“Nutrigenomics and BioactiveFood Components”

John Milner, ChiefNutritional Science Research Group

Division Cancer Prevention,National Cancer Institute

milnerj@mail.nih.govmilnerj@mail.nih.gov

Many Important

Unprecedentedopportunities

exist for use toachieve genetic

potential,increase

InterestingMany ImportantConstituents increase

productivity andreduce the risk

of disease,including cancer

InterestingInteractions

1 HIV/AIDS 9227

2 Ischemic Heart Disease 1332

3 Tuberculosis 1036

1 Ischemic heart disease 5825

2 Cerebrovascular disease 4689

3 Chron obste pulmon disease 2399

Worldwide Leading Causes of MortalityMany of Which are Linked with Eating Behaviors

(2002)Aged 15-59 Aged 60>

Death

Rank Cause (000)

Death

Rank Cause (000)

Charts, Maps, Tables, The World Health Report, WHO 2003

3 Tuberculosis 1036

4 Road traffic injuries 814

5 Cerebrovascular disease 783

6 Self-inflicted injuries 672

7 Violence 473

8 Cirrhosis of the liver 382

9 Lower respiratory infections 352

10 Chron obstr pulmon disease 343

3 Chron obste pulmon disease 2399

4 Lower respiratory infections 1396

5 Trachea, bronchus, lung cancer 928

6 Diabetes mellitus 754

7 Hypertensive heart disease 735

8 Stomach cancer 605

9 Tuberculosis 495

10 Colon and rectum cancers 477

Population Growth Will Also Influence Health Issues

Percent Change in Population, Selected Countries: 2008-2050

Haub and Mederios Kent, (2008) World Population Data Sheet.

Worldwide Public Health Approaches Center on Foods

Dr. Lee Jong-wook, Director General WHO

If Prevent Do not needto treat! Diet is clearly

importantin this strategy

OtherOther

Evidence Based Report Showcases Inconsistenciesin the Effect of Diet on Cancer Prevention

TobaccoTobacco30%30%

DietDiet35%35%

OtherOtherFactorsFactors35%35%

The Causes of Cancer– Richard Doll & Richard Peto, 1981

WCRF/AICR Report- Released Nov. 1-2, 2007

• Essential Nutrients- Ca, Zn,

Se, Folate, Vitamin D, C,

• Non-EssentialPhytochemicals Carotenoids,Flavonoids, Indoles,

Isothiocyanates, Allyl Sulfur

Exceedingly Complex Area Since Many Bioactive FoodComponents

Isothiocyanates, Allyl SulfurZoochemicals - Conjugated

linoleic acid, n-3 fatty acidsFungochemicals -- Several

compounds in mushroomsBacteriochemical -- Those

formed from foodfermentations and thoseresulting from intestinalflora

Bacterial Formed Equol MayAccount for Part of the

Anticancer Properties from Soyin Asian-Americans

Human Microbiome: New NIH Roadmap Initiative!

In addition to genistein microorganisms in subpopulations formequol which possesses anticancer properties (J Nutr. 2006Apr;136(4):946-52. Arch Microbiol. 2005;183:45–55). Themechanism by which equol may offer protection remainsunresolved but gene expression differences are evident in equolproducers (Niculescu et al (2006) J Nutr. Biochem)

Fundamental Question Remains About True Site of Actionof Functional Foods and Their Active Constituents

BioactiveFood Component(s)

Lee ‘92 (total soy protein)Lee ‘92 (total soy protein)

p < 0.001 Premenopausalp < 0.001 Premenopausal

NS PostmenopausalNS Postmenopausal

Hirose ‘95 (bean curd, miso)Hirose ‘95 (bean curd, miso)

Yuan ‘95 (tofu, soymilk)Yuan ‘95 (tofu, soymilk)

NS PremenopausalNS Premenopausal

NS PostmenopausalNS Postmenopausal

NS p = 0.44NS p = 0.44––0.79 Shanghai, Tianjin0.79 Shanghai, Tianjin

Wu ‘96 (tofu)Wu ‘96 (tofu)

p < 0.01 Premenopausalp < 0.01 Premenopausal

p < 0.05 Postmenopausalp < 0.05 Postmenopausal

Key ’99 (soy)Key ’99 (soy)

TofuTofu

MisoMiso

Zheng ’99 (urinary isoflavonoids)Zheng ’99 (urinary isoflavonoids)

Dai ‘01 (soy)Dai ‘01 (soy)

NS All Breast CancerNS All Breast Cancer.46 .66 .95

.1 .3 .6

.6 1.3 2.6

.61

.6

.6 .8 1.0

.7 1.0 1.3

AsianAsian

.78 1.07 1.47

.68 .87 1.12

.18 .65 2.37

Literature is Full of InconsistenciesBreast Cancer: Soy Intake

NS All Breast CancerNS All Breast Cancer

S Just ERS Just ER++/PR/PR++

Wu ’02 (soy)Wu ’02 (soy)

Yamamoto ’03 (isoflavonoid consumption)Yamamoto ’03 (isoflavonoid consumption)

PremenopausalPremenopausal

PostmenopausalPostmenopausal

Wu ’04 (soy)Wu ’04 (soy)

Ingram ‘97 (urinary isoflavones)Ingram ‘97 (urinary isoflavones)

NS DiadzeinNS Diadzein

p = 0.009 Equolp = 0.009 Equol

Witte ’97 (soy)Witte ’97 (soy)

den Tonkelaar ‘01 (urinary phytoestrogens)den Tonkelaar ‘01 (urinary phytoestrogens)

NS PostmenopausalNS Postmenopausal

HornHorn--Ross ’01 (phytoestrogen intake)Ross ’01 (phytoestrogen intake)

KeinanKeinan--Boker ‘02 (food content)Boker ‘02 (food content)

NS IsoflavonesNS Isoflavones

S LignansS Lignans

Linseisen ’04 (isoflavone intake)Linseisen ’04 (isoflavone intake)

daidzein and genisteindaidzein and genistein

.25 .44 .78

.17 .47 1.33

.1 .27 .69

.34 .58 .98

.79 1.08 1.59

00 11 22.5.5 1.51.5

.46 .83 1.3

WesternWestern

.2 .5 1.1

.4 .6 1.2

.22 .48 1.1

.25 .47 .90

.36 .53 .78

.79 1.0 1.3

.36 .57 .83

Comparing People Is a Little LikeComparing Apples and Oranges!

What Is Nutrigenomics About? Basically variation in the response

due to the “Omics” coupled with a Molecular Target

DNANutrigenetics

NutritionalEpigenetics

NutritionalTranscriptomics

BioactiveFood

RNA

Phenotype

Nutrigenomic Transcriptomics

Proteomics

Metabolomics

FoodComponent

Protein

Metabolite

Phenotypecs

“Nutritional Preemption”Concept that bioactive food components can be introduced at points of initiation &progression for pathway leading to an unhealthy or lethal phenotype

Future Is To Focus on the Process Needing Modification

Credentialing of nutrients and molecular targets is likely the future?Credentialing is defined as “omic” changes that bring about a phenotypic change

AbsorbedDose Biologically

Effective Dose

Inactive Metabolite

Three Types Biomarkers Needed To IdentifyResponders (Both Positive and Negative)

MolecularTarget

EarlyBiologic

Effect

SusceptibilityFactors

DietaryExposure

AlteredAlteredStructureStructure/FunctionFunction

Health Effects+ and -

• Body weight(p < 0.02):F = 6.8; R = 0.74

• Fat mass(p < 0.01):

Body Weight Change in MZ Twins in Response toNegative Energy Balance

Twin A

Wei

gh

t,k

g-6

-4

-2

Bouchard et al, Obes Res, 1994

(p < 0.01):F = 14.1; R = 0.87

• AVF(p < 0.01):F = 11.7; R = 0.84

Twin B

Wei

gh

t,k

g

-10 -8 -4 -2

-10

-8

-6

-6Weight, kgBouchard et al, New Engl J

Med, 1990 (Positive Wt Gain)

Food preference

Food tolerance

Absorption

Transport

Genomics Can Influence the Responseto Diet at Multiple Points

Transport

Metabolism

Effect in targettissue

Lampe and Potter, in Gene-Envir Interactions (2006)

Modified from Lampe (per communication)

Glucose Transporter Type-2 (GLUT2)

GlucoseGlucoseHomeostasis

110Thr IleGLUT2 Protein

Sugar Intake and GLUT 2 Polymorphism

Visit 1 Visit 2

125

100

125

100

Su

gar

(g/d

)

Su

gar

(g/d

)

**

Eny et al. (2008) Physiol. Genomics, 33:355-60.

Thr/ThrThr/Ille + Ille/Ille

75

50

75

50

Su

gar

(g/d

)

Thr/ThrThr/Ille + Ille/Ille

Su

gar

(g/d

)

Calcium Supplementation & DecreasedColorectal Cancer (7 cohort studies)

Summary Effect Estimate 0.78

Genetic Information May Assist in Identifying

Those Who Must Assure Adequate Intakes

Dietary Calcium

OR

for

Colo

nC

an

cer

*

*

2

2.5

3

< 388 mg/day

>388 mg/day

Wong et al. Carcinogenesis, 24: 1091-1095, 2003

OR

for

Colo

nC

an

cer

P for trend=0.004

0

0.5

1

1.5

FF Ff ff

VDR Genotype

VDR FokI Polymorphism Affects CalciumHomeostasis in Adolescence

Cal

cium

Acc

reti

on

toS

kel

eton

(mg/d

)300

200 * *

Abrams et al. (2005) J. Bone Mineral Res. 20: 945-953.

Cal

cium

Acc

reti

on

toS

kel

eton

(mg/d

)

100

0ff Ff FF ff Ff FFBalance Method Bone Density Method

DNA Strand Breaks and Diet

Pool-Zobel et al. Carcinogenesis1997;18:1847-50

XRCC1 (Arg399Gln) Polymorphisms MayInfluence the Response to Lycopene

n=77 CaP pts; n=174 controls

Lycopene

Intake

(g/day)

Arg/Gln +Gln/Gln

“low-risk”

Arg/Arg

“high-risk”

High 0.82 0.21

Goodman et al., (2006) Nutr. CA, 55(1):13-20.

*P trend < 0.01

(0.33-2.01) (0.06-0.71)

Medium 0.97

(0.39-2.44)

0.59

(0.23-1.50)

Low 1.0 1.0

Genetic Information May Help Identify Those LimitIntakes of Specific Foods

Od

ds

Rat

io:

Co

lon

Can

cer

*

* P = 0.001

Red Meat Intake

France: 1,023 CRC cases and 1,121 controls

** P < 0.001

< 5 Times/Wk5 > Times/Wk

**50

40

Kury et al. (2007) CEBP, 16: 1460-7.

Od

ds

Rat

io:

Co

lon

Can

cer

Diet

Multi-CYP SNPs:†

CYP1A2 -163A>C1548T>C

CYP2E1 -1293G>C-1053C>T

CYP1B1 1294C>GCYP2C9 430C>T

** P < 0.001

SNPInter + Diet

10

30

† Population 4.4%

New Funding Opportunity on theStreet. PA 08-221

Balance of Foods Important High Amylose MaizeStarch and Red Meat Ameliorates DNA Damage

Toden et al. (2007) Carcinogenesis. 28(11):2355-62

< 300 mg Caffeine > 300 mg

6

0

Genetic Information May Help Identify Those At Riskand to Formulate Appropriate Interventions

-12

-6

Vitamin D Receptor GenotypeTT Tt tt TT Tt tt

Rapuri et al. Am J Clin Nutr 2001 Nov;74(5):694-700

*

*

Coffee Intake and Risk of Myocardial Infarction

< 1 cup/d1 cup/d2-3 cups/d4+ cups/d

Od

ds

Ra

tio

2.0

1.5 4X for

* P<0.05

Cornelis et al. (2006) JAMA 295: 1135-41

CYP1A2 Genotype*1A/*1A *1A/*1F + *1F/*1F

Od

ds

Ra

tio

1.5

1.0

0.5 A/A A/C + C/C

4X forUnder50 yr

23 and MeGenotyping

-$999 /salva kit

-Results in 2-4 weeks

-Illumina HumanHap550 + BeadChip readsnearly 600,000 SNP’s

-Uploads information to online databaseSample Kit

-Uploads information to online database

-Has odds calculator to suggest common health concernsfor person with specific genetic composition

Compare your genes with friends, family and theworld!!

Sample Kit

IlluminaHumanHap

550 + BeadChip

Exceeding complex area since about 30, 000Genes, 8-10 Million SNPs

Nutrigenomic TestingPromises vs. Reality!

• Commercial Nutrition-Gene Test

– Sciona 19 genes determines whether your DNAcontains gene variants that have been associated with 5key health areas: bone health, heart health,antioxidant/detoxification, insulin sensitivity, andinflammation.inflammation.

– Cost about $300.

• Genelex The panel examines 19 genes that play majorroles in your body's detoxification capacity, antioxidantcapacity, heart health, bone health, insulin sensitivity, andtissue repair.

– About $500.

The overall contribution of Copy Number Variance(CNV) to complex phenotypes expression levels of

14,925 transcripts with SNPs and CNVs in individualswho are part of the International HapMap project was

evaluated.

SNPs and CNVs captured 83.6% and 17.7% of theSNPs and CNVs captured 83.6% and 17.7% of thetotal detected genetic variation in gene expression,

respectively.

The signals from the two types of variation had littleoverlap

Stranger et al (2007) Science. 315(5813):848-53

Diet and Human Amylase Gene Copy Number

Perry et al. (2007) Nature Genetics 39(10):1256-60.

Foods Can Also Influence Epigenetics

MaternalSupplements

withzinc, methioninebetaine, choline,

folate, B12

LTR Hypomethylated LTR Hypermethylated

Yellow Mouse Agouti Mouse

Cooney et al. J Nutr 132:2393S (2002); Dolinoy et al. Envir. Health Perspect 114: 567 (2006)

folate, B12

OrGenistein

High riskcancer, diabetes, obesity &reduced lifespan

Lower risk of cancer, diabetes,obesity and prolonged life

Bis Phenol A

Maternal Methyl Supplements* Reduce TailKinking Phenotype and Increase Offspring DNA

Methylation at Axin Fused

Waterland RA, et al.Genesis 44:401-6, 2006.

*Folic acid, vitaminB12, betaine, and choline

Oxidation of either a singleguanine to 8-oxoguanine or of asingle 5mC to 5-hydroxymethylcytosine

significantly inhibits binding ofthe methyl-CpG bindingproteins to the oligonucleotideduplex, reducing the bindingaffinity by at least an order of

Valinluck et al. Nucleic Acids Res.(2004) 32:4100.

affinity by at least an order ofmagnitude.

Oxidative damage to DNAcould therefore result inheritable, epigenetic changes inchromatin organization.

Folate Reverses Hyperhomocysteinemia andInfluences Epigenetic Control of Gene Expression

Ingrosso, D., et al. Lancet. 361:1693-9,2003.

SFN-rich Broccoli Sprouts Inhibit HDAC inHuman Volunteers.

Dashwood RH, Ho E. Semin. Cancer Biol. 17:363-9, 2007.

Dietary Intervention Can Cause Shifts inTranscriptomic Expression

Low-Fat Feeding and Gene Expression in Human Prostate Epithelium

Liu et al. Cancer Epidemiol Biomarkers Prev. 2007;16:2150-4

Garlic, fish, broccoli, tomatoes

keap-1 nrf2

HS SH S Skeap-1

Agent

Multiple Food Constituents Can InfluenceOne Nuclear Transition Factor

Active Intermediate (radical??)

Increased GST, QRARE

nrf2smallmaf Nucleus

“antioxidant responsive element”

keap-1 nrf2 keap-1

nrf2

CytoplasmCaloricRestriction

Lack of Protection Against Induced Tumors in40% Caloric Restricted KO mice

Mic

eP

rese

nti

ng

Tum

ors

(%)

100

80

60

40

AL WT

AL KO

Pearson et al. (2008) Proc Natl Acad Sci U S A. 105(7):2325-30.

Mic

eP

rese

nti

ng

Tum

ors

(%)

40

20

0

0 5 10 15 20 25 30 35 40

CR WT

CR KO

EnergyEnergyExpenditureExpenditure

Nutrition, Cell Proliferation, Obesity and CancerPreventionGene-Nutrient

Imbalance

EnergyEnergyIntakeIntake

(Carcinogen)(Carcinogen)

(Phytochemicals)(Phytochemicals)

High Fat/CaloriesHigh Fat/CaloriesHighHigh nn --6 fats6 fats

Growth FactorsGrowth FactorsGeneticGenetic

SusceptibilitySusceptibility(Polymorphisms)(Polymorphisms)

Physical ActivityPhysical ActivityFruitsFruits

VegetablesVegetablesWhole GrainsWhole Grains

Soy ProteinSoy Protein

Additional Strategies: Tea Increases Fat Oxidation

Venables et al. (2008) Am J Clin Nutr 87: 778-84

Recent News Releasesuggests:Herceptin is a Novel Pioneering Drug forPersonalized Medicine Approach Based onPharmacogenomics to block Her2-neuexpression.

A Molecular Approach to Medicine

Evidence Has Existed forYears that:In addition to EGCG from Green Tea, Apigeninfrom parsley, thyme, and peppermint cansignificantly influence HER2neu expression!

Herceptin and Dietary fish oil increased the latencytime to mammary gland tumor development in the

HER-2 transgenic mice

free

Su

rviv

al

0.8

1.0

Herceptin

free

Su

rviv

al

0.8

1.0

Fish Oil

Dis

ease

-fre

eS

urv

ival

4 6 8 10 12 14

Months

0.0

0.2

0.4

0.6

Finkle D et al Clin Cancer Res 10: 2499-511, 2004

Control

Yee LD et al J Nutr 135: 983-8, 2005

Dis

ease

-fre

eS

urv

ival

4 6 8 10 12 14

Months

0.0

0.2

0.4

0.6

Corn Oil

Not All Cells Are Equally Sensitive and Antioxidant Properties May Not beMechanism of Action!

SW620

HCT116

Ban et al. J Pharmacol Sci 104, 374 –383 (2007)

Normal

Herman-Antosiewicz et al (2007)Acta Pharmacol Sin. 28(9):1355-64.

Lycopene Influences Multiple Pathways, which are linked in crosstalk inprostate cancer development, resulting in anti-inflammation, growth reduction

& apoptosis induction

E CdhCell adhesion

gapjunction

neighbouring cellLPSbacterialinfection

IL-6inflammation

TLR4

IL6R/gp 130

Jak Jak

STAT3

IGF-Ior EGF, PDGF..

Wnt

R-PTK Frz

MAPK

PI3K AKT

GSK3

AxinAR

DHT

T

Bad

DSH

Lyc Lyc

Lyc

Lyc

Lyc

Lyc

NFBNFB STAT3

IL-6(and others)

AP1

APC

Axin

TCF -catenin

-catenin

AR

AR ARDHTDHT

STAT3 ARAR -catenin

Badcaspase 9

FHKRFOXO

inhibition of cell growthby G1/S cell cycle delay

Induction ofapoptosis

inhibition ofinflammation

Lyc Lyc

Wentz 2008 Personal Communication

DNANutrigenetics

NutritionalEpigenetics

NutritionalTranscriptomics

BioactiveFood

RNA

Nutrigenom Cellular

Needs&

Insults

Insults Such As Radicals, Bacteria and VirusesMay Modify the Response to Food Components

Transcriptomics

Proteomics

Metabolomics

FoodComponent

Protein

Metabolite

Phenotype

mics

CellularProcess(es)

“Nutritional Preemption”Concept that bioactive food components can be introducedat points of initiation & progression for pathway leading toan unhealthy or lethal phenotype

Could Obesity Be Reflecting Some Other Metabolic Effect??

Snijder, et. al. (2005) J Clin Endo & Metab 90:4119–4123

Total Body Fat (%)

15 25 35 45 55

The Foundation of Nutrition and Cancer Prevention AreControlled Dietary Interventions Studies in Humans

Lappe et al. Am J Clin Nutr 2007;85:1586–91

0 Genistein

10 mM Genistein

100 mM Genistein

Swami et al. ( 2005) Mol Cell Endocrinol. 241(1-2):49-61.

Tota

lCancer

Incid

ence

*30

40

50

60Selenium

Placebo

The Benefits of Selenium Are Likely Dose Dependent

Likely same response in Dogs!Waters et al. Carcinogenesis.2005: 26(7):1256-62

Plasma Selenium (ng/mL)

Tota

lCancer

Incid

ence

0

10

20

<105.2 105.3-

121.6

>121.6

Duffield-Lillico et al., (2002) Cancer, Epidem.Biomarkers & Prev., 11: 630

Dietary Interventions May Not Always beProtective

Today Concern AboutExcess

CalciumIron

Folic AcidSelenium

Albanes et al (1995) AJCN 62:1427S-1430S

SeleniumVitamin AVitamin DOthers???

Why does thisdifferential responseoccur? Normal vsneoplastic cells??

Linxian Nutrition Intervention TrialEsophageal cancer mortality by factor D (N=1515)

Factor D= Selenium, b-carotene, vitamin E

Log-rank P=0.024RR=0.83

<55 years

Placebo

Log-rank P=0.045RR=1.14

55+ years

Factor D

Timing of Use Also Important!!

Esophageal Cancer Death Time (Year) Esophageal Cancer Death Time (Year)Taylor, P. et al., Gastroenterology 2005 (abstract)

Factor D Placebo

Foods AreFUNdamental

For Health

However, all maynot benefit equally

While I have raise lots of Concerns:There Is Light At the End of the Tunnel!

WCRF/AICR Recommendations

1. Be as lean as possible without beingunderweight

2. Be physically active.3. Avoid Sugary drinks.4. Eat more of a variety of

vegetables, fruits, whole grains, and legumes.vegetables, fruits, whole grains, and legumes.5. Limit consumption of red meats6. If consume alcohol do so in moderation7. Limit consumption of salty foods8. Don’t use supplement to protect against cancer

We Must Be Careful About Messages to Public

One Size Does Not Fit All! More is NotAlways Better

SoySoy

TomatoesTomatoes

SpinachSpinach

BroccoliBroccoli

GarlicGarlic

NutsNuts

Suspect Functional Foods With Health Benefits

NutsNuts

SalmonSalmon

OatsOats

BlueberriesBlueberries

CurcuminCurcumin

Green teaGreen tea

Red wineRed wineModified Time Magazine: January 21, 2002Modified Time Magazine: January 21, 2002

The Future

PreemptivePersonalizedPredictive

Participatory