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* Vitamins and Carcinogenesis Laboratory and
Vitamin Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston MA 02111;
** Divisions of Clinical Nutrition and Gastroenterology, Tufts University School of Medicine, Boston, MA 02111; and
Department of Clinical and Experimental Medicine, Policlinico G.B. Rossi, University of Verona, 37134 Verona, Italy
2To whom correspondence should be addressed. E-mail: sang.choi{at}tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • vitamin B-12 • DNA methylation • uracil misincorporation • rats • colon
One-carbon metabolism is comprised of a network of biochemical reactions that are involved in the transfer of one-carbon units. Nutrients such as folate, vitamin B-12, vitamin B-6, riboflavin, choline, betaine, and methionine each play important roles in this biochemical scheme (1–3). Increasing evidence from epidemiologic studies (4,5) as well as controlled experiments in the laboratory (6–8) indicates that interference with one-carbon metabolism enhances the risk of colorectal cancer because one-carbon metabolism has critical functions in biological methylation reactions and in DNA synthesis and repair (9).
Until recently, depletion of vitamin B-12 was not often invoked as a causative factor in colorectal carcinogenesis, largely because a vitamin B-12 deficiency was thought to be distinctly uncommon in the general population. However, with the development of more sensitive measures of vitamin B-12 deficiency (such as homocysteine and methylmalonic acid), it has now become apparent that a physiologically and clinically significant degree of vitamin B-12 deficiency is relatively common in the elderly population; this is of particular interest because age is perhaps the strongest risk factor for colorectal cancer (10). Several recent nutritional surveys in ambulatory elderly populations identified a prevalence of vitamin B-12 deficiency of up to 16% (11–13), and the prevalence seems to be even higher among those who do not consume any vitamin supplements containing vitamin B-12 (14).
Population-based observations that implicate diminished vitamin B-12 status as a potential risk factor for certain cancers are generally of recent origin (15,16), although a connection between pernicious anemia and stomach cancer was noted intermittently for some decades (17,18) and corroborated by a recent study (19). Lower plasma levels of vitamin B-12 were identified as an independent risk factor in large epidemiologic studies of breast cancer (15,16); in addition, in an examination of lung tissue from squamous cell carcinomas compared with adjacent normal tissue, tissue concentrations of the vitamin were diminished in the carcinoma (20).
Evidence to date for such a relation in the colorectum is modest, but suggestive. A population-based cohort study observed that patients with pernicious anemia have a fourfold increased risk for developing colorectal adenocarcinoma, but only in the 5 y after their initial diagnosis (i.e., that period of time immediately after correction of their vitamin B-12 deficiency) (21). More recently, a large prospective cohort study observed that the occurrence of cancer in the proximal colon was significantly lower among those with both high folate and high vitamin B-12 intake, an effect not observed with high folate intake alone (22).
We were therefore interested in determining whether isolated vitamin B-12 deficiency in a laboratory animal, in the absence of other impediments in one-carbon metabolism, would produce molecular anomalies of the colonic mucosa that are both functions of one-carbon metabolism and mechanistically involved in colorectal carcinogenesis. We chose to examine DNA methylation, in which abnormalities are a constant and early phenomenon in human colonic neoplasia (23) and are thought to play an important role in the evolution of colonic cancers by altering gene expression through pathways that circumvent the necessity for a mutation or deletion (9). We also chose to examine uracil incorporation into DNA; excessive amounts are incorporated when thymidylate synthesis is inadequate to meet the cellular requirements for DNA synthesis and repair, leading to inappropriate nucleotide sequences, and the increased likelihood of chromosomal breakage, deletions, and mutations (24,25).
Depletion of one-carbon nutrients is not considered to be carcinogenic by itself in the colon; rather, the data from animal and human studies indicate that such conditions enhance carcinogenesis when there exists an underlying predisposition to cancer (9,26). Consequently, our intent in this study was not to demonstrate an increase in carcinogenesis but instead to determine whether the physiologic state of vitamin B-12 depletion creates a molecular milieu that could enhance carcinogenesis if an underlying predisposition were to be present.
To accomplish this, we chose a previously described method of inducing vitamin B-12 deficiency in rats. Rats were fed diets that either produced a modest degree of vitamin B-12 deficiency or a similar control diet that provided the basal requirement of vitamin B-12. Rats were killed after 6 and 10 wk of this regimen and the colonic mucosa and other tissues were examined.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The animal protocol was approved by the Animal Use Committee at the Human Nutrition Research Center. Male Sprague-Dawley rats (n = 30; 1 mo old; Charles River Breeding Laboratories) were housed in suspended stainless steel wire cages in a climate controlled room with free access to water. After 3 d of acclimation to the facility, the rats were randomly assigned to be fed either a vitamin B-12–deficient diet (Table 1) or a control diet. The diets were based on those described by Cullen and Oace (27), although the control diet was modified to contain only 2 mg folic acid/kg diet, as recommended by the AIN (28), and vitamin B-12 (as cyanocobalamin) was increased to 50 µg/kg diet, as recommended by the NRC (29). The deficient diet was identical except that it contained no cyanocobalamin. The diet contained 50 g pectin/kg diet, which binds vitamin B-12 in the intestine, making it less bioavailable (27). Due to the enterohepatic circulation of vitamin B-12, pectin thereby promotes depletion of endogenous vitamin B-12. The dietary intake of rats was mildly restricted so that all rats were fed, and consumed, the same amount of diet. After receiving the assigned diet for 6 wk, 8 rats from each group were killed. The remaining 7 rats from each group were killed after 10 wk. Rats were anesthetized with isoflurane after overnight food deprivation and the abdomen was then opened. Blood was immediately withdrawn by a needle-tipped syringe from the aorta, and the rats were then killed by cross-cutting the aorta. Complete blood counts and mean corpuscular volume (MCV) were analyzed immediately (System 9000, Serono Baker Diagnostic). After harvesting, the colorectum was excised and opened longitudinally on an ice-cold glass plate. After being washed with ice-cold saline, the mucosa was scraped off gently with glass microscope slides, as previously described (30). Aliquots of mucosal scrapings were wrapped in aluminum foil plackets, flash-frozen in liquid nitrogen, and stored at -70°C. DNA from colonic mucosal scrapings was extracted by a conventional technique using a lysis buffer containing proteinase K followed by phenol, chloroform, and isoamyl alcohol organic extraction. Residual RNA was treated with both RNase A and T1 (Invitrogen). The DNA was reprecipitated with 7.5 mol/L ammonium acetate, dissolved in TE buffer (10 mmol/L tris-HCl, 1 mmol/L EDTA pH 8.0) and dialyzed on 0.025-µm filter paper (Millipore). The concentration of DNA was determined at A260 by UV spectrophotometry.
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Plasma vitamin B-12 concentrations were determined using a commercially available competitive binding assay using 57Co (Quantaphase II B-12/folate radioassay, Bio-Rad). Plasma folate concentrations were determined by a conventional microbiological microtiter plate assay using Lactobacillus casei (31). Plasma total homocysteine was determined by HPLC using the fluorometric method of Vester and Rasmussen (32). Plasma methylmalonic acid was determined by the GC-MS (Hewlett-Packard 5890 Series II Gas Chromatograph, 5971A Mass Selective Detector) (33).
Colonic folate form distribution.
Folate distribution analysis of the colonic mucosa was determined by the affinity/HPLC method with Four Channel Coularray Detector (ESA), which gives a unique, characteristic electrochemical response for different pteridine ring structures of folate (34). Quantitation and identification of individual folates were done by comparison with external folate standards of known concentrations, as previously described (34).
Uracil misincorporation.
The amount of uracil in DNA was measured by a previously described GC-MS method (24). Uracil was removed from 15 µg DNA by incubation with 4 U uracil DNA glycosylase (New England Biolab) in TE buffer for 3 h at 37°C. 4,5-13C2 Uracil (200 pg; Cambridge Isotope Laboratories) was added as an internal standard after the glycosylase digestion, and the samples were dried in a speed vacuum concentrator. The residue was resuspended in a mixture of 50 µL acetonitrile, 10 µL triethylamine, and 1 µL 3,5-bis(trifluoromethyl)benzyl bromide, and shaken to derivatize for 25 min at 37°C, followed by the addition of 50 µL water (HPLC grade) to improve the extractability. N1,N3-(3,5-bis[trifluoromethyl]benzyl)uracil, a derivatized form, was extracted into 100 µL isooctane. The sample (5 µL) was injected onto a Series 6890 GC equipped with a 7683 automated liquid sampler (Agilent Technology) in the splitless mode. Adequate separation of N1,N3-(3,5-bis[trifluoromethyl]benzyl)uracil from interfering compounds was achieved on a capillary column (HP-5MS, Hewlett-Packard). A 5973N mass selective detector (Agilent Technology) using negative chemical ionization MS was used for analysis with elected ion monitoring at m/z 337 and 339. The amount of uracil in DNA is presented as pg uracil/µg DNA.
DNA methylation.
Liquid chromatography/electrospray ionization MS (Hewlett-Packard/Bruker) was used to analyze genomic DNA methylation. A detailed method was described and validated previously (35,36).
Briefly, 1 µg of DNA was hydrolyzed by sequential digestion with three enzymes, nuclease P1 (Roche Molecular Biochemicals), venom phosphodiesterase I (Sigma Chemical), and alkaline phosphatase (Sigma Chemical) (37). The hydrolyzed DNA solution was delivered directly onto the analytical column (Supelco) in isocratic mode. This allowed the separation of the four DNA bases as well as the identification of 5-methylcytosine. Electrospray ionization MS was performed in positive ion mode. Identification of cytosine and 5-methylcytosine was obtained by MS analysis of chromatographic peaks. The isotopomers 15N3 2'-deoxycytidine and methyl-D3, ring-6-D1 5-methyl-2'-deoxycytidine (Cambridge Isotope Laboratories) were used as internal standards allowing the quantitation of absolute amounts of the deoxycytidine and methylated cytosine residues in genomic DNA. DNA methylation status is presented as ng 5-methylcytosine/µg DNA.
Statistical analysis.
Data were analyzed by 2-way ANOVA for the effects of time of treatment and dietary folate. When the F-test was significant, means that differed were identified by a 2-tailed Students t test. Logarithmic transformation was performed on all skewed variables to normalize their distributions, and statistical comparison was based on logarithmically transformed data. The level of significant difference was set at P < 0.05 for all analyses. Statistical analysis was performed using Systat 7.0.1 for Windows (SPSS Inc.).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The control and vitamin B-12–deficient rats did not differ in body weight, blood hemoglobin, MCV, and plasma folate concentrations during the experiment (data not shown). However, platelet counts were lower at 10 wk in the deficient group compared with the control group: 0.97 ± 0.40 x 1012/L vs. 1.39 ± 0.19 x 1012/L, respectively (P < 0.05).
Plasma vitamin B-12 concentrations were undetectable at both 6 and 10 wk in the deficient rats, whereas concentrations in the control rats were 0.46 ± 0.07 and 0.42 ± 0.10 nmol/L at those times. Plasma total homocysteine concentrations were three- to fourfold greater in the deficient rats compared with the controls at both time points (P < 0.05). Plasma methylmalonic acid concentrations were 
20-fold greater in deficient rats at 6 wk (P < 0.05), and >60-fold greater at 10 wk compared with controls. This suggests that the magnitude of cellular vitamin B-12 depletion progressed between the 6- and 10-wk time points. The plasma folate concentrations did not differ between the groups, although values tended to be greater in the vitamin B-12–deficient group (Table 2).
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Total folate concentrations in the colonic mucosa did not differ between the vitamin B-12–deficient and control groups. However, important qualitative changes occurred in the distribution of folate coenzymes. Vitamin B-12–deficient rats had a lower proportion of tetrahydrofolate at both the 6 and 10 wk (P < 0.05), and the proportion of methylfolate was marginally greater in the deficient rats at 10 wk (P = 0.05) (Table 3).
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By the end of the experiment, the vitamin B-12–deficient rats had developed genomic DNA hypomethylation in the colon compared with controls (P < 0.05), as well as increased uracil incorporation into DNA (P < 0.05) (Fig. 1). Very similar patterns were observed at 6 wk, although the differences between the deficient and control groups were not significant (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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One-carbon metabolism is a network of interrelated biochemical reactions in which a one-carbon unit from a donor is transferred to tetrahydrofolate for subsequent reduction or oxidation and/or transfer of the one-carbon moiety to biochemical pathways essential for DNA synthesis and repair (thymidylate and purine synthesis) or biological methylation (1). In one-carbon metabolism, the methionine synthase reaction has a central role because it provides a means by which homocysteine can be remethylated, thereby synthesizing methionine. Methionine is then adenylated to form S-adenosylmethionine, which in turn donates its labile methyl group to >80 methylation reactions, including the methylation of DNA, RNA, and protein.
The methionine synthase reaction is also critical for the regeneration of tetrahydrofolate from methyltetrahydrofolate because the latter form of the coenzyme cannot enter into the pathways by which thymidine and the purines are synthesized, whereas the former can. The methionine synthase contains a cobalamin (vitamin B-12) cofactor and the reaction proceeds via a methylcobalamin intermediate (38). Consequently, when insufficient vitamin B-12 is available, cellular folates accumulate in the methylfolate form but cannot be utilized metabolically due to the block in the methionine synthase reaction, creating a conditional form of folate deficiency commonly referred to as the "methylfolate trap" (39).
Earlier studies demonstrated that dietary folate inadequacy can induce genomic DNA hypomethylation in both animal models (40) and humans (41,42). Similarly, folate depletion has been shown to increase uracil incorporation into DNA in cell culture models (43,44), animal models (45), and human subjects (24). This study demonstrates that the induction of these molecular anomalies occurs solely by restricting the availability of the cobalamin cofactor in the absence of folate depletion. Pertinent to the potential importance of such findings to human colon cancer is the fact that such phenomena were observed with a degree of vitamin B-12 depletion that was insufficient to produce macrocytosis, anemia, or weight loss, and that the molecular anomalies occurred in the colonic tissue; this is of interest because the colon seems to be particularly sensitive to the procarcinogenic effects of folate depletion.
These effects were of substantial magnitude, amounting to a 35% drop in methylcytosine and a 105% increase in uracil in colonic DNA. DNA hypomethylation is thought to increase susceptibility to carcinogenesis by enhancing the mutagenicity of cytosine (46), by activating endogenous retroviral sequences (47,48) and causing chromosome rearrangements (49), and by increasing the expression of protooncogenes (50,51). A decrease in genomic DNA methylation also appears to induce a compensatory increase in DNA methylation in a site-specific manner by activating DNA methyltransferase (52), which may induce gene silencing of tumor suppressor genes (51).
Uracil in DNA is also procarcinogenic because the repair system by which it is excised leaves behind double-stranded DNA strand breaks, which increase the possibility of translocations, deletions, and gene amplification (24,25).
Although previous cross-sectional studies have suggested a possible cause and effect relation between vitamin B-12 status, methylation, and uracil incorporation in lung and bone marrow (20,53,54), this is the first direct demonstration that the induction of vitamin B-12 depletion produces hypomethylation of DNA and excessive levels of uracil incorporation, and furthermore, that it occurs in the colonic mucosa. Whether such effects of vitamin B-12 depletion exist in noncolonic tissues is not known. Some tissues, such as the liver and kidney, contain an alternative pathway for synthesizing methionine from homocysteine that is independent of vitamin B-12; thus, this effect may be highly tissue specific (2).
DNA hypomethylation and excess uracil incorporation were noticeably present only after the rats had consumed a deficient diet for 10 wk. This is consistent with the observation that cellular depletion of vitamin B-12 worsened between the 6- and 10-wk time points, as evidenced by the progressive rise in methylmalonic acid. Thus, the duration of the depletion is an important factor, one that should be taken into account in the design of future studies utilizing this animal model. Nevertheless, it is important to note that these molecular sequelae evolved before the degree of depletion became of sufficient magnitude to produce anemia and other systemic effects, which in this model occurs only at time points beyond 10 wk.
We presume that the molecular anomalies produced by vitamin B-12 depletion in this study arose from inhibition of the same pathways that produce these effects in the setting of folate depletion, even though dietary folate was adequate throughout and total folate concentrations were normal in the colon of the vitamin B-12–deficient rats. The evidence supporting this presumption relates to our folate distribution data. There is clear evidence of a "methylfolate trap" produced by the vitamin B-12 deficiency in this study, as demonstrated by the accumulation of methylfolate in the colonic tissue, consistent with the concept that vitamin B-12 depletion produces a conditional deficiency of folate.
In summary, we observed in this animal model that vitamin B-12 deficiency of a degree that is insufficient to produce anemia, illness, or death can perturb one-carbon metabolism sufficiently to produce abnormalities in both biological methylation and DNA synthesis in the colonic mucosa. These anomalies are of particular interest because they are thought to be pathways that enhance the likelihood of carcinogenesis when other procarcinogenic factors are present. These observations may provide some understanding of the epidemiologic link between vitamin B-12 depletion and some common types of cancer, including cancer of the colorectum (15,19,21,22). The biochemical pathways by which these phenomena occurred were almost undoubtedly the same as those by which folate depletion causes similar effects. This may be clinically relevant because a depletion of folate and vitamin B-12 may therefore act synergistically in enhancing certain procarcinogenic processes.
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FOOTNOTES |
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Manuscript received 10 October 2003. Initial review completed 7 December 2003. Revision accepted 26 January 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Selhub, J. (2002) Folate, vitamin B12 and vitamin B6 and one carbon metabolism. J. Nutr. Health Aging 6:39-42.[Medline]
2. Millian, N. S. & Garrow, T. A. (1998) Human betaine-homocysteine methyltransferase is a zinc metalloenzyme. Arch. Biochem. Biophys. 356:93-98.[Medline]
3. Zeisel, S. H., Mar, M. H., Howe, J. C. & Holden, J. M. (2003) Concentrations of choline-containing compounds and betaine in common foods. J. Nutr. 133:1302-1307.
4. Giovannucci, E., Stampfer, M. J., Colditz, G. A., Rimm, E. B., Trichopoulos, D., Rosner, B. A., Speizer, F. E. & Willett, W. C. (1993) Folate, methionine, and alcohol intake and risk of colorectal adenoma. J. Natl. Cancer Inst. 85:875-884.
5. Giovannucci, E., Rimm, E. B., Ascherio, A., Stampfer, M. J., Colditz, G. A. & Willett, W. C. (1995) Alcohol, low-methionine-low-folate diets, and risk of colon cancer in men. J. Natl. Cancer Inst. 87:265-273.
6. Cravo, M. L., Mason, J. B., Dayal, Y., Hutchinson, M., Smith, D., Selhub, J. & Rosenberg, I. H. (1992) Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats. Cancer Res. 52:5002-5006.
7. Komatsu, S. I., Watanabe, H., Oka, T., Tsuge, H., Nii, H. & Kato, N. (2001) Vitamin B-6-supplemented diets compared with a low vitamin B-6 diet suppress azoxymethane-induced colon tumorigenesis in mice by reducing cell proliferation. J. Nutr. 131:2204-2207.
8. Yamamoto, R. S. (1980) Effect of vitamin B12-deficiency in colon carcinogenesis. Proc. Soc. Exp. Biol. Med. 163:350-353.[Medline]
9. Choi, S. W. & Mason, J. B. (2002) Folate status: effects on pathways of colorectal carcinogenesis. J. Nutr. 132(suppl. 8):2413S-2418S.
10. Winawer, S. J. (1988) Neoplasms of the large and small intestine. Wyngaarden, J. B. Smith, L. H. eds. Cecil Textbook of Medicine 1988:766-774 W. B. Saunders Philadelphia, PA. .
11. Lindenbaum, J., Rosenberg, I. H., Wilson, P. W., Stabler, S. P. & Allen, R. H. (1994) Prevalence of cobalamin deficiency in the Framingham elderly population. Am. J. Clin. Nutr. 60:2-11.
12. Pennypacker, L. C., Allen, R. H., Kelly, J. P., Matthews, L. M., Grigsby, J., Kaye, K., Lindenbaum, J. & Stabler, S. P. (1992) High prevalence of cobalamin deficiency in elderly outpatients. J. Am. Geriatr. Soc. 40:1197-1204.[Medline]
13. Stabler, S. P., Allen, R. H., Fried, L. P., Pahor, M., Kittner, S. J., Penninx, B. W. & Guralnik, J. M. (1999) Racial differences in prevalence of cobalamin and folate deficiencies in disabled elderly women. Am. J. Clin. Nutr. 70:911-919.
14. Rajan, S., Wallace, J. I., Beresford, S. A., Brodkin, K. I., Allen, R. A & Stabler, S. P. (2002) Screening for cobalamin deficiency in geriatric outpatients: prevalence and influence of synthetic cobalamin intake. J. Am. Geriatr. Soc. 50:624-630.[Medline]
15. Wu, K., Helzlsouer, K. J., Comstock, G. W., Hoffman, S. C., Nadeau, M. R. & Selhub, J. (1999) A prospective study on folate, B12, and pyridoxal 5'-phosphate (B6) and breast cancer. Cancer Epidemiol. Biomark. Prev. 8:209-217.
16. Zhang, S. M., Willett, W. C., Selhub, J., Hunter, D. J., Giovannucci, E. L., Holmes, M. D., Colditz, G. A. & Hankinson, S. E. (2003) Plasma folate, vitamin B6, vitamin B12, homocysteine, and risk of breast cancer. J. Natl. Cancer Inst. 95:373-380.
17. Hsing, A. W., Hansson, L. E., McLaughlin, J. K., Nyren, O., Blot, W. J., Ekbom, A & Fraumeni, J. F., Jr. (1993) Pernicious anemia and subsequent cancer: a population-based cohort study. Cancer 71:745-750.[Medline]
18. Toh, B. H., van Driel, I. R. & Gleeson, P. A. (1997) Pernicious anemia. N. Engl. J. Med. 337:1441-1448.
19. Karlson, B. M., Ekbom, A., Wacholder, S., McLaughlin, J. K. & Hsing, A. W. (2000) Cancer of the upper gastrointestinal tract among patients with pernicious anemia: a case-cohort study. Scand. J. Gastroenterol. 35:847-851.[Medline]
20. Piyathilake, C. J., Johanning, G. L., Macaluso, M., Whiteside, M., Oelschlager, D. K., Heimburger, D. C. & Grizzle, W. E. (2000) Localized folate and vitamin B-12 deficiency in squamous cell lung cancer is associated with global DNA hypomethylation. Nutr. Cancer 37:99-107.[Medline]
21. Talley, N. J., Chute, C. G., Larson, D. E., Epstein, R., Lydick, E. G. & Melton, L. J., 3rd. (1989) Risk for colorectal adenocarcinoma in pernicious anemia. A population-based cohort study. Ann. Intern. Med. 111:738-742.
22. Harnack, L., Jacobs, D. R., Jr., Nicodemus, K., Lazovich, D., Anderson, K. & Folsom, A. R. (2002) Relationship of folate, vitamin B-6, vitamin B-12, and methionine intake to incidence of colorectal cancers. Nutr. Cancer 43:152-158.[Medline]
23. Goelz, S. E., Vogelstein, B., Hamilton, S. R. & Feinberg, A. P. (1985) Hypomethylation of DNA from benign and malignant human colon neoplasms. Science (Washington, DC) 228:187-190.
24. Blount, B. C., Mack, M. M., Wehr, C. M., MacGregor, J. T., Hiatt, R. A., Wang, G., Wickramasinghe, S. N., Everson, R. B. & Ames, B. N. (1997) Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc. Natl. Acad. Sci. U.S.A. 94:3290-3295.
25. Ames, B. N. (2001) DNA damage from micronutrient deficiencies is likely to be a major cause of cancer. Mutat. Res. 475:7-20.[Medline]
26. Fuchs, C. S., Willett, W. C., Colditz, G. A., Hunter, D. J., Stampfer, M. J., Speizer, F. E. & Giovannucci, E. L. (2002) The influence of folate and multivitamin use on the familial risk of colon cancer in women. Cancer Epidemiol. Biomark. Prev. 11:227-234.
27. Cullen, R. W. & Oace, S. M. (1989) Dietary pectin shortens the biologic half-life of vitamin B-12 in rats by increasing fecal and urinary losses. J. Nutr. 119:1121-1127.
28. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
29. National Research Council (1995) General considerations for feeding and diet formulation. Nutrient Requirements of Laboratory Animals 1995:3-101 National Academy Press Washington, DC.
30. Perret, V., Lev, R. & Pigman, W. (1977) Simple method for the preparation of single cell suspensions from normal and tumorous rat colonic mucosa. Gut 18:382-385.
31. Tamura, T. (1990) Microbiological assay of folate. Picciano, M. F. Stokstad, E.L.R. Gregory, J. F., III. eds. Folic Acid Metabolism in Health and Disease 1990:121-137 Wiley-Liss New York, NY. .
32. Vester, B. & Rasmussen, K. (1991) High performance liquid chromatography method for rapid accurate determination of homocysteine in plasma and serum. Eur. J. Clin. Chem. Clin. Biochem. 29:549-554.[Medline]
33. Rasmussen, K. (1989) Solid-phase sample extraction for rapid determination of methylmalonic acid in serum and urine by a stable-isotope-dilution method. Clin. Chem. 35:260-264.
34. Bagley, P. J. & Selhub, J. (2000) Analysis of folate form distribution by affinity followed by reversed-phase chromatography with electrical detection. Clin. Chem. 46:404-411.
35. Friso, S., Choi, S. W., Dolnikowski, G. G. & Selhub, J. (2002) A method to assess genomic DNA methylation using high-performance liquid chromatography/electrospray ionization mass spectrometry. Anal. Chem. 74:4526-4531.[Medline]
36. Friso, S., Choi, S. W., Girelli, D., Mason, J. B., Dolnikowski, G. G., Bagley, P. J., Olivieri, O., Jacques, P. F., Rosenberg, I. H., Corrocher, R. & Selhub, J. (2002) A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc. Natl. Acad. Sci. U.S.A. 99:5606-5611.
37. Crain, P. F. (1990) Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. Methods Enzymol. 193:782-790.[Medline]
38. Shane, B. (2000) Folic acid, vitamin B12, and vitamin B6. Stipanuk, M. H. eds. Biochemical and Physiological Aspects of Human Nutrition 2000:483-518 W. B. Saunders Philadelphia, PA. .
39. Shane, B. & Stokstad, E.L.R. (1985) Vitamin B12-folate interrelationship. Annu. Rev. Nutr. 5:115-141.[Medline]
40. Balaghi, M. & Wagner, C. (1993) DNA methylation in folate deficiency: use of CpG methylase. Biochem. Biophys. Res. Commun. 193:1184-1190.[Medline]
41. Jacob, R. A., Gretz, D. M., Taylor, P. C., James, S. J., Pogribny, I. P., Miller, B. J., Henning, S. M. & Swendseid, M. E. (1998) Moderate folate depletion increases plasma homocysteine and decreases lymphocyte DNA methylation in postmenopausal women. J. Nutr. 128:1204-1212.
42. Rampersaud, G. C., Kauwell, G. P., Hutson, A. D., Cerda, J. J. & Bailey, L. B. (2000) Genomic DNA methylation decreases in response to moderate folate depletion in elderly women. Am. J. Clin. Nutr. 72:998-1003.
43. Duthie, S. J., Narayanan, S., Blum, S., Pirie, L. & Brand, G. M. (2000) Folate deficiency in vitro induces uracil misincorporation and DNA hypomethylation and inhibits DNA excision repair in immortalized normal human colon epithelial cells. Nutr. Cancer 37:245-251.[Medline]
44. Duthie, S. J. & Hawdon, A. (1998) DNA instability (strand breakage, uracil misincorporation, and defective repair) is increased by folic acid depletion in human lymphocytes in vitro. FASEB J. 12:1491-1497.
45. Choi, S. W., Friso, S., Dolnikowski, G. G., Bagley, P. J., Edmondson, A. N., Nadeau, M. R., Smith, D. E. & Mason, J. B. (2003) Biochemical and molecular indications that the elder rat colon is particularly susceptible to folate depletion. J. Nutr. 133:1206-1212.
46. Shen, J. C., Rideout, W. M., III. & Jones, P. A. (1992) High frequency mutagenesis by a DNA methyltransferase. Cell 71:1073-1080.[Medline]
47. Florl, A. R., Lower, R., Schmitz-Drager, B. J. & Schulz, W. A. (1999) DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas. Br. J. Cancer 80:1312-1321.[Medline]
48. Cooney, C. A., Dave, A. A. & Wolff, G. L. (2002) Maternal methyl supplements in mice affect epigenetic variation and DNA methylation of offspring. J. Nutr. 132(suppl. 8):2393S-2400S.
49. Ehrlich, M. (2003) The ICF syndrome, a DNA methyltransferase 3B deficiency and immunodeficiency disease. Clin. Immunol. 109:17-28.[Medline]
50. Tsujiuchi, T., Tsutsumi, M., Sasaki, Y., Takahama, M. & Konishi, Y. (1999) Hypomethylation of CpG sites and c-myc gene overexpression in hepatocellular carcinomas, but not hyperplastic nodules, induced by a choline-deficient L-amino acid-defined diet in rats. Jpn. J. Cancer Res. 90:909-913.[Medline]
51. Friso, S. & Choi, S. W. (2002) Gene-nutrient interactions and DNA methylation. J. Nutr. 132(suppl. 8):2382S-2387S.
52. Pogribny, I. P., Miller, B. J. & James, S. J. (1997) Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat. Cancer Lett. 115:31-38.[Medline]
53. Wickramasinghe, S. N. & Fida, S. (1994) Bone marrow cells from vitamin B12- and folate-deficient patients misincorporate uracil into DNA. Blood 83:1656-1661.
54. Wickramasinghe, S. N. & Fida, S. (1993) Misincorporation of uracil into the DNA of folate- and B12-deficient HL60 cells. Eur. J. Haematol. 50:127-132.[Medline]
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