Dietary phytate lowers K-ras mutational frequency, decreases DNA-adduct and hydroxyl radical formation in azoxymethane-induced colon cancer

Document Type : Original Article


1 Department of Biotechnology, Krishna University, Machilipatnam, Andhra Pradesh, India

2 Department of Biochemistry, Kakatiya University, Warangal, Telangana, India

3 Institute of Genetics and Hospital for Genetic Diseases, Begumpet, Osmania University, Hyderabad, Telangana, India


Objective(s): Dietary phytate is known to protect against azoxymethane (AOM)-induced preneoplastic lesions.  The present study was designed to determine whether dietary phytate affects mutation frequency in colon epithelial cells challenged with azoxymethane in vivo, through lowering the formation of O6-methyl guanosine (O6-MeG) and 8-hydroxy deoxyguanosine (8-OHdG) adducts.
Materials and Methods: We used Fisher F344 rats induced with AOM for 20 weeks and undertook 1% or 2% phytate supplementation for subsequent 16 weeks to monitor the mutation frequencies of one of the candidate genes, K-ras, along with DNA adduct load.
Results: Dietary phytate significantly suppressed aberrant crypt foci formation and effectively inhibited colon tumor formation in a dose-dependent manner. DNA sequencing results demonstrated that 60% of the colon tumors from AOM-treated and control diet fed animals showed GGT to GAT transition and 40% of the tumors showed GGT to GTT transversion at codon 12, along with 18% of the tumors showing GGC to CGC transversion at codon 13. Phytate supplementation at 1 and 2% lowered the frequency of GGT > GAT to 30 and 10%, respectively. Phytate supplementation also nullified the codon 13 mutations. No mutations were observed at codon 61 in any of the experimental groups.
Conclusion: The lowered frequency of K-ras mutations correlated with decreased formation of hydroxyl radicals, O5-meG and 8-OH-dG levels in phytate-supplemented animals with lowered tumor burden.


1. Shamsuddin AM. Phytate and colon-cancer risk. Am J Clin Nutr 1992; 55:478.
2. Norazalina S, Norhaizan ME, Hairuszah I, Norashareena MS. Anticarcinogenic efficacy of phytic acid extracted from rice bran on azoxymethane-induced colon carcinogenesis in rats. Exp Toxicol Pathol 2010; 62:259-268.
3. Challa A, Rao DR, Reddy BS. Interactive suppression of aberrant crypt foci induced by azoxymethane in rat colon by phytic acid and green tea. Carcinogenesis 1997; 18:2023-2026.
4. Khatiwada J, Verghese M, Davis S, Williams LL. Green tea, phytic acid, and inositol in combination reduced the incidence of azoxymethane-induced colon tumors in Fisher 344 male rats. J Med Food 2011; 14:1313-1320.
5. Saad N, Esa NM, Ithnin H. Suppression of beta-catenin and cyclooxygenase-2 expression and cell proliferation in azoxymethane-induced colonic cancer in rats by rice bran phytic acid (PA). Asian Pac J Cancer Prev 2013; 14:3093-3099.
6. Stevens RG, Jones DY, Micozzi MS, Taylor PR. Body iron stores and the risk of cancer. N Engl J Med 1988; 319:1047-1052.
7. Reizenstein P. Iron, free radicals and cancer. Med Oncol Tumor Pharmacother 1991; 8:229-233.
8. Nelson RL. Dietary iron and colorectal cancer risk. Free Radic Biol Med 1992; 12:161-168.
9. Kucharzewski M, Braziewicz J, Majewska U, Gozdz S. Iron concentrations in intestinal cancer tissue and in colon and rectum polyps. Biol Trace Elem Res 2003; 95:19-28.
10. Weinberg ED. Association of iron with colorectal cancer. Biometals 1994; 7:211-216.
11. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989; 49:4682-4689.
12. Winter J, Nyskohus L, Young GP, Hu Y, Conlon MA, Bird AR, et al. Inhibition by resistant starch of red meat-induced promutagenic adducts in mouse colon. Cancer Prev Res (Phila) 2011; 4:1920-1928.
13. Kuhnle GG, Bingham SA. Dietary meat, endogenous nitrosation and colorectal cancer. Biochem Soc Trans 2007; 35:1355-1357.
14. Margison GP, Santibanez Koref MF, Povey AC. Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine. Mutagenesis 2002; 17:483-487.
15. Floyd RA. The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis 1990; 11:1447-1450.
16. Pfohl-Leszkowicz A, Grosse Y, Carriere V, Cugnenc PH, Berger A, Carnot F, et al. High levels of DNA adducts in human colon are associated with colorectal cancer. Cancer Res 1995; 55:5611-5616.
17. Hall CN, Badawi AF, O’Connor PJ, Saffhill R. The detection of alkylation damage in the DNA of human gastrointestinal tissues. Br J Cancer 1991; 64:59-63.
18. Shibutani S, Takeshita M, Grollman AP. Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 1991; 349:431-434.
19. Shivapurkar N, Tang Z, Ferreira A, Nasim S, Garett C, Alabaster O. Sequential analysis of K-ras mutations in aberrant crypt foci and colonic tumors induced by azoxymethane in Fischer-344 rats on high-risk diet. Carcinogenesis 1994; 15:775-778.
20. Stopera SA, Murphy LC, Bird RP. Evidence for a ras gene mutation in azoxymethane-induced colonic aberrant crypts in Sprague-Dawley rats: earliest recognizable precursor lesions of experimental colon cancer. Carcinogenesis 1992; 13:2081-2085.
21. Ahnen DJ. Are animal models of colon cancer relevant to human disease. Dig Dis Sci 1985; 30:103S-106S.
22. Fiala ES. Investigations into the metabolism and mode of action of the colon carcinogens 1,2-dimethylhydrazine and azoxymethane. Cancer 1977; 40:2436-2445.
23. Feinberg A, Zedeck MS. Production of a highly reactive alkylating agent from the organospecific carcinogen methylazoxymethanol by alcohol dehydrogenase. Cancer Res 1980; 40:4446-4450.
24. Halliwell B, Aruoma OI. DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian systems. FEBS Lett 1991; 281:9-19.
25. Kasai H, Nishimura S. Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents. Nucleic Acids Res 1984; 12:2137-2145.
26. Umemura T, Sai K, Takagi A, Hasegawa R, Kurokawa Y. Formation of 8-hydroxydeoxyguanosine (8-OH-dG) in rat kidney DNA after intraperitoneal administration of ferric nitrilotriacetate (Fe-NTA). Carcinogenesis 1990; 11:345-347.
27. Kamiya H, Murata-Kamiya N, Fujimuro M, Kido K, Inoue H, Nishimura S, et al. Comparison of incorporation and extension of nucleotides in vitro opposite 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene. Jpn J Cancer Res 1995; 86:270-276.
28. Ellis CA, Clark G. The importance of being K-Ras. Cell Signal 2000; 12:425-434.
29. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene 2007; 26:3291-3310.
30. Bird RP. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett 1987; 37:147-151.
31. Bodiga VL, Boindala S, Putcha U, Subramaniam K, Manchala R. Chronic low intake of protein or vitamins increases the intestinal epithelial cell apoptosis in Wistar/NIN rats. Nutrition 2005; 21:949-960.
32. Shigenaga MK, Park JW, Cundy KC, Gimeno CJ, Ames BN. In vivo oxidative DNA damage: measurement of 8-hydroxy-2’-deoxyguanosine in DNA and urine by high-performance liquid chromatography with electrochemical detection. Methods Enzymol 1990; 186:521-530.
33. Khan N, Afaq F, Mukhtar H. Apoptosis by dietary factors: the suicide solution for delaying cancer growth. Carcinogenesis 2007; 28:233-239.
34. Bode AM, Dong Z. Signal transduction pathways: targets for chemoprevention of skin cancer. Lancet Oncol 2000; 1:181-188.
35. Shamsuddin AM, Vucenik I, Cole KE. IP6: a novel anti-cancer agent. Life Sci 1997; 61:343-354.
36. Dong Z, Huang C, Ma WY. PI-3 kinase in signal transduction, cell transformation, and as a target for chemoprevention of cancer. Anticancer Res 1999; 19:3743-3747.
37. Huang C, Ma WY, Hecht SS, Dong Z. Inositol hexaphosphate inhibits cell transformation and activator protein 1 activation by targeting phosphatidylinositol-3’ kinase. Cancer Res 1997; 57:2873-2878.
38. Graf E, Eaton JW. Antioxidant functions of phytic acid. Free Radic Biol Med 1990; 8:61-69.
39. Midorikawa K, Murata M, Oikawa S, Hiraku Y, Kawanishi S. Protective effect of phytic acid on oxidative DNA damage with reference to cancer chemoprevention. Biochem Biophys Res Commun 2001; 288:552-557.
40. Pretlow TP, Cheyer C, O’Riordan MA. Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity. Int J Cancer 1994; 56:599-602.
41. Al-Numair KS, Waly MI, Ali A, Essa MM, Farhat MF, Alsaif MA. Dietary folate protects against azoxymethane-induced aberrant crypt foci development and oxidative stress in rat colon. Exp Biol Med (Maywood) 2011; 236:1005-1011.
42. Lai CS, Li S, Liu CB, Miyauchi Y, Suzawa M, Ho CT, et al. Effective suppression of azoxymethane-induced aberrant crypt foci formation in mice with citrus peel flavonoids. Mol Nutr Food Res 2013; 57:551-555.
43. Waly MI, Al-Rawahi AS, Al Riyami M, Al-Kindi MA, Al-Issaei HK, Farooq SA, et al. Amelioration of azoxymethane induced-carcinogenesis by reducing oxidative stress in rat colon by natural extracts. BMC Complement Altern Med 2014; 14:60.
44. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39:44-84.
45. Sudheer Kumar M, Sridhar Reddy B, Kiran Babu S, Bhilegaonkar PM, Shirwaikar A, Unnikrishnan MK. Antiinflammatory and antiulcer activities of phytic acid in rats. Indian J Exp Biol 2004; 42:179-185.
46. Foksinski M, Rozalski R, Guz J, Ruszkowska B, Sztukowska P, Piwowarski M, et al. Urinary excretion of DNA repair products correlates with metabolic rates as well as with maximum life spans of different mammalian species. Free Radic Biol Med 2004; 37:1449-1454.
47. Oberreuther-Moschner DL, Rechkemmer G, Pool-Zobel BL. Basal colon crypt cells are more sensitive than surface cells toward hydrogen peroxide, a factor of oxidative stress. Toxicol Lett 2005; 159:212-218.
48. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 2006; 160:1-40.
49. Markowitz SD, Bertagnolli MM. Molecular origins of cancer: Molecular basis of colorectal cancer. N Engl J Med 2009; 361:2449-2460.
50. Pretlow TP, Brasitus TA, Fulton NC, Cheyer C, Kaplan EL. K-ras mutations in putative preneoplastic lesions in human colon. J Natl Cancer Inst 1993; 85:2004-2007.
51. Jen J, Powell SM, Papadopoulos N, Smith KJ, Hamilton SR, Vogelstein B, et al. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994; 54:5523-5526.
52. Smith AJ, Stern HS, Penner M, Hay K, Mitri A, Bapat BV, et al. Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons. Cancer Res 1994; 54:5527-5530.
53. Shivapurkar N, Huang L, Ruggeri B, Swalsky PA, Bakker A, Finkelstein S, et al. K-ras and p53 mutations in aberrant crypt foci and colonic tumors from colon cancer patients. Cancer Lett 1997; 115:39-46.
54. Takayama T, Ohi M, Hayashi T, Miyanishi K, Nobuoka A, Nakajima T, et al. Analysis of K-ras, APC, and beta-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis. Gastroenterology 2001; 121:599-611.
55. Singh J, Kulkarni N, Kelloff G, Reddy BS. Modulation of azoxymethane-induced mutational activation of ras protooncogenes by chemopreventive agents in colon carcinogenesis. Carcinogenesis 1994; 15:1317-1323.
56. Takahashi M, Nakatsugi S, Sugimura T, Wakabayashi K. Frequent mutations of the beta-catenin gene in mouse colon tumors induced by azoxymethane. Carcinogenesis 2000; 21:1117-1120.
57. Takahashi M, Wakabayashi K. Gene mutations and altered gene expression in azoxymethane-induced colon carcinogenesis in rodents. Cancer Sci 2004; 95:475-480.
58. Jackson PE, Cooper DP, O’Connor PJ, Povey AC. The relationship between 1,2-dimethylhydrazine dose and the induction of colon tumours: tumour development in female SWR mice does not require a K-ras mutational event. Carcinogenesis 1999; 20:509-513.
59. O’Toole SM, Pegg AE, Swenberg JA. Repair of O6-methylguanine and O4-methylthymidine in F344 rat liver following treatment with 1,2-dimethylhydrazine and O6-benzylguanine. Cancer Res 1993; 53:3895-3898.
60. Rimbach G, Pallauf J. Phytic acid inhibits free radical formation in vitro but does not affect liver oxidant or antioxidant status in growing rats. J Nutr 1998; 128:1950-1955.
61. Fujikawa K, Kamiya H, Kasai H. The mutations induced by oxidatively damaged nucleotides, 5-formyl-dUTP and 5-hydroxy-dCTP,in Escherichia coli. Nucleic Acids Res 1998; 26:4582-4587.
62. Wallace SS. Biological consequences of free radical-damaged DNA bases. Free Radic Biol Med 2002; 33:1-14.
63. Gushima M, Hirahashi M, Matsumoto T, Fujita K, Fujisawa R, Mizumoto K, et al. Altered expression of MUTYH and an increase in 8-hydroxydeoxyguanosine are early events in ulcerative colitis-associated carcinogenesis. J Pathol 2009; 219:77-86.
64. Eadie JS, Conrad M, Toorchen D, Topal MD. Mechanism of mutagenesis by O6-methylguanine. Nature 1984; 308:201-203.
65. Pegg AE. Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res 1990; 50:6119-6129.
66. Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998; 396:643-649.
67. Luceri C, De Filippo C, Caderni G, Gambacciani L, Salvadori M, Giannini A, et al. Detection of somatic DNA alterations in azoxymethane-induced F344 rat colon tumors by random amplified polymorphic DNA analysis. Carcinogenesis 2000; 21:1753-1756.