October 2006: (I)468 – 475
Brief Critical Review
Does a High Folate Intake Increase the Risk of Breast
Cancer?
Young-In Kim, MD, FRCP(C)
Although not uniformly consistent, epidemiologic
studies generally suggest an inverse association be-
tween dietary intake and blood measurements of folate
and breast cancer risk. However, the Prostate, Lung,
Colorectal, and Ovarian (PLCO) Cancer Screening
trial has recently reported for the first time a potential
harmful effect of high folate intake on breast cancer
risk. In this study, the risk of developing breast cancer
was significantly increased by 20% in women report-
ing supplemental folic acid intake ⱖ 400 ␮g/d com-
pared with those reporting no supplemental intake.
Furthermore, although food folate intake was not
significantly related to breast cancer risk, total folate
intake, mainly from folic acid supplementation, signif-
icantly increased breast cancer risk by 32%. The data
from the PLCO trial support prior observations made
in epidemiologic, clinical, and animal studies suggest-
ing that folate possesses dual modulatory effects on
the development and progression of cancer depending
on the timing and dose of folate intervention. Based on
the lack of compelling supportive evidence, routine
folic acid supplementation should not be recom-
mended as a chemopreventive measure against breast
cancer at present.
Key words: breast cancer, folate, folic acid
© 2006 International Life Sciences Institute
doi: 10.1301/nr.2006.oct.468 – 475
Folate is a water-soluble B vitamin and important
cofactor in one-carbon metabolism.1 Folic acid is the
fully oxidized monoglutamyl form of this vitamin that is
used commercially in supplements and in fortified
foods.1 The role of folate in human health and disease
has rapidly been expanding. Folate deficiency appears to
Dr. Kim is with the Department of Medicine and
Nutritional Sciences, University of Toronto, Division of
Gastroenterology, St. Michael’s Hospital, Toronto,
Canada.
Please address all correspondence to: Dr. Young-In
Kim, Room 7258, Medical Sciences Building, University
of Toronto, 1 King’s College Circle, Toronto, Ontario,
Canada, M5S 1A8; Phone: 416-978-1183; Fax: 416-
978-8765; E-mail: youngin.kim@utoronto.ca
468
play an important pathogenetic role in the development
of anemia, atherosclerosis, neural tube defects, adverse
pregnancy outcomes, and neuropsychiatric disorders.2
Folate has generally been regarded as safe and has long
been presumed to be purely beneficial3 and an ideal
functional food component for disease prevention.4 For
example, an overwhelming body of evidence for a pro-
tective effect of periconceptional folic acid supplemen-
tation against neural tube defects led to mandatory folic
acid fortification in the United States and in Canada in
1998.2 Preliminary reports suggest a significant reduc-
tion (15%–50%) in the incidence of neural tube defects
in the United States and Canada associated with this
mandatory fortification.2 In addition to the drastic in-
crease in dietary intake from mandatory folic acid forti-
fication,5-7 30% to 40% of the North American popula-
tion consume supplemental folic acid for several possible
but as yet unproven health benefits, including prevention
of atherosclerosis, improvement in cognitive function,
and cancer prevention.8
Folate deficiency has also been linked to the risk of
several malignancies in humans, including cancer of the
colorectum, oropharynx, esophagus, stomach, pancreas,
lungs, cervix and ovary, and neuroblastoma and leuke-
mia.9,10 Although not uniformly consistent, dietary in-
take and blood levels of folate have generally been
shown to be inversely related to the risk of these malig-
nancies in a large body of epidemiologic studies.9,10
However, the precise role of folate deficiency in cancer
development and progression and of folate supplemen-
tation in cancer prevention remain highly speculative and
provocative to date.
Folate and Breast Cancer: Epidemiologic
Evidence
An accumulating number of epidemiologic studies
have suggested an inverse association between folate
status and the risk of breast cancer. However, epidemi-
ologic evidence available thus far has not been consis-
tent, nor has it provided unequivocal support for the
purported inverse relationship between folate status and
breast cancer risk. The majority of 13 published case-
Nutrition Reviews姞, Vol. 64, No. 10
control studies investigating the relationship between
dietary folate intake and breast cancer risk showed either
a significant or equivocal inverse relationship that was
not statistically significant, became nonsignificant after
adjustment, or could not be distinguished from other
factors in relation to risk.11-23 Overall, these studies
suggested a 30% to 35% reduction in the risk of devel-
oping breast cancer in individuals with the highest di-
etary intake of folate compared with those with the
lowest intake.11-23 Two of these studies also examined
the association between total folate intake (dietary plus
supplemental) and breast cancer risk and found no sig-
nificant relationship.14, 22
The interpretation of results from case-control stud-
ies are often limited because of inherent problems asso-
ciated with retrospective analyses, including the accu-
racy with which intake of dietary factors or
supplementation can be established and the inability to
adequately control or correct potential confounders. The
relationship between dietary and total intake of folate
and breast cancer risk has also been examined in seven
prospective studies that avoid most of the methodologi-
cal problems of case-control studies and that can control
and correct confounding factors more adequately than
can case-control studies.24-30 None of these prospective
studies found a significant association between either
dietary or total folate intake and breast cancer risk.24-30
With respect to actual blood (serum or plasma) levels of
folate and breast cancer risk, one case-control study
reported a significant inverse association,31 two nested
case-control studies found a nonsignificant inverse asso-
ciation,32,33 and another nested case-control study ob-
served no association.34
Folate-Alcohol Interactions in Breast Cancer
Risk Modifications
Overall, the portfolio of epidemiologic evidence
supporting the relationship between folate status and
breast cancer risk in women is tenuous at best. However,
a clearer picture emerges when studies examining the
joint effects of folate and alcohol are considered. Con-
sumption of alcohol, a well-established folate antago-
nist,35 has consistently been shown to increase the risk of
breast cancer in epidemiologic studies, and is accepted as
a well-established risk factor for breast cancer develop-
ment.36,37 All prospective studies24-26,29,30 except one28
and three case-control studies16-18 that examined this
issue have found a significant interaction between alco-
hol and folate intake in modifying the risk of breast
cancer. These studies collectively suggest that low folate
intake increases, whereas high intake decreases, breast
cancer risk among women who regularly consume mod-
erate or high amounts of alcohol, but not among women
Nutrition Reviews姞, Vol. 64, No. 10
with low or no alcohol consumption. Furthermore, these
studies also suggest that the increased risk of breast
cancer associated with alcohol consumption is more
pronounced in or limited to women with low folate
intake. Two prospective studies have since shown that
the increased risk of breast cancer associated with low
folate and high alcohol intake is limited to estrogen
receptor-negative breast cancer.38,39
Folate-Gene Interactions in Breast Cancer Risk
Modifications
Recent molecular epidemiologic studies have also sug-
gested that the inverse association between folate status and
breast cancer risk, as well as the interaction between folate
and alcohol in modifying this risk, are further modulated by
polymorphisms of critical genes (e.g., C667T polymor-
phism of the methylenetetrahydrofolate reductase [MTHFR
C677T] gene) that are involved in the folate metabolic
pathway. A recent meta-analysis has shown that the
MTHFR C677T polymorphism may increase the risk of
breast cancer in premenopausal women and in women with
low folate intake.40
Folate and Breast Cancer: Evidence from
Animal Studies
Although direct evidence from human intervention
trials is lacking at present, three animal studies41-43
conducted in the well-established N-methyl-N-nitro-
sourea (MNU) rat model of mammary tumorigenesis
provide a vastly different picture of the role of folate in
breast carcinogenesis than that from human epidemio-
logic studies. These studies collectively suggest that
dietary folate deficiency of a mild degree significantly
inhibits, whereas folic acid supplementation (4 –20 times
above the basal dietary requirement) does not signifi-
cantly modulate, the development and progression of
mammary tumors in this model.41-43 Although MNU-
induced mammary tumorigenesis in rat is different from
the human disease in several important aspects, this
model is generally considered to be the best animal
model of breast cancer currently available because of
histological, clinical, and certain molecular similarities
to human breast cancer.44
Prostate, Lung, Colorectal, and Ovarian Cancer
Screening Trial
In contrast to the inverse association, albeit not
uniformly consistent, between folate intake and breast
cancer risk reported in epidemiologic studies thus far, a
recently published prospective study suggested for the
first time a potential harmful effect of high folate intake
469
on breast cancer risk.45 The Prostate, Lung, Colorectal,
and Ovarian (PLCO) Cancer Screening Trial is a pro-
spective study that investigated the association between
folate intake, alcohol consumption, and postmenopausal
breast cancer risk. Women 55 to 74 years of age between
November 1993 and July 2001 were recruited in 10 US
centers. Women with a personal history of one of the
prostate, lung, colorectal, and ovarian cancers, with a
recent history of screening procedures for one of these
cancers, or with current treatment for any cancer except
non-melanoma skin cancer were excluded from the trial.
Women randomly assigned to the intervention arm un-
derwent period cancer screening tests for these four
cancers, where those assigned to the control were in-
structed to follow their usual medical practice. Of the
77,376 women enrolled in the PLCO trial, only those in
the intervention arm were given the food frequency
questionnaire at baseline (n ⫽ 38,660), which was self-
administered and characterized usual dietary intake over
the preceding 12 months prior to the study. After appro-
priate exclusion, 25,400 women remained for analysis.
Between September 1993 and June 2003, 691 incident
breast cancer cases were identified and confirmed. The
risk of developing breast cancer was adjusted for age,
education, age at menarche, parity, age at first birth, oral
contraceptive use, age at menopause, hormonal replace-
ment therapy, mammography screening history, history
of benign breast disease, family history, and energy.
The main, and novel, finding of this study is that the
risk of developing breast cancer was significantly increased
by 20% (hazard ratio [HR] ⫽ 1.19; 95% confidence interval
[CI] ⫽ 1.01-1.41; P trend ⫽ 0.04) in women reporting
supplemental folic acid intake ⱖ 400 ␮g/d compared with
those reporting no supplemental intake. Furthermore, al-
though food folate intake was not significantly related to
breast cancer risk (HR for highest [⬎412 ␮g/d] versus
lowest [ⱕ261 ␮g/d] intake ⫽ 1.04; 95% CI ⫽ 0.83–1.31; P
trend ⫽ 0.56), total folate intake, mainly from folic acid
supplementation, significantly increased breast cancer risk
by 32% (HR for highest [⬎853 ␮g/d] versus lowest [ⱕ336
␮g/d] ⫽ 1.32; 95% CI ⫽ 1.04 –1.68; P trend ⫽ 0.03).
Consistent with previous findings, alcohol consumption
was positively associated with breast cancer risk and the
risk was greatest in women with lower total folate intake.
This study is therefore the first to suggest that high folate
intake, generally attributable to supplemental folic acid,
may increase the risk of breast cancer in postmenopausal
women.
Commentary on the PLCO Cancer Screening
Trial
The PLCO Cancer Screening Trial is a well-de-
signed and conducted prospective study with obvious
470
strengths and weaknesses usually associated with the
prospective epidemiologic design. One of the weak-
nesses associated with this study was that folate intake at
baseline was correlated to subsequent incidence of breast
cancer. In other words, folate intake at baseline was
assumed to reflect past and subsequent consumption.
Therefore, whether the subjects in these studies changed
their diet during the follow-up period and how this might
have affected the study outcome could not be deduced. In
this regard, the most limiting factor in interpreting the
data in this study is mandatory folic acid fortification in
the United States beginning in 1998.
The effectiveness of folic acid fortification in
improving folate status has already been shown to be
quite striking, with a dramatic increase in blood mea-
surements of folate concentrations in the United States
and Canada.2 In these two countries, the average
post-fortification total folate intake is estimated to be
approximately 400 ␮g/d in supplement non-users,
with approximately 200 ␮g/d consumed as naturally
occurring folate food and approximately 200 ␮g/d as
folic acid provided in enriched products.2 For those
taking multivitamins containing folic acid, the esti-
mated total folate intake is approximately 800 ␮g/d.
Several studies assessing food composition and di-
etary intakes have suggested that the increased post-
fortification folate intake in the US population may be
about twice that originally anticipated.5-7 Further-
more, these estimates of folate intake based on food
composition databases are likely to be low because of
limitations in the analytic methods previously used to
analyze food folate.5-7 The PLCO Cancer Screening
Trial only estimated folate intake at baseline (most
subjects were recruited in the pre-fortification era) and
did not measure any change in folate intake during the
study period. Therefore, this study did not correct for
possible confounding effects of folic acid fortification
in data analysis.
Another shortcoming is that this study did not con-
sider and incorporate interactions between folate and
several genetic polymorphisms in the folate metabolic
pathway in the study design and data analysis. Advances
in molecular epidemiology have added another dimen-
sion to the already complex field of nutrition and cancer.
Recently identified and characterized single nucleotide
polymorphisms and other genetic and epigenetic variants
of genes that are involved in the absorption, transport,
metabolism, and excretion of nutrients have been shown
not only to modify cancer risk but also to significantly
modulate the effect of nutrients and related compounds
on cancer risk.46 In this regard, there is an emerging body
of evidence suggesting that several genetic polymor-
phisms in the folate metabolic pathway (e.g., MTHFR
C677T) interact with folate and other related nutrients
Nutrition Reviews姞, Vol. 64, No. 10
(e.g., alcohol) in modulating the risk of cancers including
breast cancer.40,47 This emerging important topic in the
field of nutrition and cancer, termed “gene-nutrient in-
teractions” in carcinogenesis, has significant implications
in designing and interpreting data from observational
epidemiologic and intervention studies. Although indi-
viduals are subjected to the same level of nutritional
exposure, systemic and target tissue bioavailability of
nutrients and their metabolites, as well as their functional
effects in the target tissue, might be vastly different
because of genetic and epigenetic variations.
Folate and Colorectal Cancer: A Complex Role
of Folate in Cancer Risk Modification
The puzzling role of folate in breast tumorigenesis is
closely mirrored in colorectal carcinogenesis. An over-
whelming body of epidemiologic evidence from case-
control and prospective studies, including recent meta-
analyses, suggests an inverse association between folate
status and the risk of colorectal cancer.47,48 Furthermore,
small clinical trials have demonstrated that folic acid
supplementation can improve or reverse surrogate end-
point biomarkers of colorectal cancer.9,48 Animal studies
generally support a causal relationship between folate
depletion and colorectal cancer risk and an inhibitory
effect of modest levels of folate supplementation on
colorectal carcinogenesis.9,48 However, these animal
studies have also shown that the dose and timing of
folate intervention are critical in providing safe and
effective chemoprevention; exceptionally high supple-
mental folate levels and folate intervention after micro-
scopic neoplastic foci are established in the colorectal
mucosa promote, rather than suppress, colorectal carci-
nogenesis.9,48
The Aspirin-Folate Polyp Prevention Study (N ⫽
1021) reported that folic acid supplementation (1 mg/d)
for up to 6 years in subjects with previous colorectal
adenomas (well-established precursor of colorectal ade-
nocarcinoma) did not significantly prevent the recurrence
of colorectal adenomas (rate ratio [RR] ⫽ 1.04).49 How-
ever, folic acid supplementation significantly increased
the number of adenomas by 44% (RR ⫽ 1.44; 95% CI ⫽
1.03–2.02) and nonsignificantly increased the incidence
of advanced adenomas with a high malignant potential
compared with placebo.49 One explanation for this un-
expected observation is that folic acid supplementation
might have promoted the progression of already existing,
undiagnosed preneopalstic lesions (e.g., aberrant crypt
foci, the probable earliest precursor of colorectal adeno-
carcinoma or microscopic adenomas) or adenomas
missed on initial colonoscopy in these genetically pre-
disposed subjects at high risk of developing colorectal
cancer.
Nutrition Reviews姞, Vol. 64, No. 10
DUAL MODULATORY ROLE OF FOLATE IN
CARCINOGENESIS
Folate appears to possess dual modulatory effects on
carcinogenesis depending on the timing and dose of
folate intervention.2,9,48 Folate deficiency has an inhibi-
tory effect, whereas folate supplementation has a pro-
moting effect, on the progression of established neo-
plasms.2,9,48 In contrast, folate deficiency in normal
tissues appears to predispose them to neoplastic transfor-
mation, and modest supplemental levels suppress,
whereas supraphysiologic doses of supplementation en-
hance, the development of tumors in normal tissues.2,9,48
Are there biologically plausible explanations for these
seemingly paradoxical and contradictory epidemiologic,
animal, and clinical observations concerning the role of
folate in cancer development and progression?
As an essential cofactor for the de novo biosynthesis
of purines and thymidylate, folate plays an important role
in DNA synthesis, stability, integrity, and repair, aberra-
tions of which have been implicated in carcinogene-
sis.9,50,51 Folate may also modulate DNA methylation,
which is an important epigenetic determinant in gene
expression, in the maintenance of DNA integrity and
stability, in chromosomal modifications, and in the de-
velopment of mutations.9,10 The potential for a dual
modulatory role for folate on carcinogenesis is illustrated
in Figure 1. In normal tissues, folate deficiency is asso-
ciated with DNA strand breaks, impaired DNA repair,
increased mutations, and aberrant DNA methylation,
thereby predisposing them to neoplastic transformation,
and folate supplementation can correct some of these
defects induced by folate deficiency, thereby preventing
or suppressing neoplastic transformation.9,10,50,51 In con-
trast, in neoplastic cells, where DNA replication and cell
division are occurring at an accelerated rate, folate de-
pletion causes ineffective DNA synthesis, resulting in
inhibition of tumor growth and progression, which is the
basis for antifolate-based cancer chemotherapy.9,10,50,51
Folic acid supplementation promotes the progres-
sion of rapidly replicating preneoplastic and neoplastic
cells by providing nucleotide precursors for an acceler-
ated proliferation and growth.9,10,50,51 Another mecha-
nism of the folic acid supplementation-associated tumor
promoting effect may be de novo methylation of pro-
moter CpG islands of tumor suppressor genes with con-
sequent gene inactivation leading to tumor progres-
sion.2,10 This potential epigenetic mechanism of tumor
progression is supported by recent animal studies using
viable yellow agouti mice that unequivocally have dem-
onstrated that maternal dietary methyl group supplemen-
tation with a modest amount of folic acid permanently
alters phenotypic coat color of the offspring via in-
471
 Tumor promoting mechanisms:
1. DNA strand breaks
2. Impaired DNA repair
3. Increased mutagenesis
4. Genomic DNA hypomethylation
Tumor inhibitory mechanism:
Ineffective DNA synthesis leading to
inhibition of tumor growth and progression
Folate deficiency

risk of neoplatic
transformation
? progression of
early IEN
progression of
late IEN to cancer
progression of
cancer

Normal Intraepithelial Neoplasia Cancer

risk of neoplatic progression of progression of progression of

transformation ? early IEN late IEN to cancer cancer

Folate supplementation

Tumor inhibitory mechanisms: Tumor promoting mechanisms

1. DNA stability and integrity 1. Provision of nucleotide precursors for
2. Optimal DNA repair proliferation and growth of neoplastic cells
3. Decreased mutagenesis 2. De novo methylation of promoter CpG islands of
4. Prevention of aberrant DNA methylation tumor suppressor genes leading to gene inactivation

Figure 1. Dual modulatory role of folate in carcinogenesis. Cancer develops over decades, if not a lifetime, through different stages
of premalignant lesions (intraepithelial neoplasia, IEN) in the target organ. Folate deficiency in normal tissues predisposes them to
neoplastic transformation and modest supplemental levels suppress, whereas supraphysiologic doses of supplementation enhances,
the development of tumors in normal tissues. In contrast, folate deficiency has an inhibitory effect, whereas folate supplementation
has a promoting effect, on the progression of established neoplasms. The effect of folate deficiency and supplementation on the
progression of early precursor or preneoplastic IEN lesions to more advanced stages of IEN and to frank cancer is unknown at present.
The mechanisms by which folate exerts dual modulatory effects on carcinogenesis depending on the timing and dose of folate
intervention relate to its essential role in one-carbon transfer reactions involved in DNA synthesis and biological methylation
reactions.

creased methylation at the promoter CpG site of the
agouti gene.52,53
Effects of Folate Supplementation and
Fortification on Cancer Risk in Humans
In addition to the Aspirin-Folate Polyp Prevention
Study,49 which observed a possible tumor-promoting
effect of folic acid supplementation, several uncontrolled
or randomized studies have investigated the effect of
folic acid supplementation on cancer risk as either the
primary or secondary endpoint. Two recently published
large, randomized, placebo-controlled intervention trials
designed to test the effect of folic acid supplementation
in conjunction with other B vitamins on primary and
secondary prevention of cardiovascular events have re-
ported a nonsignificant trend toward an increased risk of
total cancer (RR ⫽ 1.22; 95% CI ⫽ 0.88 –1.70) in the
Norwegian Vitamin Trials (N ⫽ 3749; 800 ␮g folic
acid/d for 40 months)54 and of colon cancer (RR ⫽ 1.36;
95% CI ⫽ 0.89 –2.08) in the Heart Outcomes Prevention
Evaluation II Trial (N ⫽ 5522; 2.5 mg folic acid/d for 5
years).55
Among participants in a large (N ⫽ 2928) trial of
folic acid supplementation during pregnancy, women
who received 5 mg folic acid/d had a 70% increased risk
of total cancer mortality compared with those not on
supplementation (HR ⫽ 1.70; 95% CI ⫽ 1.06 –2.72; P ⫽
0.02).56 The risk of death from breast cancer in women
taking 5 mg folic acid/d was twice that of women taking
no supplementation in this study, albeit nonsignificant
(HR ⫽ 2.02; 95% CI ⫽ 0.88 – 4.72; P ⫽ 0.10).56 The
tumor-promoting effect of folate supplementation on
established neoplasm is actually not a new observation;

472 Nutrition Reviews姞, Vol. 64, No. 10

in the 1940s, children with acute leukemia treated with
folate supplementation experienced an accelerated pro-
gression of the disease.57
In contrast, a recent Canadian study reported the
effect of folic acid fortification on the incidence of
neuroblastoma among children ⱕ 17 years of age using
the database of the Pediatric Oncology Group of Ontario,
which captures 95% of all pediatric cancers in Ontario.58
This study showed that folic acid fortification was asso-
ciated with a significant 60% reduction in the incidence
of neuroblastoma (from 1.57 cases per 10,000 births in
1996 to 0.62 cases per 10,000 births after 1997, when
folic acid fortification of food became mandatory in
Canada).58 The incidence of infant acute lymphoblastic
leukemia and hepatoblastoma, however, remained al-
most the same in this study.58
HIGH FOLATE INTAKES: BENEFICIAL OR
HARMFUL?
Whether possible deleterious effects of folic acid
fortification and supplementation outweigh the known
and potential health benefits is essentially unknown at
present. In addition to masking of vitamin B12, especially
in the elderly, an emerging body of evidence suggests
that folate supplementation may possess potentially se-
rious adverse effects, including resistance or tolerance to
anti-folate-based chemotherapy and anti-inflammatory
drugs; epigenetic instability; decreased natural killer cell
cytotoxicity; and genetic selection of disease alleles (e.g.,
MTHFR C677T).2,4,59,60 Furthermore, recent epidemio-
logic, animal, and intervention studies suggest that folate
supplementation may promote the progression of estab-
lished preneoplastic and neoplastic lesions.2,48,49,61,62
The potential adverse effects, including the cancer-pro-
moting effect, from a drastic increase in folate intake
from mandatory fortification and supplementation in the
vast majority of the North American population need to
be determined in long-term longitudinal studies as well
as in appropriate animal studies.
CONCLUSION
What can we conclude about the role of folate in
breast cancer development and progression? Based on
the lack of compelling supportive evidence, routine folic
acid supplementation should not be recommended as a
chemopreventive measure against breast cancer at
present. In fact, folate supplementation may promote the
progression of already existing, undiagnosed pre-malig-
nant lesions in the breast. However, women consuming
moderate to high amounts of alcohol and women with
the MTHFR C677T genotype may benefit from folate
supplementation, provided that they are free of preneo-
Nutrition Reviews姞, Vol. 64, No. 10
plastic or neoplastic foci in the breast and other target
organs. Given the incidence and mortality of breast
cancer in the United States, determining the overall
benefits of folic acid fortification and supplementation
has significant public health implications. As such, the
role of folate in the development and progression of
breast cancer needs to be clarified with additional future
studies.
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