Will mandatory folic acid fortification prevent or promote cancer? 1 3

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1 Commentary Will mandatory folic acid fortification prevent or promote cancer? 1 3 Young-In Kim ABSTRACT An overwhelming body of evidence for a protective effect of periconceptional folic acid supplementation against neural tube defects (NTDs) led to mandatory folic acid fortification in the United States. The effectiveness of folic acid fortification in improving folate status has already been shown to be quite striking, with a dramatic increase in blood measurements of folate in the United States. Preliminary reports also suggest a significant reduction ( 15 50%) in NTDs in the United States. The success of folic acid fortification in improving folate status and in reducing NTD rates is truly a public health triumph and provides a paradigm of collaboration between science and public health policy. Although folic acid is generally regarded as safe, there continues to be concern that folic acid fortification may have adverse effects in subpopulation groups not originally targeted for fortification. In this regard, an emerging body of evidence suggests that folic acid supplementation may enhance the development and progression of already existing, undiagnosed premalignant and malignant lesions. Over the past few years, the US population has been exposed to a significant increase in folate intake, for which essentially no data on safety exist. The potential cancer-promoting effect of folic acid supplementation needs to be considered in carefully monitoring the long-term effect of folic acid fortification on the vast majority of the US population, who are not at risk of NTDs. Am J Clin Nutr 2004;80: KEY WORDS Folic acid fortification, folate, cancer, carcinogenesis, DNA methylation, neural tube defects Folate is a water-soluble B vitamin that appears to play an important role in the pathogenesis of several disorders in humans, including anemia, cardiovascular disease, thromboembolitic processes, neural tube defects (NTDs) and other congenital defects, adverse pregnancy outcomes, neuropsychiatric disorders, and cancer (1). Folic acid is the fully oxidized monoglutamyl form of this vitamin and is used commercially in supplements and in fortified foods. The expanding role of folate nutrition in health and disease has major public health implications. For example, evidence from intervention trials (2 4) and observational studies (5) for a protective effect of periconceptional folic acid supplementation against NTDs was considered to be sufficiently conclusive and led public health policy makers, including the US Public Health Service in 1992 and the Institute of Medicine in 1998, to recommend that all women who were of reproductive age or were capable of becoming pregnant consume daily 400 g folic acid from supplements or fortified foods in conjunction with consumption of folate-rich foods (6, 7). This recommendation was followed by a US Food and Drug Administration regulation in 1996 requiring that all flour and uncooked cereal-grain products in the United States be fortified with folic acid (140 g/100 g) by January 1998 (8). Mandatory folic acid fortification was also implemented in Canada in 1998 (9) and in Chile (10), and limited voluntary folic acid fortification of specified foods was implemented in Western Australia in 1995 (11). The effectiveness of folic acid fortification in improving folate status has already been shown to be quite striking, with a dramatic increase in blood measurements of folate (serum, plasma, and red blood cell) and a substantial decrease in plasma homocysteine (an accurate inverse indicator of folate status) concentrations in the United States and Canada (10 17). Preliminary reports suggest a significant reduction ( 15 50%) in the prevalence and incidence of NTDs in the United States, Canada, and Western Australia (18 23). However, it is impossible to definitely attribute the decrease in the incidence of NTDs in the United States solely to fortification (24) because NTD rates in the United States (and worldwide) were decreasing even before fortification began, possibly as a result of factors such as improved nutrition or prenatal diagnosis and termination. Despite the observed beneficial effects of folic acid fortification on folate status and NTDs in countries that adopted either mandatory or limited voluntary fortification, there continues to be some concern that folic acid fortification may have adverse effects in subpopulation groups not originally targeted for fortification. Although folate is safe and almost free of toxicity (25), concerns that folic acid fortification may mask symptoms of vitamin B-12 deficiency, primarily in the elderly population, have been raised (7). Vitamin B-12 deficiency has been estimated to affect up to 10 15% of the population over 60 y of age (7). Because of this concern, the amount of fortification chosen was estimated to provide on average 100 g additional folic acid/d, with only a very small proportion of the population receiving 1 mg (7). The upper limit of 1 mg was a round number 1 From the Departments of Medicine and Nutritional Sciences, University of Toronto, and the Division of Gastroenterology, St Michael s Hospital, Toronto. 2 Supported by the Canadian Institutes for Health Research and the American Institutes for Cancer Research. 3 Address reprint requests to Y-I Kim, Room 7258, Medical Sciences Building, University of Toronto, 1 King s College Circle, Toronto, Ontario, Canada M5S 1A8. youngin.kim@utoronto.ca. Received April 27, Accepted for publication July 13, Am J Clin Nutr 2004;80: Printed in USA American Society for Clinical Nutrition 1123

2 1124 KIM chosen by the Institute of Medicine as unlikely to produce masking (7), although the folate intake that produces masking is controversial, with some arguing that intakes 1 mg may cause this effect (24). Several European countries decided not to adopt mandatory folic acid fortification, and the United Kingdom s Food Standards Agency Board and the Dutch Health Council even recommended against mandatory folic acid fortification, in part because of the potential for masking the diagnosis of a vitamin B-12 deficiency (5). The debate on the folic acid fortification controversy has become highly emotional; delaying folic acid fortification in some European countries was even labeled as public health malpractice (26). A more logical alternative to generalized mandatory folic acid fortification might be targeted folic acid supplementation in a specific group at risk of NTDs (eg, women with a previous NTD-affected pregnancy). However, efforts to increase periconceptional folic acid supplements have been proven to be disappointing, and public health efforts to influence those persons most at risk are generally perceived to have been a failure. Surveys taken in the United States, Puerto Rico, Netherlands, and Western Australia have shown that the public health policy recommendations and massive folic acid education and promotion programs advocating daily consumption of supplements containing folic acid among women of childbearing age fail to substantially increase the proportion of women of reproductive age who take a daily supplement containing folic acid (5). What is most disturbing in these surveys is that although most of these women had knowledge of the importance of folic acid, only 30 35% of women of reproductive age reported taking a daily supplement containing folic acid (5). Knowing that folic acid awareness does not necessarily translate into behavior change and that the neural tube closes during the fourth week of gestation, a time when many women are unaware of their pregnancy, public health policy makers opted for generalized folic acid fortification instead of targeted folic acid supplementation in subpopulations at risk of NTDs. However, there is evidence of success regarding reductions in NTDs and increased compliance with recommendations to take folic acid supplements in targeted geographic areas (27). For example, in response to the NTD Intervention Awareness Campaign in South Carolina, overall NTD rates decreased significantly, and no NTD recurrences were reported in women with a previous NTD-affected pregnancy who periconceptionally consumed supplements containing folic acid (27). The drop in overall NTD rates preceded fortification and coincided with higher reported supplemental folic acid intakes (27). Implicit in the folic acid fortification recommendation and policy was that improving folate status in the general population may provide other health benefits in addition to the reduction in NTD rates. Over the next few years, there will likely be many epidemiologic studies that will attempt to elucidate the long-term effect of folic acid fortification on the incidence of anemia, cardiovascular disease, thromboembolitic processes, congenital defects, adverse pregnancy outcomes, neuropsychiatric disorders, and cancers in countries that adopted mandatory and limited folic acid fortification. In this regard, a recent Canadian study (28) reported evidence supporting other potential health benefits of folic acid fortification. Using the database of the Pediatric Oncology Group of Ontario, which captures 95% of all pediatric cancers in Ontario, this study determined the effect of folic acid fortification on the incidence of neuroblastoma among children aged 17 y (28). The study showed that folic acid fortification was associated with a significant (60%) reduction in the incidence of neuroblastoma (from 1.57 cases per births in 1996 to 0.62 cases per births after 1997, when folic acid fortification of food became mandatory in Canada) (28). However, the incidence of infant acute lymphoblastic leukemia and hepatoblastoma remained almost the same (28). The results of this study corroborate those of previous epidemiologic studies, which reported a protective effect of the prenatal and perinatal maternal use of folic acid against the incidence of brain tumors in offspring (29 31). Therefore, the findings of this Canadian study suggest that, in addition to reducing NTD rates, mandatory folic acid fortification may prevent the development of certain cancers. Because of significant public health implications, the potential health benefits, including cancer prevention, of improved folate status in the general population make a strong case for mandatory folic acid fortification and for providing even higher amounts of folic acid as argued for by some proponents of mandatory generalized folic acid fortification. Often neglected and lost in public health policy making and debate concerning folic acid fortification is the effect of folate on cancer development and progression. Perhaps one of the most speculative and provocative new medical applications of folate nutrition is the potential role of folate as a cancer preventive agent (1, 32). The concept that folate deficiency enhances, whereas folate supplementation reduces, the risk of neoplastic transformation appears counterintuitive and contradictory to our conventional understanding of folate biochemistry. Folate is an essential cofactor for the de novo biosynthesis of purines and thymidylate, and in this capacity, folate plays an important role in DNA synthesis and replication. Consequently, folate deficiency in tissues with rapidly replicating cells results in ineffective DNA synthesis. In neoplastic cells, in which DNA replication and cell division occur at an accelerated rate, interruption of folate metabolism causes ineffective DNA synthesis, resulting in inhibition of tumor growth (32, 33). Indeed, this has been the basis for cancer chemotherapy with several antifolate agents (eg, methotrexate) and 5-fluorouracil (32, 33). Furthermore, folate deficiency has been shown to induce regression and suppress the progression of preexisting neoplasms in experimental models (34 36). In contrast to the inhibitory and promoting effect of folate deficiency and supplementation, respectively, on established neoplasms, folate status appears to have the opposite effect in normal tissues. An accumulating body of epidemiologic, clinical, and experimental evidence over the past decade suggests that folate deficiency in normal tissues appears to predispose them to neoplastic transformation, and folate supplementation suppresses the development of tumors in normal tissues (1, 32). The potential causal relation between folate status and cancer risk has been further strengthened by the existence of several biologically plausible mechanisms relating to the sole biochemical function known for folate (mediating the transfer of one-carbon moieties), by which folate status may modulate the development and progression of cancer in normal tissues (32, 33). As an essential cofactor for the de novo biosynthesis of purines and thymidylate, folate plays an important role in DNA synthesis, stability and integrity, and repair, aberrations of which have been implicated in colorectal carcinogenesis (32, 33). Folate may also modulate DNA methylation, which is an important epigenetic determinant in gene expression, maintenance of DNA integrity and stability, chromosomal modifications, and the development of mutations (32, 33). A growing body of evidence from in vitro,

3 FOLIC ACID FORTIFICATION AND CANCER RISK 1125 animal, and human studies indicates that folate deficiency is associated with DNA strand breaks, impaired DNA repair, increased mutations, and aberrant DNA methylation and that folate supplementation can correct some of these defects induced by folate deficiency (32, 33). Epidemiologic studies have suggested an inverse association of folate with the risk of cancer of the colorectum, lungs, pancreas, esophagus, stomach, cervix, ovary, and breast and the risk of neuroblastoma and leukemia (1, 32). The precise nature and magnitude of the inverse relation between folate status and the risk of these malignancies, however, have not yet been clearly established (1, 32). The role of folate in carcinogenesis has been best studied for colorectal cancer (CRC) in the general population and in persons with chronic ulcerative colitis (1, 5, 32, 37). Most of the published epidemiologic and clinical studies found either a significant inverse relation between folate status (assessed by using dietary folate intakes or measurement of blood folate concentrations) and the risk of CRC or its precursor, adenoma, or an equivocal inverse relation that was not significant. In the case of equivalent inverse relations, either those relations became nonsignificant after adjustment, or folate status could not be distinguished from other factors in its relation to the risk of CRC or adenoma (1, 5, 32, 37). A recent meta-analysis of 11 prospective epidemiologic studies from the United States, Canada, Netherlands, and Sweden that included male and female subjects showed a significant inverse association between folate intake (dietary and supplemental) and the risk of CRC (DJ Hunter, personal communication, 2003). This metaanalysis also showed a 20% reduction in the risk of CRC in subjects with the highest folate intake compared with those with the lowest intake. In some epidemiologic studies, the observed inverse association between folate status and CRC risk was further modified by the intake of alcohol and other nutritional factors (eg, methionine and vitamins B-6 and B-12) that are involved in the folate metabolic pathway and by polymorphisms of critical genes (eg, methylene tetrahydrofolate reductase gene 677C3 T) that are involved in folate metabolism (1, 5, 32, 37). At present, human intervention trials provide no conclusive evidence supporting the protective effect of folate supplementation against CRC, although several small pilot studies have shown that folate supplementation may improve or reverse surrogate endpoint biomarkers of CRC (1, 32), and some epidemiologic studies have shown a beneficial effect of multivitamin supplements containing 400 g folic acid on CRC risk and mortality (38, 39). Although animal studies performed in chemical carcinogen and genetically engineered murine models of CRC generally support a causal relation between folate depletion and CRC risk, these studies have shown that the dose and timing of folate intervention are critical in providing safe and effective chemoprevention; exceptionally high supplemental folate doses and folate intervention after microscopic neoplastic foci are established in the colorectal mucosa promote rather than suppress colorectal carcinogenesis (1, 32). For example, in a standard chemical carcinogen rodent model of CRC, supraphysiologic doses of folate ( 20 times the basal daily dietary requirement) were shown to increase the development and progression of CRC (40 42). Furthermore, in 2 genetic models of CRC (Apc Min and Apc / x Msh2 / mice), moderate folate deficiency enhanced, whereas modest doses of folate supplementation suppressed, the development and progression of CRC if folate intervention was started before the establishment of neoplastic foci in the intestine (43, 44). If, however, folate intervention is started after the establishment of neoplastic foci, dietary folate has the opposite effect on the development and progression of CRC (43, 44). Some animal studies have also shown that dietary folate deficiency inhibits rather than enhances the development of breast cancer in rats (45, 46), which is in contrast to the inverse association between folate status and breast cancer risk observed in epidemiologic studies (46). In conjunction with some clinical observations, these animal studies suggest that folate possesses dual modulatory effects on carcinogenesis depending on the timing and dose of folate intervention (1). Folate deficiency has an inhibitory effect, whereas folate supplementation has a promoting effect on the progression of established neoplasms (1, 32). In contrast, folate deficiency in normal epithelial tissues appears to predispose them to neoplastic transformation, and modest amounts of folate supplementation (4 10 times the basal dietary requirement) suppress, whereas supraphysiologic doses enhance, the development of tumors in normal tissues (1, 32). Although some similarities do exist, tumor development in chemical and genetically engineered animal models of CRC differs in several important histologic, clinical, and molecular genetic aspects from that observed in humans. Therefore, any extrapolation of observations from these models to human situations should be made cautiously. Notwithstanding these limitations, however, the data from animal studies suggest that the optimal timing and dose of folate intervention should be established before folate supplementation can be used as a safe and effective chemopreventive agent against CRC. Although folate appears to be an ideal candidate for chemoprevention because of its proven safety and cost (25), the safe and effective dose range of folate supplementation and optimal timing of folate chemoprevention have not been clearly established in humans. Animal studies and some clinical studies have suggested that folate supplementation may increase cancer risk and accelerate tumor progression if too much is given or if it is provided after neoplastic foci are established in the target organ. Therefore, modest doses of folate supplementation should apparently be implemented before the development of precursor lesions in the target organ or in persons free of any evidence of neoplastic foci. However, determining the presence of neoplastic foci in the general population is obviously an almost impossible task. Furthermore, folate might prevent the progression of certain precursor or preneoplastic lesions to frank malignancy but promote the progression of other lesions. What constitutes safe precursor or preneoplastic lesions on which folate may exert a protective effect has not yet been established. For example, should folate chemoprevention be started before there is evidence of established premalignant lesions, such as aberrant crypt foci or microscopic adenomatous lesions in the colorectum, or should folate chemoprevention be started even after these lesions are present? It is apparent from the above discussion that folic acid supplementation should not be adopted as a chemopreventive agent against CRC and other cancers until definitive evidence indicates that such supplementation is indeed safe and effective. However, what are the effects of folic acid fortification on the risk of CRC and other malignancies? Is there any reason to believe that folic acid fortification may actually increase the risk of certain cancers? Although folic acid fortification may prevent NTDs as evidenced by preliminary reports suggesting a significant reduction ( 15 50%) in the prevalence and incidence of NTDs in the

4 1126 KIM United States, Canada, and Western Australia (18 23), the longterm effect of folic acid fortification on the risk of CRC and other malignancies may not be as clear as that observed for NTDs. Although folic acid fortification may prevent the development of new cancers in persons without preexisting premalignant lesions or neoplastic foci, it may promote the progression of these lesions in persons harboring them. The addition of folate to established tumors has previously been shown to cause an acceleration phenomenon in humans. For example, children with acute leukemia treated with folate supplementation experienced an accelerated progression of leukemia (47). Analogous to this situation, -carotene supplementation has been shown to promote the development of lung cancer in smokers who likely harbored preneoplastic or neoplastic foci before supplementation (48, 49). The success of folic acid fortification in improving folate status and in reducing NTD rates is truly a public health triumph and provides a paradigm of collaboration between science and public health policy. Improved folate status in the general population resulting from folic acid fortificaion may also lead to reduction in anemia, cardiovascular disease, thromboembolitic processes, congenital defects, adverse pregnancy outcomes, and neuropsychiatric disorders. However, an emerging body of evidence suggests that folic acid supplementation may promote the progression of preexisting, undiagnosed premalignant and malignant lesions. Over the past few years, the US and Canadian populations have been exposed to a significant increase in folate intake, for which essentially no data on safety exist (24). No studies have been done to look directly or even indirectly for the adverse effects of greatly increased folate intakes (24). Several studies that assessed food composition and dietary intakes suggested that the increased postfortification folate intake in the US population may be about twice that originally anticipated (14, 50 52). However, these estimates of folate intake based on foodcomposition databases are likely underestimates because of limitations in the analytic methods previously used to analyze food folate (7). Although the potential masking effect of folate on vitamin B-12 deficiency, especially in the elderly, has been the major concern of folic acid fortification, other adverse effects, such as the potential cancer-promoting effect of folic acid supplementation, need to be considered in carefully monitoring the long-term effect of folic acid fortification on health and disease in the vast majority of the US and Canadian populations, who are not at risk of NTDs. Furthermore, because intracellular and systemic folate concentrations are important determinants of the sensitivity of cancer cells to chemotherapy (53) and in the treatment of inflammatory and seizure disorders with the use of antifolates (25), the occurrence of resistance or tolerance to antifolate chemotherapy and antiinflammatory and antiseizure drugs should be added to the list of potential unwanted complications of folic acid fortification. Another potential harmful consequence of folic acid fortification in relation to cancer risk modification concerns DNA methylation. DNA methylation of cytosine located within the cytosine-guanine (CpG) dinucleotide sequences is an important epigenetic determinant in gene expression (inverse relation), maintenance of DNA integrity and stability, chromatin modifications and remodeling, and the development of mutations (54). Neoplastic cells simultaneously harbor widespread genomic hypomethylation and more specific regional areas of hypermethylation (54). Genomic hypomethylation is an early and consistent event in carcinogenesis and is associated with genomic instability and increased mutations. In addition, site-specific hypermethylation at promoter CpG islands of tumor suppressor and mismatch repair genes is an important mechanism in gene silencing in carcinogenesis (54). Folate, in the form of 5-methyltetrahydrofolate, is involved in remethylation of homocysteine to methionine, which is a precursor of S-adenosylmethionine, the primary methyl group donor for most biological methylations, including that of DNA (55). Although the effect of folate deficiency on DNA methylation is highly variable and complex, folate supplementation appears to significantly increase the extent of genomic and site-specific DNA methylation in certain situations (55). This then begs a question: can folic acid fortification methylate normally unmethylated promoter CpG islands of tumor suppressor or mismatch repair genes and inactivate these genes, thereby promoting the development of cancer? This is a theoretical possibility and warrants investigation. In this regard, a recent animal study using viable yellow agouti (A vy ) mice showed that maternal dietary methyl group supplementation with a modest amount of folic acid, vitamin B-12, choline, and betaine may permanently alter the phenotype of the offspring via increased CpG methylation at the promoter CpG site of the agouti gene (56). The investigators found that the methylaton status of the promoter CpG region of the agouti gene was highly correlated with the methylaton status of the adjacent transposon gene (56). Transposons are common and potentially mobile sequences of DNA that are scattered throughout the genome (57). More than 35% of human DNA is estimated to be derived from transposons (57). Depending on where they are inserted in DNA, transposons can end up silencing neighboring genes (57). Therefore, this study by Waterland and Jirtle (56) showed that there is a localized epigenetic instability in methylation that arises from an interaction between the transposon and its nearby genetic region and that genes that manifest a transposon region adjacent to a promoter region of DNA could be influenced by early nutrition containing methyl group donors, including folic acid. These investigators speculated that population-based supplementation with folic acid, intended to reduce the incidence of NTD and long presumed to be purely beneficial, may have unintended deleterious influences on the establishment of epigenetic gene-regulatory mechanisms during human embryonic development (57). Some proponents of mandatory folic acid fortification have labeled the delay in folic acid fortification in some European countries as public health malpractice (26). However, a reasonable conclusion from the above discussion is that inertia on folic acid fortification in these European countries should not be construed as public health malpractice but should be regarded as public health prudence. The effect of folic acid fortification on cancer risk has a greater effect on public health than on NTDs because of the incidence and prevalence of cancer and premalignant precursors in the general US population. For example, in the United States, CRC is the fourth most frequently diagnosed and the second most common cause of cancer-specific death for both men and women (58). In 2004 alone, new cases of CRC are expected to be diagnosed, and 40% of these are expected to die within 5 y (58). In 2004 an estimated deaths will have been caused by CRC (58). The lifetime risk of developing CRC is 6% (59), and treatment costs nearly $6 billion annually (60). Colorectal adenomas, the well-established precursor of CRC (59), are found in 25% of people by 50 y of age in the United States, and the prevalence increases with age

5 FOLIC ACID FORTIFICATION AND CANCER RISK 1127 (59). On the basis of autopsy series, which are probably less susceptible to selection and detection bias than are clinical series, the prevalence of adenomas is estimated to reach 50% by 50 y of age (59). It has been estimated that 25% of adenomas progress to CRC over 5 10 y (59). In contrast, NTDs occur in 1 of every 1000 births in the United States, and spina bifida and anencephaly, the most common NTDs, together affect 4000 pregnancies resulting in US births annually (61). It is evident from this statistic that the potential effect of folic acid fortification on adenoma progression to CRC and on CRC progression to metastasis far outweighs the effect on NTD risk reduction. Given the prevalence and incidence of colorectal adenomas and CRC in the general population in the United States, therefore, whether or not folic acid fortification promotes the progression of adenomas to CRC in the colorectum is a legitimate public health concern and needs careful monitoring. A recent study showing that folic acid fortification significantly reduced the incidence of neuroblastoma in Ontario (28) is an encouraging piece of information in this regard. However, long-term follow-up studies are urgently warranted to determine the effect of folic acid fortification on the incidence of cancer and on DNA methylation and other epigenetic regulatory machinery in countries that have adopted mandatory generalized folic acid fortification. Furthermore, safe and effective amounts of folic acid fortification need to be scientifically determined by using relevant animal, experimental, and clinical models. The potential cancer-promoting effect of folic acid fortification in the vast majority of the US population, who are not at risk of NTDs but have been unintentionally exposed to high amounts of folic acid, is a legitimate public health concern and needs careful monitoring. The author had no conflicts of interest in connection with this article. REFERENCES 1. Kim YI. Role of folate in colon cancer development and progression. J Nutr 2003;133:3731S 9S. 2. Czeizel AE, Dudas I. Prevention of the first occurrence of neural-tube defects by periconceptional vitamin supplementation. N Engl J Med 1992;327: MRC Vitamin Study Research Group. Prevention of neural tube defects: results of the Medical Research Council Vitamin Study. Lancet 1991; 338: Berry RJ, Li Z, Erickson JD, et al. Prevention of neural-tube defects with folic acid in China. China-U.S. Collaborative Project for Neural Tube Defect Prevention. N Engl J Med 1999;341: (Published erratum appears in N Engl J Med 1999;341:1864.) 5. Bailey LB, Rampersaud GC, Kauwell GP. Folic acid supplements and fortification affect the risk for neural tube defects, vascular disease and cancer: evolving science. J Nutr 2003;133:1961S 8S. 6. Centers for Disease Control and Prevention. Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. MMWR Morb Mortal Wkly Rep 1992;41: Institute of Medicine. Folate. In: Dietary reference intakes for thiamin, roboflavin, niacin, vitamin B6, folate, vitamin B12, pantothenic acid, biotin, and choline. Washington, DC: National Academy Press, 1998: Food and Drug Administration. Food standards: amendment of standards of identity for enriched grain products to require addition of folic acid. Final rule. 21 CFR Parts 136, 137, and 139. Fed Regist 1996;61: Health Canada. Regulations amending the Food and Drug Regulations (1066). Can Gaz Part ;131: Freire WB, Hertrampf E, Cortes F. Effect of folic acid fortification in Chile: preliminary results. Eur J Pediatr Surg 2000;10(suppl): Metz J, Sikaris KA, Maxwell EL, Levin MD. Changes in serum folate concentrations following voluntary food fortification in Australia. Med J Aust 2002;176: Centers for Disease Control and Prevention. Folate status in women of childbearing age United States. MMWR Morb Mortal Wkly Rep 2000;49: Choumenkovitch SF, Jacques PF, Nadeau MR, Wilson PW, Rosenberg IH, Selhub J. Folic acid fortification increases red blood cell folate concentrations in the Framingham study. J Nutr 2001;131: Choumenkovitch SF, Selhub J, Wilson PW, Rader JI, Rosenberg IH, Jacques PF. Folic acid intake from fortification in United States exceeds predictions. J Nutr 2002;132: Jacques PF, Selhub J, Bostom AG, Wilson PW, Rosenberg IH. The effect of folic acid fortification on plasma folate and total homocysteine concentrations. N Engl J Med 1999;340: Lawrence JM, Petitti DB, Watkins M, Umekubo MA. Trends in serum folate after food fortification. Lancet 1999;354: Ray JG, Vermeulen MJ, Boss SC, Cole DE. Increased red cell folate concentrations in women of reproductive age after Canadian folic acid food fortification. Epidemiology 2002;13: Honein MA, Paulozzi LJ, Mathews TJ, Erickson JD, Wong LY. Impact of folic acid fortification of the US food supply on the occurrence of neural tube defects. JAMA 2001;285: Williams LJ, Mai CT, Edmonds LD, et al. Prevalence of spina bifida and anencephaly during the transition to mandatory folic acid fortification in the United States. Teratology 2002;66: Gucciardi E, Pietrusiak MA, Reynolds DL, Rouleau J. Incidence of neural tube defects in Ontario, CMAJ 2002;167: Persad VL, Van den Hof MC, Dube JM, Zimmer P. Incidence of open neural tube defects in Nova Scotia after folic acid fortification. CMAJ 2002;167: Bower C, Ryan A, Rudy E, Miller M. Trends in neural tube defects in Western Australia. Aust NZJPublic Health 2002;26: De Wals P, Rusen ID, Lee NS, Morin P, Niyonsenga T. Trend in prevalence of neural tube defects in Quebec. Birth Defects Res Part A Clin Mol Teratol 2003;67: Shane B. Folate fortification: enough already? Am J Clin Nutr 2003;77: Campbell NR. How safe are folic acid supplements? Arch Intern Med 1996;156: Oakley GP Jr. Inertia on folic acid fortification: public health malpractice. Teratology 2002;66: Stevenson RE, Allen WP, Pai GS, et al. Decline in prevalence of neural tube defects in a high-risk region of the United States. Pediatrics 2000; 106: French AE, Grant R, Weitzman S, et al. Folic acid food fortification is associated with a decline in neuroblastoma. Clin Pharmacol Ther 2003; 74: Bunin GR, Kuijten RR, Buckley JD, Rorke LB, Meadows AT. Relation between maternal diet and subsequent primitive neuroectodermal brain tumors in young children. N Engl J Med 1993;329: Preston-Martin S, Pogoda JM, Mueller BA, et al. Prenatal vitamin supplementation and risk of childhood brain tumors. Int J Cancer Suppl 1998;11: Olshan AF, Smith JC, Bondy ML, Neglia JP, Pollock BH. Maternal vitamin use and reduced risk of neuroblastoma. Epidemiology 2002;13: Kim YI. Folate and carcinogenesis: Evidence, mechanisms, and implications. J Nutr Biochem 1999;10: Choi SW, Mason JB. Folate status: effects on pathways of colorectal carcinogenesis. J Nutr 2002;132:2413S 8S. 34. Little PA, Sampath A, Paganelli V. The effect of folic acid and its antagonists on rous chicken sarcoma. Trans N Y Acad Sci Ser II 1948; 10: Rosen F. Inhibition of the growth of an amethopterin-refractory tumor by dietary restriction of folic acid. Cancer Res 1962;22: Bills ND, Hinrichs SH, Morgan R, Clifford AJ. Delayed tumor onset in transgenic mice fed a low-folate diet. J Natl Cancer Inst 1992;84: Giovannucci E. Epidemiologic studies of folate and colorectal neoplasia: a review. J Nutr 2002;132:2350S 5S. 38. Giovannucci E, Stampfer MJ, Colditz GA, et al. Multivitamin use, folate, and colon cancer in women in the Nurses Health Study. Ann Intern Med 1998;129: Jacobs EJ, Connell CJ, Patel AV, et al. Multivitamin use and colon

6 1128 KIM cancer mortality in the Cancer Prevention Study II cohort (United States). Cancer Causes Control 2001;12: Kim YI, Salomon RN, Graeme-Cook F, et al. Dietary folate protects against the development of macroscopic colonic neoplasia in a dose responsive manner in rats. Gut 1996;39: Le Leu RK, Young GP, McIntosh GH. Folate deficiency reduces the development of colorectal cancer in rats. Carcinogenesis 2000;21: Wargovich MJ, Chen CD, Jimenez A, et al. Aberrant crypts as a biomarker for colon cancer: evaluation of potential chemopreventive agents in the rat. Cancer Epidemiol Biomarkers Prev 1996;5: Song J, Medline A, Mason JB, Gallinger S, Kim YI. Effects of dietary folate on intestinal tumorigenesis in the apcmin mouse. Cancer Res 2000;60: Song J, Sohn KJ, Medline A, Ash C, Gallinger S, Kim YI. Chemopreventive effects of dietary folate on intestinal polyps in Apc / Msh2 / mice. Cancer Res 2000;60: Baggott JE, Vaughn WH, Juliana MM, Eto I, Krumdieck CL, Grubbs CJ. Effects of folate deficiency and supplementation on methylnitrosoureainduced rat mammary tumors. J Natl Cancer Inst 1992;84: Kotsopoulos J, Sohn KJ, Martin R, et al. Dietary folate deficiency suppresses N-methyl-N-nitrosourea induced mammary tumorigenesis in rats. Carcinogenesis 2003;24: Farber S. Some observations on the effect of folic acid antagonists on acute leukemia and other forms of incurable cancer. Blood 1949;4: The Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N Engl J Med 1994;330: Omenn GS, Goodman GE, Thornquist MD, et al. Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 1996;334: Lewis CJ, Crane NT, Wilson DB, Yetley EA. Estimated folate intakes: data updated to reflect food fortification, increased bioavailability, and dietary supplement use. Am J Clin Nutr 1999;70: Rader JI, Weaver CM, Angyal G. Total folate in enriched cereal-grain products in the United States following fortification. Food Chem 2000; 70: Quinlivan EP, Gregory JF III. Effect of food fortification on folic acid intake in the United States. Am J Clin Nutr 2003;77: Kamen B. Folate and antifolate pharmacology. Semin Oncol 1997; 24(suppl):S Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002;3: Kim YI. Folate and DNA methylation: a mechanistic link between folate deficiency and colorectal cancer? Cancer Epidemiol Biomarkers Prev 2004;13: Waterland RA, Jirtle RL. Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 2003;23: Yoder JA, Walsh CP, Bestor TH. Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 1997;13: Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, CA Cancer J Clin 2004;54: Winawer SJ, Fletcher RH, Miller L, et al. Colorectal cancer screening: clinical guidelines and rationale. Gastroenterology 1997;112: Winawer SJ. A quarter century of colorectal cancer screening: progress and prospects. J Clin Oncol 2001;19:6S 12S. 61. Botto LD, Moore CA, Khoury MJ, Erickson JD. Neural-tube defects. N Engl J Med 1999;341: