maternal one-carbon nutrient intake and cancer risk in offspring

Maternal one-carbon nutrient intake and cancer riskin offspringnure_424 561..571

Eric D Ciappio, Joel B Mason, and Jimmy W Crott

Dietary intake of one-carbon nutrients, particularly folate, vitamin B2 (riboflavin),vitamin B6, vitamin B12, and choline have been linked to the risk of cancers of thecolon and breast in both human and animal studies. More recently, experimentaland epidemiological data have emerged to suggest that maternal intake of thesenutrients during gestation may also have an impact on the risk of cancer in offspringlater in life. Given the plasticity of DNA methylation in the developing embryo andthe established role of one-carbon metabolism in supporting biological methylationreactions, it is plausible that alterations in maternal one-carbon nutrient availabilitymight induce subtle epigenetic changes in the developing embryo and fetus thatpersist into later life, altering the risk of tumorigenesis throughout the lifespan. Thisreview summarizes the current literature on maternal one-carbon nutrient intakeand offspring cancer risk, with an emphasis on cancers of the colon and breast, anddiscusses specific epigenetic modifications that may play a role in theirpathogenesis.© 2011 International Life Sciences Institute

INTRODUCTION

Epidemiological and laboratory data have repeatedlyimplicated one-carbon nutrients such as folate, vitaminB6, riboflavin (vitamin B2), vitamin B12, and choline asbeing protective against various cancers, most notablythose of the colorectum and to a lesser degree, thebreast.1–6 Mechanistically, it is thought that these nutri-ents may have an impact on carcinogenesis through theirrole in providing one-carbon moieties for the synthesis ofnucleotides and of S-adenosyl methionine (SAM), theuniversal donor for nearly all methylation reactions,including that of DNA. Inadequacies of folate cause anintracellular accumulation of deoxyuridylate (dUMP),7,8

which promotes uracil misincorporation into DNA9–13

and results in double-stranded DNA breakage.14,15

Related to their role as one-carbon donors, deficiencies offolate and possibly other related nutrients are reported tocause epigenetic instability by promoting genomic

hypomethylation16–18 and the seemingly paradoxicalhypermethylation of specific gene promoters.19–21 Impor-tantly, the interconversion of different biological forms offolate is dependent on vitamins B2, B6, and B12 as fourfolate-metabolizing enzymes require one of thesevitamins as cofactors.

In the case of folate, timing has emerged as an impor-tant element that determines the polarity of the relation-ship between intake and carcinogenesis.22 For example,animal models suggest that an abundant intake of folatebefore the appearance of neoplastic lesions appears toreduce the risk of tumorigenesis, whereas supplementa-tion after the development of neoplastic foci promotestumorigenesis.23 Whether a similar promotional effectoccurs in humans remains controversial; although, insupport of this concept is a recent high-profile interven-tion trial which found that folic acid supplementationincreased both the multiplicity of recurrent colonicadenomas as well as the likelihood of a “high-risk” lesion

Affiliations: ED Ciappio, JB Mason, and JW Crott are with the Vitamins and Carcinogenesis Laboratory, Jean Mayer USDA Human NutritionResearch Center on Aging at Tufts University, Boston, Massachusetts, USA, and the Friedman School of Nutrition Science and Policy, TuftsUniversity, Boston, Massachusetts, USA.

Correspondence: JW Crott, Vitamins and Carcinogenesis Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at TuftsUniversity, 711 Washington St., Boston, MA 02111, USA. E-mail: [email protected], Phone: +1-617-556-3117, Fax: +1-617-556-3234.

Key words: breast cancer, colorectal cancer, epigenetics, maternal diet, one-carbon metabolism

Lead Article

doi:10.1111/j.1753-4887.2011.00424.xNutrition Reviews® Vol. 69(10):561–571 561

among those who had previously had an adenomaremoved.24 A secondary analysis of the same trial revealeda nearly threefold increased incidence of prostate canceramong the men who received folic acid supple-mentation.25 Four other trials of shorter duration,however, have failed to detect such a harmful effectof supplementation.26–29 Nevertheless, the prevailingconcept is that an insufficiency of folate promotes car-cinogenesis by causing DNA breakage and/or aberrationsin biological methylation, while supplemental quantitiesof the vitamin may, among those already harboring neo-plastic foci, promote tumorigenesis by supporting cellularproliferation of existing lesions through the provision ofan abundant supply of nucleotides for DNA synthesis.30

Current awareness of the critical importance oftiming in determining the outcome of nutrient intake hasprompted consideration of windows of exposure to theseone-carbon nutrients other than those in adult life.Indeed, the window of time during which cancer risk maybe modified by diet could extend back into childhood andinfancy and even during in utero life. As mentioned pre-viously, both genomic and gene-specific DNA methyla-tion patterns in various tissues have been shown to besensitive to one-carbon nutrient availability. Duringmammalian embryogenesis, DNA methylation patternsare highly labile and experience a wave of genome-widedemethylation, followed by a period of controlled andprecise remethylation.31,32 Therefore, the integrity of thedeveloping epigenome may be especially sensitive to fluc-tuations in one-carbon nutrient and methyl group supplyduring this early stage of life. In support of these mecha-nistic considerations is the increased recognition ofmaternal nutrition as a possible determinant of chronicdisease in offspring, particularly in regard to the develop-ment of obesity and diabetes.33,34

Both experimental and epidemiological evidence arebeginning to accrue that suggest cancer among offspringmay also be one of the chronic diseases whose risk isdetermined, in part, by a mother’s intake of one-carbonnutrients. The concept of optimizing maternal diets tominimize carcinogenesis in offspring is certainly enticingbecause, as demonstrated by the remarkable reduction inoffspring with neural tube defects at birth since folic acidfortification,35 modification of maternal diet has thepotential to be an extremely effective public health tool. Itis already recommended by the Centers for DiseaseControl and Prevention and the US Public Health Servicethat women of childbearing age who are seeking tobecome pregnant take a folic acid supplement for theprevention of neural tube defects. As evidence continuesto accumulate regarding the contribution of maternaldiet to offspring health, it may be worthwhile to revisitthis very effective public health measure with the goal ofmodifying it to include the optimal timing, dosage, and

combination of one-carbon nutrients to minimize therisk of cancer and possibly other chronic diseases in off-spring. Highlighting the importance of such initiatives isthe fact that, although frank deficiencies are rare in indus-trialized nations, mild inadequacies (so-called “subclini-cal deficiencies”) of vitamins B2

36, B637, and B12

38 occur in10–50% of the population. Furthermore, it is likely thatthe very earliest periods of embryogenesis represent thewindow in which intervention has the greatest potentialeffect and that B-vitamin status during these periods isthe most critical for maximizing the integrity of theembryo’s genome and epigenome. Thus, one challengecurrently envisaged is to ensure that initiation of anymaternal supplementation regimen begins before con-ception. Currently, it is estimated that only between 31%and 37% of women of childbearing age in the UnitedStates begin folate supplementation prior to concep-tion,39,40 and this proportion may be even lower amongcertain ethnic groups.41

In order to reach the point at which intelligent andeffective public health strategies can be constructed,however, more work is needed to prove that particularmaternal diets have a cancer-preventive effect in humans.Careful attention also needs to be paid to studies thatdefine the mechanistic basis of the cancer preventiveeffect, since such insights almost invariably help in arriv-ing at the most rational strategy. The purpose of thisreview is to discuss the current state of knowledge regard-ing maternal dietary interventions with one-carbonnutrients for the purpose of limiting offspring tumori-genesis, with an emphasis on cancers of the colon andbreast. To the best extent possible, this review alsodiscusses the mechanistic basis underlying theserelationships.

EPIDEMIOLOGY

Several epidemiological studies support the concept thatvarying the intake of one-carbon nutrients may impactthe risk for tumorigenesis in offspring (Table 1). Forexample, a reduced incidence of acute lymphocytic leu-kemia (ALL) has been observed among children born tomothers who used folic acid-containing multivitaminsperi-conceptionally in most42–44 but not all case-controlstudies.45

A similar relationship between maternal folic acid-containing multivitamin use and risk of several othertypes of childhood cancers in offspring has beenobserved. Three such case-control studies found thatmaternal multivitamin use was associated with a signifi-cantly reduced risk of pediatric brain tumors inoffspring,46–48 while three others found modest reductionsin risk that failed to attain statistical significance.49–51 Adecreased risk of retinoblastoma in offspring has been

Nutrition Reviews® Vol. 69(10):561–571562

Tabl

e1

Epid

emio

logi

cals

tudi

esex

amin

ing

the

role

ofm

ater

nalo

ne-c

arbo

nnu

trie

ntin

take

onoff

spri

ngca

ncer

risk

.Re

fere

nce

Stud

ypo

pula

tion

loca

tion

Dat

esD

ieta

ryex

posu

reO

ffspr

ing

canc

erou

tcom

eRe

sult

Wen

etal

.42U

S,Ca

nada

,Aus

tral

ia19

89–1

993

Mat

erna

lmul

tivita

min

use

befo

rean

d/or

durin

gpr

egna

ncy

All

Amon

gch

ildre

nbo

rnto

expo

sed

mot

hers

:OR

0.70

(95%

CI0.

5–1.

0)N

=18

42(c

ases

),19

86(c

ontr

ols)

Thom

pson

etal

.43Au

stra

lia19

84–1

992

Mat

erna

lfol

icac

idsu

pple

men

tatio

nw

ithor

with

outi

ron

supp

lem

ents

All

Amon

gch

ildre

nbo

rnto

expo

sed

mot

hers

:OR

0.40

(95%

CI0.

21–0

.73)

N=

83(c

ases

),16

6(c

ontr

ols)

Ross

etal

.44N

orth

Amer

ican

child

ren

with

Dow

nSy

ndro

me

1997

–200

2M

ater

nalm

ultiv

itam

inus

edu

ring

peric

once

ptio

nAl

lAm

ong

child

ren

born

toex

pose

dm

othe

rs:O

R0.

63(9

5%CI

0.39

–1.0

)N

=17

3(c

ases

),15

8(c

ontr

ols)

Miln

eet

al45

Aust

ralia

2003

–200

7M

ater

nalm

ultiv

itam

inus

edu

ring

peric

once

ptio

nAl

lAm

ong

child

ren

born

toex

pose

dm

othe

rs:O

R0.

83(9

5%CI

0.73

–0.9

4)N

=41

6(c

ases

),13

61(c

ontr

ols)

Pres

ton-

Mar

tinet

al.46

Uni

ted

Stat

es19

84–1

991

Mat

erna

lmul

tivita

min

use

thro

ugho

utpr

egna

ncy

Pedi

atric

brai

ntu

mor

sAm

ong

child

ren

born

toex

pose

dm

othe

rs:O

R0.

54(9

5%CI

0.39

–0.7

5)N

=54

0(c

ases

),80

1(c

ontr

ols)

Pres

ton-

Mar

tinet

al.47

Nor

thAm

eric

a,Eu

rope

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ael

1976

–199

4M

ater

nalm

ultiv

itam

inus

efo

ratl

east

2tr

imes

ters

ofpr

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ncy

Pedi

atric

brai

ntu

mor

sAm

ong

child

ren

born

toex

pose

dm

othe

rs:O

R0.

70(9

5%CI

0.50

–0.9

0)N

=10

51(c

ases

),19

19(c

ontr

ols)

Buni

net

al.48

Nor

thAm

eric

a19

86–1

989

Mat

erna

lfoo

dfo

late

inta

kedu

ring

preg

nanc

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imiti

vene

uro-

ecto

derm

altu

mor

Hig

hest

vers

uslo

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ntile

:OR

0.38

(95%

CI0.

20–0

.73)

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tren

d=

0.00

5N

=16

6(c

ases

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6(c

ontr

ols)

Buni

net

al.49

Nor

thAm

eric

a19

86–1

989

Mat

erna

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min

use

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Astr

ocyt

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iom

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child

ren

born

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pose

dm

othe

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60(9

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–1.5

)N

=15

5(c

ases

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6(c

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Lubi

net

al.50

Isra

el19

84–1

993

Mat

erna

lfol

icac

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men

tuse

Pedi

atric

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mor

sAm

ong

child

ren

born

toex

pose

dm

othe

rs:O

R1.

05(9

5%CI

0.76

–1.4

4)N

=30

0(c

ases

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4(c

ontr

ols)

Sara

sua

etal

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enve

r,Co

lora

do19

76–1

983

Mat

erna

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tivita

min

use

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Astr

ocyt

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ong

child

ren

born

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pose

dm

othe

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70(9

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0.26

–1.8

6)N

=22

3(c

ases

),20

6(c

ontr

ols)

Orju

ela

etal

.52M

exic

o19

95–1

998

Mat

erna

lfol

ate

inta

kedu

ring

preg

nanc

y(fr

ompl

antf

ood

sour

ces)

Retin

obla

stom

aM

othe

rsw

ithlo

wfo

late

inta

ke:O

R2.

6(9

5%CI

1.2–

5.4)

N=

101

(cas

es),

172

(con

trol

s)

Buni

net

al.53

Nor

thAm

eric

a19

82–1

985

Mat

erna

lmul

tivita

min

use

durin

gfir

sttr

imes

ter

Retin

obla

stom

aAm

ong

child

ren

born

toex

pose

dm

othe

rs:O

R0.

40(9

5%CI

0.20

–0.9

0)N

=20

1(c

ases

),20

1(c

ontr

ols)

Schü

zet

al.54

Ger

man

y19

92–1

997

Mat

erna

luse

ofm

ultiv

itam

ins,

folic

acid

,and

/ori

ron

supp

lem

ents

durin

gpr

egna

ncy

Vario

uspe

diat

ricca

ncer

sAL

L:(O

R0.

84,9

5%CI

0.69

–1.0

1);N

on-H

odgk

in’s

lym

phom

a:(O

R0.

68,9

5%CI

0.48

–0.9

7);W

ilms’

tum

or(O

R0.

66,9

5%CI

0.45

–0.9

5)N

=18

67(c

ases

),20

57(c

ontr

ols)

Mic

hale

ket

al.55

New

York

Stat

e19

76–1

987

Mat

erna

lmul

tivita

min

use

durin

gpr

egna

ncy

Neu

robl

asto

ma

Amon

gch

ildre

nbo

rnto

expo

sed

mot

hers

:OR

0.50

(95%

CI0.

30–0

.70)

N=

183

(cas

es),

372

(con

trol

s)O

lsha

net

al.56

Nor

thAm

eric

a19

92–1

994

Dai

lym

ater

nalm

ultiv

itam

inus

edu

ring

diffe

rent

trim

este

rsof

preg

nanc

y

Neu

robl

asto

ma

1sttr

imes

ter(

OR

0.70

,95%

CI0.

5–1.

0);2

ndtr

imes

ter

(OR

0.60

,95%

CI0.

40–0

.90)

;3rd

trim

este

r(O

R0.

60,9

5%CI

0.40

–0.9

0)N

=53

8(c

ases

),50

4(c

ontr

ols)

Gru

ppet

al.57

Ont

ario

,Can

ada

1985

–200

6In

cide

nce

ofpe

diat

ricca

ncer

spr

e-an

dpo

st-fo

licac

idfo

rtifi

catio

nin

Ont

ario

Wilm

s’tu

mor

,ALL

,em

bryo

nal

canc

ers,

brai

nca

ncer

sPo

st-fo

rtifi

catio

nre

lativ

eto

pre-

fort

ifica

tion:

Wilm

s’tu

mor

(IRR

0.74

,95%

CI0.

57–0

.95)

,P=

0.02

.No

sign

ifica

ntch

ange

sin

inci

denc

efo

roth

erca

ncer

sex

amin

ed.

Fren

chet

al.58

Ont

ario

,Can

ada

1985

–200

0In

cide

nce

ofpe

diat

ricca

ncer

spr

e-an

dpo

st-fo

licac

idfo

rtifi

catio

nin

Ont

ario

Neu

robl

asto

ma,

ALL,

hepa

tobl

asto

ma

Post

-fort

ifica

tion

rela

tive

topr

e-fo

rtifi

catio

n:N

euro

blas

tom

a(IR

R0.

40,9

5%CI

0.25

–0.6

4).N

osi

gnifi

cant

chan

ges

inin

cide

nce

foro

ther

canc

ers

exam

ined

.Ab

brev

iatio

ns:A

LL,a

cute

lym

phoc

ytic

leuk

aem

ia;O

R,od

dsra

tio;I

RR,i

ncid

ence

rate

ratio

;CI,

confi

denc

ein

terv

al.

Nutrition Reviews® Vol. 69(10):561–571 563

associated with high maternal intakes of folate fromdietary52 as well as from supplemental sources.53 Onereport also demonstrates an association between mater-nal folate and iron supplementation and a reduced risk ofnon-Hodgkin lymphoma.54 Additionally, maternal multi-vitamin use has been implicated as being protectiveagainst neuroblastoma in offspring.55,56 Finally, ecologicalstudies indicate that, along with Wilms’ tumor,57 the inci-dence of neuroblastoma58 declined significantly followingthe introduction of mandatory folic acid fortification inCanada in the late 1990s.

As suggested by a recent meta-analysis,59 the poten-tial protective effect bestowed upon children exposed topericonceptional multivitamins during gestation may bequite substantial. Among children born to motherssupplementing their diets with multivitamins containingfolic acid, the odds of having pediatric brain tumors wereapproximately 30% lower (OR 0.73; 95% CI 0.60–0.88),the odds of having ALL were roughly 40% lower(OR 0.61; 95% CI 0.50–0.74), and the odds of having neu-roblastoma were nearly 50% lower (OR 0.53; 95%CI 0.42–0.68) as compared to children born to mothersnot using these vitamins. One important point of which itis important to remain cognizant is the following: becausemany of the studies have looked at supplements in whichfolate is only one of many nutrients present, a role for theadditional nutrients in modulating carcinogenesis in off-spring cannot be excluded. However, the observation thatpolymorphisms in genes involved in folate metabolism,such as MTHFR60 and methionine synthase,61 can modu-late the risk of developing ALL in adulthood, as well as theincreasing appreciation for a role of DNA methylation inthe pathogenesis of childhood cancers,62,63 lend support tothe notion that alterations in one-carbon metabolismmay serve as a major driver behind the apparent chemo-preventive effect of maternal multivitamin intake.

It is apparent that associations between maternalone-carbon intake and cancer in offspring have, to date,been limited to cancers that affect children, and that epi-demiological evidence is not yet available to support orrefute the idea that maternal one-carbon nutrient intakecan impact the risk for developing adult cancers, such asthose of the colorectum or breast. This is likely becausesuitable databases that attempt to link maternal diet todiseases in the latter decades of the offspring’s adulthoodare not yet available. Nevertheless, the epidemiologicalobservations summarized above provide support for thenotion that early intervention with one-carbon nutrientsin the maternal diet is associated with a decreased risk ofseveral pediatric cancers in offspring. Given that tumori-genesis in the adult colorectum and breast has repeatedlybeen shown to be sensitive to one-carbon nutrient status,it is reasonable to postulate that maternal diet mightimpact these cancers as well.

ANIMAL INTERVENTION STUDIES – TUMORIGENESIS

Although all of the epidemiology regarding maternalone-carbon intake and cancer pertains to pediatriccancers, to date most of the preclinical investigations havefocused on adult cancers, specifically those of the colorec-tum and breast (Table 2). Furthermore, unlike the epide-miological data, there has been little agreement in thefindings of these studies and careful attention must bepaid to both the experimental design and the character-istics of the animal model utilized in order to reconcilethe seemingly contradictory results.

For example, maternal folic acid supplementation(alone or in combination with vitamins B2, B6, and B12)has been shown to suppress intestinal tumorigenesis inoffspring both by our group64 and Sie et al.,65 whileLawrance et al.66 observed no such protective effect.Rather, the latter group reported that maternal folic aciddeficiency, not supplementation, conferred relative pro-tection against intestinal tumorigenesis in offspring.66 Incontrast, our own studies show that combined maternal Bvitamin deficiency, compared to provision of the basalrequirement, does not increase tumor incidence in off-spring. This agrees with the maternal folic acid depletionstudies of McKay et al.,67 although our group additionallyshowed that despite an unchanged tumor incidence, asignificantly higher proportion of tumors that developedin pups of deficient dams were invasive relative to tumorsfrom pups born to replete dams.64

Given the purported dual role of folate in carcinogen-esis, it is suggested here that important determinants of theoutcome of such maternal supplementation or depletionstudies are the severity of the tumor phenotype in theanimal model used as well as the timing of the folateintervention. When studies with dietary interventionslimited to the periconceptional and suckling period areconsidered, it is apparent that when a model with a mildtumor burden and long latency period is used, such as theazoxymethane (AOM)65 and Apc+/1638N models,64 folatewith or without additional B-vitamins acts in a protectivefashion. If dietary interventions continue into the off-spring’s adolescence (thereby exposing neoplastic lesionsto varied folate concentrations), then there is an opportu-nity for the dual role to be expressed.Such is the case in thestudies of Lawrance et al., who observed a suppression oftumorigenesis in offspring with maternal folate deficiencycarried through to the pup’s adolescence.66 Additionally,when an animal with a severe tumor phenotype is usedsuch as the Apc+/min mouse,66 the ability of folate to act in itsprotective capacity may be overwhelmed, thus biasing theoutcome towards the promoting effect of folate by supply-ing nucleotides for proliferation.

The second tissue in which the relationship betweenmaternal one-carbon nutrients and cancer in offspring


Tabl

e2

Ani

mal

inte

rven

tion

stud

ies

exam

inin

gth

ero

leof

mat

erna

lone

-car

bon

nutr

ient

inta

keon

offsp

ring

canc

erri

sk.

Refe

renc

eTi

ssue

Mod

elM

ater

nald

iets

Feed

ing

prot

ocol

Canc

erre

sult

Ciap

pio

etal

.64Sm

alli

ntes

tine

Apc+/

1638

Nm

ouse

Defi

c:B 2

(2m

g/kg

),B 6

(2m

g/kg

),B 1

2

(10

mg/k

g),F

A(0

.5m

g/kg

)Fr

om4

wee

kspr

iort

om

atin

gun

tilw

eani

ng(3

wee

ksaf

terb

irth)

↓Tum

ors

inpu

psbo

rnto

supp

.da

ms;

↑Inv

asiv

enes

sin

tum

ors

from

pups

born

tode

ficie

ntda

ms

Allp

ups

give

nre

plet

edi

et(A

IN-9

3)un

tilsa

crifi

ceat

32w

eeks

ofag

eRe

plet

e:B2

(6m

g/kg

),B6

(7m

g/kg

),B12

(50

mg/k

g),F

A(2

mg/

kg)

Supp

l:B2

(24

mg/

kg),

B6(2

8m

g/kg

),B1

2(2

00mg

/kg)

,FA

(8m

g/kg

)

Sie

etal

.65Co

lon

AOM

;rat

Repl

ete:

FA(2

mg/

kg)

Dam

sfe

dfr

om3

wee

kspr

iort

om

atin

gun

tilw

eani

ng↓T

umor

sin

pups

born

tosu

ppl.

dam

s;no

effec

tofp

ostw

eani

ngdi

eton

tum

orig

enes

is

Supp

l:FA

(5m

g/kg

)

Pups

rand

omiz

edto

rece

ive

eith

ersu

ppl.

orre

plet

eFA

diet

sun

til28

wee

ksof

age

Law

ranc

eet

al.66

Smal

lint

estin

eAp

c+/m

inm

ouse

Defi

c:FA

(0.3

mg/

kg)

Dam

sfr

om4

wee

kspr

iort

oco

ncep

tion

until

pups

reac

hed

10w

eeks

ofag

e

↓Tum

ors

inpu

psbo

rnto

defic

ient

dam

sRe

plet

e:FA

(2m

g/kg

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has been explored using an animal model is the breast.Similar to the intestine, it appears that the severity of themodel used is an important consideration. This assertionis supported by comparing two studies from the samegroup of investigators involving maternal folic acidsupplementation; one studying the spontaneous appear-ance of terminal end buds,68 a reliable biomarker ofmammary tumor risk in rodents, and the second studyingthe incidence of tumors following exposure with thepotent carcinogen DMBA.69 Consistent with observationsfrom the intestinal cancer studies discussed above, folicacid supplementation was protective when there was aweak proclivity towards tumorigenesis, but it fueled tum-origenesis in the model with a severe phenotype – par-ticularly when folate supplementation was continuedthrough the pup’s post-weaning life.69

The DMBA model has also been used to test themammary tumor modulatory capacity of choline, a one-carbon nutrient that provides methyl groups for thefolate-independent remethylation of homocysteine tomethionine. Kovacheva et al.70 fed pregnant rats dietsthat were deficient, replete, or supplemented withcholine between embryonic days 11 and 17 beforereturning them to replete diets. In this setting, a signifi-cant relationship between maternal choline intake andoffspring tumor growth rate was observed, i.e., a greateramount of time was required for tumors to reach a spe-cific size (3 cm) with increasing maternal choline intake.Folate is involved in both nucleotide synthesis and DNAmethylation and it is the former process that is thoughtto mediate the cancer-promoting effect. In contrast,choline does not play a direct role in nucleotidesynthesis; rather, it is a participant in the synthesis ofmethioine, the precursor for biological methylation.This might explain, in part, why choline conveys protec-tion even in a highly procarcinogenic model such asDMBA.

Overall, the above studies clearly show that, withinthe confines of the models used, maternal intake of one-carbon nutrients can impact intestinal and mammarytumorigenesis in offspring. However, it is apparent thatthe relative proclivity of the model towards tumorigen-esis is an important determinant of outcome, and must,therefore, be taken into consideration. Our interpreta-tion of this body of literature is that, especially for folate,when a model with a relatively weak tumorigenic phe-notype and long latency period is used, the potentialchemoprotective potential of one-carbon nutrients isobserved, and may even be enhanced in the presence ofother related nutrients such as vitamins B2, B6, and B12.In contrast, in models in which tumor incidence is highand growth is fast, this chemoprotective potential offolate and other one-carbon nutrients is overwhelmed,leaving only their capacity to fuel cell proliferation. In

this latter case, and especially in situations wheresupplementation is continued into the pup’s adoles-cence, the resulting exposure of neoplastic lesions to anabundance of one-carbon nutrients would be expectedto fuel tumorigenesis.

MECHANISTIC INSIGHTS

Just as we are still accumulating enough data to synthesizea coherent understanding of the effects of maternal one-carbon supplementation on tumorigenesis in offspring,we are only just beginning to gain an appreciation of themechanisms involved. Evidence is accumulating,however, that the modulation of DNA methylation pat-terns is a key process in this regard.

Landmark studies utilizing the Agouti viable yellow(Avy/a) mouse are, perhaps, the most widely recognizeddemonstration of the potential for maternal diet toimpact gene-specific methylation and phenotype in off-spring. In this model, feeding dams a methyl-donor-richdiet during gestation shifted the coat color phenotype ofoffspring from being predominantly yellow (agouti), tobeing brown in color (pseudo-agouti).71,72 Importantly,this increase in the proportion of pups born with a browncoat color coincided with an elevated methylation of spe-cific sequences within the Avy promoter.71,72 Furthermore,it is reported that mice with the yellow coat color have anelevated propensity towards adult-onset obesity, hyper-tension, and insulin resistance compared to mice withbrown coats.73 More pertinent to the current topic,however, Wolff et al.74 demonstrated that following pro-longed exposure to the chemical carcinogen lindane, asignificant reduction in hepatic tumor multiplicity, aswell as tumor incidence in the lung, was observed inpseudo-agouti pups relative to their agouti littermates.These studies demonstrate that supplemental one-carbonnutrients in the maternal diet can shift the phenotype ofthe offspring to one that has elevated resistance totumorigenesis.

Similar to the Avy/a model, the potential for maternalmethyl group consumption to alter the phenotype of theoffspring has also been demonstrated in the Axin Fused(AxinFu), or“kinky-tail” mouse. In this mouse, one-carbonnutrient supplementation of dams during gestation sup-presses the kinky-tail phenotype, which is evident in theoffspring of control fed dams.75 Furthermore, the pres-ence of tail kinks was inversely related to the methylationdensity of a retrotransposon within exon 6 of the Axingene.

Proof of concept that maternal one-carbon intakecan impact the methylation and expression of specificgenes in offspring was established in Avy/a and AxinFu

mice; models in which an inserted retrotransposoncreates a cryptic promoter that is sensitive to CpG methy-


lation. More recently, however, studies are beginning toidentify genes with clear cancer relevance as being sensi-tive to maternal one-carbon nutrient intake.

One such gene whose methylation is modified bymaternal diet is Igf2. Expression of Igf2 is heavily depen-dent on imprinting with the maternal allele beingcompletely silenced, leaving only the paternal allele tobe expressed. However, the methylation density ofimprinted maternal alleles can diminish, a processknown as loss of imprinting, resulting in biallelicexpression of the gene. Loss of Igf2 imprinting has beenimplicated in several pathologic conditions, includingcancers of the prostate and colorectum in both animalmodels and human subjects.76,77 Evidence that perturba-tions in the methylation of this gene may occur duringdevelopment comes from a cohort of Dutch citizenswho were exposed to famine in utero during World WarII. Remarkably, those exposed to the famine during peri-conception had significantly lower methylation of theIgf2 “differentially-methylated region” compared to theirsame-sex siblings who were not exposed to famine whenassessed some 60 years after the exposure.78 In afollow-up to this study, several additional genes infamine-exposed subjects, including Il10 and Abca1, werereported to have modest methylation changes relative tothose of their unexposed siblings.79 In a separate study,periconceptional maternal supplementation with folicacid was associated with significantly higher methylationof the same differentially-methylated region of Igf2 inthe offspring.80 Several preclinical studies in rodentshave also reported an effect of one-carbon nutrientintake on Igf2 methylation and expression, both duringearly post-weaning life81 as well as during in uterodevelopment.82

One additional pathway focused on as a potentialmediator of the effect of maternal and individual diet oncolorectal carcinogenesis is the canonical Wnt signalingpathway. This pathway is important for the growth anddevelopment of several tissues, including the gastrointes-tinal mucosa.83 This pathway is also integrally involved incolorectal carcinogenesis, as activating mutations in theWnt pathway are observed in more than 80% of sporadiccolorectal tumors, and activation results in a number ofpro-transformational changes in cellular behavior.84 Theloss of Apc, a negative regulator of the Wnt pathway, isconsidered to be a major initiating event in the develop-ment of colorectal tumors.84,85 Furthermore, silencing ofgenes through promoter hypermethylation has beenreported for several Wnt pathway inhibitory genes,including Apc, Axin2, Dkk1, Sfrp1, 2, 4, and 5, andWif1.86–88

Several components of the Wnt pathway have beenpreviously shown to be responsive to B-vitamin availabil-ity both in vitro and in vivo. The expression of both Apc

and b-catenin have been shown to be responsive to folatedelivery in cancer cell lines.89 In wild-type mice, a com-bined mild B-vitamin deficiency has been shown to resultin several changes consistent with colonic Wnt signalingactivation including diminished expression of Apc,increases in nuclear b-catenin localization and CyclinD1gene expression, and diminished apoptosis.18,90 Morerecent data from a Wnt-reporter mouse model crossedwith the Apc+/1638N mouse (BAT-LacZ x Apc+/1638N) hasprovided a clear demonstration of in vivo colonic Wntactivation as a result of mild deficiency of multipleB-vitamins.91

In our own maternal intervention study a significantstepwise reduction was observed in the expression ofmultiple Wnt pathway-negative regulators, includingSfrp1, Wif1, Wnt5a, and Apc in the small intestinalmucosa of pups, with decreasing maternal B-vitaminintake. Furthermore, the promoter methylation densitywithin specific regions of the Sfrp1 gene was significantlyand inversely correlated with Sfrp1 expression, which isconsistent with the hypothesis that maternal diet inducesgene-specific epigenetic alterations in the offspring.64

Sfrp1, an extracellular inhibitor of Wnt signaling, is par-ticularly interesting as it is commonly silenced by methy-lation in human colorectal carcinogenesis,88 as well asmurine cancer models.92 Importantly, these changes wereconsistent with our tumor data, i.e., maternal deficiencyincreased the likelihood of offspring tumors being inva-sive while supplementation markedly suppressed tumorincidence.

Similarly, the existing mechanistic data regardingmaternal one-carbon nutrient intake and offspring breastcancer also suggests an epigenetic mechanism. Ly et al.69

reported that pups born to folate-supplemented mothershad significantly diminished genome-wide DNA methy-lation in mammary tissue. Furthermore, Kovachevaet al.70 identified a number of differentially expressedgenes within the tumors of offspring of rats born tomothers consuming different amounts of choline.Amongthese genes was Stratifin (Sfn), a gene frequently silencedby methylation in breast carcinogenesis. The researchersreported an inverse relationship between maternaldietary choline intake and Sfn expression at the mRNAand protein levels within tumor tissue, as well as a directrelationship between Sfn promoter methylation andmaternal dietary choline. The degree of promoter methy-lation was significantly and inversely correlated with Sfn.As is true for the relationship between maternal one-carbon nutrients and intestinal tumorigenesis in off-spring, these data are consistent with the idea that alteringmaternal one-carbon nutrient intake can lead to gene-specific methylation changes in cancer-relevant genes andthat these alterations may impact later breast cancer riskin the offspring.


CONCLUSION

There is a provocative body of literature suggesting thatmaternal intake of one-carbon nutrients has a bona fideimpact on determining the risk of cancer in offspring.This field of investigation is still very much in a nascentstage, but it offers exciting new opportunities for cancerprevention in ways that have never before been consid-ered. Going forward, however, there are several pointsthat need to be kept in mind both by researchers in thefield as well as interested observers.

First and foremost, much of this field still operateswithin the realm of animal models; it is, therefore, abso-lutely critical to pay attention to the specifics of theexperimental protocol of each study since the idiosyncra-sies of each model, as well as the timing of the specificdietary protocol, can result in marked differences inoutcome. Some studies opt for varying the mothers’ dietduring certain periods of gestation coinciding with spe-cific periods of embryonic development,70,93 others varythe maternal diet from preconception through weaning,64

and still others vary both the maternal diet as well as thediet of offspring post weaning.66 Given the pivotal signifi-cance of timing in the field of folate and carcinogenesis,22

these differences in feeding protocols almost certainlyexplain some of the disparate results among these studies.

Second, as inferred from the studies using the Avy

mouse, the prevailing concept regarding the mechanisticbasis underlying the effect of maternal one-carbon nutri-ent intake is thought to be epigenetic and, more specifi-cally, on alterations in promoter methylation in cancer-relevant genes. Nevertheless, this remains a presumptionand it is important to be aware of other possible mecha-nistic avenues by which the delivery of one-carbon nutri-ents might impact cancer risk, such as double-strandedDNA breakage events related to the role of these nutrientsin nucleotide synthesis.10,12,94 Moreover, even if oneassumes that the changes in promoter methylation are theprimary driver of the effect, there remains a need to iden-tify specific loci within the genome that are responsiblefor these effects. This task becomes more complicatedwhen one considers that other epigenetic features, such ashistone modifications, may play a role. Indeed, dietaryintake of one-carbon nutrients has been shown to impacthistone modifications in both the adult mouse95 and inoffspring when consumed in the maternal diet.96,97

Third, it is possible, if not likely, that the effect ofmaternal supplementation on tumorigenesis in offspringis tissue-specific. Careful studies of such effects arerequired in order to ensure that, should public healthmessages be developed in the future regarding maternaldietary changes to limit offspring carcinogenesis, unin-tended deleterious consequences of supplementation areavoided.

Finally, it is important to remember that each of therodent cancer models have limited applicability to thehuman condition. It is only by remaining cognizant ofboth the strengths and weaknesses of each model, par-ticularly regarding the mechanistic basis behind anyobserved effects, and by effectively translating the resultsfrom animal studies into clinical research, that it will bepossible to reach informed conclusions regarding mater-nal one-carbon nutrient intake during gestation for thepurposes of reducing the burden of cancer in humans.

The maternal diet and exposures are increasinglyrecognized as being an important determinant of thehealth of children. Taken together, the human and rodentstudies discussed here indicate that maternal intake ofone-carbon nutrients can indeed impact an offspring’srisk for tumors that appear in childhood and in the laterdecades of life. Concurrently, a growing body of evidenceindicates that maternal intake of these nutrients canimpact the methylation of specific genes in offspring. Thechallenge now is to put these two findings together andidentify which differentially methylated genes mediateobserved changes in cancer risk. As evidence continues toaccumulate in this regard, it may be found that such infor-mation can be integrated into public health initiativesthat tailor maternal one-carbon nutrient intake to notonly minimize the risk for birth defects, but to also mini-mize the risk of cancer throughout the offspring’s life.

Acknowledgments

Declaration of interest. The authors have no relevantinterests to declare.

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