Assessment of the Potential Health Risks of the Folic Acid Fortification Program on Acute Lymphoblastic Leukemia and Colorectal Cancer

Transcription

1 Assessment of the Potential Health Risks of the Folic Acid Fortification Program on Acute Lymphoblastic Leukemia and Colorectal Cancer by Deborah A. Kennedy A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy University of Toronto Copyright by Deborah Kennedy 2013

2 Assessment of the Potential Health Risks of the Folic Acid Fortification Program on Acute Lymphoblastic Leukemia and Colorectal Cancer Deborah A. Kennedy Doctor of Philosophy Department of Pharmaceutical Sciences, University of Toronto, 2013 Abstract Neural tube defects (NTD) result from the failure of the neural tube to close properly very early in gestation. A child born with an NTD may experience an early death or life-long disability. In the 1990s, the critical role of folic acid in the prevention of NTDs was confirmed and as a strategy to increase blood folate concentrations of women of childbearing age, folic acid fortification programs were mandated in Canada and the US. However, this change impacted the entire population not just women of childbearing age and not everyone may benefit from the increased folate intake. The objective of this research was to investigate the impact of higher intakes of folates on the mortality rates of children with acute lymphoblastic leukemia (ALL) and the risk of colorectal cancer (CRC) in adult populations. To address the impact in children with ALL, a comparison of the mortality rates between the preand post-fortification time periods in Ontario was performed using data from the Pediatric Oncology Group of Ontario. A second comparison between the mortality rates in these children in non-folic acid fortifying countries and the US was also completed. These analyses suggest ii

3 that folic acid fortification is not negatively impacting mortality. With respect to CRC, one systematic review and two meta-analyses were conducted investigating folate intake and the risk of CRC or adenoma recurrence. The first analysis, in observational studies, compared high versus low folate intake and the risk of CRC. The second examined folate intake within the various polymorphisms of the methylene tetrahydrofolate reductase enzyme. The final study examined the impact of supplementation of 1 milligram or more per day of folic acid and the risk of colorectal adenoma recurrence in those adults with a history of colorectal adenomas. The findings from the completed observational studies suggest that there is an associated risk reduction in colorectal cancer from the intake of higher levels of folates. The investigations into the impact of the folic acid fortification program suggest that the program is not associated with having a negative impact on mortality of children with ALL or on the risk of colorectal cancer. iii

4 Acknowledgments One does not undertake and complete a project such as an advanced degree without support and guidance from many wonderful people. I am grateful to my supervisor, Dr. Gideon Koren, for his guidance and mentorship through this process of completing my PhD. Dr. Koren, thank you for sharing your insight and wisdom with me, thank you for the guidance that you have provided to me along this journey. Without the financial support in the form of a three year career development grant from SickKids Foundation, pursuing a PhD would have been but a dream rather than a reality. Many thanks to the SickKids Foundation for their support and investment in my future as a researcher. Thank you, Dr. Dugald Seely, my co-supervisor on the SickKids Foundation grant, for your support and guidance. I would like to thank my Advisory Committee members: Dr. Tom Einarson, Dr. Deborah O Connor and Dr. Bhushan Kapur for their guidance, ideas, constructive criticism and knowledge. Thank you to the graduate students/co-authors and fellows at Motherisk and the Division of Clinical Pharmacology and Toxicology for their support and assistance and to the other researchers at the Canadian College of Naturopathic Medicine. Thank you to my family for their on-going and consistent support. Thank you to my extended Reiki family; Antia, Thomas, Rolande, Eric, Fran, David and the others of the Reiki Support Line for your unwavering support, enthusiastic voices, and additional energy sent my way in the more chaotic times in the past three years. iv

5 Table of Contents Contents ACKNOWLEDGMENTS... IV TABLE OF CONTENTS... V LIST OF ABBREVATIONS... XI LIST OF TABLES... XIII LIST OF FIGURES... XIV CHAPTER INTRODUCTION GENERAL INTRODUCTION NEURAL TUBE DEFECTS Incidence of neural tube defects Types of neural tube defects Causes of neural tube defects Public health initiatives to reduce the incidence of neural tube defects FOLATES The Structure and absorption of folates Naturally occurring folates Folic acid The cellular folate cycle methyl-tetrahydrofolate Folic acid Recommended daily allowance of folate Specific folic acid intake guidelines for women contemplating pregnancy Current intake of folate in Canadian and US populations COLORECTAL CANCER Incidence of colorectal cancer Pathways to colorectal cancer Model of colorectal cancer pathogenesis The chromosomal instability pathway The CpG island methylator phenotype pathway v

6 Microsatellite instability pathway Serrated polyp pathway The controversy: folate s link to colorectal cancer Epigenetic theories Genetic mechanisms The Dual Effect of Folate theory Specific concerns regarding folic acid intake Theories on folic acid s promotion of carcinogenesis Folic acid s impact on the immune system CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA Treatment of childhood acute lymphoblastic leukemia Remission/induction Intensification/consolidation Continuation/maintenance Methotrexate Methotrexate s mechanism of action Rescue of cells from methotrexate toxicity Folic acid supplementation/fortification and childhood acute lymphoblastic leukemia Folic acid fortification and folate intake in children CHAPTER OVERVIEW OF THESIS RESEARCH: RATIONALE, HYPOTHETICAL FRAMEWORK, AND OBJECTIVES RATIONALE OVERALL HYPOTHESIS SPECIFIC HYPOTHESES AND OBJECTIVES OF THE RESEARCH Colorectal cancer Hypothesis Objective of the research Childhood acute lymphoblastic leukemia Hypothesis Objective of the research CHAPTER PREAMBLE CHAPTER vi

7 4 FOLATE INTAKE AND THE RISK OF COLORECTAL CANCER: A SYSTEMATIC REVIEW AND META-ANALYSIS ABSTRACT INTRODUCTION MATERIALS AND METHODS Inclusion criteria Search strategy Data extraction Statistical analysis RESULTS Subgroup analyses Case control studies Cohort studies Sensitivity analysis Interaction between alcohol and folate Publication bias DISCUSSION STATEMENT OF SIGNIFICANCE AND IMPACT CHAPTER FOLIC ACID INTAKE, MTHFR POLYMORPHISMS AND THE RISK OF COLORECTAL CANCER: A SYSTEMATIC REVIEW AND META-ANALYSIS ABSTRACT INTRODUCTION METHODS AND MATERIALS Inclusion criteria Search strategy Data extraction Statistical analysis RESULTS Colorectal cancer risk and genotype Colorectal cancer risk and MTHFR C677T genotype Subgroup analysis Sensitivity analysis Publication bias vii

8 Correlation between study quality versus results Colorectal cancer risk and MTHFR A1298C genotype Sensitivity analysis Subgroup analysis Publication bias Colorectal cancer risk and combinations of the MTHFR C677T and A1298C genotypes Colorectal cancer risk, comparison of high versus low folate intake by genotype DISCUSSION ACKNOWLEDGEMENTS STATEMENT OF SIGNIFICANCE AND IMPACT FOLIC ACID AND COLORECTAL ADENOMA RECURRENCE: A SYSTEMATIC REVIEW OF RANDOMIZED CONTROLLED TRIALS ABSTRACT INTRODUCTION METHODS Search strategy Inclusion criteria Data extraction Quality assessment Statistical analysis RESULTS Summary of included studies Publication bias Quality of studies DISCUSSION STATEMENT OF SIGNIFICANCE AND IMPACT CHAPTER IMPACT OF FOLIC ACID SUPPLEMENTATION IN ANTIFOLATE CHEMOTHERAPY: A SYSTEMATIC REVIEW BACKGROUND METHODS Inclusion criteria Search strategy RESULTS viii

9 7.4 DISCUSSION CONCLUSION CHAPTER FOLATE FORTIFICATION AND SURVIVAL OF CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA ABSTRACT BACKGROUND MATERIALS AND METHODS Study design Data sources and analysis RESULTS Pediatric Oncology Group of Ontario database analysis World Health Organization database analysis DISCUSSION CONCLUSIONS STATEMENT OF SIGNIFICANCE AND IMPACT OVERALL DISCUSSION AND CONCLUSIONS SUMMARY AND DISCUSSION OF RESEARCH FINDINGS SYSTEMATIC REVIEWS AND META ANALYSES Strengths of systematic reviews and meta-analyses Weaknesses of systematic review and meta-analyses FOLIC ACID S IMPACT IN ACUTE LYMPHOBLASTIC LEUKEMIA FOLATE FORTIFICATION AND THE MTHFR POLYMORPHISM TOO MUCH OF A GOOD THING OR HOMEOSTASIS? ANOTHER SOURCE OF FOLATE? FOLATE STATUS IN WOMEN OF CHILDBEARING AGE Barriers to adequate periconceptional folate intake How is risk perceived? Cancer fear Awareness and knowledge regarding folic acid in women of childbearing age Practitioner s practices regarding periconceptional folic acid supplementation Strategies to address the barriers to periconceptional folic acid supplementation Ongoing, continuous education and outreach programs Leveraging routine care visits with family physicians and obstetricians/gynecologists ix

10 Dispelling the fear of cancer associated with folic acid intake LIMITATIONS AREAS OF FURTHER DEVELOPMENT CONCLUSIONS REFERENCES x

11 LIST OF ABBREVATIONS 5,10-methylene-THF 5-formyl-THF 5-methyl-THF ALL APC BMI BRAF CDC CI df DFE DHF DHFR DNA dtmp dump EAR FDA FFQ formyl-thf FPGS HDMTX hfr HR IOM Mcg, mcg/day Mg, mg/day MS MTHFD MTHFR 5,10-methylenetetrahydrofolate 5-formyltetrahydrofolate, leucovorin 5 methyl tetrahydrofolate Acute lymphoblastic leukemia Adenomatous polypolis coli Body mass index BRAF gene Center for Disease Control and Prevention Confidence interval Degrees of freedom Dietary folate equivalent Dihydrofolate Dihydrofolate reductase Deoxyribonucleic acid deoxythymidine monophosphate deoxyuridine monophosphate Estimated Average Requirement Food and Drug Administration Food frequency questionnaire formyltetrahydrofolate folylpolyglutamate synthase High dose MTX Human folate receptor Hazards Ratio Institute of Medicine Micrograms, micrograms/day Milligrams, milligrams per day Methionine synthase methylene tetrahydrofolate dehydrogenase Methylene tetrathydrofolate reductase xi

12 MTHFS MTX MTXG n NHANES nmol/l NTD Ob/Gyn OR PCFT RDA RFC RNA RR SHMT THF TS UL USA or US WBC WHO methylene tetrahydrofolate synthase Methotrexate Polyglutamated methotrexate, up to 6 glutamate molecules can be added to methotrexate National Health and Nutrition Examination Survey Nanomol per liter Neural tube defect Obstetricians and/or gynecologists Odds Ratio Proton coupled folate transporter Recommended daily allowance Reduced folate carrier Ribonucleic acid Risk Ratio serine hydroxymethyl transferase Tetrahydrofolate Thymidylate synthase Tolerable upper intake level United States of America White blood cell World Health Organization xii

13 LIST OF TABLES Table 1: Recommended daily allowance (RDA) of dietary folate equivalents (DFE) in the US and Canada Table 2: Dietary folic acid and folate intakes in Canada and the US Table 3: Mean folate intake per day, pre-fortification, in Canada Table 4: Dietary folic acid and folate intake in Canada and US in children Table 5: Characteristics of studies included in the systematic review Table 6: Subgroup and sensitivity analysis summary effect estimates Table 7: Characteristics of the studies included in the systematic review and meta-analysis Table 8: Subgroup analysis for the MTHFR C677T polymorphism Table 9: Subgroup analysis for the MTHFR A1298C polymorphism Table 10: Summary effect estimate results for the MTHFR C677T and A1298C polymorphism combinations Table 11: Characteristics of case control studies: comparison of high versus low folate intake.. 92 Table 12: Characteristics of the studies included in the review xiii

14 LIST OF FIGURES Figure 1: Diagrammatic representation of various neural tube defects Figure 2: Structures of the various folate related compounds... 9 Figure 3: Pathways of folate utilization within the cell Figure 4: Conversion of folic acid to tetrahydrofolate Figure 5: Methotrexate's site of inhibition within the folate cycle of the cell Figure 6: Conversion of 5-formyl-tetrahydrofolate to 5-methyl-tetrahydrofolate Figure 7: Search strategy flow chart Figure 8: Total folate intake summary effect estimate for case control studies Figure 9: Dietary folate intake case control and cohort studies Figure 10: Dietary folate intake, women only Figure 11: Forest plot of the sensitivity analysis on dietary folate high versus low dietary folate intake in case control studies Figure 12: Forest plot of the high versus low dietary folate intake for colon cancer Figure 13: Forest plot of the high versus low dietary folate intake for rectal cancer Figure 14: Plot of the highest level of dietary folate intake versus adjusted odds ratio for case control studies Figure 15: Plot of the highest level of total folate intake versus adjusted odds ratio for case control studies Figure 16: Plot of the highest level of dietary folate intake versus adjusted hazards ratio for cohort studies Figure 17: Funnel plot of all case control studies reporting total folate intake xiv

15 Figure 18: Funnel plot of all case control studies reporting dietary folate intake Figure 19: Funnel plot of all cohort studies reporting dietary folate intake Figure 20: Search strategy flow chart Figure 21: Forest plot of risk of colorectal cancer for MTHFR 677CT vs CC Figure 22: Forest plot of risk of colorectal cancer for MTHFR 677TT vs CC Figure 23: Forest plots of risk of colorectal cancer for MTHFR 1298AC vs AA Figure 24: Forest plot of the risk of colorectal cancer for MTHFR 1298CC vs AA Figure 25: Forest plot of the risk of colorectal cancer comparing high versus low folate intake within each MTHFR C677T polymorphism Figure 26: Forest plot of the sensitivity analysis for MTHFR 1298 CC versus AA polymorphism Figure 27: Search strategy flow chart Figure 28: Funnel plot of results of the studies included in the systematic review Figure 29: Search strategy flow chart Figure 30: mortality data for children aged 0 9 years with the International Classification of Diseases, 10th edition (ICD-10) code C91.0 Acute lymphoblastic leukemia in selected European countries Figure 31: mortality data for children aged 0 9 years with the International Classification of Diseases, 10th edition (ICD-10) code C91.0 Acute lymphoblastic leukemia in the US Figure 32: mortality rate per 1 million in children aged 0 9 years with the International Classification of Diseases, 10th edition (ICD-10) code C91.0 Acute lymphoblastic leukemia in the US and selected European countries xv

16 LIST OF APPENDICES Appendix A - Additional figures not included in the original journal article Appendix B - Additional figures not included in the original journal article Appendix C - Further statistical analysis Appendix D - List of publications xvi

17 CHAPTER 1 Introduction 1

18 2 1 Introduction 1.1 General introduction In the last hundred years, public health policies have permitted the enhancement of regularly consumed food products with specific vitamins and minerals designed to address possible nutrient deficiencies and prevent health problems in the general population. Iodine deficiency, during pregnancy and early childhood, can result in a form of severe mental retardation called cretinism and, in older child and adults, the development of thyroid goiters. Late in the 1800 s it was recognized that a lack of iodine was the critical element contributing to cretinism and the formation of goiters. In 1922, Switzerland introduced an iodized salt program resulting in reductions in the incidence of both cretins and goiters. 1 Most countries permit iodized salt, resulting in the elimination of iodine deficiency disorder and as a low cost, effective approach to eliminating preventable mental retardation in children. 1 The addition of fluoride to drinking water is another example of a public health policy designed to improve the health of the general population by reducing dental carie formation. Fluoride assists with the mineralization of the tooth with calcium, preventing tooth decay by the microorganisms that naturally inhabit the oral cavity. 2 Based on several studies conducted in the early 1990 s, it was determined that adequate folate intake by women of childbearing age would be able to address a significant percentage of neural tube defects. A public health policy was implemented in 1998 in both the US and Canada that mandated the fortification of white wheat flour and cereal grains with 140 mcg and 150 mcg of folic acid per 100 gram of flour, respectively. 3, 4 Folic acid is a synthetic form of folate that can be used by the body to increase both plasma and red blood cell folate concentrations. Recent publications have provided strong indications that the folic acid fortification of foods has indeed reduced the incidence of neural tube defects, as well as other congenital malformations In addition, there is good evidence that demonstrates that folic acid fortification programs have been effective in increasing folate intake and red blood cell folate concentrations in the

19 general population. 5 detractors. However, the folic acid fortification policy has both its supporters and 3 Folates serve as critical co-factors in DNA nucleotide synthesis and DNA methylation which raises questions and causes concerns regarding both folic acid fortification and/or supplementation and its potential role in carcinogenesis The objective of this thesis is to explore the impact of folate intake through the diet, including folic acid intake from folic acid fortified foods and/or folic acid supplementation, as it relates to colorectal cancer in adult populations and in childhood acute lymphoblastic leukemia. The following sections are designed to provide the background information for the investigations undertaken herein. 1.2 Neural tube defects Neural tube defects (NTD) occur during embryogenesis and result from the failure of the neural tube to close successfully by the 28 th day after conception; these defects can involve the skull, 26, 27 brain and spinal cord to varying degrees. The degree of disability in a child with an NTD can range from minimal to an inability to survive for more than a day or two. 26 In 2002 dollars, the lifetime financial cost to provide medical care, developmental and educational services to 26, 28 support a child with spina bifida was estimated at $620,484 per child. Ten is no doubt that this cost has risen. years later, there Incidence of neural tube defects Prior to the North American implementation of mandatory folate fortification, the incidence of NTDs varied from a low of 0.5 per 1000 to a high of 4.0 per 1000 births with regional variations, 7, higher rates in the eastern region versus the west. While in the European Union, approximately 4,500 pregnancies are affected by NTDs each year. 27 Internationally, the prevalence of neural tube defects ranges from 0.8 to 3.0 per 1,000 births, with those of Celtic and 26, 29, Sikh heritage exhibiting a higher risk, as do women from northern China.

20 1.2.2 Types of neural tube defects 4 Neural tube defects are classified as either open, where the affected region is open to the body surface; or closed, where the affected region is covered by skin. Open NTDs include anencephaly and myelomeningocele. Anencephaly results from the failure of anterior portion of the neural tube to close and therefore the brain, skull and scalp does not form. 36 Children born with these malformations are either stillborn or die shortly after birth. Myelomeningocele, also known as spina bifida, results from the failure of the closure of the caudal or tail end of the neural tube, leaving the spinal cord open to the exterior , 37 severe disabilities in their lower torso and limbs. Children born with this defect have Closed NTDs involve a variety of deficits and include: encephalocele, meningocele and lipomeningocele, also known as spina bifida occulta. 36 tube defects are presented in Figure 1. Diagrammatic representations of neural Causes of neural tube defects The mixed incidence patterns associated with NTDs led researchers to conclude that NTDs are 27, 38 caused by a combination of environmental and genetics factors. Researchers in the early 1970 s speculated that perhaps a woman s nutritional environment may contribute to NTDs as the incidence differences were evident based on social class. Smithells and colleagues studied the blood concentration of specific vitamins (red blood cell folate, serum folate, vitamin C, vitamin A and riboflavin) in pregnant women and stratified the results by social class and also by outcome. 39 Results of the study found differences in mean vitamin concentration between the social classes. Further stratification by outcome revealed that women who delivered a child with a NTD had significantly lower concentrations of red blood cell folate and vitamin C versus those who delivered a healthy baby. Based on results of this study, Smithells and colleagues investigated the impact of supplementation with a multivitamin containing 0.36 mg of folic acid during the periconceptional period through the first 8 weeks of pregnancy in women with a history of an

21 5 40, 41 Figure 1: Diagrammatic representation of various neural tube defects. This reproduction is a copy of an official work that is published by the Government of Canada and this reproduction has not been produced in affiliation with, or with the endorsement of the Government of Canada. 40

22 NTD affected pregnancy. The NTD recurrence rate was substantially lower in the supplemented group versus the unsupplemented group A second study on the risk of recurrence of NTDs, conducted by Laurence et al., randomly allocated women with a history of a NTD pregnancy to either supplementation with 4 mg of folic acid or placebo. No differences between the two groups were found until supplementation compliance was taken into consideration, at which point a reduced incidence in the recurrence of NTDs was found in the supplemented group. 43 Despite these striking outcomes, the results were not well received as the Smithells et al. study was criticized for the lack of randomization in its study design. In 1983, the Medical Research Council supported a large scale randomized double blind placebo controlled trial of the recurrence of NTDs in women with a history of an NTD affect pregnancy. Women were allocated to one of four groups: 1) supplementation of 4 mg per day of folic acid, 2) a multivitamin without folic acid, 3) a multivitamin and 4 mg per day folic acid, and 4) placebo. This study, reported on in 1991 by Wald et al., demonstrated a 72% reduction in the recurrence of NTDs in the folic acid supplemented groups and included two recommendations: 1) that women with a previous NTD affected pregnancy supplement with 4 mg/day of folic acid per day, and 2) that all women of childbearing age have adequate blood concentration of folate prior to conceiving. 44 While the Wald et al. study also had striking outcomes, not all NTDs were eliminated by folic acid supplementation. Further research has identified additional maternal factors that have been associated with an increased risk of NTD; these include hyperthermia, diabetes, hyperinsulinemia, obesity, folate antagonist medications, histone deacetylase inhibitor 27, medications, micronutrient deficiencies and hyperhomocysteinemia Public health initiatives to reduce the incidence of neural tube defects In 1992, recommendations from both the Expert Advisory Group in the United Kingdom and the Center for Disease Control and Prevention (CDC) in the US, advised that women with a history of a prior pregnancy affected by a NTD supplement with 4-5 mg per day of folic acid prior to conception and in the early months of pregnancy to prevent recurrence and that all women of

23 53, 54 childbearing age supplement with an extra 0.4 mg of folic acid per day. The CDC report also indicated that the Food and Drug Administration (FDA) would undertake to determine the most appropriate method(s) to ensure that these target dosages of folic acid would be delivered to the general population In 1996, as a strategy to assist women of childbearing years to achieve the target of 400 mcg per day of folic acid, the FDA mandated that as of January 1, 1998 cereal grain products would be enriched with 140 mcg per 100 grams of flour and ready-to-eat cereals enriched with 100 mcg per serving of folic acid. 3 Health Canada followed suit and on November 1, 1998 mandated that white wheat flour, enriched pasta and corn meal products be fortified with 150 mcg of folic acid per 100 grams. 4 With the implementation of these levels of folic acid fortification, it was anticipated that the incidence of NTDs would decrease by 20%. 55 Recent studies on the NTD incidence in Canada and the US have demonstrated a decline since the implementation of these 7-9, 56 programs. In Canada, the overall prevalence of NTDs since the implementation of folic acid fortification has been reduced by 46% from 1.58 to 0.86 per 1,000 births; while in the US, the overall reduction in the prevalence of NTDs has been 19%, with a 31% decrease in the 7, prevalence of spina bifida. 1.3 Folates Folates are water-soluble B vitamins that are important cofactors in key DNA synthesis and methylation pathways. Humans are not able to synthesize folate and therefore are reliant upon exogenous sources to support these critical functions. Folates are available in two forms: naturally occurring, usually found in food as 5-methyl-tetrahydrofolate (5-methyl-THF) or formyl-tetrahydrofolate (formyl-thf); and synthetic, folic acid, found in folic acid fortified foods and supplements.

24 The Structure and absorption of folates Naturally occurring folates Naturally occurring food folates are primarily found as the polyglutamyl forms of 5-methyl-THF and in smaller amounts, formyl-tetrahydrofolate. These are found in leafy green vegetables, 59, 60 legumes, yeast extracts, citrus fruit, liver, and kidney. The structure of the naturally occurring folates is composed of a pteridine residue linked by a methyl group to p-aminobenzoic acid with up to six glutamic acid moieties linked together via a 59, 61 series γ-glutamyl links (Figure 2). Both the reduced folate carrier (RFC) and proton coupled folate transporter (PCFT) are 62, 63 expressed on the luminal aspect of small intestine. The RFC functions optimally at physiological ph and so is considered the primary mechanism of cellular folate transport into cells. 63 The PCFT functions at low ph and is primarily responsible for folate absorption across 62, 63 the intestinal mucosa. Prior to absorption on the luminal aspect of the cells of the small intestine the polyglutamyl folates are hydrolysed by brush border enzymes to monoglutamates Folic acid Folic acid (pterolymonoglutamate), the oxidized synthetic form of folate, occurs in the 59, 64 monoglutamyl form (Figure 2) and is found in fortified food products and supplements. Absorption of folic acid occurs in the small intestine via the PCFT. The PCFT has equal affinity for both naturally occurring folates and folic acid , 64 as it crosses the intestinal mucosa. There Folic acid is metabolized to 5-methyl-THF is evidence that the transformation process is saturable, such that a dose of greater than 200 mcg/day results in the appearance of 64, 65 unmetabolized folic acid in blood.

25 9 Figure 2: Structures of the various folate related compounds 5 methyl tetrahydrofolate (5-methyl- THF) (source: sid=2049&loc=es_rss) folic acid (source: id=148901&width=400&height=400) tetrahydrofolate (THF) (Source: id=668192&width=400&height=400) methotrexate (MTX) (source: sid= &loc=es_rss)

26 The cellular folate cycle methyl-tetrahydrofolate 5-methyl-THF is the most common form of folate found in blood. The RFC transports 5-methyl- THF into the cell (Figure 3). 5-methyl-THF is acted upon by methionine synthase (MS), recycling homocysteine to methionine and converting 5-methyl-THF to tetrahydrofolate (THF). The enzyme folylpolyglutamate synthase (FPGS) adds additional glutamate residues to THF to 61, 66 retain the reduced folate within the cell. THF is the preferred substrate for FPGS. THF is converted to 5,10-methylene-THF via the action of serine hydroxymethyl transferase (SHMT). As 5,10-methylene-THF, it can then participate in one of three reactions (Figure 3): (a) (b) (c) It is irreversibly converted to 5-methyl-THF via the enzymatic action of methylene tetrahydrofolate reductase (MTHFR). In this reaction, 5-methyl-THF acts as a methyl donor for the regeneration of methionine from homocysteine. In the first step in pyrimidine synthesis, 5,10-methylene-THF act as a cofactor in the thymidylate synthase (TS) driven reaction converting dump (deoxyuridine monophosphate) to dtmp (deoxythymidine monophosphate), resulting in the oxidation of 5,10-methylene-THF to dihydrofolate (DHF). It can be converted to 10-formyl-THF by the enzymatic action of methylene tetrahydrofolate dehydrogenase (MTHFD) and continue along the purine synthesis pathway.

27 11 Figure 3: Pathways of folate utilization within the cell Abbreviations: DHF: Dihydrofolate, DHFR: Dihydrofolate reductase, dtmp: deoxythymidine monophosphate, dump: deoxyuridine monophosphate, MS: Methionine synthase, MTHFD: methylene tetrahydrofolate dehydrogenase, MTHFR: methylene tetrahydrofolate reductase, SHMT: serine hydroxymethyl transferase, THF: Tetrahydrofolate, TS: Thymidylate synthase. Source: Hubner & Houlston, 2009; Ulrich, Robien & Sparks, 2002; Bailey, ed

28 Folic acid The human folate receptor (hfr) transports folic acid into the cell (Figure 3). Once inside the cell, folic acid is reduced to dihydrofolate (DHF) and then THF by the enzymatic activity of dihydrofolate reductase (DHFR). 64 As either DHF or THF, it is then polyglutamated by FPGS in order to be retained within the cell. THF can be converted to 5,10-methylene-THF via the action of SHMT and participate in the same reactions described in Section DHFR has a low affinity for folic acid and therefore its conversion to DHF is slow Recommended daily allowance of folate The content of naturally occurring folates in food is reduced through the cooking process and 66, 70 through the leaching of folate into the cooking water. Bioavailability studies conducted on food folates indicate that approximately 50 percent of the available food folate is absorbed, while folic acid s bioavailability is estimated to be percent. 60 As a result of the different bioavailabilities, the Institute of Medicine (IOM) has determined a method to calculate total folate intake from both naturally occurring folate and folic acid as dietary folate equivalents (DFE). 60 The formula is: mcg of DFEs provided = mcg of food folate + (1.7 * mcg folic acid) Recommended daily allowances (RDAs) of DFEs for various age ranges and health conditions have been established by the IOM (Table 1). The adult tolerable upper limit (UL) for folic acid from fortified foods and supplements is set at 1,000 mcg per day so as to not mask a vitamin B12 deficiency. 60

29 13 Table 1: Recommended daily allowance (RDA) of dietary folate equivalents (DFE) in the US 60, 71 and Canada. Age RDA of Dietary Folate equivalents (DFE) 0-6 months 65 mcg/day 7-12 months 80 mcg/day 1-3 years 150 mcg/day 4-8 years 200 mcg/day 9-13 years 300 mcg/day years 400 mcg/day years 400 mcg/day years 400 mcg/day years 400 mcg/day >70 years 400 mcg/day Pregnancy Lactation 600 mcg/day 500 mcg/day

30 Specific folic acid intake guidelines for women contemplating pregnancy The Public Health Agency of Canada recommends that women who could become pregnant take a daily multivitamin-multimineral supplement containing 0.4 mg of folic acid with beta carotene rather than Vitamin A. 40 It is recommended that this daily supplement be started at least 2-3 months prior to conception. For women with a previous NTD affected pregnancy or with epilepsy who are taking carbamezapine or valproic acid, a dose of 4.0 mg folic acid daily in the periconceptional period is recommended. 40 The Society of Obstetricians and Gynaecologists of Canada s folic acid intake recommendations for women contemplating pregnancy are that: a) Women who have no personal health risks and a healthy diet of folate rich foods, should consume a daily multivitamin with mg of folic acid per day for two to three months prior to conception and then through the pregnancy and lactation periods. 29 b) Women with health risks such as epilepsy, diabetes, obesity (BMI >35), a family history of neural tube defects, or from an ethnicity associated with a high risk of NTDs (e.g. Sikh, Celtic), should have an increased intake of folate rich foods along with a multivitamin with 5 mg of folic acid per day for at least three months prior to conception and through the first trimester of pregnancy. For the remainder of the pregnancy and lactation periods, the recommendation is a multivitamin with mg of folic acid per day. 29 In the US, the U.S. Public Health Service and the CDC recommends that all women of childbearing age consume 400 mcg of folic acid daily. 53 When women with a history of an NTD affected pregnancy are contemplating another child, they should consult with their health care provider. 53 The recommendation is that these women consume 4 mg of folic acid daily beginning at least one month prior to pregnancy and continuing through the first trimester. 53

31 1.3.5 Current intake of folate in Canadian and US populations. 15 Table 2 summarizes the most recent results from surveys on population intake of folates. A comparison of the Canadian data in Tables 2 & 3 provides evidence for an increase in folate intake between the pre- and post-fortification time periods. A more recent report on the folate status of Canadians using the Canadian Health Measures Survey results, suggests that folate deficiency, based on red blood cell folate concentration of less than 305 nmol/l, is no longer a concern in the Canadian population. 72 Of the population surveyed, 40% had red blood cell folate concentrations in excess of 1,360 nmol/l; however, 22% of the women of childbearing age in the Canadian Health Measure Survey had red blood cell folate concentrations 55, 72 less than the optimal, 900 nmol/l, recommended to minimize the risk of neural tube defects. Often vitamin and mineral supplements contain folic acid in the range of 400 mcg to 1 mg per tablet. A 2010 report from Statistics Canada based on the 2004 Canadian Community Health Survey (cycle 2.2) data found that 60% of women and 40% of men over the age of 50 years used a daily vitamin/mineral supplement. 73 The prevalence of vitamin/mineral supplementation overall in the survey was higher for women, at 47%, versus men, at 34%. 73 If this information were incorporated into the data in Table 2, dietary folate intakes would be substantially higher than reported. An evaluation of the folate content in various folic acid fortified products was conducted in 2007 for the Canadian marketplace. The researchers found that the mean folate content was 50% higher in the studied products than what is reported on food labels. 74 In the US, data from the National Health and Nutrition Examination Survey (NHANES) for the period found that 5% of men and women had folic acid intakes greater than the UL of 1 mg per day established by the IOM. 75 While approximately 17% of individuals, both men and women, between the ages 14 and 50 years did not meet the estimated average requirement (EAR) of 320 DFEs per day of folates. 75

32 16 Table 2: Dietary folic acid and folate intakes in Canada and the US. Age Range Years of age RDA of 60, 71 DFEs (mcg/day) Canada (2004) 76 Mean Dietary folate (mcg/day ± SE) NHANES ( ) 75 Dietary folates (DFEs mcg/day ± SE) NHANES ( ) 75 Dietary folic acid (DFEs mcg/day± SE) NHANES ( ) 75 Total folate intake (DFEs mcg/day± SE) Men ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 34 Women ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 26 Note: Dietary folates are defined as naturally occurring folates plus folic acid from fortified foods. Dietary folic acid is defined as folates from fortified foods only. Total folate intake includes dietary folate plus folic acid from dietary supplements. SE: standard error Table 3: Mean folate intake per day, pre-fortification, in Canada Age Range Years of age Canada (1990s) 77 Mean Folate (mcg/day) Canada (1990s) 77 Mean Folate (mcg/day) Men Women

33 17 Folic acid fortification was estimated to contribute an additional 100 mcg/day of folic acid to the diet. 55 From the US data included in Table 2, it is evident that the contribution of folate intake from fortified foods (dietary folic acid) is higher than originally estimated, particularly for men over 50 years of age. In both Canada and the US, the policy of folic acid fortification has had a positive impact on the folate intake of the population. 1.4 Colorectal cancer Incidence of colorectal cancer Worldwide, colorectal cancer is the third most frequently diagnosed cancer in males and the second in females. 78 cancer diagnosis overall In Canada and US, colorectal cancer represents the fourth most common The developed countries (Australia and New Zealand, Europe and North America) have the highest incidence rates worldwide, while Africa and South-Central 78, 82 Asia have the lowest. Over 75% of colorectal cancers occur sporadically, with only 25% of CRC patients having a family history of colorectal cancer Pathways to colorectal cancer Approximately 98% of all colorectal cancers are adenocarcinomas, having progressed through the adenoma-carcinoma sequence as described by Fearon and Vogelstein The remaining 2% are hyperplastic polyps which, at one time, were thought to be benign; however, recent evidence 84, 85 suggests otherwise Model of colorectal cancer pathogenesis The progression of neoplastic cellular changes is a multi step process characterized by mutations in tumor suppressor genes and oncogenes. 83 In 1990, Fearon and Vogelstein proposed a genetic

34 model for colorectal tumorigenesis, the adenoma-carcinoma sequence, involving the following 80, 86 steps: Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene resulting in the loss of the cell s ability to degrade ß-catenin, a protein involved in cell-cell 80, 86 adhesion. 2. Point mutations in the K-ras oncogene, resulting in amino acid substitutions and inappropriate stimulation signals within the cell And later, the loss of tumor suppressor gene p53, promoting invasion of the epithelium. 80 Fearon and Vogelstein noted that it was the accumulation of these changes that were important rather than the specific order. 86 It is now known that there is far greater genetic heterogeneity within colorectal tumors than was first proposed by Fearon and Vogelstein s model; however, not all of these differences contribute to neoplastic changes and drive tumorigenesis. 88 research has identified alternate pathways that characterize colorectal tumor pathogenesis. The following sections describe the different pathways that are understood today regarding colorectal tumor pathogenesis; three of these pathways involve genetic instability while the fourth involves epigenetic changes. Further The chromosomal instability pathway This pathway closely follows the model proposed by Fearon and Vogelstein in 1991 and is observed in 80-85% of colorectal tumors. 83, 84 The chromosomal instability pathway is characterized by the inactivation of the APC gene, followed by mutations in the K-ras gene. The mutations in the K-ras gene predisposes the cells to proliferate towards the development of polyps. 84 This is then followed by the loss in function of p53, one of the genes responsible for directing cell growth, proliferation and apoptosis. 89

35 The CpG island methylator phenotype pathway 19 CpG islands are short lengths of DNA, frequently containing CpG dinucleotides (cytosine joined by a phosphodiester bond to guanosine) with either a low amount of methylation or no methylation. 90 CpG islands are located close to the promoter region of a gene and when unmethylated, permit the transcription of the gene , 91 are associated with silencing of gene expression. When hypermethylated, these CpG islands This pathway is characterized by a hypermethylation of CpG islands of the promoter regions 84, 92 associated with tumor suppressor genes, and overall, DNA hypomethylation Microsatellite instability pathway A microsatellite is a small run of DNA repeat sequences. 90 The microsatellite instability pathway is characterized by random changes in the DNA of tumor cells that occurs during DNA 84, 90 replication resulting in repeat copy numbers of microsatellites. This repetition can occur in one of two ways: through hypermethylation of the CpG promoter region(s) of the proteins of the DNA Mismatch Repair system, of which there are seven, or through mutation(s) in the one or more of the genes for the proteins of the DNA Mismatch Repair system Serrated polyp pathway Serrated polyps are characterized by a saw-toothed in-folding of the crypt epithelium and are classified as hyperplastic polyps. 85 Three categories have been identified: traditional serrated 84, 85 adenomas, sessile serrated adenomas and true hyperplastic polyps. Traditional serrated adenomas appear to be microsatellite stable; however, mutations in the K-ras gene have been identified in a large percentage of these tumors. 84 Sessile serrated adenomas tumors are characterized by microsatellite instability and have mutations in the BRAF gene. BRAF is involved in signal transduction for cell proliferation, survival and inhibition of apoptosis. 85

36 1.4.3 The controversy: folate s link to colorectal cancer. 20 To date, no direct link has been identified between folates and colorectal cancer. Rather the link is speculative and based on biological plausibility due to folates critical role in DNA synthesis 93, 94 and methylation. How remains to be clarified. these alterations in DNA methylation and synthesis are mediated Epigenetic theories Epigenetic changes are those that result from heritable changes in gene expression that do not 83, 88, 92, 94, 95 result from changes in DNA sequence. One such epigenetic change involves DNA 83, 96 methylation, which is an important regulatory process in gene transcription. Alterations DNA methylation, specifically hypomethylation, is an early and consistent event in 94, 96, 97 carcinogenesis and colorectal cancer, specifically. Some in of the consequences of DNA hypomethylation include: chromosomal instability, increased mutations, mitotic recombination leading to loss of heterozygosity, and aneuploidy. 94 are important determinants of DNA methylation. 98 Age, alcohol consumption, and folate intake Investigations into the impact of DNA methylation in both animal models and human studies have yielded conflicting results. Under conditions of folate depletion, studies in animal models and humans have identified hypomethylation of the DNA. 93 However, these results have not consistently been reproducible, which could be due to differences in the methods used to assess methylation status or timing in tissue sampling. 93 Alternatively, supplementation with folic acid has been found to increase DNA methylation in the colorectal mucosa of patients with colorectal ademonas Genetic mechanisms Genomic/genetic instability refers to the characteristics of DNA mutations, chromosomal breaks and aneuploidy. 89 Such characteristics are evident in tumorigenesis. Mutations and chromosomal breaks can activate oncogenes and deactivate tumor suppressor genes. 89

37 The reliability of DNA synthesis is dependent upon the availability of purines and pyrimidines. 100 Under conditions of low folate supply, the use of folate coenzymes for methylation and nucleotide synthesis would appear to compete Folate deficiency may result in uracil misincorporations in the DNA due to the lack of folate coenzymes required by TS to convert dump to dtmp. To replace the uracil molecule, DNA repair mechanisms within the cell create a break in the DNA; if insufficient thymine is available, 93, 100 there is an inability to replace the uracil, resulting in a break in the DNA strand. Double strand breaks result from single strand breaks on each DNA strand within 14 base pairs of each other and are highly mutagenic. 93 In animal models, under conditions of folate deficiency, DNA 93, 100, 101 strand breaks have been identified in the coding area of both the APC and p53 genes. The mechanism by which higher folate intake can promote the progression of preneoplastic or neoplastic lesions is thought to occur through the provision of folates for pyrimidine and purine synthesis in these rapidly dividing cells. 94 Conversely, folate deficiency can result in ineffective DNA synthesis in these cells and halt the progression of the neoplasm The Dual Effect of Folate theory The dual effect of folate, a term coined by YI Kim, summarizes the results of the evidence on folates and colorectal cancer with respect to timing and dose. 13 It suggests, based on evidence from animal models and human studies, that in normal colonic mucosa: folate depletion can predispose the normal colonic mucosa to neoplastic transformations; with modest supplementation, it can suppress neoplastic transformations; and in supraphysiologic doses, it can enhance the development of neoplasms. 13 Whereas in preexisting colonic neoplastic lesions, folate deficiency inhibits the progression of these neoplasms; while folate supplementation promotes the progression/proliferation of established neoplasms Specific concerns regarding folic acid intake There is evidence from dosing studies that suggests that the intestinal conversion of folic acid to 5-methyl-THF is a saturable process such that doses over 200 mcg/day result in folic acid circulating in plasma. 102 In the post folate fortification era, researchers have investigated the

38 22 plasma folic acid, 5-methyl-THF and total folate concentrations in the blood of participants in the Framingham Heart Study, in both supplemented and non-supplemented participants. 103 These researchers concluded that both supplemental use and folic acid fortification contributed to increased folic acid in circulation. The impact, if any, on the body as a result of circulating concentrations of folic acid is unknown and requires further investigation. Several researchers have developed theories to explain how folic acid might promote carcinogenesis. These are detailed in the next section Theories on folic acid s promotion of carcinogenesis There is concern that folic acid can provide a direct supply of reduced folates for thymidine synthesis, bypassing the regulating processes in the folate cycle. 16 Folic acid enters the cell via the hfr and, prior to entering the folate cycle, is converted to DHF and then THF by the enzymatic action of DHFR (Figure 4). As THF, it can be acted upon by SHMT, converted to 5,10-methylene-THF and provide a direct supply of reduced folates for nucleotide synthesis. This is a concern since rapidly proliferating cells have a high demand for nucleotide synthesis. Alternatively, high intracellular concentrations of DHF can impact the folate cycle in three ways: 1) inhibit the enzyme TS; preventing the conversion of dump to dtmp and reducing the formation of thymidine, 2) inhibit the enzyme MTHFR; preventing the formation of 5-methyl- THF, and 3) inhibit the enzymes involved in purine synthesis. As folic acid is first converted to DHF, it is possible for high concentrations folic acid to produce elevated levels of DHF and therefore have an antagonistic effect on these three enzymes and on DNA synthesis and 17, 104 methylation.

39 23 Figure 4: Conversion of folic acid to tetrahydrofolate. 67 Folic acid DHFR DHF DHFR THF Abbreviations: DHF: Dihydrofolate, DHFR: Dihydrofolate reductase, THF: Tetrahydrofolate;

40 Folic acid s impact on the immune system A small observational study conducted by Troen and colleagues evaluated the association between total plasma folates, unmetabolized plasma folic acid, and 5-methyl-THF concentrations in the blood of 105 postmenopausal women and immune function. 105 Dietary intake was collected via a Food Frequency Questionnaire (FFQ) and information on dietary supplementation was obtained via a face to face interview. They found that when there was less than 233 mcg/day of dietary folate intake, modest supplementation of folic acid up to 400 mcg/day was associated with increased natural killer cell cytotoxicity. When dietary folate intake was greater than 233 mcg/day with folic acid supplementation of more than 400 mcg/day, there was an associated reduction in natural killer cell cytotoxicity. Further, natural killer cell cytotoxicity was reduced when there was unmetabolized folic acid in the plasma. In summary, there are several theories that have hypothesized folic acid s potential role in the transformation of preneoplastic lesions and in the progression of neoplasia. However, no definitive link has yet been established. 1.5 Childhood acute lymphoblastic leukemia Acute lymphoblastic leukemia (ALL) is the most commonly diagnosed childhood cancer, representing approximately 25% of childhood cancers in Canada. 81 Treatment for ALL is based, in part, on the assessed risk of the cancer, with age and white blood cell (WBC) count deemed as important factors in the risk assessment. 106 Children between the ages of 1 and 9.9 years with a WBC count < 50,000 cells/ L at diagnosis are characterized as being at standard risk, while children greater than 10 years and/or with a WBC count > 50,000 cells/ L are categorized as high risk Treatment of childhood acute lymphoblastic leukemia Treatment for ALL generally involves three main stages: remission induction, intensification/consolidation and continuation/maintenance therapy. The combination of drugs

41 25 used in each treatment stage varies depending on the phenotype, genotype and assessed risk of the child s ALL. 107 However, methotrexate (MTX), an antifolate chemotherapy drug, plays a central role through all treatment phases. 107 The following paragraphs provide a brief overview of the treatments provided in each stage Remission/induction The goal of this stage of treatment is to eradicate the leukemic cells and restore normal hematopoiesis. In most cases, the drugs used include: a glucocorticoid, vincristine, and one other agent, usually asparaginase, or an anthracycline, for three to four weeks Intensification/consolidation In this stage, the goal is to prevent leukemic regrowth and the emergence of drug resistance in the remaining leukemic cells. 108 This stage usually lasts four to six months and different drug combinations are used based on the degree of assessed risk. These include intermediate or highdose MTX (HDMTX) (1-5 grams/m 2 ) with leucovorin rescue or escalating doses of MTX. Mercaptopurine, asparaginase, cytarabine, anthracycline and/or alkylating agents are also used Continuation/maintenance This stage generally lasts from 2 to 2.5 years and involves daily oral mercaptopurine and weekly 107, 108, 112 low dose MTX therapy Methotrexate Methotrexate (MTX), an antifolate chemotherapy drug, was first developed in the 1950 s. It is used in the treatment of a wide array of hematological and solid neoplasms. 113 Structurally, MTX is similar to 5-methyl-THF (Figure 2).

42 Methotrexate s mechanism of action MTX enters the cell via the RFC, competing with 5-methyl-THF for entry. If extracellular concentrations of MTX are high, MTX will also enter via passive diffusion , 113, 115, 116 cell, MTX is slowly polyglutamated (MTXG n ) by the enzyme FPGS. Having Once inside the equal affinity for DHFR, both MTX monoglutamates and MTXG n inhibit DHFR causing a depletion of 69, 114, 117, 118 THF and an accumulation of DHF. The resulting accumulation of DHF polyglutamates inhibits the enzyme MTHFR and TS. While MTXG n inhibits the enzyme TS, as well as the enzymes involved in purine synthesis (Figure 5). 69, 113, 116 The inhibition of these key enzymes effectively halts thymidine, and purine synthesis. The lack of nucleotides results in DNA strand breaks which DNA repair mechanisms are unable to repair, RNA synthesis is also inhibited and cellular death ensues. 113 The polyglutamation of MTX traps MTXGn s within the cell and it is the resulting increase in intracellular MTX concentrations that prolongs the suppression of thymidine and purine 116, 117 synthesis. Intracellular concentrations of MTX must substantially exceed the binding capacity of DHFR in order to prevent the competitive interaction between DHF and MTX for DHFR; otherwise, the elevated concentrations of DHF will displace MTX from DHFR. 119 a small amount of DHFR enzyme activity (5%) is required to sustain DHF conversion to THF 114, 117, 119 and nucleotide synthesis. High MTXG n DHFR complexes. 114 DHF concentrations are not believed to displace Only

43 , 113, 116 Figure 5: Methotrexate's site of inhibition within the folate cycle of the cell. Abbreviations: DHF: dihydrofolate, DHF glu n : polyglutamated dihydrofolate, DHFR: Dihydrofolate reductase, dtmp: deoxythymidine monophosphate, dump: deoxyuridine monophosphate, MS: Methionine synthase, MTHFD: methylene tetrahydrofolate dehydrogenase, MTHFR: methylene tetrahydrofolate reductase, MTX: methotrexate, MTXG n : polyglutamated methotrexate, SHMT: serine hydroxymethyl transferase, THF: tetrahydrofolate, TS: Thymidylate synthase. Source: Hubner & Houlston, 2009; Ulrich, Robien & Sparks, 2002; Bailey, ed. 2010, McGuire, 2003 and Jolivet, 1991.