comparative carcinogenic effects of folic acid vs. 5

COMPARATIVE CARCINOGENIC EFFECTS OF FOLIC ACID VS. 5-

METHYLTETRAHYDROFOLATE SUPPLEMENTATION ON COLON CANCER

PROGRESSION IN A RODENT MODEL

by

Baljit Kaur

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Department of Nutritional Sciences

University of Toronto

© Copyright Baljit Kaur 2021

ii

COMPARATIVE CARCINOGENIC EFFECTS OF FOLIC ACID VS. 5-

METHYLTETRAHYDROFOLATE SUPPLEMENTATION ON COLON CANCER

PROGRESSION IN A RODENT MODEL

Baljit Kaur

Master of Science

Department of Nutritional Sciences

University of Toronto

2021

Abstract

A growing body of evidence has linked high folic acid (FA) intake to adverse health outcomes,

including tumour promotion. 5-methyltetrahydrofolate (5MTHF) has been proposed to be a safer

alternative form of folate supplementation. However, its effects on tumor promotion are

unknown. We investigated the comparative effects of FA versus 5MTHF supplementation on the

progression of aberrant crypt foci (ACF; earliest precursor of colon cancer). Equimolar doses of

FA and MTHF (control=1mg/kg diet; supplemental=10mg/kg diet) were provided to rats injected

a colorectal carcinogen, azoxymethane, following ACF formation. At necropsy, colorectal

lesions and tumour parameters were assessed histologically and compared. 5MTHF resulted in

higher plasma folate concentration compared to FA (p<0.05). Tumor incidence (adenoma, p=0.5;

adenocarcinoma, p=0.60) did not differ between folate forms. However, 5MTHF resulted in a

greater increase in tumor burden (sum of tumor diameters) compared to FA (p<0.05). Our

findings suggest 5MTHF may have a higher tumor promoting effect than FA.

iii

Acknowledgements

I’d like to start off by thanking my supervisor, Dr. Young-In Kim for providing me with

the opportunity to pursue a Masters. Thank you for your endless support and guidance. I am

especially appreciative of your interest towards my education and career and am thankful for all

of the help and insight you have provided.

Thank you to Dr. Debbie O’Connor and Dr. Elena Comelli for your support throughout

my project. I am beyond appreciative of your insight and advice towards my research but also

your compassion and understanding throughout my project.

A sincere thank you to Kyoung-Jin Sohn for your direction throughout my project. I am

grateful for your lab expertise, and support. Thank you for being so willing to lend a helping

hand, even if it meant coming to the lab on weekends to help me conduct my experiments.

To the entire vivarium and histology staff at Li Ka Shing – I am truly grateful for your

insight and knowledge. Thank you for your endless support throughout my project.

To Dr. Medline, thank you for your enthusiasm and insight towards this project and for

taking the time to thoroughly assist in the histological analysis of my many many slides.

To everyone that I’ve met and worked with in the Kim Lab, thank you. To the summer

students and volunteers, Carina Chan, David Park and Samantha Yim – you guys were

absolutely amazing to work with! I could not have completed this project without you guys.

Last but not least, my family and friends – I know the rat stories weren’t your favourite

but thank you for listening. To my cousins, Arshi, Neetu and Kena – thank you for the rides

home, the 2am trips downtown, and most importantly the coffee runs. To my friends Amrita and

Gurmeet – thank you for your endless encouragement especially during the most stressful of

times. I couldn’t have done it without you guys.

iv

These past few years have gone by so quickly, and I am so grateful to have worked with, learned

from and experienced this journey with each and every one of you.

v

Table of Contents

I. Table of Contents………………………………………………………………………….v

II. List of Tables…………………………………………………………………………….vii

III. List of Figures…………………………………………………………………………...viii

IV. List of Abbreviations……………………………………………………………………..ix

Chapter 1: Introduction

Chapter 2: Literature Review

2.1 Colon Cancer………………………………………………………………………………..5

2.1.1 An Overview of Colorectal Cancer ………………………………………………..5

2.1.2 Colon Cancer Risk Factors ………………………………………………………..7

2.1.3 Colon Cancer Carcinogenesis …………………………………………………….8

2.1.4 Molecular Genetics of Colorectal Cancer …………………………………………9

2.2 Folate Overview …………………………………………………………………………11

2.2.1 Chemistry of Folate ……………………………………………………………12

2.2.2 Folate Absorption and Metabolism ……………………………………………...15

2.2.3 Folate Metabolism, nucleotide synthesis and the methionine cycle …………….18

2.2.3.1 Thymidylate and purine synthesis ……………………………………19

2.2.3.2 SAM Regeneration, Methionine Cycle and DNA Methylation ………20

2.2.4 Biomarkers of Folate Status ……………………………………………………..21

2.2.5 Folate Intake ……………………………………………………………………..22

2.2.5.1 Folic Acid Fortification ………………………………………………25

2.2.5.2 Folic Acid Supplementation ………………………………………….26

2.2.6 Folate and Health ………………………………………………………………..26

2.2.6.1 Low Folate Status …………………………………………………….29

2.2.6.2 High Folate Status ……………………………………………………30

Folate and Masking of B12 Deficiency ………………………30

Folate and Unmetabolized Folic Acid ………………………..31

Folate and Cancer …………………………………………….32

2.3 Folate and Colon Cancer ………………………………………………………………….36

2.3.1 Evidence from Epidemiological Studies ………………………………………...36

2.3.2 Evidence from Intervention Studies ……………………………………………..42

2.3.3 Evidence from Animal Studies ………………………………………………….47

2.3.4 Summary of Folate and Colon Cancer Risk ……………………………………..49

2.4 5-methyltetrahydrofolate ………………………………………………………………….51

2.4.1 5-methyl-Ca+ ……………………………………………………………………51

2.4.2 Arguments for 5MTHF supplementation ………………………………………..52

2.4.3 5MTHF and 5MTHFR SNP ……………………………………………………..52

2.4.4 5MTHF and B12 ………………………………………………………………...53

vi

2.5 Folic acid vs. 5MTHF ……………………………………………………………………..54

Chapter 3: Rationale, Objectives, Hypothesis and Significance

3.1 Rationale ………………………………………………………………………………….55

3.2 Study Objective …………………………………………………………………………...56

3.3 Study Hypothesis …………………………………………………………………………56

3.4 Significance ……………………………………………………………………………….56

Chapter 4: The Effects of Folic Acid and 5MTHF Supplementation on the Progression

of Colon ACF to Adenomas and Adenocarcinomas in the AOM Rat Mode

4.1 Introduction ……………………………………………………………………………….58

4.2 Materials and Methods ……………………………………………………………………60

4.2.1 The AOM Rodent Model ………………………………………………………..60

4.2.2 Study Design …………………………………………………………………….61

4.2.3 AOM Administration ……………………………………………………………61

4.2.4 Experimental Diets ………………………………………………………………62

4.2.5 Sample Collection ……………………………………………………………….63

4.2.6 Folate Concentrations ……………………………………………………………64

4.2.7 Determination of Aberrant Crypt Foci …………………………………………..66

4.2.8 Histology ………………………………………………………………………...67

4.2.9 Statistics …………………………………………………………………………67

4.3 Results …………………………………………………………………………………….68

4.3.1 Animal Health and Body Weight ………………………………………………..68

4.3.2 Plasma Folate Concentrations …………………………………………………...69

4.3.3 Aberrant Crypt Foci ……………………………………………………………..70

4.3.4 Tumor Incidence and Multiplicity ………………………………………………71

4.3.5 Size of Tumors …………………………………………………………………..74

4.4 Discussion …………………………………………………………………………………76

4.5 Conclusion ………………………………………………………………………………...85

Chapter 5: Summary

5.1 Summary ………………………………………………………………………………….86

5.2 Future Directions ………………………………………………………………………….88

References ………………………………………………………………………………………89

Appendices ……………………………………………………………………………………107

A Nutrient composition of experimental L-amino acid defined diets for FA …….107

B Nutrient composition of experimental L-amino acid defined diets for 5MTHF

…………………………………………………………… ……………………..108

C Salt mix and vitamin mix compositions of experimental L-amino acid defined diet

…………………………………………………………………………………..109

vii

LIST OF TABLES

Table 2.1: Dietary and Lifestyle factors associated with decreased or increased CRC risk ……..7

Table 2.2: Characteristic of genes often associated with CRC …………………………….……10

Table 2.3 Localization and Affinity of main folate carrier, transporter and receptor ……………16

Table 2.4: Recommended daily allowance for folate …………………………………………...23

Table 2.5: Expression of FRα in normal and malignant human tissues …………….…………..35

Table 2.6: Summary of case-control studies of folate and colon cancer across populations ……37

Table 2.7: Summary of case-control studies of folate and adenoma risk ……………………….39

Table 2.8: Summary of cohort studies of folate and colon cancer risk …………………………40

Table 2.9: Summary of RCT of FA supplementation and biomarkers of CRC risk …………….43

Table 2.10: Summary of RCT of FA supplementation and colorectal adenoma recurrence ……44

Table 2.11: Summary of RCT of FA supplementation and cancer incidence as secondary

endpoint …………………………………………………………………………………………45

Table 2.12: Summary of meta-analyses consisting of both epidemiological and intervention-

based studies. ……………………………………………………………………………………46

Table 2.13. Summary of animal studies of FA supplementation and adenoma or colorectal cancer

incidence ………………………………………………………………………………………...50

Table 4.1: Effect of FA and 5MTHF supplementation on the development of colorectal adenoma

and adenocarcinomas. …………………………………………………………………………...71

Table 4.2: Summary of Results ………………………………………………………………….75

viii

LIST OF FIGURES

Figure 2.1: Chemical structure of folic acid and 5MTHF ……………………………………….14

Figure 2.2: Summary of folate absorption and metabolism …..…………………………………17

Figure 2.3: Biochemical functions of folate. …………………………………………………….18

Figure 2.4: Cumulative percentile distribution of red blood folate concentrations by age group

among female participants in the Canadian Health Measures Survey …………………………..24

Figure 2.5: Mean percentiles of dietary and total FA intake in the United States. ………………27

Figure 2.6: The dual modulatory role of folate …………………………………………………..48

Figure 4.1: Experimental Study Design …………………………………………………………64

Figure 4.2: Effect of dietary folate supplementation on body weight …………………………...68

Figure 4.3: Effects of FA and 5MTHF supplementation on plasma folate concentrations. ……..69

Figure 4.4: Effect of FA and 5MTHF supplementation on total number of ACF. ……………...70

Figure 4.5: Effect of FA and 5MTHF supplementation on incidence of colonic neoplasms …..72

Figure 4.6: Distribution of signet ring cell carcinoma among the four dietary groups with respect

to total number of adenocarcinomas. ……………………………………………………………72

Figure 4.7: Effect of FA and 5MTHF supplementation on the mean number of adenomas …….74

Figure 4.8: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in all

animals. ………………………………………………………………………………………….74

Figure 4.9: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in

tumor bearing animals. …………………………………………………………………………..75

ix

LIST OF ABBREVIATIONS

5-MTHF 5-methyltetrahydrofolate

ACF Aberrant Crypt Foci

APC Adenomatous Polyposis Coli

ANO VA analysis of variance

BDR Basal dietary requirement

CpG Cytosine preceding guanine

CRC Colorectal cancer

DFE Daily Folate Equivalent

DHF Dihydrofolate

DHFR Dihydrofolate reductase

DNMT DNA methyltransferase

dTMP Ddeoxythymidine monophosphate or thymidylate

dUMP Deoxyuridine monophosphate

FA Folic acid

FAP Familial Adenomatous Polyposis

FR Folate receptor

GCPII Glutamate carboxypeptidase II

GGH γ-glutamyl hydrolase

GMP Guanosine monophosphate

Hcyst Homocysteine

MMR Mismatch Repair

MAT Methionine S-adenosyltransferase or SAM synthase

MS Methionine synthase

MTHFR Methylene tetrahydrofolate reductase

NTD Neural tube defect

PABA Para-aminobenozic acid

PCFT Proton-coupled folate transporter

RBC Red blood cell

RFC Reduced folate carrier

SAM S-adenosylmethionine

SHMT Serine hydroxymethyltransferase

SNP Single nucleotide polymorphism

THF Tetrahydrofolate

TS Thymidylate synthase

UL Upper tolerable intake

UMFA Unmetabolized folic acid

1

CHAPTER 1: INTRODUCTION

Folate is a water-soluble B vitamin (B9) that is found naturally in green leafy vegetables,

citrus fruits, legumes and organ meats (1). Folates are essential for human health and

development through their role in one-carbon transfer reactions involved in nucleotide

biosynthesis and biological methylation reactions (1, 4). As a result, folates play a critical role in

processes such as DNA replication, integrity and repair as well as DNA methylation (1, 4).

Folate deficiency in humans has been associated with a number of adverse health outcomes

including megaloblastic anemia, cognitive impairments, adverse pregnancy outcomes including

neural tube defects (NTD) and development of certain cancers (2, 9, 10). As such,

supplementation with folic acid (FA), the synthetic form of folate, has been suggested as an

effective way to prevent and treat the aforementioned disorders (3). One example of health

benefits of FA supplementation is the protective effect of periconceptional FA supplementation

on NTD risk (59) as well as the drastic decrease of NTDs in Canada following the mandated

public health initiative of FA fortification in 1998 (3).

FA fortification was intended to provide an additional 100-200 μg/day, but population

data suggest otherwise. Data from the Canadian Health Measures Survey conducted between

2007 and 2009 revealed folate deficiency in Canada was virtually non-existent and that

approximately 40% of the population exhibited red blood cell (RBC) folate concentrations well

above the high cut-off of 1360 nmol/L (97th percentile of RBC folate concentrations) (5). While

fortified foods have contributed to this overall increase in folate status to some degree, a number

2

of studies have shown that widespread supplement use is the most significant predictor of high

folate status post-fortification (5, 12).

It has been estimated that about 30-40% of the general North American population take

FA supplements containing a minimum of 400 μg and up to 1 mg FA, which is the tolerable

upper limit (UL) set by the Institute of Medicine (8, 21-25). Additionally, about 64-81% of

cancer patients and survivors take a dietary supplement in the range of 1-5 mg FA, well above

the tolerable UL (9). Although the protective effects of FA supplementation on NTDs are well

established (6-8), a growing body of evidence suggests that FA, particularly at high doses may

have adverse outcomes on human health, such as masking B12 deficiency, presence of

unmetabolized FA (UMFA) in circulation as well as promotion of existing pre-cancerous lesions

and cancers (13).

Perhaps the most studied relationship between folate and cancer risk is colorectal cancer

(CRC). CRC studies provide the most compelling epidemiological, intervention and animal

evidence supporting the role of folate in carcinogenesis. Animal studies in particular, have

demonstrated that folate plays a dual modulatory role, dependent on dose and stage of cell

transformation at the time of folate intervention (13). In normal cells, folate deficiency

predisposes cells to neoplastic transformation, whereas modest levels of FA supplementation

may prevent neoplastic transformation (13, 15). Once preneoplastic lesions are established, folate

deficiency suppresses, whereas FA supplementation promotes, progression of those lesions (13,

3

16). Whether the observed tumor promoting effect is specific to FA or high folate status in

general remains unresolved (3).

5-methyletetrahydrofolate (5MTHF), the predominant circulating form of folate, has been

proposed to be a safer means of providing supplemental folates (3). Clinical studies have

suggested that 5MTHF supplementation is, at least, as effective as FA in increasing blood folate

levels and reducing plasma concentrations of homocysteine, an inverse indicator of folate status

(3, 10, 13-18). A few studies have compared the effects of FA and 5MTHF supplementation on

folate metabolism and intracellular one carbon reactions (128, 129). One study in particular,

conducted on colon cancer cell lines, found greater cellular proliferation and higher intracellular

folate concentrations with 5MTHF supplementation compared to FA (129). It should also be

noted that there is currently no established UL for 5MTHF as high intakes of naturally occurring

folates were generally thought to be improbable and supplemental MTHF was unavailable due to

its unstable chemical structure. However, now that 5MTHF is commercially available in a more

stable form and speculated to provide a safer means of supplementation over FA, it is important

to define safe parameters of use.

Currently, no studies have been conducted to evaluate the comparative effects of FA and

5MTHF supplementation on colon cancer progression. Although animal studies have

demonstrated the effects of FA supplementation on the promotion of pre-existing lesions (2, 3,

58, 59, 105), the effects of 5MTHF are unknown. This is especially problematic considering the

dramatic increase in population folate status, particularly among colon cancer patients and

4

survivors (13). Additionally, about 35-50% of the population above the age of 50 have adenomas

and many more have aberrant crypt foci (ACF; earliest colon cancer precursor), many of which

are asymptomatic (61, 130). Given the dual modulatory role of FA supplementation, this poses a

risk. 5MTHF has been proposed to be a safer alternative supplemental form of folate, however

this is largely without proof and may not be true. This study aims to determine and compare the

effects of 5MTHF versus FA supplementation on the progression of colon cancer. A rodent

model using a chemical carcinogen, AOM, will be used to establish colon cancer precursors,

following which, supplemental diets will be administered, and colorectal tumours and their

parameters will be examined and compared. This study will help to elucidate the potential

differential effects of FA and 5MTHF at various equimolar doses and may provide a framework

for future studies aimed at assessing safe parameters of use.

5

CHAPTER 2: LITERATURE REVIEW

2.1 COLORECTAL CANCER

2.1.1 AN OVERVIEW OF COLORECTAL CANCER

Colorectal cancer (CRC) is the second most commonly diagnosed cancer in Canada

(131). It is the second leading cause of death from cancer in men and third leading cause of death

from cancer in women in Canada (131). According to the Canadian Cancer Society, in 2017

alone, there was an estimated 26, 800 new cases and 8, 400 deaths from colon cancer (1). It is

estimated that 1 in 13 Canadian men and 1 in 16 Canadian women will develop CRC during

his/her lifetime (131).

Since 1985, age-standardized incidence and mortality rates have steadily declined (131)

However, due to the growth and aging of the Canadian population, the number of new cases

continues to increase (130). Mortality rates, however, have steadily declined in both males and

females, likely due to a combination of improved prevention, early detection and treatment of

CRC (131).

The etiology of CRC involves a complex interaction between genetics and environmental

factors and can be classified as either familial or sporadic. Heritable CRC include familial

adenomatous polyposis, hereditary nonpolyposis CRC, Peutz-Jegher syndrome, MYH-associated

polyposis, and juvenile polyposis syndrome (24). Individuals with a family history of these

syndromes are at an increased risk of inheriting germline mutations in genes associated with

mismatch repair (MMR), APC (adenomatous polyposis coli), MYH and overall DNA

6

maintenance and integrity (38). Approximately 25% of CRC patients have a family history of

CRC, however the remaining 75% of CRC cases are said to be sporadic. These are more

spontaneous in nature and are thought to arise from an interaction between genetics, age and

environmental factors.

7

2.1.2 COLON CANCER RISK FACTORS

Non-modifiable risk factors involve genetic predisposition (CRC syndromes and family

history of CRC or other cancers), age, previous history of adenomas or CRC, and chronic

inflammatory bowel disease (21-23). Dietary factors are also thought to play a significant role in

the causation of CRC (13). Other life-style factors, such as physical activity and smoking have

also been noted to impact risk of developing CRC (23, 101).

Table 2.1: Dietary and Lifestyle factors associated with decreased or increased CRC risk

(132, 133)

Evidence Decrease Risk Increase Risk

Convincing Physical activity, dietary fiber Red meat, processed meat,

alcohol (men), body fatness,

abdominal fatness, adult

attained height

Probable Garlic, milk and calcium Alcohol (women)

Limited Non-starchy vegetables, fruit,

vitamin D, folate, selenium,

fish

Iron, cheese, animal fats,

sugars

No Conclusion Cereals, poultry, shellfish and other seafood, total fat, fatty

acid composition, caffeine, vitamin A, retinol, vitamin C,

vitamin E, meal frequency

8

2.1.3 COLON CANCER CARCINOGENESIS

CRC pathogenesis is characterized as a multi-step process. It starts with increased

proliferation and/or decreased apoptosis of epithelial cells, followed by adenoma formation and

dysplasia, invasion and finally metastasis (102).

Most human adenocarcinomas evolve from ACF (aberrant crypt foci), which are the

earliest known precursors of colon cancer (100). These were first observed in rodents as lesions

consisting of large, thick crypts (34). Similar ACF have been reported in human colonic mucosa

(36-38). Being able to quantify these ACF in animals provides useful information into

mechanisms associated with disease progression.

ACF have been classified as either dysplastic or nondysplastic (103, 104). Dysplastic

ACF are equivalent to microadenomas and are estimated to account for about 5% of all ACF

(104). Nondysplastic ACF have the histological appearance of crypt serration in the epithelium

folds which is a recognizable morphological alteration arising as a consequence of inhibition of

apoptosis. However, both dysplastic and nondysplastic ACF confer a high risk of developing

colon cancer with increases in size and crypt multiplicity.

9

2.1.4. MOLECULAR GENETICS OF COLORECTAL CANCER

The transformation of normal human colon epithelial cells to advanced cancer involves a

multitude of molecular genetic changes. In general, patients with familial adenomatous polyposis

inherit APC mutations and develop numerous dysplastic ACF, some of which progress as they

acquire further mutations. Tumors from patients with hereditary nonpolyposis CRC go through a

similar series of mutations. MMR deficiency accelerates the process of tumor progression and

causes microsatellite instability and chromosomal instability. Progression of sporadic CRC is the

result of the accumulation of accidental and sequential genetic events. Genes commonly mutated

in human cancer belong to one of three different classes: oncogenes, tumor suppressor genes and

mismatch repair genes. Table 2.2 outlines some of the characteristics of genes associated with

CRC.

Microsatellite instability and chromosomal instability are the two main types of genomic

instability (68). Chromosomal instability involves the loss or gain of chromosomes which leads

to abnormal DNA content (aneuploidy). This is characteristic of familial adenomatous polyposis

associated cancers due to germline mutations of the APC gene as well as about 80% of sporadic

CRC. The microsatellite instability pathway involves the loss of function of genes that repair

base pair mismatches occurring during the normal process of DNA replication. Hereditary

nonpolyposis CRC develops as a result of germline mutations of the MMR genes that promote

replication errors throughout the genome.

10

Table 2.2: Characteristics of genes associated with different stages of CRC

Gene Function Stage of CRC

Kirsten ras oncogene (K-ras) Encodes protein bound to a

guanine-triphosphate that

plays a role in the

transduction of extracellular

growth signals (116)

Early stage of CRC (117)

APC tumor suppressor gene Encodes APC protein,

regulates cell signal and

growth (118)

Early stage of CRC (119)

P53 tumor suppressor gene Temporarily arrests

progression of G1 cell cycle

to allow for DNA repair prior

to the initiation of DNA

synthesis

Benign progression and

malignant progression (119)

Mismatch repair (MMR) Corrects errors of DNA

replication (120, 121)

Polyps begin to form with

MMR gene mutations

11

2.2 FOLATE OVERVIEW

Folate, a water-soluble B vitamin, refers to a general class of compounds sharing similar

chemical structures and nutritional properties. These compounds are essential for human

development and health through their role in one-carbon transfer reactions involved in nucleotide

biosynthesis and biological methylation reactions (1, 4). Folate is critical for processes such as

cell division, DNA integrity and maintenance, and epigenetic modifications that regulate gene

expression (1, 4). Folate deficiency in humans has been associated with a number of negative

health outcomes and diseases including megaloblastic anemia, neuropsychiatric disorders,

cognitive impairment, congenital disorders, adverse pregnancy outcomes, and development of

certain cancers (2, 9, 10, 60).

Folic acid (FA), the synthetic form of folate is found most commonly in supplements and

in fortified foods. Naturally occurring folates, such as 5-methytetrahydrofolate (5MTHF) are

found naturally in green leafy vegetables, citrus fruits, liver and other organ meats (3).

12

2.2.1 CHEMISTRY OF FOLATE

Folate, a water-soluble B vitamin, refers to a general class of compounds sharing similar

chemical structures and nutritional properties. These compounds are composed of a pteridine

ring, para-aminobenzoic acid (PABA) and varying number of glutamate residues (63) (Figure

2.1). Despite being able to synthesize all of the individual components of folate, mammals lack

the enzyme required to couple the pteridine ring to PABA and are thus are unable to synthesize

folate de novo (1). Mammals must obtain folate from the diet, in food or supplemental form

(1,2).

Most naturally occurring folates have reduced pteridine rings and are therefore

susceptible to oxidative chemical rearrangements which consequently results in a loss of activity

(64). FA (the synthetic form of folate), on the other hand, has a fully oxidized pteridine ring and

one glutamate residue, thus increasing its bioavailability and stability in light and food

processing (65). Due to its low cost and high stability, FA is used most commonly in food

fortification and in supplements (66).

In order for FA to be incorporated in the folate pathway (Figure 2.3) and utilized for

cellular processes, it must first be reduced to dihydrofolate (DHF), then tetrahydrofolate (THF)

and then is methylated to produce 5MTHF (63, 66). 5MTHF, which is a naturally occurring form

of folate, can enter the folate pathway freely without being further reduced (63). FA and

naturally occurring folates (not including 5MTHF) are metabolized to 5MTHF by the enterocytes

and to a larger degree, the liver. The 5MTHF form of folate makes up 98% of the circulating

13

folate in the blood and is also the only form of folate (3) able to cross the blood brain barrier

(16).

5MTHF is also known as L-5-MTHF, (6S)-5-MTHF, and levomefolate. It has recently

become commercially available in supplemental form as a 5MTHF Ca+ salt (Metfolin®, Merk

Eprova AG, Switzerland). A distinction between the two 5MTHF enantiomers exists: L-5MTHF

is the active form of folate whereas D-5MTHF is the inactive form. Previously, both

diasterioisomers of 5MTHF were supplied due to instability. However, the Ca+ salt allows for an

increased stability and shelf life in the active form (17). It is also important to note that the

supplemental form of 5MTHF is manufactured to be monoglutamted (17) and as such is referred

to as synthetic-5MTHF by the FDA.

14

Figure 2.1: Chemical structure of folic acid and 5MTHF. Folic acid is portrayed at the top

with oxidized pteridine rings. The bottom portrays the structure of reduced folates and positions

of one carbon substitutions. In nature, 5MTHF is polyglutamylated but synthetic 5MTHF-Ca

(used commercially in supplements) is monoglutamated. (63)

15

2.2.2 FOLATE ABSORPTION AND METABOLISM

Folates are absorbed in the small intestine, specifically the acidic cell surface of the

duodenum and jejunum (70). Before folates enter the enterocyte, they are hydrolyzed into their

monoglutamated forms by glutamate carboxypeptidase II (GCPII). These monoglutamylated

folate forms are transported into the cell via folate carriers, transporters, and receptors: reduced

folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptors (FR). The

expression of these transport systems varies with tissue, and each transport system has varying

affinities for varying folate derivatives and optimally function at varying pH levels (71).

Once in the enterocyte, the monoglutamylated forms of folate take on one of two paths.

They are either polyglutamylated by FPGS (folylpolyglutamate synthase) which traps the folates

inside the cell, or they enter the portal hepatic circulation for distribution. Polyglutamylated

folates are better retained in cells and are better substrates for intracellular folate-dependent

enzymes, compared to monoglutamated forms (72). 5MTHF and FA are poor substrates for

FPGS (72). 5MTHF and FA must be converted to THF in order to be polyglutamylated (72).

Intracellularly, folates are converted to 5MTHF, which is the primary circulating form of folate.

Metabolism of folates to 5MTHF occurs as folates pass through the enterocytes. However,

reduction and conversion to 5MTHF can also occur in the liver (73). In order to leave the cell,

polyglutamylated folates are hydrolysed by gamma-glutamyl hydrolase (GGH) to the

monoglutamylated form (74). Folate export is mediated by RFC on the basolateral membrane

16

(66). The below table outlines the affinities of FA and reduced folate (5MTHF) to folate carrier,

transporter and receptor.

Table 2.3 Localization and Affinity of main folate carrier, transporter and receptors (62,

63, 66, 70)

Localization Affinity

Reduced folate carrier (RFC) All tissues and cell lines;

specifically, in the liver,

kidney and jejunum

High affinity for reduced

folates

Low affinity for FA

Proton coupled folate

transporter

(PCFT)

Small intestine, kidney,

colon, liver, brain

High affinity for both reduced

folates and FA

Folate receptors

(FRα, FRβ, FRγ)

Uterus, kidney, liver, placenta

choroid plexus

FRα: High affinity for all

forms of folate

FRβ: High affinity for

reduced folate

17

Figure 2.2: Summary of folate absorption and metabolism. Folate hydrolysis occurs via

GCPII (glutamate carboxypeptidase II) activity. Folate uptake occurs via FR (folate receptor),

PCFT (proton coupled folate transporter) and RFC (reduced folate carrier) at the cell membrane.

Folate retention is mediated by FPGS (folypolyglutamyl synthase) while hydrolysis and efflux

are mediated by GGH (y-glutamyl hydrolase). Each green triangle represents a glutamate

residue, which is linked via a peptide bond to form a polyglutamated folate. Adapted from (137)

In addition to the aforementioned carriers, receptors and transporters, the small intestine

also expresses OATP (organic, anion-transporting polypeptide), BCRP (breast cancer resistance

protein) and MRP (multidrug-resistance-associated protein) (141). The OATP2B1 mediates

some folic acid and 5MTHF transport, however antifolates seem to be a better substrate, which

make this family of transporters a topic of increasing interest in the context of drug interaction

and drug delivery (141). The BCRP and MRP2 transporter are both ATP binding transporters

that have the capacity to oppose the inward flux of folates mediated by PCFT (142). These

transporters pump folate from the enterocytes back into the intestinal lumen (142). However, the

extent to which these transporters influence net folate absorption remains unclear (142).

18

2.2.3 FOLATE METABOLISM, NUCLEOTIDE SYNTHESIS AND THE METHIONINE CYCLE

Folates are involved in one-carbon transfer reactions, which are important for cell growth

and division. This process mediates de novo synthesis of thymidylate and purine nucleotides

which are important substrates for DNA methylation and other biological methylation reactions.

Figure 2.3 outlines the biochemical functions of folate.

Figure 2.3: Biochemical functions of folate. Within the cell, folate exists as one of three active

metabolites (red): tetrahydrofolate (THF), 5,10-methyleneTHF and 5-methylTHF, and have a

role in nucleotide biosynthesis and biological methylation reactions. Dihydrofolate reductase

(DHFR) and serine hydroxymethyltransferase (SHMT) are involved in the maintenance of the

intracellular folate pool. Thymidylate synthase (TS) is involved in nucleotide biosynthesis.

Methytetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are involved in the

methionine cycle. DNA is methylated by DNA methyltransferases (DNMTs) and unmethylated

by DNA demethylase. DHF=dihydrofolate, dUMP=deoxyuridine-5-monophosphate,

dTMP=deoxythymidine-5 monophosphate, DMG=dimethylglycine, SAM=S-

adenosylmethionine, SAH=S adenosylhomocysteine. Adapted from (137).

19

2.2.3.1 THYMIDYLATE AND PURINE SYNTHESIS

DHF (which is produced via the conversion of dUMP to dTMP) is reduced to regenerate

THF (refer to figure 2.3) by DHFR (62). THF can either re-enter the nucleotide biosynthesis

pathway or be used to regenerate 5MTHF via conversion to 5,10-methyleneTHF by serine

hydroxymethyltransferase (SHMT), which is then further reduced by 5,10-

methylenetetrahydrofolate reductase (MTHFR) to yield 5MTHF (66, 62).

5MTHF donates a carbon to the biological methylation pathway (described in section

2.2.3.2.). This yields THF, which plays an important role in the building blocks of DNA and

RNA via its role as a substrate for pyrimidylate and purine synthesis. THF and serine are

catalyzed via SHMT, in a reversible conversion, to form 5,10-methyleneTHF and glycine (66, 8).

5,10-methyleneTHF transfers a methyl group to deoxyuridine monophosphate (dUMP) yielding

dTMP (a precursor of pyrimidylate biosynthesis) and DHF by thymidylate synthase (TS) (75).

The products from the previous cycle, THF and 5,10-methyleneTHF also contribute to purine

synthesis once they are formylated. THF and serine form 5, 10-methyleneTHF and glycine by

SHMT. 5, 10-methyleneTHF can also be converted to 5,10-methyenylTHF and then to 10-

formylTHF by MTHFD1 or MTHFD2 (methylenetetrahydrofolate dehydrogenase) (75). In

purine synthesis, the carboxyl groups of two 10-formylTHFs are required to yield IMP (inosine

monophosphate), a precursor to the purines AMP (adenosine monophosphate) and GMP

(guanosine monophosphate) (75).

20

2.2.3.2 SAM REGERNATION, METHIONINE CYCLE AND DNA METHYLATION

5MTHF is the only folate form able to participate in the regeneration of S-

adenosylmethionine (SAM). SAM is a universal methyl donor for biological methylation

reactions including DNA methylation (37, 72). 5MTHF donates a methyl group for the

transmethylation reaction governing the conversion of homocysteine to methionine. This

reaction is catalyzed by methionine synthase (MS), a vitamin B12-dependent enzyme (8, 63, 72).

Methionine is then converted to SAM by methionine S-adenosyltransferase (MAT) or SAM

synthase (73, 76, 30). DNA methylation is mediated by DNA methyltransferases (DNMT1,

DNMT3a, and DNMT3b) (71). DNMT1 is believed to maintain DNA methylation patterns

following DNA replication (76,29) and DNMT 3a and 3b are capable of de novo methylation

(77). Methyl binding protein 2 (MBD2) can act as both a DNA methylation-dependent repressor

and activator of genes silenced by methylation (78). De novo de-methylation is mediated by ten

eleven translocation (TET) 1 protein which oxidizes methylated cytosines to 5-

hydroxymethylcytosine, the first step in DNA de-methylation (81, 94, 95).

21

2.2.4 BIOMARKERS OF FOLATE STATUS

Serum and RBC folate concentrations, in addition to homocysteine concentrations can be

used to determine folate status (68). Serum folate concentrations are reflective of short-term

dietary intake whereas RBC folate concentrations reflect long-term status. RBC folate

concentrations are considered more reflective of tissue folate stores (1). RBC folate is directly

related to bone marrow folate stores at the time of erythropoiesis and the 120-day turnover rate

of RBC makes this measure resistant to short-term folate variation (1, 4). This measure is also

used to diagnose clinical folate deficiency. According to WHO standards, RBC folate

concentrations of <340 nM or serum folate concentrations of <10 nM observed repeatedly over a

1-month period are indicative of folate deficiency (69). There are currently no established high

cut-offs for RBC and serum folate concentrations. The 97th percentile of RBC folate

concentrations (1360 nM) in the 1994-2004 National Health and Nutrition Examination Survey

(NHANES) has been used as an arbitrary cut off by Colapinto et al. (4, 5).

Homocysteine is a nonspecific inverse functional indicator of folate status and at

concentrations above 16 μM can indicate folate deficiency (1, 3-5). Folate, in the form of

5MTHF, is required to remethylate homocysteine to methionine. During folate deficiency, there

is reduced conversion of homocysteine to methionine, thereby increasing homocysteine

concentrations (1, 4, 5). Elevated homocysteine concentrations can also indicate inadequate

vitamin B12 and vitamin B6 status (1, 4). Renal dysfunction and aging can also raise

homocysteine concentrations (1, 4, 5).

22

2.2.5 FOLATE INTAKE

As mentioned earlier, mammals lack the enzyme required to couple the pteridine ring to

PABA and are thus unable to synthesize folate de novo. For this reason, mammals must obtain

folate from the diet, in food or supplemental form (1, 2, 4). Table 2.3 outlines the recommended

dietary allowance (RDA) as outlined by the Institute of Medicine (4, 5). DFE is a measure for

folate intake which takes into consideration the nearly 50% lower bioavailability of food folate

compared to folic acid (4). One DFE is equal to 1μg of naturally occurring dietary folate, 0.6 μg

of FA when taken with food, or 0.5 μg of FA on an empty stomach (1, 4, 5). There is currently

no tolerable upper limit for folate intake, including 5MTHF and its supplements, however the

Institute of Medicine suggests no more than 1 mg/day FA from fortified foods and supplements.

This guideline is deemed acceptable to prevent masking vitamin B12 deficiency (4, 5). Whether

or not this guideline holds true for 5MTHF supplemental use has not yet been established.

Currently, there is no published guideline for effective conversion factors governing 5MTHF

supplements to their expressed μg DFE equivalents. However, FDA suggests 5MTHF

supplements (both branded and generic calcium salts) to use the same conversion factor as that

used for folic acid (1 μg 5MTHF = 1.7 μg DFE) (142).

More recent recommendations suggest that the dietary reference intakes for

micronutrients be reconsidered to better explain risk factors for chronic disease (137, 143). This

refers to the chronic disease risk reduction index (CDRR) which takes into account the strength

of evidence for both a causal and dose dependent relationship of a micronutrient and chronic

23

disease outcome (137). Currently, there is no CDRR for folate/folic acid (137, 143). However, if

the risk of chronic disease is found to be causally related to intake, the DRI should be updated

and the CDRR should be established.

Table 2.4: Recommended daily allowance for folate.

Population Children Adults

Demographic 1-3 4-8 9-13 14-18 18+ Pregnant Lactating

RDA (μg DFE/day) 150 200 300 400 400 600 500

Note: values are defined as DFE (daily folate requirements), which accounts for the

variability in bioavailability and stability of naturally occurring folate. RDA refers to the

recommended daily allowance, which is the average daily intake sufficient to meet the nutritional

requirements of nearly all healthy individuals in a group.

Dietary intakes and blood folate measurements in the US and Canada have increased

dramatically over the past two decades owing mostly to the widespread use of FA supplements,

but also to some degree, fortification. The Canadian Health Measures Survey (CHMS), reported

that folate deficiency (red blood cell folate concentration < 305 nmol/L) is present in less than

1% of the population, and 40% have high folate concentrations (red blood cell folate

concentration > 1360 nmol/L) (5) (Figure 2.4).

24

Figure 2.4: Cumulative percentile distribution of red blood folate concentrations by age

group among female participants in the Canadian Health Measures Survey. Folate

concentrations for deficiency (305 nmol/L) and high folate concentrations (1360 nmol/L) are

indicated by vertical lines (5). Copied under license from the Canadian Medical Association and

Access Copyright.

25

2.2.5.1 FOLIC ACID FORTIFICATION

FA fortification was first mandated in 1998, after an overwhelming body of evidence

from several epidemiological and intervention studies showing the protective effects of FA

supplementation against neural tube defects (NTDs) (2, 60). The Medical Research Council

Vitamin Study; a multi-centered, double blinded, randomized control trial with pregnant women

considered high-risk of having NTDs, showed a near 72% protective effect with a daily

intervention of 4mg FA (6). As a result, the Canadian and US government-initiated fortification

of all white flours, cornmeal and pasta. In Canada, 150 ug FA per 100g product is added, and is

intended to provide an additional 100-200 ug FA daily. The effectiveness of this can be seen in

the improvements of folate status as well as the significant reductions in the incidences and

prevalence of NTDs in the US and Canada (2, 7-9, 60, 98).

26

2.2.5.2 FOLIC ACID SUPPLEMENTATION

Among the general population, the use of a daily folate acid containing multivitamin or

folate alone has become a habitual practice. According to data from the NHANES and the

Canadian Community Health Survey, up to 50% of adults regularly use a dietary supplement

(10). Data from the United States further state that 70% of the population over 70 years of age

also take at least one dietary supplement per day (11). A number of studies show that individuals

amongst the highest percentile of total folate and FA intake have greater contributions from

dietary supplements (12) (Figure 2.5). In fact, 40% over the age of 60 years have detectable

levels of unmetabolized FA (UMFA) which persist after fasting (12). Studies have also shown

that about 10% of FA supplement users in the general population have daily intake of folate

exceeding the Dietary Reference Intakes upper limit of 1 mg/day (3). A number of studies also

show that supplement use is the most significant predictor of folate status, not diet (5, 12).

27

Figure 2.5: Mean percentiles of dietary and total FA intake in the United States. Dietary FA

is from fortified foods, and total FA includes fortified foods and dietary supplements. As total

FA intake increases, the proportion contributed to by supplement use increases almost

exponentially (12).

The use of FA supplements is widespread among cancer patients and survivors. A

systematic review (13) summarized 32 studies between 1999-2006 and found that regular

supplement use ranged from 64 to 81% among cancer survivors, which is a higher prevalence

than the general population of 50% (14, 15). Furthermore, over 50% of CRC patients report

taking a daily supplement during the course of their treatment (16). Supplemental levels of folic

acid in the range of 1-5 mg/day are routinely provided to certain subgroups of patients who are

taking antifolate based medications (e.g. methotrexate) to prevent adverse effects relating to

folate depletion (3).

28

2.2.6 FOLATE AND HEALTH

Mandated FA fortification and prevalent supplemental use have increased the intake and

blood levels of folate in North America including those of cancer survivors (3). Although FA

fortification has achieved its primary objective, that is decreasing the rate of NTDs, and FA

supplementation may provide several health benefits, an emerging body of evidence suggests

that high folate status, primarily from high FA intake, may be associated with adverse health

outcomes (8, 72).

A large number of epidemiological studies report an association between low folate status

and increased risk of megaloblastic anemia, neural tube defects and other adverse birth outcomes

(3). However, there is a growing body of evidence suggesting that FA supplementation at high

doses has adverse outcomes, including masking of vitamin B12 deficiency as well as the

potential negative effects associated with detectable levels of unmetabolized folic acid (UMFA)

(1, 17, 138). Furthermore, its effects on cancer, and whether or not supplementation is cancer

preventative or promoting, remains a topic of increasing interest.

29

2.2.6.1 LOW FOLATE STATUS

Folate deficiency can occur as a result of any of the following: insufficient dietary intake,

impaired folate absorption and metabolism, and/or increased demand/utilization (1, 15). Well-

known contributors to folate deficiency include: lack of folate in diet, gastrointestinal disorders

such as celiac disease which can impair folate absorption, chronic alcoholism, conditions such as

inflammatory bowel, Crohn’s disease or pregnancy which increase the rates of cell turnover thus

increasing folate requirements, and drugs such as antifolates used in chemotherapy as well as

certain anti-epileptic and anti-inflammatory medications that can interfere with folate

absorption/metabolism (1, 8, 15, 78, 97). Folate is essential for nucleotide biosynthesis and

biological methylation reactions. Folate deficiency has been associated with many diseases such

as megaloblastic anemia, cardiovascular disease, cognitive impairments, neural tube defects

(NTDs), and cancer (66).

30

2.2.6.2 HIGH FOLATE STATUS

FA supplementation has been used to prevent and treat some of the above-mentioned

conditions associated with folate deficiency. FA supplementation is highly effective and

successful in treating megaloblastic anemia (72, 79) and especially preventing NTDs (80-82). FA

supplementation has been generally regarded as safe and has long been presumed to be purely

beneficial (83). However, supplementation, particularly at high doses has been linked to adverse

health outcomes, including the masking B12 deficiencies leading to an accelerated risk of

neurocognitive impairments, accumulation of unmetabolized folic acid (UMFA), decreased

natural killer cell cytotoxicity and cancer promotion (7, 66, 84, 99).

Folate and Masking of B12 Deficiency

Vitamin B12 is an essential cofactor, involved in the conversion of 5MTHF and

homocysteine to THF and methionine (Figure 2.3). When FA levels are high, potential B12

deficiencies can be masked. Although B12 deficiency is uniform among the Canadian population

(41), the Institute of Medicine recommends that adults over the age of 50 years achieve their B12

intake through supplements or fortified foods (1, 4). Due to this recommendation, the elderly are

more likely to be exposed to higher FA intakes from supplements and from their diets, which are

generally high in grain products (5, 66). This poses a risk, as it can lead to a delayed diagnosis

and impaired cognitive function (136). A prospective study by Morris et al showed that rates of

cognitive decline were highest among those taking additional 400 ug FA supplement (135).

31

Proposed mechanisms suggest that an abundance of FA allows for continuous production

of nucleotides (via the 5,10 methylene THF to purine and pyrimidine synthesis pathway) but

impairs DNA methylation pathway since SAM regeneration is dependent on B12, and thus

cannot occur during B12 deficiency (1, 4) (Figure 2.3). 5MTHF has been proposed to be a more

advantageous supplement, however many have also proposed that supplementing with 5MTHF

would result in a methyl folate trap during B12 deficiency (4), resulting in an accumulation of

unusable 5MTHF (Figure 2.3).

Folate and Unmetabolized Folic Acid

High doses of FA can also result in the appearance of and accumulating levels of UMFA

in circulation (19, 20, 90). When FA is metabolized, it undergoes a series of reductions, one of

which is governed by the enzyme dihydrofolate reductase (DHFR) (1). This enzyme is

responsible for converting FA to dihydrofolate (DHF) and subsequently to THF, which becomes

the metabolically active 5MTHF form (1, 4-6). The DHFR enzyme, however, has a relatively

low capacity for FA biotransformation (150). This ultimately results in an accumulation of

UMFA in the circulation which does not occur after consumption of naturally occurring food

folates (150, 138). It has been suggested that high levels of UMFA could interfere with the

metabolism, cellular transport and regulatory functions of the natural folates by competing with

the reduced forms for binding with enzymes, carrier proteins, and binding proteins (1, 4). Other

reported adverse effects associated with UMFA include epigenetic instability, decreased natural

32

killer cell cytotoxicity, progression of preneoplastic lesions and disruptions in methylation

patterns (1, 4, 90, 139). Mechanistic explanations are currently lacking, however, UMFA’s have

been suggested to be converted to the reduced forms of folates by peripheral tissues (137). It has

also been proposed that the presence of folate receptor 4 (a subtype of folate receptor) in the

immune systems’ regulatory T cells may be involved in the mechanism governing decreased

natural killer cell cytotoxicity in response to high levels of UMFA (144). However, more studies

are needed to confirm this mechanism of action.

Folate and Cancer

Perhaps the most controversial relationship between folate and health is cancer risk.

Cancers of the lung, prostate, esophagus, stomach, pancreas, breast and colorectum have been

investigated in relation to folate intake (89). In general, epidemiologic studies have suggested

folate deficiency to increase the risk of several of the aforementioned cancers, while FA

supplementation may reduce the risk (86, 3). However, counterintuitive to this idea, is the nature

of antifolate based medications for cancer treatments, which are based on the idea of folate

depletion to disrupt intracellular folate mechanisms resulting in decreased substrates for

nucleotide biosynthesis and ultimately preventing cell replication and cancer proliferation (3).

Additionally, details of this inverse relationship are not yet well established for all cancer types

(3). CRC is without a doubt the most studied cancer and provides the most compelling

epidemiologic, clinical and animal evidence supporting the role of folate in carcinogenesis.

33

There are several proposed mechanisms relating to the role of folate, its role in one-

carbon transfer reactions, DNA synthesis and epigenetic regulations all of which aim to detail the

dual effects of folate on cancer development and progression. The first is folate’s role in DNA

synthesis and repair (8, 40). In normal tissues, folate ensures the fidelity of DNA replication and

stability (8, 72). However, folate deficiency can lead to DNA strand breaks, chromosomal and

genomic instability, uracil misincorporation, increased mutations, and impaired DNA repair (8,

91-93). FA supplementation can prevent some of these defects (8, 91-93). In contrast, in

preneoplastic cells (or transformed cells) where DNA replication and cell division are occurring

at an accelerated rate, folate deficiency can result in ineffective DNA synthesis, resulting in

inhibition of tumor growth and progression (8, 93, 105-107). The most likely mechanism by

which FA supplementation may promote the progression of established preneoplastic lesions is

by providing the nucleotide precursors needed for accelerated cell proliferation and thus cancer

progression (66).

The second mechanism relates to folate’s role in DNA methylation (86). The 5MTHF

form of folate, is involved in the remethylation of homocysteine to methionine, which is a

precursor of SAM, the primary methyl group donor for most biological reactions including DNA

methylation (15). Aberrant DNA methylation due to folate status can contribute to

carcinogenesis through its effects of global DNA hypomethylation, gene-specific

hypermethylation and/or hypermutability (86). Global DNA hypomethylation is associated with

genomic instability (86). DNA hypermethylation at promotor and/or regulatory regions of tumor

34

suppressor and MMR genes can result in gene silencing (86). Hypermutability of methylated

cytosine can also contribute to carcinogenesis (86).

In normal cells, folate deficiency can result in global DNA hypomethylation resulting in

genomic instability, leading to neoplastic transformation (83, 86). In transformed cells, however,

folate deficiency can suppress tumor progression by reversing promoter cytosine-guanine (CpG)

island DNA methylation of tumor suppressors and other cancer related genes leading to the

reactivation of these epigenetically silenced genes (86, 94). In normal cells, FA supplementation

can prevent global DNA hypomethylation, thus reducing the risk of neoplastic transformation by

ensuring genomic stability (86). In transformed cells, however, FA supplementation can promote

tumor progression by inducing de novo methylation of promoter CpG islands of tumor

suppressors and cancer related genes, in turn silencing these genes (86). However, the effects of

folate deficiency and FA supplementation on DNA methylation are highly complex as they are

gene and site-specific and dependent on species, cell type and stage of transformation as well as

timing and duration of folate intervention (83, 86, 63, 94).

The role of folate receptors in cancer development, progression and treatment has

become a topic of increasing interest. Folate receptors are membrane found proteins that have

high levels of affinity for binding and transporting folate into the cell. These receptors have three

isoforms; FRα, FRβ, FRγ, however it is the alpha isoform that has been most widely studied

(122). The FRα has been suggested to have a greater growth advantage to tumors via regulation

of folate uptake or via cell signaling pathways. Increased growth and folate accumulation by

35

cancer cells with elevated FRα suggests that this may be another mechanism by which folate

may promote the progression of neoplastic cells in humans (122, 123). In vitro studies have

suggested that the expression of the FRα gene is regulated by extracellular folate depletion,

increased homocysteine accumulation, and possibly genetic mutations (122, 124). This concept

has led to the development of more targeted therapies involving the FRα. The overexpression of

this isoform in conjunction with its relatively restricted distribution in normal tissues renders it a

viable therapeutic target (125). The below table outlines the expression of FRα in human

malignant tissues.

Table 2.5: Expression of FRα in normal and malignant human tissues (126)

Normal tissues Malignant tissue

(high expression)

Malignant tissue

(low expression)

Uterus, kidney, liver, placenta

choroid plexus

Cervical carcinoma, uterine

carcinoma, metastatic

endometrial carcinoma,

pancreatic carcinoma, renal

cell carcinoma

Lung carcinoma and

adenocarcinoma, breast

carcinoma, colorectal

carcinoma, liver carcinoma,

prostate carcinoma

36

2.3 FOLATE AND COLON CANCER

2.3.1 EVIDENCE FROM EPIDEMIOLOGICAL STUDIES

Although epidemiological evidence has been inconsistent, in general, the association

between folate intake and CRC appear to be an inverse relationship where the greater the intake

the lower the risk (Table 2.5-2.7). Overall, there is a 20-40% decreased risk of CRC and its

precursor, adenoma when those with highest intake of folate are compared to those with the

lowest (21-23, 104, 105, 107). The relationship between blood levels of folate and risk of CRC

and adenoma is less well defined than that between dietary intake and risk of CRC and adenoma

(2, 3, 105, 106).

37

Table 2.6: Summary of case-control studies of folate and colon cancer across populations

Reference Characteristics Cases/Controls Folate source OR, HR (95% CI) Significance

Freudenheim et al

1991

USA

Caucasian

428/428 Dietary folate

<210 vs. >340 ug/d

M: 1.03 (0.56-1.89)

F: 0.69 (0.36, 1.30)

N.S.

N.S.

Benito et al

Majorca, Spain

50-74 yr


<141 vs. >222.3 ug/d

0.27 (N.A.)

P<0.01

Ferraroni et al

1994

Italy

20-74 yr

Median age 62 years


<162 vs. >227 ug/d

0.52 (0.40-0.68)

P<0.05

Slattery et al

1997

USA

30-79 yr

men and women


<120 vs. >210 ug/d

1.20 (0.90-1.60)

N.S.

N.S.

Jiang et al

2004

China 73/343 Dietary folate

<115.64 vs. >172 ug/d

0.91 (0.70-1.19)

N.S.

White et al

1997

USA

30-62 yr

M: 251/233

F: 193/194

Supplemental folate

0 vs. 400 ug/d

M: 0.59 (0.34-1.01)

F: 0.44 (0.24-0.80)

P=0.04

P=0.007

Le Marchand et al

2002

USA

The Multiethnic Cohort Study

45-75 yr

822/2021 Total folate

<297 vs. >2430 ug/d

Dietary folate

<252 vs. >406 ug/d

0.80 (0.58-1.10)

0.90 (0.62-1.30)

N.S.

N.S.

Lightfoot et al

2010

United kingdom

45-80 yr


267 vs. >397 ug/d

1.08 (0.78-1.50)

N.S.

Sharp et al

2008

United kingdom 255/398 Total folate

<263.9 vs. >348.6 ug/d

1.37 (0.80-2.36)

N.S.

Kim et al

2009

Korea

30-79 yr


<209.69 vs. >282.72 ug/d

0.64 (0.49-0.84)

P=0.002

Kato et al

1999

Women’s Health Study

45 yr

Female

105/523 Serum folate

12.23 vs. 31.04 nM/L

Total folate

224 vs. 626 ug/d

0.52 (0.27-0.97)

0.88 (0.46-1.69)

P=0.04

N.S.

Otani et al

2005

Japan

40-69 yr

375/750 Plasma folate

<5.6 vs. >8.6 ng/mL

M: 0.86 (0.45-1.60)

F: 1.00 (0.56-1.90)

N.S.

N.S.

38

Glynn et al

1996

Finland

ATBC Cancer Prevention

Study

50-69 yr

Male Smokers

144/276 Serum folate

2.9 vs. >5.2 ng/mL

0.51 (0.20-1.3)

4.79 (1.36-16.93)

N.S.

P<0.05

Satia-Abouta et al

2003

USA /North Carolina

30-80 yr

Caucasian

African American

C: 337/596

AA: 276/400

Total folate

<196 vs. >741 ug/d

C: 0.8 (0.5-1.2)

AA: 0.9 (0.5-1.6)

N.S.

N.S.

Van Guelpen et al

2006

Northern Sweden Health and

Disease Cohort


<5.7 vs. >13 nmol/L

3.87 (1.52-9.87)

P=0.007

Baron et al

USA

Median age 60 yr


<214 vs. >388 ug/d

Total folate

<243 vs. >391 ug/d

0.94 (0.53-1.67)

1.11 (0.69-1.78)

N.S.

N.S.

Meyer et al

1993

USA

30-62 yr

M: 238/224

F: 186/190

Total folate

M: <151 vs. >281 ug/d

F: <131 vs. 276 ug/d

1.00 (0.81-1.24)

0.81 (0.66-1.00)

N.S.

P<0.05

Pufulete et al

2005

United kingdom 28/76 Total folate

<260 vs. >348 ug/d

0.09 (0.01-0.57)

P=0.01

Levi et al

2000

Switzerland

27-74 yr


<173 vs. >403 ug/d

1.54 (0.80-3.1)

N.S.

Murtaugh et al

2007

United states

Men and women


<441 vs. >742

0.66 (0.47, 0.92)

P<0.05

39

Table 2.7: Summary of case-control studies of folate and adenoma risk

Reference Characteristics Cases/Controls Folate source OR (95% CI) p-trend

Pufulete et al United Kingdom 35/76 Total folate

<282 vs. >359 ug/d

0.98 (0.30-3.22)

N.S.

Han et al USA

Prostate, Lung,

Colorectal, Ovarian

Cancer Screening Trial

Study

55-74 yr


262 vs. >466 ug/d

1.46 (1.17-1.92)

P<0.001

Giovannucci et al

1998

USA

Nurses’ Health Study

Health Professionals’

Follow-Up Study

M: 331/9159

F: 564/15420

Total folate

M: <241 vs. >847 ug/d

F: <166 vs. >711 ug/d

0.63 (0.41- 0.98)

0.66 (0.46-0.095)

P=0.03

P=0.04

Bird et al

USA

50-75 yr

M: 180/189

F: 152/161

Erythrocyte folate

<165 vs. >315 ng/mL

Plasma folate

3 vs. 16.9 ng/mL

Total folate intake

<242 vs. >576 ug/d

M: 0.47 (0.24-

0.90)

F: 1.26 (0.65-2.43)

M: 0.65 (0.45-

0.95)

F: 0.95 (0.69-1.30)

M: 0.70 (0.36-

1.34)

F: 1.47 (0.73-2.95)

P=0.02

N.S.

P=0.04

N.S.

N.S.

N.S.

40

Boutron-Ruault et

al

1996

France

30-79 yr

1: <10mm adenoma

(n=154)

2: large adeoma (n=208)

3: polyp free (n=426)

subjects recruited after

colonoscopy

Total folate

<218 vs. >402.5 ug/d

1 vs. 3: 0.5 (0.3-

1.0)

2 vs. 1: 0.9 (0.4-

1.9)

2 vs. 3: 0.5 (0.3-

1.0)

P=0.3

N.S.

P=0.04

Benito et al Majorca 101/242 Dietary folate

<146 vs. >227 ug/d

0.27 (N.A.)

P<0.01

Table 2.8: Summary of cohort studies of folate and colon cancer risk

Reference Characteristics # Cases Outcome Folate source RR (95% CI) P-value

Su et al

2000

NHANES I Epidemiologic

Follow-up study

Men

United states

219 CC Dietary folate

103.3 vs. >249 ug/d

M: O.40 (0.18-

0.88)

F: 0.74 (0.36-1.51)

P=0.03

N.S.

Giovannucci

et al

1995

Health Professionals

Follow-up study

51 529 men

40-75 yr

205

6 yr follow

up

Dietary folate

<269 vs. >646 ug/d

0.86 (0.50-1.47)

N.S.

Zhang et al

2005

WHS

39 876 women

>45 yr

220

10.1 yr

follow up

CRC Total folate

<259 vs. 614 ug/d

Dietary folate

<244 vs. 285 ug/d

Dietary folate (excluding

supplement users)

<244 vs. 385 ug/d

1.16 (0.76-1.79)

0.67 (0.43-1.03)

0.46 (0.26-0.81)

N.S.

N.S.

P=0.02

Konings et

al

2002

Netherlands Cohort Study

120 852

55-69 yr

1171

7.3 yr

follow up

CRC Total folate

<168 vs. >266 ug/d

M: 0.73 (0.46-1.17)

F: 0.68 (0.39-1.20)

P=0.03

N.S.

Harnack et

al

2002

Iowa Women’s Health

Study

41 836

598 Total folate

32.1 vs. >2555.2 ug/d

1.12 (0.77-1.63)

N.S.

41

55-69 yr

Flood et al

2002

Breast Cancer Detection

Demonstration

Project Follow-up Cohort

45 264 women

490

8.5 yr

follow up

Total folate

<188 vs. >633 ug/d

Dietary folate

<142 vs. >272 ug/d

1.01 (0.75-1.35)

0.86 (0.65-1.13)

N.S.

N.S.

Larsson et al

2005

Swedish Mammography

Cohort

66 651 women

40-75 yr

805

14.8 yr

follow up

Dietary folate

<150 vs. 212 ug/d

0.80 (0.60-1.06)

N.S.

Roswall et

al

Diet Cancer and Health

Study

Denmark

56, 332

study duration = 10.6 years

men and women

50-64 years

465 CC

cases

283 RC

cases

CC

RC

CC: FA supplement

Per 100 mcg folate

0 vs. >0-83.2 mcg

0 vs. >83.2-142.8 mcg

0 vs. >142.8 mcg

RC: FA supplement

Per 10 mcg folate

0 vs. >0-83.2 mcg

0 vs. >83.2-142.8 mcg

0 vs. >142.8 mcg

1.01 (0.96, 1.06) 0.79 (0.55, 1.13)

0.90 (0.61, 1.30)

0.83 (0.58, 1.20)

0.98 (0.97, 1.06)

0.56 (0.34, 0.91)

0.82 (0.51, 1.33)

0.60 (0.36, 0.99)

Stevens et al

2011

Cancer Prevention Study II

Nutrition Cohort

USA

99, 528

8 year duration

Men and women

50-74 years

1023 CRC

cases

CRC FA supplementation,

fortification during the last

year, and CRC

<101 vs. 101-182 mcg/d

<101 vs. 182-<452 mcg/d

<101 vs. 452-<560 mcg/d

<101 vs. 560 mcg/d

1.02 (0.86, 1.24) 0.95 (0.78, 1.16)

0.96 (0.79, 1.18)

0.84 0.68, 1.03)

42

2.3.2 EVIDENCE FROM INTERVENTION STUDIES

In summary, the majority of small clinical trials examining the effect of FA

supplementation on colon cancer risk as the primary endpoint suggest that FA supplementation

plays a preventative role in developing surrogate end point biomarkers of CRC (Table 2.8 and

2.9). Intervention trials investigating the effects of FA supplementation in the prevention of

cardiovascular disease and/or related events have also determined CRC incidence and/or

adenoma recurrence as a secondary endpoint (Table 2.10). These studies in general do not

support a chemopreventative effect of FA supplementation and instead suggest a possible tumor

promoting effect.

43

Table 2.9: Summary of RCT of FA supplementation and biomarkers of CRC risk

Reference Characteristics FA Dosage/Duration Endpoint Outcome

Cravo et al

1994

CRC or adenoma

N=22

10mg/d

6 months

Rectal mucosa

genomic DNA

methylation

FA increased DNA

methylation

(p<0.002)

Biasco et al

1997

Chronic UC

N=24

15mg/d

3 months

Rectal cell

proliferation

FA reduced (44%

decrease) cell

proliferation in the

upper 40% of

crypts (p<0.01)

Cravo et al

1998

Adenoma

N=20

5mg/d

3 months

Rectal mucosa

genomic DNA

methylation

FA increased (37%

increase) DNA

methylation in

patients with single

adenoma (p=0.05)

Kim et al

2001

Adenoma

N=20

5mg/d

1st: 6 months

2nd: 1 year

Rectal mucosa

genomic DNA

methylation

P53 strand breaks

FA increased

genomic DNA

methylation at 1st

and 2nd follow-up

(P=0.001)

FA decreased

strand breaks at 1st

and 2nd follow up

(P<0.02)

Khosraviani

et al

2002

Adenoma

N=11

2mg/d

3 months

Rectal mucosal cell

proliferation

FA decreased cell

proliferation

(20% decrease,

most pronounced

decrease in upper

1/3 of crypt)

Pufulete et al

2005

Adenoma

N=31

400ug/d

10 weeks

Rectal mucosal

genomic DNA

methylation

FA increased (25%

increase) DNA

methylation

(P=0.09)

Bruce et al

2005

CRC or adenoma

N=98

3mg/d + calcium

carbonate + X-3 fish oil

28 days

Biomarkers of

insulin resistance,

fecal calprotectin,

C-reactive protein

18% decrease in

free fatty acid

(p=0.013)

No effects on other

biomarkers

44

Table 2.10: Summary of RCT of FA supplementation and colorectal adenoma recurrence

Reference Study/Subjects

Previous

Diagnoses

FA

dosage

Duration

Endpoint RR (95% CI) P-value

Paspatis et

al

1994

Adenoma

N=60

1mg/f FA

1st: 1 year

2nd: 2

years

Recurrence Recurrence in Tx vs.

control:

1st: 23% vs. 38%

2nd: 13% vs. 28%

N.S.

N.S.

Cole et al

2007

Aspirin/Folate

Polyp Prevention

Study

21-80 years

Adenoma

N=1021

1mg/d FA

1st: 3 years

2nd: 3-5

years

1˚: recurrence

2˚: advanced

lesions

1st/1˚: RR = 1.04

(0.90-1.20)

1st/2˚: RR = 1.32

(0.90-1.92)

2nd/2˚: RR = 1.13

(0.93-1.37)

2nd/2˚: RR = 1.67

(1.00-2.80)

N.S.

N.S.

N.S.

P=0.05

Jaszewski

et al

Adenoma 5mg/d FA

3 years

Multiplicity OR = 2.77 (0.06-

0.84)

P=0.03

Logan et

al

2008

ukCAP Trial

(United Kingdom

Colorectal

Adenoma

Prevention)

<75 years

Adenoma

0.5 mg/d

FA

3 years

Recurrence RR = 1.07 (0.85-

1.34)

N.S.

45

Table 2.11: Summary of RCT of FA supplementation and cancer incidence as secondary

endpoint

Reference Study/Subjects

Previous

Diagnoses

Intervention

Duration

Endpoint RR or HR

(95% CI)

p-value

Zhu et al Atrophic gastritis 1st year: 20 mg

FA

2nd year: 20 mg

FA

(twice weekly) +

B12

Gastrointestinal

cancer

incidence

OR = 0.12

(0.03-0.51)

P=0.004

Toole et

al

VISP (vitamin,

intervention for

stroke prevention

trial)

Preexisting CVD

20 ug FA + B6 +

B12

2.5 mg FA + B6

+ B12

1.7 years

Cancer

incidence

RR = 0.98

(0.74-1.30)

N.S.

Lonn et al HOPE-2

Preexisting CVD

2.5 mg FA + B6

+ B12

5 years

Cancer

incidence

Cancer

mortality

RR = 1.06

(0.92-1.21)

RR = 0.99

(0.75-1.31)

N.S.

N.S.

Jamison et

al

HOST

Chronic renal

disease

40 mg FA + B6

+ B12

3.2 years

Cancer

incidence

RR = 0.90

(0.65-1.24)

N.S.

Zhang et

al

WAFAC study

≥42 years, women

Preexisting CVD

or ≥3 coronary risk

factors

2.5 mg FA + B6

+ B12

7.3 years

Cancer

incidence

HR = 0.97

(0.79-1.18)

N.S.

Ebbing et

al

NORVIT,

WENBIT

Ischemic heart

disease

0.8 mg FA +

B12 + B6

3.25 years

Cancer

incidence

Cancer

mortality

HR = 1.21

(1.03-1.41)

HR = 1.38

(1.07-1.79)

P=0.02

P=0.01

Armitage

et al

SEARCH

Preexisting CVD

2.0 mg FA +

B12

6.7 years

Cancer

incidence

Cancer

mortality

RR = 1.06

(0.96-1.17)

RR = 1.03

(0.87-1.22)

N.S.

N.S.

46

Table 2.12: Summary of meta-analyses consisting of both epidemiological and intervention-

based studies.

Reference Number/type of

studies included

Outcome Measurement Inverse Positive N.S.

Kim et al

2010

13 cohort studies CRC risk Dietary folate

Total folate X

Kennedy

et al

2011

18 case control

9 cohort studies

CRC risk Dietary folate

Total folate X

Park et al 3 cross sectional

studies

4 case control

studies

4 clinical trial

studies

Adenoma Plasma folate (no

polyp)

Plasma folate

(polyp) X

Qin et al

2013

7 clinical trials Cancer

incidence

Supplement FA X

Figueredi

et al

2009

3 clinical trials Adenoma

recurrence

Plasma folate

<11 nmol/L

>29 nmol/L

X

Wien et al

2012


incidence

Supplement FA

(0.4 to 1.0 mg)

Supplement FA

(>1.0 mg)

X

Vollset et

al

2013


Incidence

Supplement FA

X

Zhou et al 16 clinical trials Cancer

incidence

Supplement FA X

47

2.3.3 EVIDENCE FROM ANIMAL STUDIES

Animal studies conducted in colorectal cancer models have shown that FA supplementation

prevents the development of cancer in normal tissues but promotes the progression of established

neoplastic lesions (2, 3). Table 2.12 summarizes animal studies looking at the effects of FA

supplementation on adenoma or colorectal cancer incidence. Collectively, these studies suggest

that folate possesses dual modulatory effects on cancer development and progression. These

studies also highlight the importance of dose and stage of cell transformation at the time of FA

supplementation (Figure 2.6).

As mentioned, folate deficiency in preneoplastic tissues results in tumor inhibition, which is

also the main rationale behind the use of antifolate medications. Limiting availability of

substrates for DNA synthesis can result in inefficient replication, thus suppressing tumor growth.

On the other hand, in tissues with neoplastic transformation, FA supplementation can promote

tumor growth by providing the substrates necessary for DNA synthesis and replication at the

accelerated rate characteristic of cancer cells. Lindzon et al, showed this promoting effect of FA

supplementation on established colon cancer precursors, using a rodent model. In this model,

aberrant crypt foci (ACF), the earliest precursors of colon cancer, were induced in the animals

using azoxymethane (AOM). Following the induction of ACFs, FA intervention was given, and a

promoting effect was seen, where supplementation promoted transformation to neoplastic cells.

The effect of FA supplementation in normal tissue, prior to preneoplastic transformation has

the opposite effect to that described above. Supplementation in normal epithelia is thought to be

48

cancer preventative due to the sufficient supply of substrates for DNA synthesis, one carbon

metabolism and biological methylation reactions (3, 25). On the other hand, folate deficiency can

result in DNA and chromosome breaks due to uracil misincorporation (3, 26). The limitation of

substrates can also lead to impairments in DNA repair mechanisms, resulting in mutations and

loss of DNA integrity (28).

Figure 2.6: The dual modulatory role of folate deficiency and folate supplementation on

colon cancer progression (3). Copied under licence from the John Wiley and Sons Copyright

Clearance Centre.

49

2.3.4 SUMMARY OF FOLATE AND COLON CANCER RISK

Evidence from animal studies, suggests dual modulatory effect where outcome of folate

supplementation is dependent on dose and time of intervention. Epidemiological and intervention

studies present inconsistent results and suggest that it is unclear whether these adverse effects are

due to high FA or high folate status. Due to the dramatic increase in folate status in the North

American population, in conjunction with the many individuals harbouring asymptomatic

colonic lesions (29), understanding the effects of both low and high dose folate forms is

necessary to interpret safer parameters of use.

50

Table 2.13: Summary of animal studies of FA supplementation and adenoma or colorectal cancer incidence

Reference N; model mg FA/kg diet Duration Endpoint Outcome

Cravo et al

1992

Male Sprague

Dawley rats

DMH injection

0

8

20

weeks

Tumor incidence

- Microadeomas

- Macroadenomas

100% fed 0 mg 29% fed

8mg

86% fed 0 mg

43% fed 8mg

Kim et al

1996

N=40

Sprague Dawley

rats

DMH injections

0

2

8

40

15

weeks

Tumor incidence

- Microscopic (%) - Macroscopic (%)

NS

0mg (70%)

2mg (40%)

8mg (10%)

20mg (50%)

(p<0.03)

Reddy et al

1996

N=72

Fischer-344 rats

AOM injection

Group 1:

control

Group 2: 2000

50

weeks

Tumor incidence

Tumor size

Tumor multiplicity

NA

Group 1 > group 2

Group 1 > group 2

Wargovich et al

1996

N=20

Fischer-344 rats

AOM injections

0

2.5

5

2

weeks

Mean # ACFs Increase vs. control

Le Leu et al

200l

AOM injection

Sprague Dawley

rats

0

8

12

weeks

Tumor incidence

Multiplicity:

Adenomas

Adenocarcinoma

Increase with FA (SI &

colon, and CI alone)

NS increase with FA

Le Leu et al

2000

AOM injection

Sprague Dawley

rats

0

8

12

weeks

ACF Increase with FA

Lindzon et al

2007

N=152

Sprague Dawley

rats

AOM injection

0

2

5

8

34

weeks

Tumor incidence

Size

Multiplicity

ACF

NS

Increase with FA

Increase with FA

Increase with FA

51

2.4 5-METHYLTETRAHYDROFOLATE

FA has been considered more advantageous in food fortification and supplementation

compared to reduced natural folate forms primarily due to its greater stability allowing for a

longer shelf-life. Until recently, 5MTHF, a reduced folate, was available as mixed

diastereoisomers (L and D forms), which allowed for a longer shelf-life but possessed half the

biological activity compared to FA (119). Now, 5MTHF is available commercially as a calcium

salt in the L (active) form (24) and has been shown to be as stable as the diasterioisomers (121).

2.4.1 5-METHYL-CA+

FA and 5MTHF are available commercially, over the counter in doses of 200 μg, 400 μg,

800 μg, and 1000 μg. As a supplement, FA is widely distributed and is easily accessible at drug

stores. 5MTHF is also found commercially, in the form of a crystalline calcium salt

(Metafolin®) and is likely to be found in organic or natural health product stores, or online,

mainly in supplemental form. Metafolin®, patented by Merck Inc., is distributed to supplement

(eg. Genestra Brands, ProThera Inc. Pure encapsulations, and Thorne) and pharmaceutical

companies (eg. PamLab and Bayer Schering Pharma). These supplement brands are available for

purchase in Canada as well as the US and are widely available online.

In the US, 5MTHF-Ca is regarded as GRAS (Generally Recognized as Safe) for its

intended use (67, 124) and can be used as a dietary ingredient (67, 24). The EFSA (European

52

Food Safety Authority) has also supported the use 5MTHF-Ca in foods for specific nutritional

uses with an upper limit of 1 mg/day for adults (125).

2.4.2 ARGUMENTS FOR 5MTHF SUPPLEMENTATION

Albeit little evidence to support, it has been proposed that 5MTHF supplementation may

be beneficial for those with MTHFR polymorphisms or those presenting with B12 deficiency.

However, other arguments for 5MTHF as a better supplemental form of folate to FA are: 1)

5MTHF is immediately available for cellular use (whereas FA must be reduced twice to become

THF) and thus making it more bioavailable (2); 2) supplementation of 5MTHF results in much

less UMFA in the blood; and 3) 5MTHF prevents masking of B12 deficiency (24). Studies have

shown the bioavailability of FA and 5MTHF to be approximately equivalent with comparable

physiological activity (24). Furthermore, long-term supplementation in human studies showed

that 5MTHF and FA similarly decreased homocysteine concentrations and similarly increased

RBC and plasma folate concentrations (74).

2.4.3 5MTHF AND 5MTHFR SNP

Studies have shown polymorphisms of several genes involved in folate metabolism to

greatly affect health (2, 61). The MTHFR C677T polymorphism has been studied extensively.

The frequency of 677TT genotype varies across ethnic groups and geographical locations.

Approximately 10% of the Caucasian and Asian population are homozygous carriers while the

53

Hispanic population has a higher frequency (24). The primary concern for those with MTHFR

C677T polymorphism is the reduced ability to recycle folate within the cell. Studies have shown

the potential for increases in total homocysteine levels, and DNA hypomethylation in those with

the MTHFR C677T polymorphism (40). Many believe that 5MTHF supplementation allows the

folate cycle to bypass the need for MTHFR, allowing for consistent SAM regeneration and DNA

methylation (Figure 2.3). However, the effects of the MTHFR C677T polymorphism on folate

biomarkers have not been consistently observed (125, 127).

2.4.4 5MTHF AND B12

The relationship between B12 and FA has been discussed earlier. In terms of B12 and

5MTHF, there is not yet convincing evidence of and advantage for 5MTHF over FA as a

supplement or fortificant. Many propose that supplementing with 5MTHF will create a ‘methyl

trap’ during B12 deficiency. The ‘methyl trap’ refers to a halt in the folate pathway at MS during

B12 deficiency. Methionine synthase requires B12 to function. Without methionine synthase

activity, 5MTHF will not be able to participate in the methylation of homocysteine to methionine

in the generation of SAM nor will it be able to be involved in nucleotide biosynthesis (Figure

2.3). This could possibly result in megaloblastic anemia, a clinical marker of B12 deficiency.

54

2.5 FOLIC ACID VS. 5MTHF

There are currently no studies that have compared the effects of FA and 5MTHF

supplementation on cancer risk including that of colon cancer. However, human studies

comparing FA and 5MTHF in the general healthy population have shown 5MTHF

supplementation to be as effective as FA supplementation at raising blood concentrations of

folate and lowering homocysteine concentrations (30). There have also been a few animal and in

vivo studies comparing the effects of FA and 5MTHF supplementation on folate absorption (30,

32). Jing et al investigated comparative effects on folate absorption in the jejunum of laying

hens. They found that 5MTHF supplementation significantly lowered RFC and PCFR mRNA

expression compared to control diet. However, this was no different from the results observed

via FA supplementation. Wang et al (33) conducted an in vitro study, comparing the effects of

FA and 5MTHF supplementation on DNA damage and cell death in human lymphocytes.

5MTHF was not more effective than FA in preventing human lymphocyte genomic instability in

vitro (33). Another in vitro study, using human colon cancer cell lines, reported a decreased RFC

and PCFT expression with increasing FA supplementation, but not with 5MTHF (129). 5MTHF

was also shown to have higher global DNA methylation compared to FA in one cell line, while

the opposite was shown in another (130). This study also found faster growth with 5MTHF

supplementation in comparison to equimolar concentrations of FA (130), which suggests that

further research is required in order to determine a safe parameter of use.

55

CHAPTER 3: RATIONALE, OBJECTIVES, HYPOTHESIS AND SIGNIFICANCE

3.1 RATIONALE

Folate status has increased dramatically in the Canadian and US population, mostly from

supplementation but also some from fortification. Many adults in the US and Canada regularly

use dietary supplements for potential health benefits, which are largely unproven. This is

especially problematic for populations at risk for developing colorectal cancer, particularly due

to the growing body of evidence suggesting that folate supplementation can promote progression

of pre-existing lesions (3, 140). Furthermore, it remains unclear whether these adverse effects are

attributable specifically to FA or to high folate status in general. Although 5MTHF is being

promoted as a safer alternative to FA supplementation, convincing evidence supporting this

claim is lacking. Only few studies have compared the effects of 5MTHF supplementation with

FA supplementation on biomarkers of folate status but no studies comparing the effects of

5MTHF versus FA supplementation on health outcomes such as colon cancer risk exit to date.

Given these considerations, this study was undertaken to address these gaps in knowledge, in

particular the potential effects of 5-MTHF supplementation in comparison to FA

supplementation on the progression of colon cancer precursors.

56

3.2 STUDY OBJECTIVE

To compare the effects of FA and 5MTHF supplementation in a rodent model on promotion of

established colon cancer precursors induced by the chemical carcinogen azoxymethane.

3.3 STUDY HYPOTHESIS

5MTHF supplementation will have a greater tumor promoting effect than FA supplementation in

a rodent model of colon cancer in which the precursors of colon cancer was induced by the

chemical carcinogen azoxymethane.

3.4 SIGNIFICANCE

Colon cancer patients and survivors are exposed to high levels of folate, particularly in

the form of FA (13). Furthermore, 35-50% of the population above the age of 50 have adenomas

and many more have ACFs (61, 130). Given the dual modulatory role of folate supplementation,

this high intake of folate poses a risk. 5MTHF has been proposed to be a safer alternative

supplemental form of folate, however the evidence to support this is lacking and this may not be

true given its supplemental effects on cellular proliferations (3). This project will determine

whether supplementation of 5-MTHF, the predominant form of naturally occurring folate,

influences the progression of colon cancer differently to FA. This study will help to elucidate the

effects of 5-MTHF in a colon cancer model to facilitate better understanding of its potential

57

adverse health effects and will also help to provide information for future research studies on

mechanisms associated with carcinogenesis.

58

CHAPTER 4: THE EFFECTS OF FOLIC ACID AND 5MTHF SUPPLEMENTATION

ON THE PROGRESSION OF COLON ACF TO ADENOMAS AND

ADENOCARCINOMAS IN THE AOM RAT MODEL

4.1 INTRODUCTION

Folate plays an important role in human health and disease. Widespread FA supplement use

and FA fortification have increased folate intake (in the form of FA) and blood folate levels in

the North American population. Although there are suggested benefits to taking FA supplements

and FA fortification such as reduced NTD rates, high folate intake, in particular from FA

supplementation have been purportedly associated with adverse health outcomes including a

potential tumor promoting effect. These effects have been well documented in animal studies,

particularly in the context of CRC (2, 9, 10, 142). The major finding from these studies is that

folate possesses a dual modulatory effect on the development of CRC, depending on both the

dose and timing of intervention (3). Folate deficiency has an inhibitory effect whereas folate

supplementation has a promoting effect on the progression of established colorectal neoplasms

(3). In contrast, folate deficiency in normal colorectal mucosa appears to predispose it to

neoplastic transformation, whereas supplementation suppresses transformation (3). In addition to

these tumor promoting effects, high levels of FA have also been shown to mask B12 deficiency

which can progress to irreversible neurological deficits and cognitive impairments as well as

result in the accumulation of UMFA which can compete with the metabolized form of folate (13,

141). For this reason, 5MTHF has been proposed to provide as a safer alternative to FA

supplementation. Some reasons for this conjecture include: 1) the idea that it would benefit

those with 5MTHFR polymorphisms as 5MTHF is immediately available for use by the cells

59

(whereas FA must be reduced), 2) supplementation of 5MTHF will result in less UMFA in the

blood and 3) 5MTHF supplementation will prevent masking B12 deficiency. Although studies

looking at bioavailability of FA and 5MTHF have shown equivalent physiological activity, there

is not yet convincing evidence for 5MTHF as an advantageous supplemental/fortificant over FA.

Additionally, no studies have compared its effects with FA on tumor progression. Thus, the

objective of this study was to elucidate the potential cancer promoting effects of 5MTHF in

comparison to FA on CRC precursors (ACFs) in an animal model to clarify the role of 5MTHF

in CRC. We hypothesized that 5MTHF would have a greater tumor promoting effect compared

to FA in the animal model of CRC.

60

4.2 MATERIALS AND METHODS

4.2.1 THE AOM RODENT MODEL

The use of azoxymethane (AOM) has been demonstrated in a number of studies, as a

potent inducer of carcinomas in the colon. AOM is a methylating agent that results in a loss of

colonic cells via apoptosis followed by an increase in proliferation and mutations of colonic

epithelial cells (133). The resulting epithelial lesions are similar to those found in the human

colorectum (134). This model, like humans, develop tumours that bear K-ras mutations (24, 58).

Although rats do not possess Apc mutations, like human Apc mutated tumours, rat tumours

accumulate beta-catenin in the nucleus (24, 58). This happens as a result of decreased beta-

catenin degradation (Ctnnb1 mutation and GSK3B phosphorylation motif mutation of the beta-

catenin gene) (133, 134). COX-2 and iNOS are also overexpressed in rat tumours, similar to

humans (24, 58, 133, 134).

AOM-induced tumours in rats share many histopathological characteristics with human

tumours (24, 58). The ACF develop 4-6 weeks post injection, followed by adenoma (often

polyps) and carcinoma (microadenocarcinomas at 12-18 weeks, macroadenocarcinomas at 12-18

weeks post injection) (24, 58). This model has been extensively used to study the relationship

between folate and colorectal carcinogenesis and has well documented results showing ACF and

adenoma development (24, 133, 134). The well-established timeline of tumour development also

allows for accurate intervention, to study nutritional factors in colon carcinogenesis.

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4.2.2 STUDY DESIGN

Three-week-old male Sprague-Dawley rats (n=124) were purchased from Charles Rivers

(St. Constant, QC). Rats were housed 2-3 per cage and cages and were changed twice weekly.

Upon arrival, the rats were tagged and placed on the control diet in the form of pellets (1mg/kg

FA diet or 5MTHF molar equivalent) (Dyets, Bethlehem, PA) for 7 weeks (Appendix A, B & C).

At 5 and 6 weeks of age, the rats were given a subcutaneous injection of Azoxymethane (AOM)

(15mg/kg body weight). The animals were randomized at 10 weeks of age to either remain on

their respective controls or receive a 10mg/kg FA diet or a 5MTHF molar equivalent. The rats

were then sacrificed at either 28 or 29 weeks of age.

4.2.3 AOM ADMINISTRATION

Animals were randomized into 5 groups (Monday-Friday) to determine which day of the

week the AOM injection would be administered. The animals received the AOM injection on the

same day at both 5 and 6 weeks. Within the fume hood, 180 mL of double distilled water

(ddH2O) was added to 20 mL of PBS solution and then shaken. From 200 mL, 50 mL was

transferred into a falcon tube to which 70 uL of AOM was added. AOM is light sensitive, thus,

the falcon tube was wrapped in tin foil and then put on ice. Animals were weighed the morning

of the injection and injected with 15 mg of AOM/ kg of their body weight.

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4.2.4 EXPERIMENTAL DIETS

Amino acid-defined diets were used throughout the study (Dyets, Bethlehem, PA). Four

weeks after the last AOM injection the rats were randomized into a total of four experimental

groups (n=30/group). Diets contained 1mg/kg FA or 5MTHF molar equivalent, or 10mg/kg FA

or 5MTHF molar equivalent. Both diet and water were provided ad libitum and diets were stored

at 4°C. Detailed composition of the diet is shown in tables 4.1, 4.2 and 4.3.

The control diet provides 1 mg FA/kg diet. Although 2 mg FA/kg diet is generally

accepted as the BDR for rodents (183, 184) and is the most commonly used control diet in rodent

studies, the 1 mg FA/kg diet provides an alternative dose in the study and has also been observed

to be equally as effective as the 2 mg FA/kg diet in preventing folate deficiency and maintaining

normal metabolic functions. The control level of FA closely approximates the recommended

dietary allowance (RDA) of 0.4 mg dietary folate equivalent (DFE) per day in humans

consuming an average of 2000 kcal per day (183). The 10 mg FA/kg diet represents FA

supplementation at 5X BDR. Folate intake levels at 5X the RDA (2.0 mg/day) can be commonly

found in individuals in the North American population taking daily multivitamin supplements

and consuming high levels of fortified food sources. Because of inherent differences in folate

metabolism between humans and rodents, the selected supplemental levels of FA may not

accurately reflect the corresponding levels in humans (11). These differences include body

weight, lifespan as well as gene regulation, all of which translate to differences in metabolism

(11). One of the major metabolic differences being the substantially lower DHFR activity in

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humans compared to rodents (11). Equimolar dose of 5-MTHF (5-MTHF Ca+ salt, Metafolin®,

Merck Eprova MW 497.5 g/mol) for each level of FA was supplemented per kg of diet to

generate 5-MTHF diets.

Food and water were provided ad libitum and diets were replenished every other day to ensure

palatability. Food intake was not measured as previously conducted studies have shown no

difference in food consumption among animals placed on similar diets containing varying

amounts of folate for similar intervention period (24).

4.2.5 SAMPLE COLLECTION

At the end of dietary intervention period (28/29 weeks of age), rats were sacrificed by

isoflurane overdose followed by T-61 injection. Blood samples were collected from the heart by

cardiac puncture using pre heparinized 16-gauge needles and sterile 1 ml syringe. Blood was

taken off of ice and left at room temperature for 30 minutes. The blood samples were then

centrifuged at 25000x g for 10 minutes at room temperature. Plasma (475µl) was aliquoted into

vacutainers containing 25µl 0.5% ascorbic acid for serum folate assays. The samples were stored

at -80°C. The liver was removed, snap frozen in liquid nitrogen and stored at -80 °C for liver

folate determination. The colon was promptly excised and flushed with PBS solution to eliminate

fecal debris. The entire length of the colon was opened longitudinally. Two centimeters from the

distal end of the colon (rectum) was cut and placed in a cassette with a foam cushion and stored

in 10% buffered formalin for immunohistochemistry. The colon was stored in 10 cm tissue

culture dishes between two pieces of Whatman filter paper and preserved in Bouin’s solution for

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ACF enumeration & tumor parameter analysis. All macroscopic lesions were identified,

measured along the longest diameter, harvested and stored in glass vials containing formalin.

Figure 4.1: Experimental Study Design. Supplemental diets were administered at Week 7 followed by

necropsy at week 24. Body weight measurements were taken weekly.

4.2.6 FOLATE CONCENTRATIONS

Folate concentrations were measured using a standard microbiological Lactobacillus

rhomnosus microtiter plate technique (136). The media becomes proportionally turbid as the

Lactobacillus rhomnosus bacteria grow (136). The bacteria growth rate is proportional to the

amount of folate in the media. Therefore, the turbidity measured by the spectrophotometer

indicates the folate concentration in the samples tested (136).

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FOLIC ACID STANDARD PREPARATION

FA was dissolved in ddH2O with 0.1 M NaOH to a concentration of 10 mg FA/ml. The

concentration was verified using a spectrophotometer at 282 nm. The solution was diluted to 2

ng/ml in sterile filtered 0.1 KPO4 buffer containing sodium ascorbate (0.5%). Standard was

stored in aliquots at -80°C.

LACTOBACILLUS RHOMNOSUS STOCK PREPARATION

Lactobacillus rhomnosus bacterial stock (250µL) was added to 200mL of Lactobacillus MRS

Broth (Difco™, BD Biosciences) and incubated at 35-37°C for 18 hours. Under aseptic

conditions, the tube was centrifuged to sediment the cells and the supernatant was removed. The

pellet was resuspended in 180mL of Lactobacillus MRS Broth and 20mL of 100% autoclaved

cold glycerol was added. The solution was mixed well, aliquoted and stored at -80°C.

DETERMINATION OF FOLATE CONCENTRATION

Lactobacillus rhomnosus stock (3µL) was inoculated in 3 mL o f Lactobacillus MRS

Broth at 37 °C for 16-18 hours. Five hundred µL of the overnight culture was then inoculated in

2.5mL of Lactobacillus MRS Broth for 5 hours (O.D. should be approximately 1.8 at this point).

Folic acid media (9.5 g of folic acid media, 0.05 g NaAscorbate, l00mL of ddH20, boiled for 1-2

minutes, cooled and filter sterilized) and potassium phosphate buffer were made fresh each day

prior to starting the assay. Potassium phosphate buffer was added to all the wells of a 96 well

microtiter plate (150 µL per well). In columns 1 and 2 of the plate, 150 µL of folic acid standard

(2µg/mL) was added and then serially diluted along the plate to create a standard curve. Plasma

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samples were thawed and 5 µL was used. Potassium phosphate buffer was added to each well

and the samples were serially diluted three times per plate. All samples were run in duplicates.

The Lactobacillus rhomnosus was diluted lmL in 24mL of folic acid media (l00x dilution) then

375 µL of the diluted inoculum was added to 15 mL of folic acid media and 150µL was used to

inoculate each of the 96 well plate. Each plate was covered with a mylar sealer, mixed and

incubated at 37°C for 16 to 18 hours allowing it to grow. The plate was then read on a

spectrophotometer at 650 nm and using SoftMax software. The standard curve of folate

concentrations plotted against the densities, as generated by the SoftMax software, was used to

estimate the folate concentrations from the samples.

4.2.7 DETERMINATION OF ABERRANT CRYPT FOCI

Colons were promptly removed, cut open along the longitudinal axis and flushed with

phosphate-buffered saline (PBS) to remove fecal debris. The colons were prepared using the

swiss roll technique and further processed using an automatic tissue processor, paraffin

embedded, and cut into sections for hematoxylin and eosin staining using a rotary microtome.

The resulting slides were evaluated for microscopic lesions, conducted by a gastrointestinal

pathologist (Dr. Alan Medline) blinded to the different experimental groups.

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4.2.8 HISTOLOGY

All macroscopic neoplasms were excised at necropsy and stored in 10% formalin. The

samples were then embedded in paraffin, and further processed using an automatic tissue

processor, paraffin embedded, and cut into sections for hematoxylin and eosin staining using a

rotary microtome. The resulting slides were evaluated for microscopic lesions, conducted by a

gastrointestinal pathologist (Dr. Alan Medline) blinded to the different experimental groups.

4.2.9 STATISTICS

Statistical significance of the effects of folate dose and form on body weight, tumor

incidence, tumor multiplicity and tumor parameters were tested using a repeated measured Two-

Way ANOVA followed by a post hoc Tukey’s multiple comparisons test to detect differences

between dietary groups at each week of dietary intervention. Spearman’s rho correlation test was

used to assess correlation between variables to help explain the observed outcome. Two-way

ANOVA was conducted to examine the effects of the dose and form of folate supplementation

on plasma folate concentrations (n=30/dietary group).

Statistical analysis was conducted using SPSS 26.0 and graphs were prepared using SPSS

output. All tests were two-sided and considered significant if the observed significance level (p-

value) was less than 0.05.

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4.3 RESULTS

4.3.1 ANIMAL HEALTH AND BODY WEIGHT

Rats were weighed weekly, on a predetermined day of the week. Growth curves among the four

dietary groups were not significantly different. The mean body weights (in grams) of the rats in each

group did not exhibit any significant differences at any time point throughout the experiment (p=0.076)

(Figure 4.1). At the time of sacrifice, there was no significant difference among the body weights of the

animals on the four diets (p=0.36). The mean weight at the end of the intervention, in each dietary group

was 744.5, 757.2, 766.6 and 772.4 for rats on the 1mg FA, 10 mg FA, 1 mg 5MTHF and 10 mg 5MTHF

per kg diet, respectively.

Figure 4.2: Effect of dietary folate supplementation on body weight (p=0.076, n=30/dietary group).

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4.3.2 PLASMA FOLATE CONCENTRATIONS

There was a significant interaction between the effects of dose and form on plasma folate

concentrations (p<0.05) (Figure 4.2). Within each folate form (FA & 5MTHF), increasing dose

(1mg/kg, 10mg/kg) resulted in significantly increased plasma folate concentrations with both FA

and 5MTHF supplementation (p<0.05). Between equimolar doses, 10 mg 5MTHF showed a

significantly higher plasma folate concentration relative to 10 mg FA (p<0.05), but no

differences were observed between the 1 mg 5MTHF and 1 mg FA. These results are comparable

to previously conducted studies comparing FA and 5MTHF.

Figure 4.3: Effects of FA and 5MTHF supplementation on plasma folate concentrations. Solid

Grey = 1 mg FA, solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10

mg 5MTHF. Significance for the effects of dose and form were determined using two-way

ANOVA. Asterisks (*) denote significant differences between folate forms within doses. Letters

(ab) denote significant differences between folate doses within forms.

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4.3.3 ABERRANT CRYPT FOCI

There was no difference in the total number of ACF developed between equimolar folate

doses. Within each folate form (FA & 5MTHF), increasing dose resulting in significantly greater

number of total ACF (p<0.05, n=30/dietary group) (Figure 4.4).

Figure 4.4: Effect of FA and 5MTHF supplementation on total number of ACF (p=0.023). .

Solid Grey = 1 mg FA, solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched

black = 10 mg 5MTHF. Means with different letters differ at p<0.05.

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4.3.4 TUMOR INCIDENCE AND MULTIPLICITY

There was no significant difference in the incidence of adenomas (p=0.57) and

adenocarcinomas (p=0.60) between folate forms (FA & 5MTHF) (Figure 4.5). Furthermore,

there was no significant difference within folate groups in the incidence of adenomas (p=0.68)

and adenocarcinoma (p=0.53) (Figure 4.5). In order to determine tumour incidence, the most

severe histological diagnosis was considered the final diagnosis. For example, if a rat presented

adenoma and adenocarcinoma, the final diagnosis for this rat was adenocarcinoma. However, if a

rat only present adenoma, then the final diagnosis for this rat was adenoma. However, out of the

total 68 adenocarcinomas observed, 8 signet ring cell carcinomas, a more advanced form of

adenocarcinoma, were identified in both the 10mg FA and 10mg 5MTHF dietary group (Figure

4.6). When comparing 10mg FA and 10mg 5MTHF, no significant difference was observed in

the incidence of signet ring cell carcinoma (p=0.074) (Figure 4.6).

Group Normal Adenoma Adenocarcinoma

1 mg FA 8 (26.7%) 7 (23.3%) 15 (50.0%)

1 mg 5MTHF 6 (20.0%) 10 (33.3%) 14 (46.7%)

10 mg FA 5 (16.7%) 6 (20.0%) 19 (63.3%)

10 mg 5MTHF 4 (13.3%) 6 (20.0%) 20 (66.7%)

Total 23 29 68

Table 4.1: The effect of folic acid (mg FA/kg diet) and 5-methyltetrahydrofolate (mg

5MTHF/kg diet) on the development of colorectal neoplasm.

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Figure 4.5: Effect of FA and 5MTHF supplementation on incidence of colonic neoplasms. Solid

bars = FA, hatched bars = 5MTHF. Tumor incidence was not significantly different between

folate forms (adenomas, p=0.57; adenocarcinomas, p=0.60). Tumor incidence was not

significantly different within folate groups (adenomas, p=0.68; adenocarcinoma, p=0.53)

Figure 4.6: Distribution of signet ring cell carcinoma among the four dietary groups with respect

to total number of adenocarcinomas. Solid bars = FA, hatched bars = 5MTHF. Incidence of

signet ring cell carcinoma was not significantly different between folate forms (p=0.074).

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The mean number of adenomas in all rats did not differ significantly among the four

dietary groups (Figure 4.7A). The mean number of adenocarcinomas in all rats did not differ

significantly between the two folate forms; however, a significant effect within each group was

observed where high dose FA and 5MTHF had significantly greater number of adenocarcinomas

compared to their respective low doses, at p<0.05 (Figure 4.7B)

Figure 4.7: Effect of FA and 5MTHF supplementation on the mean number of adenomas (A,

p=0.19) and adenocarcinomas (B). Means with different letters differ at p<0.05. Solid Grey = 1

mg FA, solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10 mg

5MTHF.

When correlation analysis was conducted between plasma folate with number of tumors in

all animals, a statistically significant, positive correlation was observed between plasma folate

and number of adenocarcinomas (r=0.076, p=0.023). However, no relation was observed with

number of adenomas in all animals (r=0.043, p=0.478). The same correlation analysis was then

conducted on tumor-bearing animals only (excluding rats without tumors). The positive

correlation between plasma folate and adenocarcinoma strengthened when only animals bearing

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tumors were considered (r=0.24, p=0.01) and no relation was observed between adenomas in

tumor bearing animals only and plasma folate (r=0.037, p=0.37).

4.3.5 SIZE OF TUMORS

There was a significant interaction between the effects of dose and form on the sum of all

tumor diameter (adenoma + adenocarcnoma). Within each folate form (FA & 5MTHF),

increasing dose (1mg/kg to 10mg/kg) resulted in significantly higher tumor diameter in both FA

and 5MTHF groups. Within folate doses, 10 mg 5MTHF showed a significantly higher sum of

tumor diameter relative to 10 mg FA (p<0.05), however no differences were observed between

the 1 mg 5MTHF and 1 mg FA.

Figure 4.8: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in all

animals. Means without a common letter differ at p<0.05. Solid Grey = 1 mg FA, solid black

bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10 mg 5MTHF.

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Figure 4.9: The Effect of FA and 5MTHF supplementation on the sum of tumor diameter in

tumor bearing animals. Means without a common letter differ at p<0.05. Solid Grey = 1 mg FA,

solid black bars = 10 mg FA, hatched grey = 1 mg 5MTHF, hatched black = 10 mg 5MTHF.

Table 4.2 outlines and summarizes the findings from this study. The novel findings here are the

significant differences presented between high dose 5MTHF and FA (10mg), where 5MTHF

supplementation resulted in a significantly greater tumour diameter relative to FA.

DOSE FORM

PLASMA FOLATE ↑ 5MTHF > FA

MEAN # ACF ↑ NS

INCIDENCE OF TUMORS Adenoma NS Adenoma NS

Adenocarcinoma NS Adenocarcinoma NS

MULTIPLICITY OF TUMORS Adenoma NS Adenoma NS

Adenocarcinoma ↑ Adenocarcinoma NS

TUMOR DIAMETER IN ALL

ANIMALS ↑ 5MTHF > FA

TUMOR DIAMETER IN TUMOR

BEARING ANIMALS ↑ (FA only) 5MTHF > FA

Table 4.2: Summary of results. “↑” indicates a significant dose effect, where increasing dose

resulted in the corresponding increased outcome. NS indicates no significant effect.

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4.4 DISCUSSION

Due to the dramatic increase in folate status in the North American population, dual role of

folate in colorectal carcinogenesis, and a significant portion of the population harbouring

asymptomatic colonic lesions with neoplastic potential, understanding the effects of different

supplemental forms of folate is necessary to interpret safer parameters of use (61, 130). A

growing body of evidence has suggested adverse health outcomes with high doses of FA,

including tumor promoting effects. 5MTHF, or metafolin®, is now being promoted as a safer

alternative supplemental form of folate compared to FA, however this is largely without proof.

Although studies have compared the effects of 5MTHF and FA metabolically, its effects on

colon cancer progression still remain unknown (128, 129). Given the growing body of evidence

showing tumor promoting effects of FA, particularly in high doses, this poses a risk. As such, we

conducted a comparative study to evaluate the effects of FA and 5MTHF supplementation on

colon cancer progression using an AOM rat model. Dietary interventions with amino-acid

defined diets containing either 1 mg FA or 10 mg FA, or their respective equimolar 5MTHF

concentrations were selected to study these effects. Measures include plasma folate status, ACF

analysis, tumor incidence and multiplicity as well as tumor size.

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Folate Status

We observed significantly higher plasma folate concentrations with increasing

supplemental levels of both FA and 5MTHF. Furthermore, 10 mg 5MTHF was associated with a

significantly higher plasma folate concentration relative to the equimolar FA dose. However,

when 1 mg FA and 1 mg 5MTHF, no differences were seen in plasma folate concentration. The

difference was only noted with the high folate diets (10 mg).

Previously conducted animal and human studies have shown similar results, in that a

positive association between supplemental levels of FA and 5MTHF and plasma folate

concentration has been recognized (30, 109, 113). The novel finding from this study, however, is

that 5MTHF resulted in a higher plasma folate concentration relative to FA at the 10 mg

supplemental level. A study conducted in mice, comparing FA and 5MTHF found similar results

where 5MTHF resulted in a higher plasma folate concentration compared to FA at the 20 mg

supplemental level (128). This study compared the folate forms at the 2mg, 10mg and 20mg

supplemental level (128). This study also found no significant difference between FA and

5MTHF on tissue folate concentrations observed in the liver and small intestine (128). The

differences in these results could be attributed to the difference in animal study model.

Clinical studies observing similar results, where 5MTHF increased plasma folate

concentrations to a greater extent than FA (163), attribute the findings to differences in

metabolism of the two folate forms (163). 5MTHF bypasses many of the metabolic steps which

are otherwise necessary for FA and can thus act directly to alter plasma folate concentrations. In

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particular, the metabolism step governed by the DHFR enzyme, which is easily saturated at

higher doses, can lead to reduced conversion of FA to 5MTHF (115). 5MTHF is a biologically

active form folate and bypasses this metabolic step and can also be stored in the body. For this

reason, it can have direct effects on plasma folate concentrations.

Aberrant Crypt Foci

This study observed an increasing number of ACF with increasing supplemental levels of

both FA and 5MTHF. However, the mean number of ACF did not differ between the FA and

5MTHF groups. Although we did not see 5MTHF to have a greater effect on increasing number

of ACF relative to FA, our results support the idea that 5MTHF is equally as effective as FA in

regard to ACF proliferation. For this reason, it is important to consider the potential

consequences of marketing 5MTHF as a safer alternative to FA.

Tumor Parameter Outcomes

Supplemental folate levels did not affect the incidence of colorectal adenoma or

adenocarcinoma. Additionally, an advanced form of adenocarcinoma known as signet ring cell

carcinoma was observed in only the 10 mg supplemental folate groups. Supplemental folate

levels did not affect the number of colorectal adenomas; however, number of colorectal

adenocarcinomas did increase with increasing level of supplemental folate in both FA and

5MTHF groups. Plasma folate concentrations were also positively correlated with number of

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adenocarcinomas in all animals and this positive association was further strengthened when only

tumor bearing animals were analyzed. Tumor diameter was seen to increase with increasing

supplemental levels of both FA and 5MTHF. The novel finding in this study, however, is that

5MTHF resulted in a significantly higher tumor diameter relative to FA at the 10 mg

supplemental level. When plasma folate concentrations were correlated with sum of tumor

diameter in all animals as well as in tumor bearing animals, a statistically significant positive

association was observed. Although plasma folate concentrations could explain the differential

effects on tumor diameter, it may not be the only explanation, as no differential effects were

observed in other tumor parameter measures between folate forms. However, the positive

correlation between plasma folate concentrations and number of adenocarcinomas within FA and

5MTHF groups suggests that 5MTHF is as effective as FA in promoting adenocarcinoma.

Additionally, although tumor incidence holds greater clinical relevance compared to tumor

diameter, exploring the potential mechanistic differences between folate forms to study the

tumor diameter results from our study warrants further research.

The present study is the first to investigate the comparative effects of FA and 5MTHF

supplementation on the progression of established preneoplastic lesions of colon cancer (ACF) to

colorectal adenomas and adenocarcinomas. Previous studies have provided a considerable

amount of data on the tumor promoting effects of FA as well as the dual modulatory effects of

FA (Table 2.12). However, there has been limited research on the effects of 5MTHF on

colorectal carcinogenesis. An in vitro study conducted in colon cancer cell lines found 5MTHF

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supplementation to have significantly greater effects on growth rates compared to FA (129). This

study highlighted the potential negative outcomes of providing equimolar doses of a more

readily available form of folate (129). Based on the aforementioned data, we hypothesized that

5MTHF would have a greater tumor promoting effect relative to FA. Although our findings are

not unequivocally in line with our hypothesis, this study does suggest that both FA and 5MTHF

provided after the establishment of colorectal ACF appear to promote the progression of ACF to

adenocarcinomas. However, 5MTHF was shown to have a greater effect on tumor diameter

relative to FA at the 10 mg supplemental level, which is supportive of our hypothesis.

Some mechanistic explanations can be applied to these results. Firstly, folate

supplementation may promote the progression of established preneoplastic lesions by facilitating

nucleotide precursors to the replicating preneoplastic cells resulting in accelerated proliferation

and growth (27, 28). Thymidylate synthase (TS), is the enzyme responsible for thymidylate

biosynthesis (Figure 2.2). TS uses the substrate 5,10-methyleneTHF and dUMP to generate DHF

and dTMP. A limited number of comparative in vivo studies have been conducted to assess the

effects of FA and 5MTHF on enzymes such as TS, and the findings have shown varying results.

A recent comparative mechanistic study conducted in mice found no difference between FA and

5MTHF supplementation on TS gene expression (128). However, another study conducted in a

regenerating rat liver found FA supplementation to reduce TS gene expression and decrease

DNA synthesis initially but upregulate DNA synthesis later (142). The study also noted that the

mRNA expression did not directly translate to protein expression suggesting post-transcriptional

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and translational modifications to have effects on TS. Although our study did not assess gene or

protein expression, it is clear that future studies are necessary to better understand the effects of

FA and 5MTHF on nucleotide biosynthesis, particularly in the context of colon cancer.

Secondly, folate supplementation may promote the progression of preneoplastic lesions via

its effects on de novo methylation of promoter CpG islands of tumor suppressor and other

critical genes involved in carcinogenesis resulting in possible gene inactivation and then tumor

progression. This hypothesis needs to be tested in future studies. Lastly, folate supplementation

may promote carcinogenesis through hypermutability of methylated cytosines in CpG

dinucleotide sequences (86). The mutations observed in these sites (majority cytosine to

thymine) are governed by the enzyme DNMT (particularly DNMT3A and DNMT3B), which is

an important enzyme for de novo methylation patterns. (34, 35). These mutational hot spots can

lead to inactivation of critical genes resulting in cancer progression. Comparative studies have

suggested 5MTHF supplementation to be associated with greater DNMT3A mRNA expression

relative to FA and have also shown a greater contribution to upregulating genes involved in the

methionine cycle (129). FA supplementation studies using in vitro, animal models and human

models have suggested that the effects of methylation tend to be site and gene specific and

depend on a variety of factors including duration of folate exposure and stage of cell

transformation (127). The mechanistic differences between FA and 5MTHF in the context of

DNA methylation, seem to be the most plausible mechanism to describe the findings from our

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study. Further research assessing DNA methylation in a colon cancer model is necessary to

elucidate and clarify these potential mechanisms of action for cancer progression.

Strengths and Limitations

One of the strengths of this study comes from the amino acid defined diets provided to the

rats in each group. The rats in each group were fed diets that were identical in nutritional content

but only differed in the supplemental amount of FA and 5MTHF. This makes sure that the results

and effects seen in the study are the result of the dietary intervention.

The AOM rat model is a well-established model used to study colon carcinogenesis. This

model has its strengths and limitations. AOM is a methylating agent used to induce colon cancer

via increased apoptosis of colonic cells, followed by an increase in proliferation of mutations in

colonic cells. The use of AOM produces colonic epithelial lesions that are similar to those

observed in humans. The AOM-induced tumors in rats share histopathological characteristics

with human tumors. They develop similarly in terms of timeline (ACF → adenoma/polyp →

carcinomas) and the regional distribution of these tumors is also comparable to that in humans as

the induced tumors are predominantly observed in the distal colon. However, there exist some

genetic differences; the rat model lacks the p53 mutation and unlike Apc mutations noted in

human colonic tumors, rats exhibit an accumulation of β-catenin in the nucleus. Given these

inherent limitations, and differences from human colon cancer, the AOM rat model is still the

best model of CRC due to the similarities in histology and the timeline of cancer progression.

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Although rats and humans share many biological functions, which is another reason that

this model is valuable for colon cancer studies, it is important to note that these findings are not

entirely translatable to the human situation. Major differences between rats and humans include

body weight, lifespan as well as gene regulation, all of which can translate to differences in

metabolism. One of the major metabolic differences between humans and rodents in the context

of this study is the substantially lower DHFR activity in humans compared to rodents. However,

this is the first study of its kind to assess the comparative effects of FA and 5MTHF on colon

cancer progression. Furthermore, our study did not report UMFA concentrations which could

highlight some potential differences between the two folate forms.

Additional limitations, that can be mended for future studies is the use of plasma folate as a

measure to assess folate status, the lack of mechanistic endpoints and the measure of dietary

folate levels in the experimental diets. Plasma concentrations are reflective of short-term dietary

intake and supplement usage (2). Red blood cell folate concentrations should be considered for

future studies as they are more reflective of tissue stores and chronic dietary intake (2). The 120-

day turnover period of red blood cells allow for a measure that is resistant to short term

variations in consumption. The lack of mechanistic endpoints in the study leaves questions

regarding the nature of the differential effects observed. Analyzing gene and protein expression,

DNA methylation and nucleotide biosynthesis could have given insight into the differential

tumor diameters observed between FA and 5MTHF groups. Dietary folate levels in the

experimental diets were not measured throughout the study period. Although similar diets have

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been used in previously conducted studies, it is possible to have slight variation between diet

batches. Future studies should assess folate levels in experimental diets to ensure consistency

between batches.

This study observed the differential effects of FA and 5MTHF on plasma folate

concentrations as well as tumor parameters. The results of this study can be used as a framework

for future studies to elucidate potential biochemical mechanisms and further study the

comparative effects of these two folate vitamers on cancer progression.

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4.5 CONCLUSION

Based on previously conducted comparative studies looking at the effects of FA and

5MTHF on the folate pathway as well as blood folate concentrations, we expected FA and

5MTHF to elicit differential effects on colon cancer progression (128, 129). Our data suggests

that FA and 5MTHF supplementation work similarly in terms of their effects on ACF

proliferation and adenocarcinoma proliferation. The novel finding of this study is their

differential effect on tumor diameter. 5MTHF supplementation resulted in a significantly greater

tumor diameter relative to FA. Therefore, the hypothesis of our study was supported. For this

reason, the findings of this study are in line with previously conducted comparative studies

which show that 5MTHF is indeed more readily available than FA. The findings of this study

also suggest that 5MTHF supplementation may have significant public health implications.

Based on previously conducted studies in conjunction with the findings of this study, it is

possible that the proliferative effects differ in their effects on biological methylation reactions.

For this reason, further studies are necessary to better understand the effects on DNA

methylation and health outcomes, to ultimately conclude on safer parameters of use.

86

CHAPTER 5: SUMMARY

5.1 SUMMARY

FA supplementation has been linked with a number of adverse health outcomes, and a

growing body of evidence supports the idea of FA supplementation having tumor promoting

effects particularly in the context of colon cancer (141). 5MTHF has been marketed as a safer

folate supplemental alternative to FA and has been speculated to have beneficial effects over FA.

This comparative study attempted to elucidate the differential effects of FA and 5MTHF

supplementation on the progression of colon cancer using an AOM rat model. The outcomes of

measure included plasma folate concentration, ACF analysis, tumor incidence and multiplicity as

well as tumor diameter. We hypothesized that 5MTHF would have a greater tumor promoting

effect relative to FA.

Our study supports the findings of previously conducted studies which suggest that

5MTHF is at least as effect as FA in raising plasma folate concentrations. However, at the higher

dose (10mg) 5MTHF increased plasma folate concentrations to a greater degree relative to FA,

which is also in line with previously conducted studies. In terms of ACF enumeration, the

number of ACF were shown to increase with dose of supplemental folate in both groups. Tumor

incidence was not affected by folate form or dose; however, the number of adenocarcinomas was

shown to increase with dose of supplemental folate in both groups. Plasma folate concentrations

were also positively correlated with the number of adenocarcinomas. The novel finding,

concerning the differential effects of FA and 5MTHF supplementation was the differences in

87

tumor diameter. Tumor diameter was seen to increase with increasing level of supplemental

folate in both groups. Plasma folate concentrations were also positively correlated with increases

in tumor diameter. The difference between the two folate forms was seen in the 10mg

supplemental folate groups, where 5MTHF showed a greater increase in tumor diameter

compared to FA. These results provide evidence to support that 5MTHF as a supplement may in

fact not be more beneficial than FA. An important implication of this is the idea that DHFR

activity in rodents is greater than that in humans, and as a results rats metabolize folates more

efficiently than do humans. Taking into consideration the findings of this study, while also

considering the idea that humans are less effective in folate metabolism compared to rodents, it is

possible that the differential effects observed in rodents may be even more adverse in humans.

As such, future studies are warranted to conclude on safe measures for use.

In previous studies, investigators hypothesized that 5MTHF is a more readily available

form of folate. It is possible that due to this, it is also a better substrate for cell proliferation and

growth. Mechanistic studies have eluted to emphasizing the differential effects of 5MTHF on

biological methylation reactions compared to FA. Increases in DNA methylation can lead to

increases in aberrant DNA methylation which can in turn result in the progression of disease (9,

104). However, these studies are quite limited, and findings presented display gene expression

data rather than protein expression data. Despite this limitation, the results of these studies in

conjunction with the results from our study provide framework for further investigations.

88

5.2 FUTURE DIRECTIONS

There are a number of ways to follow up with the findings of our study in terms of cancer

progression, protein and gene expression, and public health translatability. From this study we

were able to confirm that 5MTHF is effective as FA at increasing plasma folate concentrations,

at low doses. However, 5MTHF differentially impacted plasma folate concentrations compared

to FA at the higher supplemental level of 10mg. This study also observed increasing

adenocarcinoma with increasing supplemental dose of both folate forms as well as a significantly

greater tumor diameter shown with 10mg 5MTHF compared to equimolar FA. This is

particularly important as 5MTHF is advertised as being a safer supplemental form of folate

relative to FA. Given the widespread evidence suggesting FA as having cancer promoting

properties, it is important to establish whether or not 5MTHF acts in a similar or differential

manner. A follow up in vivo real time study monitoring the effects of FA and 5MTHF on colon

cancer progression with mechanistic endpoints, involving DNA methylation, nucleotide

biosynthesis would give further insight into the potential differential effects of FA and 5MTHF.

Since it is true that 5MTHF resulted in greater tumor diameter and was at least as effective as FA

in increasing the number of adenocarcinoma when equimolar doses were compared, further

studies are warranted to understand these effects such that safe parameters of use be established.

89

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APPENDICES

Appendix A: Nutrient composition of experimental L-amino acid defined diets for FA

Nutrient (g/kg of diet) 1 mg FA/kg 10 mg FA/kg

L-Alanine 3.5 3.5

L-Arginine free base 11.2 11.2

L-Asparagine.H20 6 6

L-Aspartic Acid 3.5 3.5

L-Cystine 3.5 3.5

L-Glutamic Acid 35.0 35.0

Glycine 23.3 23.3

L-Histidine free base 3.3 3.3

L-Isoleucine 8.2 8.2

L-Leucine 11.1 11.1

L-Lysine HCl 14.1 14.1

L-Methionine 8.2 8.2

L-Phenylalanine 11.6 11.6

L-Proline 3.5 3.5

L-Serine 3.5 3.5

L-Threonine 8.2 8.2

L-Tryptophan 1.74 1.74

L-Tyrosine 3.5 3.5

L-Valine 8.2 8.2

Total L-amino acid 171.44 171.44

Dextrin 407 407

Sucrose 194 193

Cellulose 50 50

Corn Oil (w/0.015% BHT) 100 100

Salt Mix #210006 57.96 57.96

Vitamin Mix #317756 (no Folate) 10 10

Choline Chloride 2 2

Sodium Bicarbonate 6.6 6.6

Folic Acid in sucrose premix 1mg/g 1 10

Total 1000.000 1000.000

108

Appendix B: Nutrient composition of experimental L-amino acid defined diets for 5MTHF

Nutrient (g/kg of diet) 1 mg 5-MTHF/kg 10 mg 5-MTHF/kg

L-Alanine 3.5 3.5

L-Arginine free base 11.2 11.2

L-Asparagine.H20 6 6

L-Aspartic Acid 3.5 3.5

L-Cystine 3.5 3.5

L-Glutamic Acid 35.0 35.0

Glycine 23.3 23.3

L-Histidine free base 3.3 3.3

L-Isoleucine 8.2 8.2

L-Leucine 11.1 11.1

L-Lysine HCl 14.1 14.1

L-Methionine 8.2 8.2

L-Phenylalanine 11.6 11.6

L-Proline 3.5 3.5

L-Serine 3.5 3.5

L-Threonine 8.2 8.2

L-Tryptophan 1.74 1.74

L-Tyrosine 3.5 3.5

L-Valine 8.2 8.2

Total L-amino acid 171.44 171.44

Dextrin 407 407

Sucrose 193.96 184.59

Cellulose 50 50

Corn Oil (w/0.015% BHT) 100 100

Salt Mix #210006 57.96 57.96

Vitamin Mix #317756 (no Folate) 10 10

Choline Chloride 2 2

Sodium Bicarbonate 6.6 6.6

5-MTHF in sucrose 1mg/g 1.04 10.41

Total 1000.000 1000.000

109

Appendix C : Salt mix and vitamin mix compositions of experimental L-amino acid defined

diets

Salt Mix #210006

Ingredients (g/kg of diet)

Calcium carbonate 14.60000

Calcium phosphate, dibasic 0.17000

Sodium chloride 12.37000

Potassium phosphate, dibasic 17.16000

Magnesium sulfate, anhydrous 2.45000

Magnesium sulfate, monohydrate 0.18000

Ferric citrate 0.62000

Zinc carbonate 0.05400

Cupric carbonate 0.05400

Potassium iodide 0.00058

Sodium selenite 0.00058

Chromium potassium sulfate 0.01900

Sodium fluoride 0.00230

Molybdic acid, ammonium salt 0.00120

Sucrose 10.27534

Vitamin Mix #317756

Ingredients (g/kg of diet)

Thiamin HCl 0.006

Riboflavin 0.006

Pyridoxine HCl 0.007

Nicotinic acid 0.030

Calcium pantothenate 0.016

Cyanocobalamin 0.00005

Vitamin A palmitate (500 000 IU/g) 0.008

Vitamin D3 (400 000 IU/g) 0.0025

Vitamin E acetate (500 IU/g) 0.100

Menadioine sodium bisulfate 0.00080

Biotin 0.00002

Sucrose 9.82363

comparative carcinogenic effects of folic acid vs. 5

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