguez MA, McLaughlin P, et al. Doxorubicin, bleomycin, vinblastine and dacarbazine chemotherapy with interferon for advanced stage classic Hodgkin lymphoma: a 10-year follow-up study. Leukemia & lymphoma. 2012;53(5):801-6. </Text>

<Text>3. Minuk LA, Monkman K, Chin-Yee IH, Lazo-Langner A, Bhagirath V, Chin-Yee BH, et al. Treatment of Hodgkin lymphoma with adriamycin, bleomycin, vinblastine and dacarbazine without routine granulocyte-colony stimulating factor support does not increase the risk of febrile neutropenia: a prospective cohort study. Leukemia & lymphoma. 2012;53(1):57-63. </Text>

<Text>4. Wilson KS, Robert C, Thomas L, Bondarenko I. Ipilimumab plus dacarbazine in melanoma. N Engl J Med. 2011;365(13):1256. </Text>

<Text>5. Walter T, Bruneton D, Cassier PA, Hervieu V, Pilleul F, Scoazec JY, et al. Evaluation of the combination 5-fluorouracil, dacarbazine, and epirubicin in patients with advanced well-differentiated neuroendocrine tumors. Clinical colorectal cancer. 2010;9(4):248-54. </Text>

<Text>6. Kumar SG, Narayana K, Bairy K, D'Souza UJ, Samuel VP, Gopalakrishna K. Dacarbazine induces genotoxic and cytotoxic germ cell damage with concomitant decrease in testosterone and increase in lactate dehydrogenase concentration in the testis. Mut Res./Gen Tox and Env Mut . 2006;607(2):240-52. </Text>

<Text>7. Al-Hawary B, Al-Saleh A. Cytogenetic effects of dacarbazine on mouse bone marrow cells in vivo. Mut Res/Gen Tox. 1989;223(2):259-66. </Text>

<Text>8. Psaroudi MC, Kyrtopoulos SA. Toxicity, mutation frequency and mutation spectrum induced by dacarbazine in CHO cells expressing different levels of methylguanine-DNA methyltransferase. Mut Res/Fund and Mol Mech of Mut. 2000;447(2):257-65. </Text>

<Text>9. Sanada M, Hidaka M, Takagi Y, Takano TY, Nakatsu Y, Tsuzuki T, et al. Modes of actions of two types of anti-neoplastic drugs, dacarbazine and ACNU, to induce apoptosis. Carcinogenesis. 2007;28(12):2657-63. </Text>

<Text>10. Pourahmad J, Kobarfard F, Amirmostofian M. Lysosomal Oxidative Stress Cytotoxicity Induced by Dacarbazine and It's Pyridine Derivative in Hepatocytes. Ir J of Pharm S. 2006;2(4):123-8. </Text>

<Text>11. Pourahmad J, Amirmostofian M, Kobarfard F, Shahraki J. Biological reactive intermediates that mediate dacarbazine cytotoxicity. Cancer chemo and pharm. 2009;65(1):89-96. </Text>

<Text>13. Lev DC, Onn A, Melinkova VO, Miller C, Stone V, Ruiz M, et al. Exposure of melanoma cells to dacarbazine results in enhanced tumor growth and metastasis in vivo. J of clinical oncology. 2004;22(11):2092-100. </Text>

<Text>14. Horiguchi M, Kim J, Matsunaga N, Kaji H, Egawa T, Makino K, et al. Glucocorticoid-dependent expression of O6-methylguanine-DNA methyltransferase gene modulates dacarbazine-induced hepatotoxicity in mice. J of Pharm and Exp Ther. 2010;333(3):782-7. </Text>

<Text>15. Khan F, Sherwani AF, Afzal M. Analysis of genotoxic damage induced by dacarbazine: an in vitro study. Tox Re. 2010;29(3-4):130-6. </Text>

<Text>16. Collins CM, Morgan DS, Mosse C, Sosman J. Dacarbazine induced acute myeloid leukemia in melanoma. Mel Re. 2009;19(5):337-40. </Text>

<Text>18. Merlin J-L, Marchal S, Ramacci C, Berlion M, Poullain M-G. Enhancement of fotemustine (Muphoran (R)) cytotoxicity by amifostine in malignant melanoma cell lines. Anti-cancer drugs. 2002;13(2):141-7. </Text>

<Text>19. Blasiak J, Gloc E, Pertynski T, Drzewoski J. DNA damage and repair in BCR/ABL-expressing cells after combined action of idarubicin, STI571 and amifostine. Anti-cancer drugs. 2002;13(10):1055-60. </Text>

<Text>20. Marzatico F, Porta C, Moroni M, Bertorelli L, Borasio E, Finotti N, et al. In vitro antioxidant properties of amifostine (WR-2721, Ethyol). Cancer chem and pharm. 2000;45(2):172-6. </Text>

<Text>22. Dedieu S, Canron X, Rezvani HR, Bouchecareilh M, Mazurier F, Sinisi R, et al. The cytoprotective drug amifostine modifies both expression and activity of the pro-angiogenic factor VEGF-A. BMC med. 2010;8(1):19. </Text>

<Text>23. Mozaffarieh M, Schoetzau A, Sauter M, Grieshaber M, Orgül S, Golubnitschaja O, et al. Comet assay analysis of single-stranded DNA breaks in circulating leukocytes of glaucoma patients. Mol vis. 2008;14:1584. </Text>

<Text>24. Jena G, Kaul C, Ramarao P. Genotoxicity testing, a regulatory requirement for drug discovery and development: Impact of ICH guidelines. Ind J of pharm. 2002;34(2):86-99. </Text>

<Text>25. Sardas S. Genotoxicity tests and their use in occupational toxicology as biomarkers. Indoor and Built Env. 2005;14(6):521-5. </Text>

<Text>27. De Meo M, Laget M, Castegnaro M, Dumenil G. Genotoxic activity of potassium permanganate in acidic solutions. Mut Res/Gen Tox. 1991;260(3):295-306. </Text>

<Text>28. Olive P, Banath J, Fjell C. DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay. Cytometry. 1994;16(4):305-12. </Text>

<Text>29. Etebari M, Zolfaghari B, Jafarian-Dehkordi A, Rakian R. Evaluation of DNA damage of hydro-alcoholic and aqueous extract of Echium amoenum and Nardostachys jatamansi. J Res Med Sci. 2012;17(8):782-5. </Text>

<Text>30. Etebari M, Ghannadi A, Jafarian-Dehkordi A, Ahmadi F. Genotoxicity evaluation of aqueous extracts of Cotoneaster discolor and Alhagi pseudalhagi by comet assay. J Res in Med S. 2012;17:S238-S42. </Text>

<Text>31. Etebari M, Sajjadi SE, Jafarian-Dehkordi A, Panahi M. Antigenotoxic Effects of Methanolic and Aqueous Extracts of Kelussia Odoratissima Mozaffarian against Damage Induced by Methyl Methanesulfonate. J Isf Med Sch. 2013;30(215). </Text>

<Text>32. Tavakoli M, Bateni E, Rismanchian M, Fathi M, Doostmohammadi A, Rabiei A, et al. Genotoxicity effects of nano bioactive glass and Novabone bioglass on gingival fibroblasts using single cell gel electrophoresis (comet assay): An in vitro study. Den res j. 2012;9(3):314. </Text>

<Text>33. Belpaeme K, Cooreman K, Kirsch-Volders M. Development and validation of the in vivo alkaline comet assay for detecting genomic damage in marine flatfish. Mut Res/Gen Tox and Env Mut. 1998;415(3):167-84. </Text>

<Text>34. Tice R, Vazquez M. Protocol for the application of the pH> 13 alkaline single cell gel (SCG) assay to the detection of DNA damage in mammalian cells. Sigma (x-100). 1998;503:465-8353. </Text>

<Text>35. Braybrooke JP, Houlbrook S, Crawley JE, Propper DJ, O'Byrne KJ, Stratford IJ, et al. Evaluation of the alkaline comet assay and urinary 3-methyladenine excretion for monitoring DNA damage in melanoma patients treated with dacarbazine and tamoxifen. Cancer chem and pharm. 2000;45(2):111-9. </Text>

<Text>36. Souliotis VL, Valavanis C, Boussiotis VA, Pangalis GA, Kyrtopoulos SA. Comparative study of the formation and repair of O6-methylguanine in humans and rodents treated with dacarbazine. Carcinogenesis. 1996;17(4):725-32. </Text>

<Text>37. Greene MH, Boice Jr JD, Greer BE, Blessing JA, Dembo AJ. Acute nonlymphocytic leukemia after therapy with alkylating agents for ovarian cancer: a study of five randomized clinical trials. N Eng J of Med. 1982;307(23):1416-21. </Text>

<Text>38. Esteller M, Garcia-Foncillas J, Andion E, Goodman SN, Hidalgo OF, Vanaclocha V, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Eng J of Med. 2000;343(19):1350-4. </Text>

<Text>39. Schabel Jr F, Trader M, Laster Jr W, Corbett T, Griswold Jr D. cis-Dichlorodiammineplatinum (II): combination chemotherapy and cross-resistance studies with tumors of mice. Can treat rep. 1979;63(9-10):1459. </Text>

<Text>40. Grochova D, Smardova J. The antimutagenic and cytoprotective effects of amifostine: the role of p53. J Appl Biomed. 2007;5:171-8. </Text>

<Text>41. Feng M, Smith DE, Normolle DP, Knol JA, Pan CC, Ben-Josef E, et al. A Phase I Clinical and Pharmacology Study Using Amifostine as a Radioprotector in Dose-escalated Whole Liver Radiation Therapy. I J of Rad Onc Bio Phys. 2012;83(5):1441-7. </Text>

<Text>42. Cassatt DR, Fazenbaker CA, Bachy CM, Kifle G, McCarthy MP. Amifostine (ETHYOL) protects rats from mucositis resulting from fractionated or hyperfractionated radiation exposure. I J of Rad Onc Bio Phys. 2005;61(3):901-7. </Text>

<Text>43. McCumber LM. The potential influence of cell protectors for dose escalation in cancer therapy: an analysis of amifostine. Med Dos. 2004;29(2):139-43. </Text>

<Text>44. Fuchs-Tarlovsky V. Role of antioxidants in cancer therapy. Nutrition. 2012. </Text>

<Text>45. Husseini GA, El-Fayoumi RI, O'Neill KL, Rapoport NY, Pitt WG. DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study. Can lett. 2000;154(2):211-6. </Text>

<Text>46. Buschini A, Alessandrini C, Martino A, Pasini L, Rizzoli V, Carlo-Stella C, et al. Bleomycin genotoxicity and amifostine (WR-2721) cell protection in normal leukocytes vs. K562 tumoral cells. Biochem pharm. 2002;63(5):967-75. </Text>

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